cancers Review Mycobacteria-Based Vaccines as Immunotherapy for Non-urological Cancers Estela Noguera-Ortega 1,2, * , Sandra Guallar-Garrido 1 and Esther Julián 1, * 1 Mycobacteria Research Laboratory, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; sandra.guallar@uab.cat 2 Center for Cellular Immunotherapies and, Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA * Correspondence: enoguera@pennmedicine.upenn.edu (E.N.-O.); esther.julian@uab.cat (E.J.); Tel.: +1-215-573-9848 (E.N.-O.); +34-93 5814870 (E.J.)  Received: 2 June 2020; Accepted: 2 July 2020; Published: 5 July 2020  Abstract: The arsenal against different types of cancers has increased impressively in the last decade. The detailed knowledge of the tumor microenvironment enables it to be manipulated in order to help the immune system fight against tumor cells by using specific checkpoint inhibitors, cell-based treatments, targeted antibodies, and immune stimulants. In fact, it is widely known that the first immunotherapeutic tools as immune stimulants for cancer treatment were bacteria and still are; specifically, the use of Mycobacterium bovis bacillus Calmette-Guérin (BCG) continues to be the treatment of choice for preventing cancer recurrence and progression in non-invasive bladder cancer. BCG and also other mycobacteria or their components are currently under study for the immunotherapeutic treatment of different malignancies. This review focuses on the preclinical and clinical assays using mycobacteria to treat non-urological cancers, providing a wide knowledge of the beneficial applications of these microorganisms to manipulate the tumor microenvironment aiming at tumor clearance. Keywords: BCG; non-tuberculous mycobacteria; adjuvant; immunotherapy; mycobacteria antigens 1. Introduction The use of immunotherapy for cancer treatment was reported around 130 years ago, with the use of the inactivated product from two strains of bacteria, Streptococcus pyogenes and Serratia marcenses, namely Coley’s toxin, which were mixed in order to be injected into patients affected by diverse types of cancer. In a series of experiments, Dr. Coley demonstrated the effect of the product in the reversion of cancer in treated patients [1–3]. After these first successful works, mycobacteria entered into the immunotherapy scene owing to the confluence of different facts. On the one hand, at that time, Dr. Pearl observed an inverse relationship between deaths due to tuberculosis (TB) and those due to cancer in a series of autopsies. On the other hand, during the second decade of the last century, the perseverance of doctors Albert Calmette and Camille Guérin led to the development of an attenuated strain in animal models derived from an original isolate of Mycobacterium bovis that was later called Mycobacterium bovis bacillus Calmette-Guérin (BCG). As soon as BCG was considered a safe vaccine, and along with the successful experience using Coley’s toxin, and in view of the evidence from Pearl’s research, several trials were conducted in the fight against different types of cancer using BCG. Since the 1930s, studies in gastric cancer patients [4], melanoma [5], and leukemia [6] treated with BCG showed disease remission or a non-relapsing disease. Despite the appearance and the rapid and broad implementation of chemotherapy and radiotherapy for cancer treatment, the Cancers 2020, 12, 1802; doi:10.3390/cancers12071802 www.mdpi.com/journal/cancers Cancers 2020, 12, 1802 2 of 25 study of mycobacteria as an immunotherapeutic agent was not abandoned [7]. Thus, in the 1970s and 1980s, BCG was used by injecting into the tumor or in combination with other therapies in melanoma [8,9], as well as in lung [10–12], cervical [10,13], ovarian [14,15], colon [16], and head and neck cancers [10], etc. All these years of preclinical and clinical assays finally resulted in the use of BCG as immunotherapeutic agent for the treatment of non-muscle invasive bladder cancer (NMIBC). This type of tumor only affects the mucosa or sub-mucosa of the bladder wall. When mycobacteria are intravesically instilled, the bladder cavity provides the optimal conditions for an efficacious BCG effect due to it being a closed space. Although BCG is an attenuated strain and belongs to biosafety level 2 due to its ability to cause infections in some cases, it can be instilled in its live form into the bladder because, since it is a closed space, BCG could arduously spread to the patient body. Furthermore, and as the bladder cavity enables mycobacteria to be in close contact with the tumor, this interaction seems to be necessary for a favorable outcome. After some successful trials conducted by Dr. Morales in 1976, BCG was later approved for the treatment of NMIBC by the Food and Drug Administration (FDA), and until this day is the first option of treatment for these patients. It is worth mentioning that BCG is the most efficacious treatment to avoid recurrences and progression of NMIBC, even superior to intravesically instilled chemotherapeutic drugs. 2. Use of other Species Different from BCG Although BCG has been the most studied mycobacteria for cancer treatment, other species or antigens derived from mycobacteria species have been studied for the treatment of urological and non-urological malignancies showing also promising immunotherapeutic properties. Those are, for instance, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis), Mycolicibacterium phlei (basonym Mycobacterium phlei), Mycolicibacterium vaccae (basonym Mycobacterium vaccae), Mycolicibacterium obuense (basonym Mycobacterium obuense), “Mycobacterium indicus pranii”, Mycolicibacterium brumae (basonym Mycobacterium brumae), or more recently Mycobacterium paragordonae, and an attenuated strain of Mycobacterium tuberculosis. In depth, M. smegmatis was first isolated by Lustgarten in 1885 from genital secretions (smegma) in a patient with a penile ulcer [17]. M. phlei, found in the environment [18], was first described in 1899, called the grass bacillus of Timothy bacillus. Since their discovery, M. smegmatis and M. phlei have been used as models for the study of mycobacteria, and in consequence, M. phlei was one of the first species considered for cancer treatment. Some cases of infections due to M. phlei are described in the literature [19–26], but studies using M. phlei for cancer treatment are based on non-viable mycobacteria. The formulation based on M. phlei, known as M. phlei cell wall-nucleic acid complex (MCNA), or commercially known as UrodicinTM , consists in a preparation of cell wall (CW) fractions plus DNA of the same bacterium [27]. Four more mycobacteria are used in non-viable form for cancer treatment. The M. vaccae strain used for cancer treatment is the R877R (NCTC 11659), which is a selected rough colony variant of a strain originally isolated from the Ugandan environment [28]. The commercial preparation is called IMM-201, previously SRL172. M. obuense was initially isolated from soil samples and the sputum of a patient with lung disease in 1971 [29]. M. obuense is closely related to M. vaccae [30], and the specific strain used for cancer treatment (NCTC 13365 strain) is commercially called IMM-101. Mycobacterium indicus pranii, called MIP, (DSMZ 45239T) is a saprophytic bacterium that has been extensively used as an immunotherapeutic agent for leprosy treatment but is currently under evaluation for the treatment of other infectious diseases and other kind of illnesses [31]. Finally, M. paragordonae (Mpg) is a slow-growing mycobacterium that fails to grow above 37 ◦ C and is isolated from water supply systems worldwide [32,33]. This mycobacterium has recently been proposed as a novel live vaccine candidate for the prevention of mycobacterial infections, showing enhanced protective efficacy in vaccinated mice compared to BCG [34]. Cancers 2020, 12, 1802 3 of 25 Finally, two mycobacteria have been studied in pre-clinical studies in their live form. M. brumae was isolated from soil and water samples in 1993 by M. Luquin et al. [35], and similarly to the above-mentioned species, is a rapid grower. No cases of infections due to M. brumae have been described, and its non-pathogenicity and non-toxicity have recently been demonstrated [36,37]. M. brumae has Cancers 2020, shown 12, x to inhibit tumor growth and increase survival in preclinical studies for bladder cancer3 (BC) of 25 treatment [38–42]. Finally, MTBVAC, a Spanish vaccine developed by deleting virulence factors of treatment [38–42]. Finally, MTBVAC, a Spanish vaccine developed by deleting virulence factors of M. tuberculosis and currently in clinical trials for TB prevention [43], has been analyzed for its value in M. tuberculosis and currently in clinical trials for TB prevention [43], has been analyzed for its value BC treatment [44]. in BC treatment [44]. Not only the whole mycobacteria, but also some cell extracts or purified mycobacteria antigens Not only the whole mycobacteria, but also some cell extracts or purified mycobacteria antigens from these species, have been studied in preclinical and clinical studies for cancer treatment. Despite the from these species, have been studied in preclinical and clinical studies for cancer treatment. Despite fact that the molecule or molecules of BCG responsible for its antitumor effect are still unknown, the fact that the molecule or molecules of BCG responsible for its antitumor effect are still unknown, several genus-specific antigens have been described in mycobacteria cells, and most of them are known several genus-specific antigens have been described in mycobacteria cells, and most of them are stimulators of the immune system [45,46]. Antigens belonging to different cell fractions are recognized known stimulators of the immune system [45,46]. Antigens belonging to different cell fractions are by surface-located receptors present both in professional and non-professional antigen-presenting recognized by surface-located receptors present both in professional and non-professional antigen- cells. Most of the structural molecules located in the mycobacterial cell wall (Figure 1) are agonists of presenting cells. Most of the structural molecules located in the mycobacterial cell wall (Figure 1) are immune receptors such as lectins (Mincle, Dectin 1, or mannose receptor), Toll-like receptors (TLR1, agonists of immune receptors such as lectins (Mincle, Dectin 1, or mannose receptor), Toll-like TLR2, TLR6), or T cell receptors via CD1 (detailed in reference [46]). Signaling through these receptors receptors (TLR1, TLR2, TLR6), or T cell receptors via CD1 (detailed in reference [46]). Signaling can induce the production of cytokines and/or chemokines favoring a desirable pro-inflammatory through these receptors can induce the production of cytokines and/or chemokines favoring a profile in the tumor micro-environment [47–49]. desirable pro-inflammatory profile in the tumor micro-environment [47–49]. Figure 1.1.Schematic Schematic representation representation of mycobacterium of mycobacterium immunomodulatory immunomodulatory molecules. Themolecules. The peptidoglycan– peptidoglycan–arabinogalactan–mycolic arabinogalactan–mycolic acids complex acids complex linked is covalently is covalently to thelinked to the cell membrane cell membrane and and is present is all in present in all mycobacteria. mycobacteria. Glycopeptidolipids, Glycopeptidolipids, lipooligosaccharides, lipooligosaccharides, phenolglycolipids, phenolglycolipids, phthiocerol phthiocerol dimycocerosates dimycocerosates (PDIM), and poliacyl-trehaloses (PDIM), and poliacyl-trehaloses are located in the are located outer in the of membrane outer onlymembrane some species.of only some Mycolic acids,species. Mycolic lipomannan, acids, lipomannan, and trehalose-mycolates areand trehalose-mycolates present are the in all mycobacteria, but present in all fine structure mycobacteria, differs but the fine among different structure species. differs Neither among the cell different nor the wholespecies. cell wallNeither theare molecules celldrawn nor theto whole scale. cell wall molecules are drawn to scale. Due to the success of BCG in the treatment of NMIBC, most of the research in the last thirty years for the use of mycobacteria in cancer treatment has been carried out on urological cancers (reviewed in [41,46,50]). However, in view of the comprehensive research already done, and the interest in these Cancers 2020, 12, 1802 4 of 25 Due to the success of BCG in the treatment of NMIBC, most of the research in the last thirty years for the use of mycobacteria in cancer treatment has been carried out on urological cancers (reviewed in [41,46,50]). However, in view of the comprehensive research already done, and the interest in these agents for combining with other antitumor therapies, we will focus our review on the use of Cancers 2020, 12, x 4 of 25 mycobacteria or mycobacteria components for the treatment of non-urological cancers (see Table 1). In all these cancers, there is still not a case in which mycobacteria or their components became the In all these cancers, there is still not a case in which mycobacteria or their components became the standard of care, as in the use of BCG in NMIBC. standard of care, as in the use of BCG in NMIBC. 3. Melanoma and Mycobacteria 3. Melanoma and Mycobacteria After NMIBC, melanoma is the cancer in which more mycobacterial products have been studied After NMIBC, melanoma is the cancer in which more mycobacterial products have been studied as antitumor agents. Around three dozens of these studies described purified antigens, cell fractions, as antitumor agents. Around three dozens of these studies described purified antigens, cell fractions, or whole mycobacteria as possible therapies for melanoma (summarized in Figure 2). or whole mycobacteria as possible therapies for melanoma (summarized in Figure 2). Figure 2.2.In vitro andand In vitro in vivo studies in vivo usingusing studies mycobacteria-derived agents carried mycobacteria-derived agents out for the carried treatment out for the of melanoma and lung cancer. A, alone or inA combination with chemotherapy; treatment of melanoma and lung cancer. , alone or in combination with chemotherapy; , along B, along withBMUC1- MBP MUC1-MBP with fusion protein;fusion protein; C , B16 C, B16 cells expressing mycobacterial cells expressing antigens;antigens; mycobacterial D , in combination D, in combination with monosodium with monosodiumurate crystals; E, anchored urate crystals; E to tumor proteins; , anchored to tumor , proteins; F F administered together with together , administered B16 cell with B16G, cell lysates; lysates; Gwith co-expressed herpes simplex , co-expressed with thymidine kinase thymidine herpes simplex in Salmonella typhimurium; kinase H, in in Salmonella combination with typhimurium; H , inchemotherapy combination with and radiotherapy. chemotherapyAg85—antigen 85 complex, and radiotherapy. BCG—Mycobacterium Ag85—antigen 85 complex, BCG—Mycobacterium bovis bacillus bovis bacillus Calmette-Guerin, Calmette-Guerin, BEC2—anti-idiotypic BEC2—anti-idiotypic antibody mimicking antibody the GD3 mimicking the ganglioside GD3 ganglioside expressed on mostexpressed small-cellon most lung small-cell cancer (SCLC)lung cancer cells, (SCLC) cells, CpG—cytosine-guanosine CpG—cytosine-guanosine nucleotide, CWS— nucleotide, CWS—cell cell wall skeleton, wall skeleton, HK—heat-killed, HK—heat-killed, HSP—heat-shock HSP—heat-shock proteins, IL—-Interleucin, proteins, IL-—Interleucin, L-MTP-PE— L-MTP-PE—liposomal muramylphosphatidyl liposomal muramyl tripeptide tripeptide phosphatidyl ethanolamine, ethanolamine, MBP—maltose MBP—maltose binding binding protein,protein, MIP— MIP—Mycobacterium indicus pranii, RelE—toxin of Mycobaterium tuberculosis, Mycobacterium indicus pranii, RelE—toxin of Mycobaterium tuberculosis, SMP-105—CWS-BCG Tokyo SMP-105—CWS-BCG Tokyo 172, SRL172, 172—M. SRL 172—M. vaccaevaccae commercial commercial preparation, preparation, WT1—Wilms’tumor WT1—Wilms' tumor protein, protein, Z-100—an Z-100—an arabinomannan from M. tuberculosis tuberculosis strain strain Aoyama Aoyama B. B. 3.1. Purified Antigens and Cell Extracts 3.1. Purified Antigens and Cell Extracts Many studies focused on the use of the immunogenic mycobacterial heat-shock proteins (HSP). Many studies focused on the use of the immunogenic mycobacterial heat-shock proteins (HSP). Studies on M. tuberculosis HSP70 have shown contradictory results. On the one hand, HSP70 protein Studies on M. tuberculosis HSP70 have shown contradictory results. On the one hand, HSP70 protein administered together with B16 melanoma cells lysate to tumor-bearing mice suppressed tumor growth administered together with B16 melanoma cells lysate to tumor-bearing mice suppressed tumor and metastasis, and extended mice survival [51]. Furthermore, an attenuated Salmonella typhimurium growth and metastasis, and extended mice survival [51]. Furthermore, an attenuated Salmonella that co-expresses HSP70 and herpes simplex virus thymidine kinase genes showed inhibitory effects typhimurium that co-expresses HSP70 and herpes simplex virus thymidine kinase genes showed on melanoma growth and prolonged tumor-bearing mice survival [52]. On the one hand, HSP70 was inhibitory effects on melanoma growth and prolonged tumor-bearing mice survival [52]. On the one found to be immunosuppressive in an allograft model by reducing rejection of the implants [53]. hand, HSP70 was found to be immunosuppressive in an allograft model by reducing rejection of the Moreover, the Prostate-Specific Antigen (PSA)–HSP70 fusion protein was as immunostimulating as implants [53]. Moreover, the Prostate-Specific Antigen (PSA)–HSP70 fusion protein was as PSA alone in a murine melanoma model [54]. immunostimulating as PSA alone in a murine melanoma model [54]. Another HSP tested for melanoma was BCG HSP65. Cytosine-guanosine nucleotide (CpG) enhanced the antitumor effect of BCG HSP65 when anchored to tumor antigens Her2 [55] or tumor- associated antigen Mucin 1 (MUC1) [56,57], both expressed in melanoma cells. In fact, mycobacterial DNA alone was also shown to have therapeutic properties against melanoma. CpG Cancers 2020, 12, 1802 5 of 25 Another HSP tested for melanoma was BCG HSP65. Cytosine-guanosine nucleotide (CpG) enhanced the antitumor effect of BCG HSP65 when anchored to tumor antigens Her2 [55] or tumor-associated antigen Mucin 1 (MUC1) [56,57], both expressed in melanoma cells. In fact, mycobacterial DNA alone was also shown to have therapeutic properties against melanoma. CpG oligodeoxynucleotides from M. bovis encapsulated in liposomes showed antitumor activity against B16 tumor-bearing mice when administered into the tumor [58]. Another studied combination is BCG HSP65 and melanoma cell lysates. When administered together, they induced apoptosis in different melanoma cell lines and inhibited tumor growth in tumor-bearing mice [59]. As evidence of the concept for an immunotherapy consisting of inducing the transient expression of mycobacterial HSP65 or the antigen 85 complex (Ag85) (fibronectin-binding protein) to make melanoma tumors more immunogenic, Tarrant et al., induced subcutaneous tumors using B16-F10 cells transfected with either HSP65 or Ag85 and observed a significant tumor burden reduction in the case of Ag85-B16-F10 tumors but not in the tumors that expressed HSP65, indicating that Ag85 protein is more immunogenic [60]. A completely different strategy is the use of tumor cells transfected to express Ag85 as a vaccine. The addition mycobacterial agent enhances the immune response against the parental cell line and protects mice form developing tumors [61]. The ESAT-6 antigen of M. tuberculosis has also been used in melanoma studies as prophylactic or therapeutic treatment. The prophylactic strategy consisted of transfecting B16 cells to express ESAT-6 fused to glycosyl-phosphatidylinositol (GPI) and IL-21. In these experiments, vaccinated mice showed significantly reduced tumor and lung metastasis [62]. This antitumor response was even more enhanced when animals were previously primed with an ESAT-6-GPI DNA vaccine [63]. Therapeutic efficacy was evaluated by administering ESAT-6-anchored IL-2 or IL-12 DNA vaccines into the tumor, with the first combination being able to completely remove tumors [64]. The same authors were successful in a second therapeutic strategy. When they used exosomes from B16-ESAT-6 transfected cells that contained antigens from both the tumor and the mycobacterium to treat melanoma-bearing mice, they observed a significant reduction in tumor growth [65]. The mechanism of action of these exosomes is immediate due to the activation of macrophages and dendritic cells (DCs) followed by the suppressive effects of the adaptive immunity responding against ESAT-6 [66]. Different CW structural antigens have been evaluated. Z-100, an arabinomannan from M. tuberculosis strain Aoyama B, known to establish a balance of Th1/Th2 responses in tumor-bearing mice [67], has been tested as immunotherapy in highly metastatic melanoma mouse models. Monotherapy or combined therapy with radiation inhibits lung and lymphatic metastasis [68–70]. A synthetic peptidoglycan mimicking muramyl dipeptide from BCG with an additional peptide and encapsulated in liposomes: liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE) [71] has been widely studied in different types of cancers. L-MTP-PE showed antitumor activity in dogs suffering spontaneous early-stage oral melanoma, but was not efficacious in advanced-stage melanoma [72], as had been already observed in a clinical trial in stages III and IV melanoma patients [73]. A fusion protein consisting of the maltose-binding protein (which binds to Toll-like receptor-2 (TLR2) and MUC1 demonstrated higher antitumor activity in B16 MUC1-expressing cells when BCG was present [74]. Regarding mycobacterial cell fractions, squalane emulsified cell wall skeleton (CWS) from BCG Tokyo (BCG-CWS) has been mainly used as adjuvant for melanoma vaccines. BCG-CWS significantly lowered lung metastasis events in B16-BL6 melanoma subline cells bearing mice administered both intravenously and subcutaneously, due to its effect on reducing tumor growth and inhibiting angiogenesis [75]. On the contrary, when MIP-CWS was tested in B16-F10 melanoma-bearing mice and due to its capacity to induce Th2, a worse antitumor effect was obtained compared to MIP-CW [76] or whole MIP [77] (see Section 3.2.). Cancers 2020, 12, 1802 6 of 25 3.2. Whole Mycobacteria The whole cells of M. vaccae, M. obuense, MIP, M. smegmatis, and BCG have been studied for melanoma. SRL172 showed a positive correlation between longer survival and intracellular IL-2 detection in peripheral blood lymphocytes when used as antitumor agent for the treatment of stage IV melanoma patients [78]. Then, a clinical trial was performed administering an SRL172 plus IL-2 combination therapy, but only three partial remissions were observed in a total of 16 patients. Thus, no therapeutic effect was established [79]. Nevertheless, a retrospective study concluded that this treatment could be effective in lower grade melanoma patients [80]. M.2020, Cancers obuense 12, x was unsuccessfully used in two clinical trials. Although IMM-101 had a positive effect 6 of 25 on avoiding metastasis in a melanoma mouse model, it showed no effect on the overall tumor burden patients tolerated (NCT01559818, the therapy, but NCT01559819) [81].only 15% of clinical Furthermore, in a responses were small clinical observed trial (NCT01308762) with advanced [82]. melanoma, Recently, immune checkpoint inhibitors have become the standard of care patients tolerated the therapy, but only 15% of clinical responses were observed (NCT01308762) [82]. for melanoma patients, as they show the highest long-term survival of patients with metastatic Recently, immune checkpoint inhibitors have become the standard of care for melanoma patients, melanoma that has ever seen with as theyanyshow otherthe therapy, although survival highest long-term high levels of toxicity of patients withhave been observed. metastatic melanomaDalgleish and that has ever collaborators, in a small clinical trial, showed it was more efficacious seen with any other therapy, although high levels of toxicity have been observed. Dalgleish and without added toxicity when combining IMM-101 collaborators, andclinical in a small checkpointtrial,inhibitors showed it[83]. was more efficacious without added toxicity when MIP preparation combining IMM-101 and hascheckpoint been also assayed inhibitorsin subcutaneously [83]. implanted B16-F10 melanoma-bearing miceMIPand preparation reduced tumor growth by triggering macrophage has been also assayed in subcutaneously implanted and DCs activation B16-F10 and increasing T-cell melanoma-bearing infiltration mice inside the and reduced tumor tumor [77]. by growth MIP activatesmacrophage triggering Th1 immuneand response through TLR2 DCs activation and MyD88 and increasing axis T-cell [84] and inhibits invasion of the highly invasive B16-F10 cells [85]. infiltration inside the tumor [77]. MIP activates Th1 immune response through TLR2 and MyD88 Notand axis [84] only can proteins, inhibits invasionlipids, of theand sugars highly be immunogenic: invasive insoluble salts like urate also are. B16-F10 cells [85]. Accumulation Not only can of crystallized uric acid proteins, lipids, and in the joints sugars triggers an inflammatory be immunogenic: insoluble salts response, like urateresulting also are.in an attack of gout. A combination therapy with monosodium urate crystals Accumulation of crystallized uric acid in the joints triggers an inflammatory response, resulting in an and M. smegmatis showed a delaying attack of gout.effect A in melanoma therapy combination tumor growth, unlike each monotherapy. with monosodium urate crystals and In this M. case, the antitumor smegmatis showed efficacy correlated with the presence of monocyte-derived DCs in the a delaying effect in melanoma tumor growth, unlike each monotherapy. In this case, the antitumor tumor-draining lymph nodes [86]. efficacy correlated with the presence of monocyte-derived DCs in the tumor-draining lymph nodes [86]. Antitumor effect Antitumor effect of of intra-lesional intra-lesional BCG BCG TiceTice was wasalso alsotested. tested.BCG BCG triggered triggered antitumor antitumor effect by effect changing the melanoma microenvironment due to a switch from by changing the melanoma microenvironment due to a switch from M2 to M1 macrophages [87]. M2 to M1 macrophages [87]. Moreover, this Moreover, this treatment treatment alsoalso attracted attracted important important antitumor antitumor immune immune cells,cells, such such asas ɣδ δTT cells cells [88]. [88]. Despitethese Despite thesepromising promisingresults, results, unfortunately, unfortunately, notnot even even combining combining BCG BCGwithwith checkpoint checkpoint inhibitors inhibitors [89] [89] resulted in clinical benefit in a phase I study which involved advanced resulted in clinical benefit in a phase I study which involved advanced metastatic melanoma patients. metastatic melanoma patients. Dose Dose escalation escalation was not well wastolerated not welland tolerated triggeredandimmune-related triggered immune-related adverse events adverse [90]. events [90]. Thus, after Thus, after all the attempts to find an efficacious mycobacterial immunotherapy all the attempts to find an efficacious mycobacterial immunotherapy for melanoma patients, there is for melanoma patients, still there is still no consensus no consensus treatment yet [91].treatment yet [91]. 4. Lung Cancer 4. Cancer and and Mycobacteria Mycobacteria In the In the case case of of lung cancer (LC), purified purified mycobacteria mycobacteria antigens, antigens, BCG BCG extracts, extracts, MIP, MIP,and and M. M. vaccae vaccae have been have been studied studied as as immunotherapeutic immunotherapeuticagents. agents. 4.1. 4.1. Purified Purified Antigens Antigens and and Cell Cell Extracts Extracts Mycobacteria Mycobacteriaand andpurified antigens purified have antigens beenbeen have tested in the tested inLewis lung carcinoma the Lewis (LLC) mouse lung carcinoma model. (LLC) mouse The Z-100 antigen in combination with radiotherapy significantly reduces model. The Z-100 antigen in combination with radiotherapy significantly reduces both primary both primary tumor growth and pulmonary tumor growth metastasis and pulmonary of LLC-bearing metastasis mice, being of LLC-bearing mice,more being effective than eachthan more effective therapy each individually [92]. Not [92]. therapy individually only Not polysaccharides only polysaccharides butM.also but also the tuberculosis toxin RelEtoxin the M. tuberculosis showed anshowed RelE inhibitory an effect on human A549 lung adenocarcinoma tumor cells [93]. inhibitory effect on human A549 lung adenocarcinoma tumor cells [93]. BCG BCG extracts extracts have have also also been been used used combined combined with with other other treatments treatments for for LC LC therapy. therapy. The The SMP-105 SMP-105 formulation, formulation, consisting consisting ofof CWS CWS purified purified from from BCG BCG Tokyo Tokyo172 172strain, strain, in in combination combination with with mitomycin mitomycin C-inactivated C-inactivatedLLCLLCcells, hadhad cells, a therapeutic effecteffect a therapeutic and induced an IFN-γ and induced anresponse through the IFN-γ response activation through the of TLR2, and activation of independently from TLR4, from TLR2, and independently in LLC-bearing mice [94]. Moreover, TLR4, in LLC-bearing mice [94].the intradermal Moreover, the intradermal administration of BCG-CWS, combined with Wilms' tumor protein (WT1), in mice, induced a cytotoxic immune response against LC cells expressing WT1 [95]. BCG-CWS-treated non- small-cell LC (NSCLC) patients showed prolonged survival rates without worsening their quality of life. However, in the case of small-cell LC (SCLC) patients, although a small clinical trial suggested longer survival and relapse-free survival in patients treated with an immunoadjuvant consisting of BCG extract bound to hydroxyapatite and microparticulated tuberculin called CalTUMP in Cancers 2020, 12, 1802 7 of 25 administration of BCG-CWS, combined with Wilms’ tumor protein (WT1), in mice, induced a cytotoxic immune response against LC cells expressing WT1 [95]. BCG-CWS-treated non-small-cell LC (NSCLC) patients showed prolonged survival rates without worsening their quality of life. However, in the case of small-cell LC (SCLC) patients, although a small clinical trial suggested longer survival and relapse-free survival in patients treated with an immunoadjuvant consisting of BCG extract bound to hydroxyapatite and microparticulated tuberculin called CalTUMP in combination with BEC2 (which is an anti-idiotypic antibody mimicking the GD3 ganglioside expressed on most SCLC cells) [96], two other studies demonstrated no beneficial effect of the combined therapy [97,98]. 4.2. Whole Mycobacteria Most studies in lung used MIP and SRL172, but one study used whole BCG. Although maltose- binding proteins from Escherichia coli have no direct inhibitory effect on the LLC cell line, in combination with BCG they have a synergic effect on LLC-bearing mice [99]. A recent study showed the antitumor effect of heat-killed (HK) MIP on several cancer cell lines, including A549 cells. However, neither live MIP nor supernatant from MIP cultures showed an inhibitory effect on cancer cells [100]. However, MIP showed a beneficial effect in the past as an adjuvant in the therapy of NSCLC patients. When MIP was administered along with cisplatin chemotherapy and radiotherapy therapy, patients were able to finish the treatment and experienced a regression in tumor size and improvement in the lung function compared to the control group, which did not receive MIP [101]. Furthermore, intradermally administered MIP along with paclitaxel and cisplatin treatment increased the overall survival and the progression-free survival compared to the chemotherapy alone group [102]. In view of the promising results of SRL172 in prostate cancer [103,104] and melanoma patients (see Section 3.2.), two phase II clinical trials were performed using it in LC patients in which a small group of NSCLC [105] and SCLC patients [106] received chemotherapy together with intradermal injections of SRL172. No differences regarding toxicity were observed between the chemotherapy alone or the combination treatment in any group and, in both cases, there was a trend towards the improvement of the median survival and the quality of life of the patients [107]. Only in the case of NSCLC patients, a trend towards improvement of the response rate was observed on SRL172-treated patients. The phase III trial on a cohort of more than 400 NSCLC patients confirmed that SRL172 led to the improvement in the quality of life of the patients, but without any improvement on overall survival [108]. However, the number of intradermal SRL172 injections was very variable between patients in this study. When the data obtained were re-analyzed taking into account the histological lung tumor type and the number of adjuvant inoculations, adenocarcinoma patients, but not squamous cell carcinoma patients, survived significantly longer when receiving combination therapy [109]. A more recent study on the mechanistic effect demonstrated that SRL172 showed capacity to activate γδ T-cells to be cytotoxic to A549 cell line [110]. 5. Gastrointestinal Tract Cancers and Mycobacteria Mycobacteria agents have been tested in several parts of the gastrointestinal tract from the esophagus to the rectus, including the stomach and pancreas. M. smegmatis is the only mycobacterium studied for the treatment of esophageal tumors. M. smegmatis was used as a vector to produce a recombinant bacterium containing MAGEA3 and SSX2, two important immunogenic tumor antigens. Treatment of esophageal tumor-bearing mice with the recombinant mycobacterium inhibited tumor growth and reduced the tumor weight and volume [111]. In a study of locally advanced gastric cancer in a group of 156 patients, the administration of BCG Moreau plus chemotherapeutic treatment up to two years after resection of tumors resulted in a significant increase in survival with respect to the monotherapies [112]. Several studies have been carried out in pancreatic cancer using mycobacteria, mainly in combination with chemotherapeutic agents. In fact, it has been demonstrated that gemcitabine Cancers 2020, 12, 1802 8 of 25 (the standard chemotherapeutic agent for this type of cancer) enhanced the antitumor effect of cytotoxic T cells induced by BCG-stimulated DCs, by sensitizing pancreatic tumor cells [113]. Using purified mycobacterial antigens, a vaccine composed of mycobacteria HSP65 plus Panc02 pancreatic cancer tissue lysate was inoculated in mice before implanting Panc02 cancer cells, showing prolonged survival in treated tumor-bearing mice compared to unvaccinated mice [114]. The most successful mycobacteria immunotherapy for this type of cancer was achieved using IMM-101, which was designed as an orphan drug in 2014 for the treatment of advanced pancreatic cancer patients. Repeated treatment with IMM-101 plus chemotherapy (over a year), showed significantly enhanced survival rates compared to patients receiving only chemotherapy [115]. The recent evaluation of the administration of IMM-101 plus gemcitabine in advanced pancreatic cancer patients was shown to be as safe as chemotherapy alone and suggests improved survival rates [116]. Although HK-M. obuense showed great success in the treatment of pancreatic cancer patients, little was known about its cytotoxic and immunomodulatory activity. Recently, Bazzi et al. published an extensive work on how IMM-101 modulates DC, macrophages, and granulocytes, in terms of surface markers and cytokine secretion profile [117,118]. Regarding colorectal cancer, different strategies have been designed for the use of mycobacteria components. In vitro experiments have shown that exposure of HCT116 colon cancer cells to BCG Pasteur-CWS before ionizing radiation resulted in increased cell death in a caspase-independent manner, through the induction of autophagic cell death mediated through TLR2 and TLR4 signaling [119]. Another group reported that BCG Tokyo-CWS significantly reduced lung metastasis in a colon26-M3.1 carcinoma mouse model [75]. The adjuvant DETOX (M. phlei-CW together with Salmonella minnesota lipid A) included in an allogeneic cell vaccine plus subcutaneous IL-1 treatment in metastatic colorectal carcinoma patients was shown to be safe, also suggesting a beneficial effect [120]. The M. tuberculosis Rv2299c (which belongs to the HSP90 family) has been used alone or in combination with tumor antigen-activated DC and enhanced reduced tumor burden in a colon cancer mouse model, but only the combination induced a long-term systemic response [121]. Finally, the use of IMM-101 first for priming before tumor challenge, and later as a subcutaneous therapeutic agent in colorectal mouse models, showed a significant reduction in the number of lung metastatic lesions. However, IMM-101 on its own administered subcutaneously as therapy had no significant effect on tumor growth [81]. Some clinical trials demonstrated the efficacy of repeated injections (up to a year) of IMM-101 in colorectal cancer (NCT03009058). In combination with radiation in metastatic colorectal patients in which other therapies were not effective, no synergy was observed (NCT01539824). Finally, the use of the whole HK-M. paragordonae alone, or in synergism with cisplatin in tumor-bearing mice, improved mice survival rate and enhanced a strong activation of the immune system through natural killer (NK) and DCs activation, and release of cytokines such as IL-12 or tumor necrosis factor (TNF-α) [122]. Regarding hepatoma treatment, different vaccines have been designed in which some peptides of mycobacteria, HSP70 [123,124], or M. bovis HSP65 [125,126] have been included in the composition. After treating hepatoma mice models, a beneficial effect was observed by delaying the progression of liver tumors, which was mediated by the induction of favorable antitumor immunity. Moreover, the 64-kDa protein from BCG, which shares antigenic determinants with line 10 hepatoma, Meth A, CT-26, and RL female 1 mouse tumor cells, showed antitumor activity in immunized guinea pigs [127] and also in immunized mice [128]. Studies have also been carried out on mycobacteria extracts. In vitro studies showed a growth inhibition effect of the optimized formulation of SMP-105 in Line 10 hepatoma cells [129]. SMP-105 treatment close to the tumor focus in guinea pigs bearing syngeneic line 10 hepatoma was able to eliminate an established tumor and metastasis [130]. Repeated intra-lesion injections of DNA from BCG plus poly-L-lysine resulted in a delay of tumor growth and complete cure of guinea pigs inoculated with line 10 hepatoma cells, while both monotherapies were ineffective [131]. Finally, intra-tumor vaccination using CalTUMP combined with proton-beam radiotherapy has shown to be safe and may be a promising treatment for recurrence reduction [132]. Cancers 2020, 12, 1802 9 of 25 6. Breast Cancer and Mycobacteria Different vaccines using recombinant BCG were evaluated for prevention or treatment of breast cancer. Most of those studies are based on BCG expressing MUC1 and adding or co-expressing some other immunostimulatory molecules, such as cytokines or chemokines, in order to improve the relatively poor immunogenicity of this breast-tumor-associated antigen. A human IL-2-MUC1-expressing-BCG (Pasteur strain) vaccine was used to immunize humanized mice that were later challenged with ZR75-1 human breast cancer cells, showing a reduction of palpable tumors together with CD8+ T cells immune response [133]. Immunization with recombinant BCG expressing MUC1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibits breast tumor growth in mice [134]. BCG expressing variable-number tandem repeats of MUC1 and CD80 inhibits tumors in mice, triggering the infiltration of CD4 and CD8 lymphocytes and the production of IFN-γ in splenocytes [135]. Furthermore, BCG plus allogenic tumor cells vaccine has shown to be safe in mice and patients with advanced breast cancer and, combined with chemotherapy and radiotherapy, showed to be practicable [136,137]. Other vaccines showed efficacy in breast cancer. Ag38-expressing cancer cells administered with IL-12 protected against the formation of mammary tumors [138]. Immunotherapeutic treatment with DNA vaccines that contain BCG HSP65 has been also evaluated for breast cancer treatment in mice, showing an effect on reducing tumor growth [139–141]. SRL172, BCG, and M. phlei CW extracts have also been studied. SRL172 plus anti-HER-2 antibodies stimulate the cytotoxic activity of immune cells to tumor HER2-expressing cells [142]. Intra-tumor injection of BCG (Frappier strain, named PACISMD ) or M. phlei-CW extract combined with photodynamic therapy increases the antitumor effect of photodynamic therapy alone by inducing the infiltration of immune cells, which favors the antitumor response [143]. The use of MIP has also been proposed in the literature to resolve breast cancer cases. MIP plus Survivin and alum immunization before tumor implantation seemed to induce a long protective effect [144]. More recently, MIP combined with 1’-S-1’-acetoxychavicol acetate and cisplatin reduced tumor volume of 4T1 tumors in mice with no observed toxicity, which is enough evidence to set up a clinical trial [145]. As observed in a melanoma mouse model (Section 3.2.), M. smegmatis plus monosodium urate crystals reduced the growth rates in the orthotopic 4T1 tumors and decreased the 4T1 lung metastases. [86]. 7. Ovarian and Cervix Tumors and Mycobacteria From the beginning of the 1990s, there were no studies published on the antitumor potential of mycobacteria or mycobacteria components for ovarian or cervix tumors. In 2009, a study including 73 ovarian cancer patients revealed that the efficacy of intracutaneously administered BCG-CWS in different stages of ovarian cancer patients is compromised in women who underwent lymphadenectomy compared to patients who did not [146]. In 2012, Gottschalk and collaborators demonstrated the immunostimulatory effect of the 30kDa antigen (or phosphate transport protein (Pst-1)) from M. tuberculosis on the cytotoxic function of NK on five ovarian cancer cell lines through monocyte activation in a cell–cell contact-dependent manner [147]. In 2014, Yuan et al. constructed a fusion protein consisting of HSP70 plus a single-chain antibody variable fragment which makes the protein specifically target mesothelin-expressing tumors such as mesothelioma or ovarian cancer. The construct improved DC antigen presentation, which resulted in inhibited tumor growth and prolonged survival in orthotopic mouse models of papillary ovarian cancer [148]. Using the unique opportunity of the fact that human papillomavirus type 16 (HPV-16) expresses E7 protein in HCP-linked cervical tumors, many immunotherapy strategies have been based on the combination of highly immunogenic mycobacterial proteins and E7 protein. For instance, a suicidal plasmid containing HSP70 and E7 induced significantly higher cytotoxic T cell immune responses against E7-expressing tumors than the plasmid only containing E7 [149]. A similar approach using a fusion protein between HSP70 with E7 gene induced an immune response and a prolonged survival of TC-1 cervical cancer model tumor-bearing mice [150]. Cancers 2020, 12, 1802 10 of 25 Like M. tuberculosis HSP70, HSP65 from M. bovis has been fused to E7 to treat cervical tumors. Two studies have demonstrated the efficacy of this fusion protein in mouse models, in which tumor regression was observed due to the triggered CD4-mediated immune response [151,152]. Finally, a synthetic chimeric peptide from computationally predicted HPV-16 T cell epitopes was used to immunize E6- and E7-expressing-TC-1-bearing mice along with different adjuvants including HK MIP, demonstrating therapeutic and protective activity. However, the peptide in combination with MIP showed less effect, both in vitro and in vivo, than the other adjuvants used in the study [153]. Two other posterior studies tested its efficacy in the clinics. Both of them concluded that the HSP65-E7 fusion protein (SGN-00101) confers a benefit for cervical intraepithelial neoplasia III patients [154,155]. Whole mycobacteria MIP and BCG cells have also been tested in cervical tumors. The antineoplastic potential of live BCG Tokyo was tested on HeLa cells, with an inhibitory effect being observed [156]. Similarly, whole HK MIP had a cytotoxic effect on CaSki cervical cancer cells, while not on non-cancerous cells [100]. 8. Hematopoietic and Lymphoid Malignancies and Mycobacteria There are reports in the literature on the use of specific mycobacterial antigens, cell extracts, and whole mycobacteria cells against leukemia. HSP70 transfected into L1210 mouse leukemia cells showed reduced tumorigenicity in immunocompetent mice, and HSP70-L1210 cells also reduced tumor burden when used either as vaccine or treatment in the L1210-parental mouse model [51,157]. Another approach is vaccinating using BCG-CWS plus WT1 peptide also expressed in leukemia, which demonstrated that mycobacteria components increase WT1-specific immune responses, favoring rejection of WT1-expressing leukemia cells [95]. Finally, intradermal injection of MIP in BALB/c and C57BL/6 mice with Sp2/0 (myeloma) and EL4 (thymoma) cells, respectively, inhibited tumor growth while triggering an antitumor immunity. The contribution of IFN-γ, CD4, and CD8 T cells is critical on MIP effect [158]. Many vaccines comprising mycobacteria HSPs have been studied for lymphoma treatment. Vaccination with HSP70 plus idiotype-encoding plasmids in the 38C13 murine lymphoma model enhanced survival rates in tumor-bearing mice [159]. Vaccination with A20 B cell lymphoma cells plus HSP70 together with anti-idiotype before tumor induction in the A20 murine lymphoma model triggered antitumor immunity by activating tumor cytotoxicity in T cells and increasing IFN-γ secretion and anti-A20 IgG2a levels [160]. Moreover, recombinant fusion protein of HSP65 BCG plus a variable-number tandem repeats of MUC1, inoculated both as preventive and therapeutic treatment in mice, inhibits EL4 thymus lymphoma MUC1-expressing EL4 thymus lymphoma tumors and significantly prolonged the survival compared to non-vaccinated tumor-bearing mice [56]. BCG extracts have also been tested for lymphoma treatment. Lipomannan (LM) from BCG Tokyo was able to trigger antitumor effect in A20 tumor-bearing mice, contrary to CW extract or lipoarabinomannan antigen. Effect of LM from BCG seemed to be due to both recruitment of DCs that trigger superoxide production by eosinophils and memory Th2 cells [161]. In another study, maturation of DCs, specific cytolytic T cell activity, and antitumor effect in an ovalbumin (OVA)-expressing EL4 tumor model were seen combining Tokyo BCG strain-CWS formulated in nanoparticles with OVA-loaded nanoparticles [162]. Cancers 2020, 12, 1802 11 of 25 Table 1. Promising mycobacteria and mycobacteria components studied in vivo and in clinical trials to treat several types of cancer. Type of Cancer Species Mycobacteria or Mycobacteria Components In Vivo Experiments Outcome Clinical Trials Outcome BCG /MUC1/ IL-2 Inhibit tumor growth [133] BCG /MUC1/GM-CSF Inhibit tumor growth [134] BCG /MUC1/CD80 Elicit tumor-specific immune response [135] BCG + Allogenic tumor cells Increase Survival [136] M. bovis BCG Tumor cells + BCG + Formalin Safe and no toxic [137] Safe, Increase Survival [137] Immunogenicity, induce humoral response HSP65 Breast Cancer [139,140] HSP65 + MUC1 Safe [141] M. phlei CW + Photodynamic Trigger immune response [143] Immunogenicity and tumor growth inhibition MIP + Survivin + Alum [144] M. indicus pranii (MIP) MIP + 1’-S-1’-acetoxychavicol acetate + Cisplatin Control cancer progression [145] M. smegmatis M. smegmatis + Monosodium urate crystals Trigger immune response, delay the tumor [86] Specific CD8+ T Cell Responses and Antitumor M. tuberculosis HSP70 + E7 Effect [150] Cervical Cancer Reduce carcinoma [151]; regression of palpable M. bovis BCG HSP65 + E7 Tumor regression [154,155] tumors, increase long-term survival [152] M. indicus pranii (MIP) HK-MIP + HPV16T epitope Cytotoxic T lymphocytes cytolysis [153] Rv2299c (HSP90 family) + Tumor No generation of immune-suppressive cells, M. tuberculosis Antigen-activated DC induce Th1 immune response [121] Colorectal Cancer M. paragordonae HK- M. paragordonae + cisplatin Induce antitumor immune response [122] Esophageal cancer M. smegmatis M. smegmatisMAGEA3/ SSX2 Reduce tumor volume [111] M. tuberculosis Z-100 + Radiation Inhibit tumor growth [92] Fibrosarcoma M. bovis BCG HSP65 + PSA Protect to PSA-expressing tumors [54] Gastric Cancer M. bovis BCG BCG + 5-Fluorouracil, doxorubicin, and mitomycin Improve survival [112] M. tuberculosis HSP70 + Human umbilical vein endothelial cell Inhibit tumor growth, prolong survival [124] M. bovis BCG GnRH(3)-hinge-MVP-Hsp65 Decrease tumor weight [125] BCG + Proton-beam radiotherapy Safe [132] Hepatoma HSP65-X10-beta-hCGCTP37 Inhibit tumor growth [126] BCG 64-kDa surface protein Inhibit tumor growth [127] CWS of BCG (SMP-105) Tumor-eliminating effect [129,130] DNA + Poly-L-lisine Delay of tumor growth [131] Cancers 2020, 12, 1802 12 of 25 Table 1. Cont. Type of Cancer Species Mycobacteria or Mycobacteria Components In Vivo Experiments Outcome Clinical Trials Outcome M. tuberculosis HSP70 Prolong survival [157] Leukemia M. bovis BCG CWS + WT1 Specific immune responses [95] M. tuberculosis Z-100 + Radiation Inhibit tumor growth [92] BCG + maltose-binding protein from E. coli Induce Th1 response [99] M. bovis BCG CWS Reducce metastasis [75] CWS + WT1 Reject of WT1-expressing lung cancer cells [95] Lung Cancer SMP-105 + mitomycin C-inactivated tumor cells Supress tumor growth [94] SRL172 Improve quality of life [107] M. vaccae Improve quality of life and survival SRL172 + chemotherapy [108,109] Improve quality of life, regress tumor M. indicus pranii (MIP) MIP + Cisplatin + Radiotherapy size [101] HSP70 + Idiotype Prolong survival [159] M. tuberculosis HSP70 + A20 tumor cells Regress tumor size [160] HSP65 + MUC1 Specific CTL and anti-tumor responses [56] Lymphoma M. bovis BCG CWS in nanoparticles Inhibit tumor growth, induce cytotoxic T cells [162] Inhibits tumor growth, eosinophils infiltration Lipomannan [161] Increase natural killer and CD8(+) T cells, reduce M. bovis Liposomes with CpG oligodeoxynucleotides regulatory CD4(+) T cell recruitment [58] B16F10/ESAT-6-GPI-IL-21 Inhibit tumor growth, prolong survival [62] B16F10-ESAT-6-gpi/IL-21 Inhibit tumor growth [63] M. tuberculosis ESAT-6 + IL-2 Tumor regression [64] HSP70 + B16 cells lysate Inhibit tumor growth, prolong survival [51] HSP70 + Thymidine kinase genes (HSV) + Suppress tumor growth and extend survival [52] attenuated S. typhimurium Melanoma BCG + MUC1 + MBP Inhibit tumor growth, induce Th1 response [74] M. bovis BCG CpG oligodeoxynucleotide + HSP65 + MUC1 Inhibit tumor growth, prolong survival [57] CWS Inhibited tumor metastasis [75] M. vaccae SRL172 Increase survival [80] M. obuense IMM-101 Reduce metastatic lesions [81], safe [82] Reduce tumor growth and weight [77,84], block M. indicus pranii (MIP) MIP tumor growth and inhibit metastasis [85] M. smegmatis M. smegmatis + Monosodium urate crystals Delay subcutaneous melanomas [86] Cancers 2020, 12, 1802 13 of 25 Table 1. Cont. Type of Cancer Species Mycobacteria or Mycobacteria Components In Vivo Experiments Outcome Clinical Trials Outcome Increase survival, slow tumor growth, augment M. tuberculosis HSP70 + single-chain antibody tumor-specific CD8+ T-cells [148] Mesothelioma M. vaccae SRL 172 + Chemotherapy Safe [163] Myeloma M. indicus pranii (MIP) MIP Reduce tumor growth [158] Oral melanoma M. bovis BCG L-MTP-PE Prolong survival [72] Decrease risk of recurrence and death L-MTP-PE M. bovis BCG [71] Osteosarcoma Inhibit primary osteosarcoma L-MTP-PE + Zoledronic acid progression [164] Increase survival, slow tumor growth, augment M. tuberculosis HSP70 + single-chain antibody tumor-specific CD8+ T-cells [148] Ovarian cancer M. bovis BCG CWS High quality of life [146] M. bovis BCG HSP65 + Tumor tissue lysate of pancreatic cancer Prolong survival [114] Pancreatic cancer M. obuense IMM-101 + Gemcitabine Improve overall survival [115] Sarcoma M. tuberculosis Z-100 + C. parvum Prolong concomitant antitumor immunity [165] M. bovis BCG PS1 glycan Suppress tumor cell growth [166] M. vaccae PS4A proteoglycan Decreased tumor incidence [167] Thymoma M. indicus pranii (MIP) MIP Reduce tumor growth [158] Ag85—antigen 85 complex, BCG—Mycobacterium bovis bacillus Calmette-Guerin, CpG—cytosine-guanosine nucleotide, CW—cell wall, CWS—cell wall skeleton, DC—dendritic cell, GPI—glycosyl-phosphatidylinositol, HK—heat-killed, HSP—heat-shock proteins, HSV—herpes simplex virus, HPV—human papillomavirus, IL—-Interleucin, IMM-101—heat-killed Mycolicibacterium obuense, L-MTP-PE—liposomal muramyl tripeptide phosphatidyl ethanolamine, MIP—Mycobacterium indicus pranii, MUC1—tumor-associated antigen Mucin 1, PSA—prostate-specific antigen, SRL 172—M. vaccae commercial preparation, Rv2299c—Mycobacterium tuberculosis HSP90 family member, SMP-105—CWS-BCG Tokyo 172, WT1—Wilms’ tumor protein, Z-100—an arabinomannan from M. tuberculosis strain Aoyama B. Cancers 2020, 12, 1802 14 of 25 9. Sarcoma and Mesothelioma and Mycobacteria Inoperable sarcoma was the focus of Coley’s studies back in the 19th century, and more recently, several polysaccharides from BCG have shown to have antitumor effect on murine sarcoma models. In 1994, Lou et al. formulated PS1, a complex polysaccharide derived from BCG, into gelatin particles; however, no increased antitumor activity with respect to unencapsulated PS1 was observed, neither against S180 murine sarcoma cells in vitro nor in the mice model [166]. Boiling-water extracts mainly containing glucose and high-molecular-weight polysaccharide glycans from BCG Connaught [168], BCG Tice [169], and M. vaccae [167,170] demonstrated antitumor activity against murine sarcoma model. Furthermore, products from the digestion in urea of BCG Connaught [168] and M. vaccae [170] were also active against sarcomas. Surprisingly, the fractions obtained from M. vaccae, although active in the murine model, showed no direct antitumor effect in vitro. The authors further studied this phenomenon and designated the macrophages as responsible for the positive immune response orchestrated around the tumor [167]. Meth A fibrosarcoma-bearing mice treated either with radiation or Z-100 experienced a slight tumor inhibition, but when used in combination, the inhibition was improved [92]. Another successful case of Z-100 combined therapy is the administration together with Corynebacterium parvum, which not only made tumors completely regress but also prevented tumor growth if animals were re-challenged with more tumor cells [165]. As regards mycobacterial proteins, fusion proteins between PSA and M. tuberculosis HSP70 or M. bovis HSP65 activated cytotoxic T cell responses, which prevented the implantation of PSA-expressing-MC57 fibrosarcoma cells inoculated in mice. However, no significant differences were observed between the fused plasmid and the single protein PSA plasmid; thus in this case, the presence of the mycobacterial proteins did not contribute to an enhanced cytotoxic response or give an extra tumor-protective advantage [54]. As mentioned before, live BCG or BCG extracts have been widely used as adjuvant therapy in osteosarcoma animal models and patients, and it is known that BCG Connaught exerts its antitumor activity mediated by fibronectin attachment to sarcoma cells [171]. However, a case report of cutaneous TB in a patient with pelvic osteosarcoma treated with live BCG discourages its use due to the immunocompromising status of patients who undergo chemotherapy treatment [172]. Unlike using live BCG, promising results have been obtained in the use of L-MTP-PE. L-MTP-PE lowered the risk of recurrences and death in osteosarcoma patients [71] and eradicated lung metastasis [164]. Moreover, L-MTP-PE in combination with zoledronic acid (a potent inhibitor of bone resorption) showed promising results in xenogeneic and syngeneic mice models of osteosarcoma. The combination therapy induced an additive, and in some cases synergistic, inhibition of primary tumor progression [164,173]. Clinical I and II phases demonstrated the monocyte-mediated cytotoxic activity and an increased production of TNF-α and IL-6 cytokines. Later, a phase III study corroborated the previous results, showing increased overall survival of patients with predominantly non-metastatic and resectable tumors. Due to the satisfactory results and good tolerability of the drug, this treatment was approved and is currently being used in Europe (reviewed in [173]). As regards mesothelioma, the same positive results seen in ovarian cancer (see Section 7) using HSP70 fused to an antibody fragment directed at mesothelin were also observed in malignant mesothelioma-bearing mice [148]. Two studies are based on the intradermal administration of SRL172 in combination with chemotherapy. In the first small clinical trial, a trend towards improved response rate was found in patients receiving both therapies [105]. In the second study, a phase I trial was carried out with intradermal and intratumor SRL172 injection together with chemotherapy. No toxicity was found, and the appropriate dose was established for a further phase II study [163]. It is worth noting that no more studies have been published on this issue. Cancers 2020, 12, 1802 15 of 25 10. Conclusions When mycobacterial agents used for the treatment of non-urological cancers are revised, it can be observed that many laboratories use the whole mycobacterium, mainly in the heat-killed form. Thus, the structural molecules located in the mycobacteria cell wall could be crucial to trigger a favorable antitumor immune response, but also could be relevant for hampering the interaction of other key antigens with the immune system. The presence of some molecules in the cell wall can disturb the immunomostimulatory capacity of antigenic mycobacterial components. For instance, the smooth morphotype of M. vaccae, which possesses a polyester in its cell wall, induces an anti-inflammatory response to compare to the pro-inflammatory profile triggered by the rough morphotype which devoids the polyester [30]. Similarly, BCG substrains differ in their capacity to trigger cytokine production in cancer or immune cells [41,50,174]. The use of different BCG substrains for non-urological cancers is worthy of mention as detailed through this review since it could influence on the reported outcomes as antitumor agent due to their different antigenic profile. Thus, the knowledge of the repertoire of antigenic compounds and their location in each species is relevant for the design of specific therapies for each type of cancer. Other mycobacteria molecules located inside the cell or excreted by the cell, such as Ag85 complex proteins [175] or heat-shock proteins have also been described as potent activators of the immune system. In fact, a great part of the works described in this review are based on the use of different mycobacterial HSPs as vaccine adjuvants. HSPs are able to activate antigen-presenting cells such as dendritic cells, to trigger the production of chemokines and cytokines after binding to different Toll-like receptors; and HSPs by themselves can act as adjuvants due to their capacity to deliver peptides that are recognized by the major histocompatibility complex, priming the adaptative immunity [176–179]. Previous studies have demonstrated the role of heat-killed BCG and M. vaccae to prime the immune system for diverse proteins [178,180]. Therefore, the potential to manipulate the tumor environment with mycobacterial HSPs when administrated together with tumor antigens or even with the whole heat-killed mycobacterium could be considered in the rational design of mycobacteria-based improved immunotherapies. The efficacy of mycobacterium species as immunomodulatory agents is unquestionable. The abovementioned BCG and other mycobacteria species have demonstrated a role in the treatment of different urological cancers, but, outstandingly, no effect of some of these species was reported when evaluated in other types of cancers. This detailed knowledge of the mechanism by which mycobacteria are able to modify tumor microenvironment could even enable the physician to decide the ideal treatment according to the patient conditions. Our research efforts should be addressed to the knowledge of the key immunostimulatory mycobacterial antigens or the rational combination of certain mycobacterial antigens; the study of the host immune response, including also the host genetic background; the knowledge of the tumor microenvironment in each status of the disease; and the finding of appropriate biomarkers to be able to measure the efficacy of the mycobacterial immunotherapy effect in each patient. In view of all that explained above, the link between mycobacteria and cancer treatment has a lot of room for improvement. Author Contributions: Original draft preparation, E.N.-O., S.G.-G., and E.J.; review and editing, E.N.-O., S.G.-G., and E.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Science, Innovation and Universities (RTI2018-098777-B-I00), the FEDER Funds, and the Generalitat of Catalunya (2017SGR-229). SG-G is recipient of a PhD contract (FI) from the Generalitat de Catalunya. Conflicts of Interest: The authors declare no conflict of interest. References 1. Coley, W.B. The treatment of malignant tumors by repeated inoculations of erysipelas: With a report of ten original cases. Clin. Orthop. Relat. Res. 1991, 3–11. Cancers 2020, 12, 1802 16 of 25 2. Coley, W.B. The Treatment of Inoperable Sarcoma by Bacterial Toxins (the Mixed Toxins of the Streptococcus erysipelas and the Bacillus prodigiosus). Proc. R. Soc. Med. 1910, 3, 1–48. [CrossRef] [PubMed] 3. Oelschlaeger, T.A. Bacteria as tumor therapeutics? Bioeng. Bugs 2010, 1, 146–147. [CrossRef] [PubMed] 4. Holmgren, I. Employment of BCG, especially in Intravenous Injection. Acta Med. Scand. 1936, 90, 350–361. [CrossRef] 5. Morton, D.; Eilber, F.R.; Malmgren, R.A.; Wood, W.C. Immunological factors which influence response to immunotherapy in malignant melanoma. Discussion 163–4. Surgery 1970, 68, 158–163. 6. Mathé, G.; Amiel, J.L.; Schwarzenberg, L.; Schneider, M.; Cattan, A.; Schlumberger, J.R.; Hayat, M.; De Vassal, F. Active immunotherapy for acute lymphoblastic leukaemia. Lancet 1969, 1, 697–699. [CrossRef] 7. Zbar, B.; Bernstein, I.; Tanaka, T.; Rapp, H.J. Tumor immunity produced by the intradermal inoculation of living tumor cells and living Mycobacterium bovis (strain BCG). Science 1970, 170, 1217–1218. [CrossRef] 8. Nathanson. Spontaneous regression of malignant melanoma: A review of the literature on incidence, clinical features, and possible mechanisms. Natl. Cancer Inst. Monogr. 1976, 44, 67–76. 9. MacGregor, A.B.; Falk, R.E.; Landi, S.; Ambus, U.; Langer, B. Oral bacille Calmette Guérin immunostimulation in malignant melanoma. Surg. Gynecol. Obstet. 1975, 141, 747–754. 10. Olkowski, Z.L.; McLaren, J.R.; Skeen, M.J. Effects of combined immunotherapy with levamisole and Bacillus Calmette-Guérin on immunocompetence of patients with squamous cell carcinoma of the cervix, head and neck, and lung undergoing radiation therapy. Cancer Treat. Rep. 1978, 62, 1651–1661. 11. McCracken, J.D.; Chen, T.; White, J.; Samson, M.; Stephens, R.; Coltman, C.A.; Saiki, J.; Lane, M.; Bonnet, J.; McGavran, M. Combination chemotherapy, radiotherapy, and BCG immunotherapy in limited small-cell carcinoma of the lung: A Southwest Oncology Group Study. Cancer 1982, 49, 2252–2258. [CrossRef] 12. Bekierkunst, A.; Levij, I.S.; Yarkoni, E. Suppression of urethan-induced lung adenomas in mice treated with trehalose-6,6-dimycolate (cord factor) and living bacillus Calmette Guérin. Science 1971, 174, 1240–1242. [CrossRef] [PubMed] 13. Cappello, F.; Corradi, P.; Meli, G. Use of B.C.G. as loco-regional aspecific immunostimulator in cervical carcinoma. Clin. Exp. Obstet. Gynecol. 1982, 9, 186–188. [PubMed] 14. Pattillo, R.A.; Komaki, R.; Reynolds, M.; Robles, J. Bacillus Calmette-Guérin immunotherapy in ovarian cancer. J. Reprod. Med. 1988, 33, 41–45. 15. Alberts, D.S.; Mason-Liddil, N.; O’Toole, R.V.; Abbott, T.M.; Kronmal, R.; Hilgers, R.D.; Surwit, E.A.; Eyre, H.J.; Baker, L.H. Randomized phase III trial of chemoimmunotherapy in patients with previously untreated stages III and IV suboptimal disease ovarian cancer: A Southwest Oncology Group Study. Gynecol. Oncol. 1989, 32, 8–15. [CrossRef] 16. Wolmark, N.; Fisher, B.; Rockette, H.; Redmond, C.; Wickerham, D.L.; Fisher, E.R.; Jones, J.; Glass, A.; Lerner, H.; Lawrence, W. Postoperative adjuvant chemotherapy or BCG for colon cancer: Results from NSABP protocol C-01. J. Natl. Cancer Inst. 1988, 80, 30–36. [CrossRef] 17. Lustgarten, S. THE BACILLUS OF SYPHILIS. Lancet 1885, 126, 305. [CrossRef] 18. Skerman, V.; McGowan, V.; Sneath, P. Approved Lists of Bacterial Names (Amended); ASM Press: Washington, DC, USA, 1989; ISBN 9781555810146. 19. Shojaei, H.; Heidarieh, P.; Hashemi, A.; Feizabadi, M.M.; Naser, A.D. Species identification of neglected nontuberculous Mycobacteria in a developing country. Jpn. J. Infect. Dis. 2011, 64, 265–271. 20. Khatter, S.; Singh, U.B.; Arora, J.; Rana, T.; Seth, P. Mycobacterial infections in human immuno-deficiency virus seropositive patients: Role of non-tuberculous mycobacteria. Indian J. Tuberc. 2008, 55, 28. 21. de Lima, C.A.M.; Gomes, H.M.; Oelemann, M.A.C.; Ramos, J.P.; Caldas, P.C.; Campos, C.E.D.; Pereira, M.A.d.S.; Montes, F.F.O.; de Oliveira, M.d.S.C.; Suffys, P.N.; et al. Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of rondônia, Brazil. Mem. Inst. Oswaldo Cruz 2013, 108, 457–462. [CrossRef] 22. Aguilar, J.L.; Sanchez, E.E.; Carrillo, C.; Alarcón, G.S.; Silicani, A. Septic arthritis due to Mycobacterium phlei presenting as infantile Reiter’s syndrome. J. Rheumatol. 1989, 16, 1377–1378. [PubMed] 23. Karnam, S.; Alla, V.M.; Kwon, J.; Harbert, T.; Sharma, A.; Airey, K.; Mooss, A. Mycobacterium phlei, a previously unreported cause of pacemaker infection: Thinking outside the box in cardiac device infections. Cardiol. J. 2011, 18, 687–690. [CrossRef] [PubMed] 24. Paul, E.; Devarajan, P. Mycobacterium phlei peritonitis: A rare complication of chronic peritoneal dialysis. Pediatr. Nephrol. 1998, 12, 67–68. [CrossRef] [PubMed] Cancers 2020, 12, 1802 17 of 25 25. Spiegl, P.V.; Feiner, C.M. Mycobacterium phlei Infection of the Foot: A Case Report. Foot Ankle Int. 1994, 15, 680–683. [CrossRef] 26. Tanaka, S.; Hoshino, Y.; Sakagami, T.; Fukano, H.; Matsui, Y.; Hiranuma, O. Pathogenicity of Mycolicibacterium phlei, a non-pathogenic nontuberculous mycobacterium in an immunocompetent host carrying anti-interferon gamma autoantibodies: A case report. BMC Infect. Dis. 2019, 19, 1–6. [CrossRef] 27. Li, R.; Amrhein, J.; Cohen, Z.; Champagne, M.; Kamat, A.M. Efficacy of Mycobacterium Phlei Cell Wall-Nucleic Acid Complex (MCNA) in BCG-Unresponsive Patients. Bladder Cancer 2017, 3, 65–71. [CrossRef] 28. Stanford, J.L.; Stanford, C.A. Immunotherapy of Tuberculosis with Mycobacterium vaccae NCTC 11659. Immunobiology 1994, 191, 555–563. [CrossRef] 29. Tsukamura, M.; Mizuno, S. Mycobacterium obuense, a rapidly growing scotochromogenic mycobacterium capable of forming a black product from p-aminosalicylate and salicylate. J. Gen. Microbiol. 1971, 68, 129–134. [CrossRef] 30. Rodríguez-Güell, E.; Agustí, G.; Corominas, M.; Cardona, P.J.; Casals, I.; Parella, T.; Sempere, M.A.; Luquin, M.; Julián, E. The production of a new extracellular putative long-chain saturated polyester by smooth variants of Mycobacterium vaccae interferes with Th1-cytokine production. Antonie van Leeuwenhoek, Int. J. Gen. Mol. Microbiol. 2006, 90, 93–108. [CrossRef] 31. Gupta, A.; Ahmad, F.J.; Ahmad, F.; Gupta, U.D.; Natarajan, M.; Katoch, V.M.; Bhaskar, S. Protective efficacy of Mycobacterium indicus pranii against tuberculosis and underlying local lung immune responses in guinea pig model. Vaccine 2012, 30, 6198–6209. [CrossRef] 32. Kim, B.J.; Hong, S.H.; Kook, Y.H.; Kim, B.J. Mycobacterium paragordonae sp. nov., a slowly growing, scotochromogenic species closely related to Mycobacterium gordonae. Int. J. Syst. Evol. Microbiol. 2014, 64, 39–45. [CrossRef] 33. Kim, B.-J.; Cha, G.-Y.; Kim, B.-R.; Kook, Y.-H.; Kim, B.-J. Insights From the Genome Sequence of Mycobacterium paragordonae, a Potential Novel Live Vaccine for Preventing Mycobacterial Infections: The Putative Role of Type VII Secretion Systems for an Intracellular Lifestyle Within Free-Living Environmental Predators. Front. Microbiol. 2019, 10, 1524. 34. Kim, B.J.; Kim, B.R.; Kook, Y.H.; Kim, B.J. A temperature sensitive Mycobacterium paragordonae induces enhanced protective immune responses against mycobacterial infections in the mouse model. Sci. Rep. 2017, 7, 1–14. [CrossRef] [PubMed] 35. Luquin, M.; Ausina, V.; Vincent-Levy-Frebault, V.; Laneelle, M.A.; Belda, F.; Garcia-Barcelo, M.; Prats, G.; Daffé, M. Mycobacterium brumae sp. nov., a Rapidly Growing, Nonphotochromogenic Mycobacterium. Int. J. Syst. Bacteriol. 1993, 43, 405–413. [CrossRef] 36. Bach-Griera, M.; Campo-Pérez, V.; Barbosa, S.; Traserra, S.; Guallar-Garrido, S.; Moya-Andérico, L.; Herrero-Abadía, P.; Luquin, M.; Rabanal, R.M.; Torrents, E.; et al. Mycolicibacterium brumae Is a Safe and Non-Toxic Immunomodulatory Agent for Cancer Treatment. Vaccines 2020, 8, 198. [CrossRef] [PubMed] 37. Jiménez, M.S.; Julián, E.; Luquin, M. Misdiagnosis of Mycobacterium brumae infection. J. Clin. Microbiol. 2011, 49, 1190–1192. [CrossRef] [PubMed] 38. Noguera-Ortega, E.; Blanco-Cabra, N.; Rabanal, R.M.; Sánchez-Chardi, A.; Roldán, M.; Guallar-Garrido, S.; Torrents, E.; Luquin, M.; Julián, E. Mycobacteria emulsified in olive oil-in-water trigger a robust immune response in bladder cancer treatment. Sci. Rep. 2016, 6, 1–12. [CrossRef] [PubMed] 39. Noguera-Ortega, E.; Secanella-Fandos, S.; Eraña, H.; Gasión, J.; Rabanal, R.M.; Luquin, M.; Torrents, E.; Julián, E. Nonpathogenic Mycobacterium brumae Inhibits Bladder Cancer Growth In Vitro, Ex Vivo, and In Vivo. Eur. Urol. Focus 2016, 2, 67–76. [CrossRef] 40. Noguera-Ortega, E.; Rabanal, R.M.; Secanella-Fandos, S.; Torrents, E.; Luquin, M.; Julián, E. γ Irradiated Mycobacteria Enhance Survival in Bladder Tumor Bearing Mice although Less Efficaciously than Live Mycobacteria. J. Urol. 2016, 195, 198–205. [CrossRef] 41. Guallar-Garrido, S.; Julián, E. Bacillus Calmette-Guérin (BCG) Therapy for Bladder Cancer: An Update. ImmunoTargets Ther. 2020, 9, 1–11. [CrossRef] 42. Noguera-Ortega, E.; Rabanal, R.M.; Gómez-Mora, E.; Cabrera, C.; Luquin, M.; Julián, E. Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice. Sci. Rep. 2018, 8, 1–9. [CrossRef] [PubMed] Cancers 2020, 12, 1802 18 of 25 43. Tameris, M.; Mearns, H.; Penn-Nicholson, A.; Gregg, Y.; Bilek, N.; Mabwe, S.; Geldenhuys, H.; Shenje, J.; Luabeya, A.K.K.; Murillo, I.; et al. Live-attenuated Mycobacterium tuberculosis vaccine MTBVAC versus BCG in adults and neonates: A randomised controlled, double-blind dose-escalation trial. Lancet Respir. Med. 2019, 7, 757–770. [CrossRef] 44. Alvarez-Arguedas, S.; Uranga, S.; Martín, M.; Elizalde, J.; Gomez, A.B.; Julián, E.; Nardelli-Haefliger, D.; Martín, C.; Aguilo, N. Therapeutic efficacy of the live-attenuated Mycobacterium tuberculosis vaccine, MTBVAC, in a preclinical model of bladder cancer. Transl. Res. 2018, 197, 32–42. [CrossRef] [PubMed] 45. Julián, E.; Noguera-Ortega, E. Bacteria-Derived Alternatives to Live Mycobacterium bovis Bacillus Calmette–Guerin for Nonmuscle Invasive Bladder Cancer Treatment. In Microbial Infections and Cancer Therapy; Jenny Stanford Publishing: Singapore, 2019; pp. 123–188. 46. Noguera-Ortega, E.; Julián, E. Mycobacteria-Derived Agents for the Treatment of Urological and Renal Cancers. In Mycobacterium—Research and Development; InTech: London, UK, 2018. 47. Okamoto, M.; Sato, M. Toll-like receptor signaling in anti-cancer immunity. J. Med. Invest. 2003, 50, 9–24. [PubMed] 48. Higuchi, T.; Shimizu, M.; Owaki, A.; Takahashi, M.; Shinya, E.; Nishimura, T.; Takahashi, H. A possible mechanism of intravesical BCG therapy for human bladder carcinoma: Involvement of innate effector cells for the inhibition of tumor growth. Cancer Immunol. Immunother. 2009, 58, 1245–1255. [CrossRef] [PubMed] 49. LaRue, H.; Ayari, C.; Bergeron, A.; Fradet, Y. Toll-like receptors in urothelial cells—Targets for cancer immunotherapy. Nat. Rev. Urol. 2013, 10, 537–545. [CrossRef] 50. Pettenati, C.; Ingersoll, M.A. Mechanisms of BCG immunotherapy and its outlook for bladder cancer. Nat. Rev. Urol. 2018, 15, 615–625. [CrossRef] 51. Wang, L.; Li, H.; Yu, Y.; Sun, L.; Wang, H.; Zhang, P.; Wei, H.; Weng, L.; Wan, M.; Cao, Z.; et al. Vaccination with B16 tumor cell lysate plus recombinant Mycobacterium tuberculosis Hsp70 induces antimelanoma effect in mice. Cancer Biother. Radiopharm. 2010, 25, 185–191. 52. Zeng, S.; Liu, Q.; Wang, S.; Peng, X.; Zhang, J.; Zhang, J. Intratumor injection of recombinant attenuated salmonella carrying Mycobacterium tuberculosis heat shock protein 70 and herpes simplex virus thymidine kinase genes to suppress murine melanoma growth. Nan Fang Yi Ke Da Xue Xue Bao 2012, 32, 101–105. 53. Borges, T.J.; Porto, B.N.; Teixeira, C.A.; Rodrigues, M.; Machado, F.D.; Ornaghi, A.P.; de Souza, A.P.D.; Maito, F.; Pavanelli, W.R.; Silva, J.S.; et al. Prolonged survival of allografts induced by mycobacterial Hsp70 is dependent on CD4+CD25+ regulatory T cells. PLoS ONE 2010, 5, e14264. [CrossRef] 54. Pavlenko, M.; Roos, A.-K.; Leder, C.; Hansson, L.-O.; Kiessling, R.; Levitskaya, E.; Pisa, P. Comparison of PSA-specific CD8+ CTL responses and antitumor immunity generated by plasmid DNA vaccines encoding PSA-HSP chimeric proteins. Cancer Immunol. Immunother. 2004, 53, 1085–1092. [CrossRef] [PubMed] 55. Yan, Y.; Cao, Z.; Yang, M.; Li, H.; Wei, H.; Fu, Y.; Song, D.; Wang, L.; Yu, Y. A CpG oligodeoxynucleotide potentiates the anti-tumor effect of HSP65-Her2 fusion protein against Her2 positive B16 melanoma in mice. Int. Immunopharmacol. 2012, 12, 402–407. [CrossRef] 56. Li, D.; Li, H.; Zhang, P.; Wu, X.; Wei, H.; Wang, L.; Wan, M.; Deng, P.; Zhang, Y.; Wang, J.; et al. Heat shock fusion protein induces both specific and nospecific anti-tumor immunity. Eur. J. Immunol. 2006, 36, 1324–1336. [CrossRef] [PubMed] 57. Yang, M.; Yan, Y.; Fang, M.; Wan, M.; Wu, X.; Zhang, X.; Zhao, T.; Wei, H.; Song, D.; Wang, L.; et al. MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1+ tumor immunity in mice. Int. Immunopharmacol. 2012, 13, 408–416. [CrossRef] [PubMed] 58. Kim, D.H.; Moon, C.; Oh, S.-S.; Park, S.; Jeong, J.-W.; Kim, S.; Lee, H.G.; Kwon, H.-J.; Kim, K.D. Liposome-Encapsulated CpG Enhances Antitumor Activity Accompanying the Changing of Lymphocyte Populations in Tumor via Intratumoral Administration. Nucleic Acid Ther. 2015, 25, 95–102. [CrossRef] [PubMed] 59. Dong, B.; Dai, G.; Qi, Z.; Yang, G.; Lu, J.; Zhang, Y. Effect of MHSP65-TCL anti-melanoma vaccine on the activity of immunocytes. Nan Fang Yi Ke Da Xue Xue Bao 2013, 33, 1673–1677. 60. Tarrant, J.P.; Walsh, M.J.; Blanchard, M.C.; Lee, T.D.G.; Hoskin, D.W.; Giacomantonio, C.A. Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A. Int. J. Oncol. 2004, 25, 1693–1699. [CrossRef] Cancers 2020, 12, 1802 19 of 25 61. Sfondrini, L.; Morelli, D.; Ménard, S.; Maier, J.; Singh, M.; Melani, C.; Terrazzini, N.; Colombo, M.; Colnaghi, M.; Balsari, A. Anti-tumor immunity induced by murine melanoma cells transduced with the Mycobacterium tuberculosis gene encoding the 38-kDa antigen. Gene Ther. 1998, 5, 247–252. [CrossRef] 62. He, X.; Wang, J.; Zhao, F.; Yu, F.; Chen, D.; Cai, K.; Yang, C.; Chen, J.; Dou, J. Antitumor efficacy of viable tumor vaccine modified by heterogenetic ESAT-6 antigen and cytokine IL-21 in melanomatous mouse. Immunol. Res. 2012, 52, 240–249. [CrossRef] 63. He, X.; Wang, J.; Zhao, F.; Chen, D.; Chen, J.; Zhang, H.; Yang, C.; Liu, Y.; Dou, J. ESAT-6-gpi DNA Vaccine Augmented the Specific Antitumour Efficacy Induced by the Tumour Vaccine B16F10-ESAT-6-gpi/IL-21 in a Mouse Model. Scand. J. Immunol. 2013, 78, 69–78. [CrossRef] 64. Koyama, Y.; Yoshihara, C.; Ito, T. Novel antitumor strategy utilizing a plasmid expressing a Mycobacterium tuberculosis antigen as a “danger signal” to block immune escape of tumor cells. Pharmaceutics 2015, 7, 165–174. [CrossRef] [PubMed] 65. Koyama, Y.; Ito, T.; Hasegawa, A.; Eriguchi, M.; Inaba, T.; Ushigusa, T.; Sugiura, K. Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: A novel vaccine for cancer therapy. Biotechnol. Lett. 2016, 38, 1857–1866. [CrossRef] [PubMed] 66. Ushigusa, T.; Koyama, Y.; Ito, T.; Watanabe, K.; Chambers, J.K.; Hasegawa, A.; Uchida, K.; Kanegi, R.; Hatoya, S.; Inaba, T.; et al. Innate immunity mediated by dendritic cells/macrophages plays a central role in the early period in tumor treatment using gene of Mycobacterium tuberculosis antigen. J. Vet. Med. Sci. 2018, 80, 190–196. [CrossRef] [PubMed] 67. Oka, H.; Emori, Y.; Ohya, O.; Kobayashi, N.; Sasaki, H.; Tanaka, Y.; Hayashi, Y.; Nomoto, K. An immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, Z-100, restores the balance of Th1/Th2 cell responses in tumor bearing mice. Immunol. Lett. 1999, 70, 109–117. [CrossRef] 68. Oka, H.; Emori, Y.; Sasaki, H.; Shiraishi, Y.; Yoshinaga, K.; Kurimoto, T. Anti-tumor mechanism of Z-100, an immunomodulatory Arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, on pulmonary metastases of B16F10 melanoma: Restoration of helper T cell responses via suppression of glucocorticoid-genesis. Microbiol. Immunol. 2002, 46, 343–351. [CrossRef] 69. Oka, H.; Sasaki, H.; Shiraishi, Y.; Emori, Y.; Yoshinaga, K.; Takei, M. Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, augments anti-tumor activities of X-ray irradiation against B16 melanoma in association with the improvement of type 1T cell responses. Biol. Pharm. Bull. 2004, 27, 82–88. [CrossRef] 70. Horii, T.; Yoshinaga, K.; Kobayashi, N.; Seto, K.; Orikawa, Y.; Okamoto, M.; Eta, R.; Ohira, Y.; Katsunuma, K.; Hori, Y.; et al. Z-100, an immunomodulatory extract of Mycobacterium tuberculosis strain Aoyama B, prevents spontaneous lymphatic metastasis of B16-BL6 melanoma. Biol. Pharm. Bull. 2014, 37, 642–647. [CrossRef] 71. Meyers, P.A.; Chou, A.J. Muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE) in the treatment of osteosarcoma. Adv. Exp. Med. Biol. 2014, 804, 307–321. 72. MacEwen, E.G.; Kurzman, I.D.; Vail, D.M.; Dubielzig, R.R.; Everlith, K.; Madewell, B.R.; Rodriguez, C.O.; Phillips, B.; Zwahlen, C.H.; Obradovich, J.; et al. Adjuvant therapy for melanoma in dogs: Results of randomized clinical trials using surgery, liposome-encapsulated muramyl tripeptide, and granulocyte macrophage colony-stimulating factor. Clin. Cancer Res. 1999, 5, 4249–4258. 73. Gianan, M.A.V.; Kleinerman, E.S. Liposomal Muramyl Tripeptide (CGP 19835A Lipid) Therapy for Resectable Melanoma in Patients Who Were at High Risk for Relapse: An Update. Cancer Biother. Radiopharm. 1998, 13, 363–368. [CrossRef] 74. Fang, F.; Ma, J.; Ni, W.; Wang, F.; Sun, X.; Li, Y.; Li, Q.; Xie, F.; Wang, J.; Zhai, R.; et al. MUC1 and maltose-binding protein recombinant fusion protein combined with Bacillus Calmette-Guerin induces MUC1-specific and nonspecific anti-tumor immunity in mice. Mol. Med. Rep. 2014, 10, 1056–1064. [CrossRef] [PubMed] 75. Yung, C.Y.; Hata, K.; Kyung, B.L.; Azuma, I. Inhibitory effect of BCG cell-wall skeletons (BCG-CWS) emulsified in squalane on tumor growth and metastasis in mice. Arch. Pharm. Res. 2002, 25, 522–527. 76. Saqib, M.; Khatri, R.; Singh, B.; Gupta, A.; Bhaskar, S. Cell wall fraction of Mycobacterium indicus pranii shows potential Th1 adjuvant activity. Int. Immunopharmacol. 2019, 70, 408–416. [CrossRef] [PubMed] 77. Ahmad, F.; Mani, J.; Kumar, P.; Haridas, S.; Upadhyay, P.; Bhaskar, S. Activation of anti-tumor immune response and reduction of regulatory T cells with Mycobacterium indicus pranii (MIP) therapy in tumor bearing mice. PLoS ONE 2011, 6, e25424. [CrossRef] Cancers 2020, 12, 1802 20 of 25 78. Maraveyas, A.; Baban, B.; Kennard, D.A.; Rook, G.A.W.; Westby, M.; Grange, J.M.; Lydyard, P.; Stanford, J.L.; Jones, M.; Selby, P.J.; et al. Possible improved survival of patients with stage IV AJCC melanoma receiving SRL 172 immunotherapy: Correlation with induction of increased levels of intracellular interleukin-2 in peripheral blood lymphocytes. Ann. Oncol. 1999, 10, 817–824. [CrossRef] [PubMed] 79. Nicholson, S.; Guile, K.; John, J.; Clarke, I.A.; Diffley, J.; Donnellan, P.; Michael, A.; Szlosarek, P.; Dalgleish, A.G. A randomized phase II trial of SRL172 (Mycobacterium vaccae) +/- low-dose interleukin-2 in the treatment of metastatic malignant melanoma. Melanoma Res. 2003, 13, 389–393. [CrossRef] [PubMed] 80. Cananzi, F.C.M.; Mudan, S.; Dunne, M.; Belonwu, N.; Dalgleish, A.G. Long-term survival and outcome of patients originally given Mycobacterium vaccae for metastatic malignant melanoma. Hum. Vaccin. Immunother. 2013, 9, 2427–2433. [CrossRef] [PubMed] 81. Fowler, D.; Dalgleish, A.; Liu, W. A heat-killed preparation of Mycobacterium obuense can reduce metastatic burden in vivo. J. Immunother. Cancer 2014, 2, P54. [CrossRef] 82. Stebbing, J.; Dalgleish, A.; Gifford-Moore, A.; Martin, A.; Gleeson, C.; Wilson, G.; Brunet, L.R.; Grange, J.; Mudan, S. An intra-patient placebo-controlled phase I trial to evaluate the safety and tolerability of intradermal IMM-101 in melanoma. Ann. Oncol. 2012, 23, 1314–1319. [CrossRef] 83. Dalgleish, A.G.; Mudan, S.; Fusi, A. Enhanced effect of checkpoint inhibitors when given after or together with IMM-101: Significant responses in four advanced melanoma patients with no additional major toxicity. J. Transl. Med. 2018, 16, 227. [CrossRef] 84. Kumar, P.; Das, G.; Bhaskar, S. Mycobacterium indicus pranii therapy induces tumor regression in MyD88- and TLR2-dependent manner. BMC Res. Notes 2019, 12, 1–5. [CrossRef] [PubMed] 85. Halder, K.; Banerjee, S.; Ghosh, S.; Bose, A.; Das, S.; Chowdhury, B.P.; Majumdar, S. Mycobacterium indicus pranii ( Mw ) inhibits invasion by reducing matrix metalloproteinase (MMP-9) via AKT/ERK-1/2 and PKCα signalling: A potential candidate in melanoma cancer therapy. Cancer Biol. Ther. 2015, 18, 850–862. [CrossRef] [PubMed] 86. Kuhn, S.; Hyde, E.J.; Yang, J.; Rich, F.J.; Harper, J.L.; Kirman, J.R.; Ronchese, F. Increased Numbers of Monocyte-Derived Dendritic Cells during Successful Tumor Immunotherapy with Immune-Activating Agents. J. Immunol. 2013, 191, 1984–1992. [CrossRef] [PubMed] 87. Lardone, R.D.; Chan, A.A.; Lee, A.F.; Foshag, L.J.; Faries, M.B.; Sieling, P.A.; Lee, D.J. Mycobacterium bovis Bacillus Calmette–Guérin Alters melanoma microenvironment Favoring antitumor T cell responses and improving M2 Macrophage Function. Front. Immunol. 2017, 8, 1–14. [CrossRef] [PubMed] 88. Yang, J.; Jones, M.S.; Irene Ramos, R.; Chan, A.A.; Lee, A.F.; Foshag, L.J.; Sieling, P.A.; Faries, M.B.; Lee, D.J. Insights into local tumor microenvironment immune factors associated with regression of cutaneous melanoma metastases by Mycobacterium bovis Bacille Calmette-Guérin. Front. Oncol. 2017, 7, 1–13. [CrossRef] 89. Darvin, P.; Toor, S.M.; Sasidharan Nair, V.; Elkord, E. Immune checkpoint inhibitors: Recent progress and potential biomarkers. Exp. Mol. Med. 2018, 50, 1–11. [CrossRef] 90. Duarte, J.D.G.; Parakh, S.; Andrews, M.C.; Woods, K.; Pasam, A.; Tutuka, C.; Ostrouska, S.; Blackburn, J.M.; Behren, A.; Cebon, J. Autoantibodies may predict immune-related toxicity: Results from a phase I study of intralesional Bacillus Calmette-Guérin followed by ipilimumab in patients with advanced metastatic melanoma. Front. Immunol. 2018, 9, 411. [CrossRef] 91. Lotem, M.; Merims, S.; Frank, S.; Hamburger, T.; Nissan, A.; Kadouri, L.; Cohen, J.; Straussman, R.; Eisenberg, G.; Frankenburg, S.; et al. Adjuvant Autologous Melanoma Vaccine for Macroscopic Stage III Disease: Survival, Biomarkers, and Improved Response to CTLA-4 Blockade. J. Immunol. Res. 2016, 2016, 1–12. [CrossRef] 92. Hayashi, Y.; Sasaki, H.; Emori, Y.; Nomoto, K. The effect of combination therapy of radiation and Z-100, an arabinomannan on tumor growth in mice. Biotherapy 1993, 7, 63–69. [CrossRef] 93. Shi, Y.-L.; Bao, L.; Shang, Z.-L.; Yao, S.-X. RelE toxin protein of Mycobacterium tuberculosis induces growth inhibition of lung cancer A-549 cell. Sichuan Da Xue Xue Bao. Yi Xue Ban 2008, 39, 368–372. 94. Murata, M. Activation of Toll-like receptor 2 by a novel preparation of cell wall skeleton from Mycobacterium bovis BCG Tokyo (SMP-105) sufficiently enhances immune responses against tumors. Cancer Sci. 2008, 99, 1435–1440. [CrossRef] [PubMed] Cancers 2020, 12, 1802 21 of 25 95. Nakajima, H.; Kawasaki, K.; Oka, Y.; Tsuboi, A.; Kawakami, M.; Ikegame, K.; Hoshida, Y.; Fujiki, F.; Nakano, A.; Masuda, T.; et al. WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone. Cancer Immunol. Immunother. 2004, 53, 617–624. [CrossRef] [PubMed] 96. Grant, S.C.; Kris, M.G.; Houghton, A.N.; Chapman, P.B. Long survival of patients with small cell lung cancer after adjuvant treatment with the anti-idiotypic antibody BEC2 plus Bacillus Calmette-Guérin. Clin. Cancer Res. 1999, 5, 1319–1323. 97. Giaccone, G.; Debruyne, C.; Felip, E.; Chapman, P.B.; Grant, S.C.; Millward, M.; Thiberville, L.; D’addario, G.; Coens, C.; Rome, L.S.; et al. Phase III Study of Adjuvant Vaccination With Bec2/Bacille Calmette-Guerin in Responding Patients With Limited-Disease Small-Cell Lung Cancer (European Organisation for Research and Treatment of Cancer 08971-08971B.; Silva Study). J. Clin. Oncol. 2005, 23, 6854–6864. [CrossRef] [PubMed] 98. Bottomley, A.; Debruyne, C.; Felip, E.; Millward, M.; Thiberville, L.; Addario, G.D.; Rome, L.; Zatloukal, P.; Coens, C.; Giaccone, G. Symptom and quality of life results of an international randomised phase III study of adjuvant vaccination with Bec2/BCG in responding patients with limited disease small-cell lung cancer. Eur. J. Cancer 2008, 44, 2178–2184. [CrossRef] 99. Zhang, Q.; Ni, W.; Zhao, X.; Wang, F.; Gao, Z.; Tai, G. Synergistic antitumor effects of Escherichia coli maltose binding protein and Bacillus Calmette-Guerin in a mouse lung carcinoma model. Immunol. Lett. 2011, 136, 108–113. [CrossRef] [PubMed] 100. Subramaniam, M.; In, L.L.A.; Kumar, A.; Ahmed, N.; Nagoor, N.H. Cytotoxic and apoptotic effects of heat killed Mycobacterium indicus pranii (MIP) on various human cancer cell lines. Sci. Rep. 2016, 6, 19833. [CrossRef] 101. Sur, P.K.; Dastidar, A.G. Role of mycobacterium w as adjuvant treatment of lung cancer (non-small cell lung cancer). J. Indian Med. Assoc. 2003, 101, 120. 102. Belani, C.P.; Chakraborty, B.; Desai, D.; Khamar, B.M. Randomized multicenter phase ii clinical trial of a toll like receptor-2 (TLR-2) agonist mycobacterium w (CADI-05) in combination with paclitaxel plus cisplatin versus paclitaxel plus cisplatin in advanced nonsmall cell lung cancer (NSCLC). J. Thorac. Oncol. 2011, 6, S468–S469. 103. Hrouda, D.; Baban, B.; Dunsmuir, W.D.; Kirby, R.S.; Dalgleish, A.G. Immunotherapy of advanced prostate cancer: A phase I/II trial using Mycobacterium vaccae (SRL172). Br. J. Urol. 1998, 82, 568–573. [CrossRef] 104. Eaton, J.D.; Perry, M.J.A.; Nicholson, S.; Guckian, M.; Russell, N.; Whelan, M.; Kirby, R.S. Allogeneic whole-cell vaccine: A phase I/II study in men with hormone-refractory prostate cancer. BJU Int. 2008, 89, 19–26. [CrossRef] 105. O’Brien, M.E.R.; Saini, A.; Smith, I.E.; Webb, A.; Gregory, K.; Mendes, R.; Ryan, C.; Priest, K.; Bromelow, K.V.; Palmer, R.D.; et al. A randomized phase II study of SRL172 (Mycobacterium vaccae) combined with chemotherapy in patients with advanced inoperable non-small-cell lung cancer and mesothelioma. Br. J. Cancer 2000, 83, 853–857. [CrossRef] [PubMed] 106. Assersohn, L.; Souberbielle, B.E.; O’Brien, M.E.R.; Archer, C.D.; Mendes, R.; Bass, R.; Bromelow, K.V.; Palmer, R.D.; Bouilloux, E.; Kennard, D.A.; et al. A randomized pilot study of SRL172 (Mycobacterium vaccae) in patients with small cell lung cancer (SCLC) treated with chemotherapy. Clin. Oncol. R. Coll. Radiol. 2002, 14, 23–27. [CrossRef] [PubMed] 107. Harper-Wynne, C.L.; Sumpter, K.; Ryan, C.; Priest, K.; Norton, A.; Ross, P.; Ford, H.E.R.; Johnson, P.; O’Brien, M.E.R. Addition of SRL 172 to standard chemotherapy in small cell lung cancer (SCLC) improves symptom control. Lung Cancer 2005, 47, 289–290. [CrossRef] 108. O’Brien, M.E.R.; Anderson, H.; Kaukel, E.; O’Byrne, K.; Pawlicki, M.; Von Pawel, J.; Reck, M. SR-ON-12 Study Group SRL172 (killed Mycobacterium vaccae) in addition to standard chemotherapy improves quality of life without affecting survival, in patients with advanced non-small-cell lung cancer: Phase III results. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. 2004, 15, 906–914. [CrossRef] 109. Stanford, J.L.L.; Stanford, C.A.A.; O’Brien, M.E.R.; Grange, J.M.M.; O’Brien, M.E.R.; Grange, J.M.M. Successful immunotherapy with Mycobacterium vaccae in the treatment of adenocarcinoma of the lung. Eur. J. Cancer 2008, 44, 224–227. [CrossRef] 110. Fowler, D.W.; Copier, J.; Wilson, N.; Dalgleish, A.G.; Bodman-Smith, M.D. Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: A mechanism of action for cancer immunotherapy. Cancer Immunol. Immunother. 2012, 61, 535–547. [CrossRef] Cancers 2020, 12, 1802 22 of 25 111. Jian, W.; Li, X.; Kang, J.; Lei, Y.; Bai, Y.; Xue, Y. Antitumor effect of recombinant Mycobacterium smegmatis expressing MAGEA3 and SSX2 fusion proteins. Exp. Ther. Med. 2018, 16, 2160–2166. [CrossRef] 112. Popiela, T.; Kulig, J.; Czupryna, A.; Szczepanik, A.M.; Zembala, M. Efficiency of adjuvant immunochemotherapy following curative resection in patients with locally advanced gastric cancer. Gastric Cancer 2004, 7, 240–245. [CrossRef] 113. Pei, Q.; Pan, J.; Ding, X.; Wang, J.; Zou, X.; Lv, Y. Gemcitabine sensitizes pancreatic cancer cells to the CTLs antitumor response induced by BCG-stimulated dendritic cells via a Fas-dependent pathway. Pancreatology 2015, 15, 233–239. [CrossRef] 114. Xuan, W.; Yan, Y.; Wan, M.; Wu, X.; Ji, D.; Wang, L.; Lin, C.; Chen, Y.; Yu, Y.; Zhang, X. Antitumor activity of mHSP65-TTL enhanced by administration of low dose cyclophosphamide in pancreatic cancer-bearing mice. Int. Immunopharmacol. 2015, 27, 95–103. [CrossRef] [PubMed] 115. Bilyard, H.; Mines, C.; Brunet, L.; Dalgleish, A.; Macintosh, F. IMM-101, an immunotherapeutic agent in clinical development as an adjunctive treatment for pancreatic cancer. J. Immunother. Cancer 2014, 2, P83. [CrossRef] 116. Dalgleish, A.G.; Stebbing, J.; Adamson, D.J.; Arif, S.S.; Bidoli, P.; Chang, D.; Cheeseman, S.; Diaz-Beveridge, R.; Fernandez-Martos, C.; Glynne-Jones, R.; et al. Randomised, open-label, phase II study of gemcitabine with and without IMM-101 for advanced pancreatic cancer. Br. J. Cancer 2016, 115, 789–796. [CrossRef] [PubMed] 117. Bazzi, S.; Modjtahedi, H.; Mudan, S.; Achkar, M.; Akle, C.; Bahr, G.M. Immunomodulatory effects of heat-killed Mycobacterium obuense on human blood dendritic cells. Innate Immun. 2017, 23, 592–605. [CrossRef] 118. Bazzi, S.; El-Darzi, E.; McDowell, T.; Modjtahedi, H.; Mudan, S.; Achkar, M.; Akle, C.; Kadara, H.; Bahr, G.M. Defining genome-wide expression and phenotypic contextual cues in macrophages generated by granulocyte/macrophage colony-stimulating factor, macrophage colony-stimulating factor, and heat-killed mycobacteria. Front. Immunol. 2017, 8, 1253. [CrossRef] 119. Yuk, J.-M.; Shin, D.-M.; Song, K.-S.; Lim, K.; Kim, K.-H.; Lee, S.-H.; Kim, J.-M.; Lee, J.-S.; Paik, T.-H.; Kim, J.-S.; et al. Bacillus Calmette-Guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy. Autophagy 2010, 6, 46–60. [CrossRef] 120. Woodlock, T.J.; Sahasrabudhe, D.M.; Marquis, D.M.; Greene, D.; Pandya, K.J.; McCune, C.S. Active specific immunotherapy for metastatic colorectal carcinoma: Phase I study of an allogeneic cell vaccine plus low-dose interleukin-1 alpha. J. Immunother. 1999, 22, 251–259. [CrossRef] 121. Vo, M.-C.; Lee, H.-J.; Kim, J.-S.; Hoang, M.-D.; Choi, N.-R.; Rhee, J.H.; Lakshmanan, V.-K.; Shin, S.-J.; Lee, J.-J. Dendritic cell vaccination with a toll-like receptor agonist derived from mycobacteria enhances anti-tumor immunity. Oncotarget 2015, 6, 33781–33790. [CrossRef] 122. Lee, S.Y.; Yang, S.B.; Choi, Y.M.; Oh, S.J.; Kim, B.J.; Kook, Y.H.; Kim, B.J. Heat-killed Mycobacterium paragordonae therapy exerts an anti-cancer immune response via enhanced immune cell mediated oncolytic activity in xenograft mice model. Cancer Lett. 2020, 472, 142–150. [CrossRef] 123. Gao, J.; Luo, S.M.; Peng, M.L.; Deng, T. Enhanced immunity against hepatoma induced by dendritic cells pulsed with Hsp70-H22 peptide complexes and CD40L. J. Cancer Res. Clin. Oncol. 2012, 138, 917–926. [CrossRef] 124. Xu, M.; Zhou, L.; Zhang, Y.; Xie, Z.; Zhang, J.; Guo, L.; Wang, C.; Yang, X. A Fixed Human Umbilical Vein Endothelial Cell Vaccine With 2 Tandem Repeats of Microbial HSP70 Peptide Epitope 407-426 As Adjuvant for Therapy of Hepatoma in Mice. J. Immunother. 2015, 38, 276–284. [CrossRef] [PubMed] 125. Zhang, Y.; Xu, J.; Zhao, R.; Liu, J.; Wu, J. Inhibition effects on liver tumors of BALB/c mice bearing H22 cells by immunization with a recombinant immunogen of GnRH linked to heat shock protein 65. Vaccine 2007, 25, 6911–6921. [CrossRef] 126. Xiangbing, H.; Yankai, Z.; Ming, L.; Yong, L.; Yu, Z.; Huiyong, Z.; Yingying, C.; Jing, H.; Yun, X.; Liang, J.; et al. The fusion protein of HSP65 with tandem repeats of beta-hCG acting as a potent tumor vaccine in suppressing hepatocarcinoma. Int. Immunopharmacol. 2010, 10, 230–238. [CrossRef] [PubMed] 127. Ahsan, C.R.; Sasaki, J. A 64 kDa protein from Mycobacterium bovis BCG shares the same antigenic determinants with line 10 hepatoma cells and has anti-line 10 tumor activity. FEBS Lett. 1991, 288, 77–80. [CrossRef] 128. Ahsan, C.R.; Sasaki, J. ichi The Mycobacterium bovis BCG 64-kDa surface protein is antigenically shared with different mouse tumor cells and has anti-tumor activity in immunized mice. Immunol. Lett. 1993, 36, 235–238. [CrossRef] Cancers 2020, 12, 1802 23 of 25 129. Miyauchi, M.; Murata, M.; Fukushima, A.; Sato, T.; Nakagawa, M.; Tetsuya, F.; Koseki, N.; Chiba, N.; Kashiwazaki, Y. Optimization of cell-wall skeleton derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105) emulsion in delayed-type hypersensitivity and antitumor models. Drug Discov. Ther. 2012, 6, 218–225. [CrossRef] 130. Kashiwazaki, Y.; Murata, M.; Sato, T.; Miyauchi, M.; Nakagawa, M.; Fukushima, A.; Chiba, N.; Azuma, I.; Yamaoka, T. Injection of cell-wall skeleton of Mycobacterium bovis BCG draining to a sentinel lymph node eliminates both lymph node metastases and the primary transplanted tumor. Drug Discov. Ther. 2008, 2, 168–177. 131. Kataoka, T.; Yamamoto, S.; Yamamoto, T.; Tokunaga, T. Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose. Jpn. J. Med. Sci. Biol. 1990, 43, 171–182. [CrossRef] 132. Abei, M.; Okumura, T.; Fukuda, K.; Hashimoto, T.; Araki, M.; Ishige, K.; Hyodo, I.; Kanemoto, A.; Numajiri, H.; Mizumoto, M.; et al. A phase I study on combined therapy with proton-beam radiotherapy and in situ tumor vaccination for locally advanced recurrent hepatocellular carcinoma. Radiat. Oncol. 2013, 8, 239. [CrossRef] 133. Chung, M.A.; Luo, Y.; O’Donnell, M.; Rodriguez, C.; Heber, W.; Sharma, S.; Chang, H.R. Development and preclinical evaluation of a Bacillus Calmette-Guérin-MUC1-based novel breast cancer vaccine. Cancer Res. 2003, 63, 1280–1287. 134. Yuan, S.; Shi, C.; Ling, R.; Wang, T.; Wang, H.; Han, W. Immunization with two recombinant Bacillus Calmette-Guérin vaccines that combine the expression of multiple tandem repeats of mucin-1 and colony stimulating-factor suppress breast tumor growth in mice. J. Cancer Res. Clin. Oncol. 2010, 136, 1359–1367. [CrossRef] [PubMed] 135. Yuan, S.; Shi, C.; Lv, Y.; Wang, T.; Wang, H.; Han, W. A Novel Bacillus Calmette-Guérin -based Breast Cancer Vaccine that Coexpresses Multiple Tandem Repeats of MUC1 and CD80 Breaks the Immune Tolerance and Inhibits MUC1-Positive Breast Cancer Growth. Cancer Biother. Radiopharm. 2009, 24, 607–613. [CrossRef] 136. Wiseman, C.L. Inflammatory breast cancer: 10-year follow-up of a trial of surgery, chemotherapy, and allogeneic tumor cell/BCG immunotherapy. Cancer Invest. 1995, 13, 267–271. [CrossRef] 137. Montesinos, J.C.H.; Romero, H.O.G. Autologous tumor lysate / BacillusCalmette–Guérin immunotherapy as an adjuvant to conventional breast cancer therapy. Clin. Transl. Oncol. 2015, 17, 3–6. 138. Sfondrini, L.; Rodolfo, M.; Singh, M.; Colombo, M.P.; Colnaghi, M.I.; Ménard, S.; Balsari, A. Cooperative effects of Mycobacterium tuberculosis Ag38 gene transduction and interleukin 12 in vaccination against spontaneous tumor development in proto-neu transgenic mice. Cancer Res. 2000, 60, 3777–3781. [PubMed] 139. Yi, H.; Rong, Y.; Yankai, Z.; Wentao, L.; Hongxia, Z.; Jie, W.; Rongyue, C.; Taiming, L.; Jingjing, L. Improved efficacy of DNA vaccination against breast cancer by boosting with the repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65. Vaccine 2006, 24, 2575–2584. [CrossRef] [PubMed] 140. Wang, X.J.; Gu, K.; Xu, J.S.; Li, M.H.; Cao, R.Y.; Wu, J.; Li, T.M.; Liu, J.J. Immunization with a recombinant GnRH vaccine fused to heat shock protein 65 inhibits mammary tumor growth in vivo. Cancer Immunol. Immunother. 2010, 59, 1859–1866. [CrossRef] [PubMed] 141. Huo, Y.; Li, B.; Zhang, Y.; Wang, S.; Bao, M.; Gao, X.; Li, D.; Wang, L.; Yu, Y.; Wang, J. Pre-clinical safety evaluation of heat shock protein 65-MUC1 peptide fusion protein. Regul. Toxicol. Pharmacol. 2007, 49, 63–74. [CrossRef] 142. Altundag, K.; Mohamed, A.-S.; Altundag, O.; Silay, Y.S.; Gunduz, E.; Demircan, K. SRL172 (killed Mycobacterium vaccae) may augment the efficacy of trastuzumab in metastatic breast cancer patients. Med. Hypotheses 2005, 64, 248–251. [CrossRef] 143. Korbelik, M.; Cecic, I. Enhancement of tumour response to photodynamic therapy by adjuvant mycobacterium cell-wall treatment. J. Photochem. Photobiol. B Biol. 1998, 44, 151–158. [CrossRef] 144. Garg, H.; Gupta, J.C.; Talwar, G.P.; Dubey, S. Immunotherapy approach with recombinant survivin adjuvanted with alum and MIP suppresses tumor growth in murine model of breast cancer. Prep. Biochem. Biotechnol. 2018, 48, 264–269. [CrossRef] [PubMed] 145. Subramaniam, M.; Arshad, N.M.; Mun, K.S.; Malagobadan, S.; Awang, K.; Nagoor, N.H. Anti-Cancer Effects of Synergistic Drug-Bacterium Combinations on Induced Breast Cancer in BALB/c Mice. Biomolecules 2019, 9, 626. [CrossRef] [PubMed] Cancers 2020, 12, 1802 24 of 25 146. Hayashi, A.; Nishida, Y.; Yoshii, S.; Kim, S.Y.; Uda, H.; Hamasaki, T. Immunotherapy of ovarian cancer with cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guérin: Effect of lymphadenectomy. Cancer Sci. 2009, 100, 1991–1995. [CrossRef] [PubMed] 147. Gottschalk, N.; Lang, S.; Kimmig, R.; Singh, M.; Brandau, S. Monocytes and the 38kDa-antigen of Mycobacterium tuberculosis modulate natural killer cell activity and their cytolysis directed against ovarian cancer cell lines. BMC Cancer 2012, 12, 451. [CrossRef] [PubMed] 148. Yuan, J.; Kashiwagi, S.; Reeves, P.; Nezivar, J.; Yang, Y.; Arrifin, N.; Nguyen, M.; Jean-Mary, G.; Tong, X.; Uppal, P.; et al. A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma. J. Hematol. Oncol. 2014, 7, 15. [CrossRef] [PubMed] 149. Hsu, K.-F.; Hung, C.-F.; Cheng, W.-F.; He, L.; Slater, L.A.; Ling, M.; Wu, T.-C. Enhancement of suicidal DNA vaccine potency by linking Mycobacterium tuberculosis heat shock protein 70 to an antigen. Gene Ther. 2001, 8, 376–383. [CrossRef] 150. Zong, J.; Peng, Q.; Wang, Q.; Zhang, T.; Fan, D.; Xu, X. Human HSP70 and modified HPV16 E7 fusion DNA vaccine induces enhanced specific CD8+ T cell responses and anti-tumor effects. Oncol. Rep. 2009, 22, 953–961. 151. Daniel, D.; Chiu, C.; Giraudo, E.; Inoue, M.; Mizzen, L.A.; Chu, N.R.; Hanahan, D. CD4+ T Cell-Mediated Antigen-Specific Immunotherapy in a Mouse Model of Cervical Cancer. Cancer Res. 2005, 65, 2018–2025. [CrossRef] 152. Chu, N.R.; Wu, H.B.; Wu, T.C.; Boux, L.J.; Mizzen, L.A.; Siegel, M.I. Immunotherapy of a human papillomavirus type 16 E7-expressing tumor by administration of fusion protein comprised of Mycobacterium bovis BCG Hsp65 and HPV16 E7. Cell Stress Chaperones 2000, 5, 401–405. [CrossRef] 153. Sharma, C.; Khan, M.A.; Mohan, T.; Shrinet, J.; Latha, N.; Singh, N. A synthetic chimeric peptide harboring human papillomavirus 16 cytotoxic T lymphocyte epitopes shows therapeutic potential in a murine model of cervical cancer. Immunol. Res. 2014, 58, 132–138. [CrossRef] 154. Roman, L.D.; Wilczynski, S.; Muderspach, L.I.; Burnett, A.F.; O’Meara, A.; Brinkman, J.A.; Kast, W.M.; Facio, G.; Felix, J.C.; Aldana, M.; et al. A phase II study of Hsp-7 (SGN-00101) in women with high-grade cervical intraepithelial neoplasia. Gynecol. Oncol. 2007, 106, 558–566. [CrossRef] [PubMed] 155. Einstein, M.H.; Kadish, A.S.; Burk, R.D.; Kim, M.Y.; Wadler, S.; Streicher, H.; Goldberg, G.L.; Runowicz, C.D. Heat shock fusion protein-based immunotherapy for treatment of cervical intraepithelial neoplasia III. Gynecol. Oncol. 2007, 106, 453–460. [CrossRef] [PubMed] 156. Kitamura, A.; Mastumoto, S.; Asahina, I. Growth inhibition of HeLa cell by internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Tokyo. Cancer Cell Int. 2009, 9, 30. [CrossRef] [PubMed] 157. Li, X.-L.; Zhao, C.-L.; Dong, Q.; Sun, L.-R. Enhancement of immunogenicity of murine lymphocytic leukemia cells by transfection with BCG heat shock protein 70 gene. Int. Immunopharmacol. 2013, 15, 1–5. [CrossRef] 158. Rakshit, S.; Ponnusamy, M.; Papanna, S.; Saha, B.; Ahmed, A.; Nandi, D. Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: Critical roles of IFNγ. Int. J. Cancer 2012, 130, 865–875. [CrossRef] [PubMed] 159. Liso, A.; Benedetti, R.; Fagioli, M.; Mariano, A.; Falini, B. Modulatory effects of mycobacterial heat-shock protein 70 in DNA vaccination lymphoma. Haematologica 2005, 90, 60–65. 160. Karyampudi, L.; Ghosh, S.K. Mycobacterial HSP70 as an adjuvant in the design of an idiotype vaccine against a murine lymphoma. Cell. Immunol. 2008, 254, 74–80. [CrossRef] 161. Ito, T.; Hirahara, K.; Onodera, A.; Koyama-Nasu, R.; Yano, I.; Nakayama, T. Anti-tumor immunity via the superoxide-eosinophil axis induced by a lipophilic component of Mycobacterium lipomannan. Int. Immunol. 2017, 29, 411–421. [CrossRef] 162. Masuda, H.; Nakamura, T.; Noma, Y.; Harashima, H. Application of BCG-CWS as a Systemic Adjuvant by Using Nanoparticulation Technology. Mol. Pharm. 2018, 15, 5762–5771. [CrossRef] 163. Mendes, R.; O’Brien, M.E.R.; Mitra, A.; Norton, A.; Gregory, R.K.; Padhani, A.R.; Bromelow, K.V.; Winkley, A.R.; Ashley, S.; Smith, I.E.; et al. Clinical and immunological assessment of Mycobacterium vaccae (SRL172) with chemotherapy in patients with malignant mesothelioma. Br. J. Cancer 2002, 86, 336–341. [CrossRef] Cancers 2020, 12, 1802 25 of 25 164. Biteau, K.; Guiho, R.; Chatelais, M.; Taurelle, J.; Chesneau, J.; Corradini, N.; Heymann, D.; Redini, F. L-MTP-PE and zoledronic acid combination in osteosarcoma: Preclinical evidence of positive therapeutic combination for clinical transfer. Am. J. Cancer Res. 2016, 6, 677–689. [PubMed] 165. Sasaki, H.; Schmitt, D.A.; Kobayashi, M.; Hayashi, Y.; Pollard, R.B.; Suzuki, F. Prolongation of concomitant antitumor immunity in mice treated with Z-100, an arabinomannan extracted from Mycobacterium tuberculosis. Nat. Immun 1993, 12, 152–164. [PubMed] 166. Lou, Y.; Groves, M.J.; Klegerman, M.E. In-vivo and in-vitro targeting of a murine sarcoma by gelatin microparticles loaded with a glycan (PS1). J. Pharm. Pharmacol. 1994, 46, 863–866. [CrossRef] [PubMed] 167. Tian, X.X.; Li, A.; Zhou, W.; Farrugia, I.V.; Groves, M.J. Isolation and biological activities of an antineoplastic protein- polysaccharide complex (PS4A) obtained from Mycobacterium vaccae. Anticancer Res. 1999, 19, 237–243. [PubMed] 168. Garrido, J.L.; Klegerman, M.E.; Reyes, H.R.; Groves, M.J. Antineoplastic activity of BCG: Location of antineoplastic glycans in the cellular integument of Mycobacterium bovis, BCG vaccine, Connaught substrain. Cytobios 1997, 90, 47–65. [PubMed] 169. Wang, R.; Klegerman, M.E.; Marsden, I.; Sinnott, M.; Groves, M.J. An anti-neoplastic glycan isolated from Mycobacterium bovis (BCG vaccine). Biochem. J. 1995, 867–872. [CrossRef] 170. Tian, X.X.; Groves, M.J. Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccae in chitosan nanoparticles. J. Pharm. Pharmacol. 1999, 51, 151–157. [CrossRef] 171. Klegerman, M.E.; Zeunert, P.L.; Lou, Y.; Devadoss, P.O.; Groves, M.J. Inhibition of murine sarcoma cell adherence to polystyrene substrata by bacillus Calmette-Guérin: Evidence for fibronectin-mediated direct antitumor activity of BCG. Cancer Invest. 1993, 11, 660–666. [CrossRef] 172. Zoccali, G.; Cota, C.; Marolda, G.; Ferraresi, V.; Giuliani, M.; Zoccali, C. Chemotherapeutically induced cutaneous tuberculosis after BCG injection in a patient with pelvic osteosarcoma. Surg. Infect. 2012, 13, 406–408. [CrossRef] 173. Frampton, J.E.; Anderson, P.M.; Chou, A.J.; Federman, N.C.; Ferrari, S.; Kim, H.J.; Loeb, D.M.; Mori, K. Mifamurtide: A review of its use in the treatment of osteosarcoma. Pediatr. Drugs 2010, 12, 141–153. [CrossRef] 174. Secanella-Fandos, S.; Luquin, M.; Julián, E. Connaught and russian strains showed the highest direct antitumor effects of different bacillus calmette-guérin substrains. J. Urol. 2013, 189, 711–718. [CrossRef] [PubMed] 175. Babaki, M.K.Z.; Soleimanpour, S.; Rezaee, S.A. Antigen 85 complex as a powerful Mycobacterium tuberculosis immunogene: Biology, immune-pathogenicity, applications in diagnosis, and vaccine design. Microb. Pathog. 2017, 112, 20–29. [CrossRef] [PubMed] 176. Calderwood, S.K.; Murshid, A.; Gong, J. Heat shock proteins: Conditional mediators of inflammation in tumor immunity. Front. Immunol. 2012, 3, 75. [CrossRef] [PubMed] 177. Stewart, G.R.; Young, D.B. Heat-shock proteins and the host-pathogen interaction during bacterial infection. Curr. Opin. Immunol. 2004, 16, 506–510. [CrossRef] 178. Qazi, K.R.; Qazi, M.R.; Julián, E.; Singh, M.; Abedi-Valugerdi, M.; Fernández, C. Exposure to mycobacteria primes the immune system for evolutionarily diverse heat shock proteins. Infect. Immun. 2005, 73, 7687–7696. [CrossRef] 179. Bulut, Y.; Michelsen, K.S.; Hayrapetian, L.; Naiki, Y.; Spallek, R.; Singh, M.; Arditi, M. Mycobacterium tuberculosis heat shock proteins use diverse toll-like receptor pathways to activate pro-inflammatory signals. J. Biol. Chem. 2005, 280, 20961–20967. [CrossRef] 180. Skinner, M.A.; Prestidge, R.; Yuan, S.; Strabala, T.J.; Tan, P.L.J. The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein. Immunology 2001, 102, 225–233. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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