cancers Article Immunotherapy with Monoclonal Antibodies in Lung Cancer of Mice: Oxidative Stress and Other Biological Events Jun Tang 1,2,3 , Daniel Ramis-Cabrer 1,4 , Xuejie Wang 1,3 and Esther Barreiro 1,2,4, * 1 Pulmonology Department-Muscle Wasting & Cachexia in Chronic Respiratory Diseases & Lung Cancer Research Group, IMIM-Hospital del Mar, Parc de Salut Mar, Biomedical Research Park (PRBB), C/Dr. Aiguader, 88, E-08003 Barcelona, Spain 2 Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Biomedical Research Park (PRBB), C/Dr. Aiguader, 88, E-08003 Barcelona, Spain 3 Department of Medicine, Autonomous University of Barcelona (UAB), Biomedical Research Park (PRBB), C/Dr. Aiguader, 88, E-08003 Barcelona, Spain 4 Health and Experimental Sciences Department (CEXS), Pompeu Fabra University (UPF), Biomedical Research Park (PRBB), C/Dr. Aiguader, 88, E-08003 Barcelona, Spain * Correspondence: ebarreiro@imim.es; Tel.: +34-9-3316-0385; Fax: +34-9-3316-0410  Received: 7 June 2019; Accepted: 30 August 2019; Published: 4 September 2019  Abstract: Background: Lung cancer (LC) is a major leading cause of death worldwide. Immunomodulators that target several immune mechanisms have proven to reduce tumor burden in experimental models through induction of the immune microenvironment. We hypothesized that other biological mechanisms may also favor tumor burden reduction in lung cancer-bearing mice treated with immunomodulators. Methods: Tumor weight, area, T cells and tumor growth (immunohistochemistry), oxidative stress, apoptosis, autophagy, and signaling (NF-κB and sirtuin-1) markers were analyzed (immunoblotting) in subcutaneous tumor of BALB/c mice injected with LP07 adenocarcinoma cells treated with monoclonal antibodies (CD-137, CTLA-4, PD-1, and CD-19, N = 9/group) and non-treated control animals. Results: Compared to non-treated cancer mice, in tumors of monoclonal-treated animals, tumor area and weight and ki-67 were significantly reduced, while T cell counts, oxidative stress, apoptosis, autophagy, activated p65, and sirtuin-1 markers were increased. Conclusions: Immunomodulators elicited a reduction in tumor burden (reduced tumor size and weight) through decreased tumor proliferation and increased oxidative stress, apoptosis, autophagy, and signaling markers, which may have interfered with the immune profile of the tumor microenvironment. Future research should be devoted to the elucidation of the specific contribution of each biological mechanism to the reduced tumor burden. Keywords: experimental lung cancer; immunomodulators; oxidative stress; autophagy; tumor growth; sirtuin-1 1. Introduction Lung cancer is the most prevalent cancer worldwide that affects both sexes and has a very high mortality [1]. Despite the development of new therapeutic strategies, patients with lung cancer have an overall survival rate lower than 15% in five years [1–3]. Respiratory conditions such as chronic obstructive pulmonary disease (COPD) and lung fibrosis predispose patients to a greater risk to develop lung cancer, especially non-small cell lung cancer (NSCLC) type [1,2,4–6]. The underlying biology of lung cancer is complex, as several mechanisms may interplay at different stages. For instance, inflammation, which is key in host protection, may promote lung cancer initiation Cancers 2019, 11, 1301; doi:10.3390/cancers11091301 www.mdpi.com/journal/cancers Cancers 2019, 11, 1301 2 of 16 and malignancy in chronic inflammatory processes such as in patients with COPD [2,5,7–11]. Moreover, oxidative stress was also shown to participate in tumor initiation, promotion, and progression of carcinogenesis in patients with lung cancer, particularly in those with COPD [2,9,12,13]. Besides, inflammatory events and oxidative stress may drive the release of a cascade of cytokines and growth factors, which may favor lung tumorigenesis [2,9,12,13] through interference with biological processes such as apoptosis and autophagy [11,13–15]. In the last few years, the implications of biological mechanisms such as increased oxidative stress, inflammatory events, particularly a Th1-predominant response, and epigenetic events were demonstrated to be differentially expressed in the lung tumors of patients with COPD compared to patients without this respiratory condition [8,11,13,16]. These results are important, since they may help establish a differential profile of patients that may be more or less susceptible to certain therapies. The immune system defends the host against diseases, including neoplastic transformation. However, cancer cells may evade the host immune system through a process defined as cancer immunoediting [17]. Cancer immune scape results from the action of immunosuppressive pathways that involve membrane receptors that are located in immune cell types along different steps of the cancer-immunity cycle [17–21]. Immune checkpoints enable immune tolerance to prevent autoimmunity events in the host [17–22]. Several immune checkpoints have been identified so far. As such, programmed cell death protein 1 (PD1) is a membrane receptor that promotes immune tolerance through T cell inactivation [18,19,22]. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a disruptor of antigen presentation upon T cell activation [17]. Cluster of differentiation 137 (CD137) is located in several immune cells such as T regulatory cells (Treg) that are responsible for repressing T cell activity [21]. Additionally, B cells present cluster of differentiation 19 (CD19), which can trigger pro- and anti-tumorigenic responses (Table 1) [20]. Table 1. Monoclonal antibodies for the treatment of lung tumors in mice. Monoclonal Antibodies Targets Function PD-1 receptor is expressed in activated T cells and induces Anti-PD-1 PD-1 immune tolerance by repressing T cell effector function [23,24]. CTLA-4 receptor is expressed in T cells and induces immune Anti-CTLA-4 CTLA-4 tolerance by repressing antigen presentation [23,25]. Anti-CD19 CD-19 CD-19 activates B cells [23,26]. Anti-CD137 CD-137 CD-137 receptor activates CD8+ T and NK cells [21,23]. Definition of abbreviations: PD-1, programmed cell death-1; CTLA-4, cytotoxic T-lymphocyte associated protein-4; CD-137, TNF receptor superfamily member 9; CD-19; B-lymphocyte antigen; NK, natural killer.Specific immune checkpoint inhibitors have been designed, namely monoclonal antibodies that specifically act against these membrane receptors in order to boost the immune microenvironment. The blockade of these inhibitory pathways has been shown to restore the anti-tumor activity of the immune system [17,22,27–32]. The therapeutic efficacy of the combination of different immunomodulatory monoclonal antibodies has been recently demonstrated in animal models of lung cancer, in which the tumor immune microenvironment was specifically explored [18,19]. Furthermore, in previous studies from our group [8,11–13,16,33], the contribution of inflammation and signaling pathways [e.g., nuclear factor (NF)-kB and Sirtuin-1], oxidative stress, autophagy, and apoptosis in response to several pharmacological strategies was shown in mice bearing lung tumors. Oxidative stress was also shown to mediate the response to immunotherapy in colorectal cancer in mice [34] and the chemoresistance in ovarian cancer of patients [35]. Whether similar biological mechanisms can be observed in the tumors of mice treated with a combination of several immunomodulators remains to be identified. Thus, we reasoned that immunomodulators may also exert beneficial effects on tumor burden through biological events other than the immune microenvironment. Cancers 2019, 11, 1301 3 of 16 On this basis, we hypothesized that treatment of a combination of specific immunomodulatory monoclonal antibodies that included anti-PD1, anti-CTLA-4, anti-CD137, and anti-CD19 may have an effect on tumor progression through several biological mechanisms such as oxidative stress, autophagy, and apoptosis through specific signaling pathways in wild-type lung adenocarcinoma cells of mice [33]. Accordingly, in the current investigation, the main objectives were two-fold: (1) to assess the immune tumor microenvironment (T cells) and (2) to quantify levels of oxidative stress, antioxidant enzymes, apoptosis, autophagy, signaling, and cell proliferation rates in the subcutaneous lung adenocarcinoma tumors of BALB/c mice treated with a combination of immunomodulators (anti-PD1, anti-CTLA-4, anti-CD137, and anti-CD19 monoclonal antibodies). A group of tumor-bearing mice that did not receive treatment with the cocktail of monoclonal antibodies was the control group in the study. This experimental model of NSCLC has been previously well-validated in our group [12,33,36–38]. 2. Methods 2.1. Animal Experiments 2.1.1. Experimental Design The study protocol is illustrated in Figure 1. An animal model with lung cancer was developed through the inoculation of cancer cells from LP07 stable adenocarcinoma cell line derived from P07 lung tumor that spontaneously appeared in BALB/c mice [39–41]. Eighteen female BALB/c mice (8 weeks old, 20 g weight) acquired from Harlan Interfauna Ibérica SL (Barcelona, Spain) received a subcutaneous inoculation of LP07 cells (4 × 105 ) resuspended in 0.2 mL of minimal essential medium (MEM) in the left flank (Figure 1). After tumor cell inoculation on day 0 of all the mice, they were randomly divided into two independent groups (N = 9/group) to be thereafter followed for 30 days: (1) experimental control group in which mice received an intraperitoneal administration of 0.2 mL phosphate-buffered saline (PBS) every 72 h (non-treated controls group) and (2) mice treated with a combination of monoclonal antibodies (treated lung cancer group) that included anti-PD1 (RMP1-14; Cat. #BE0146, BioXCell, West Lebanon, NH, USA), anti-CTLA-4 (9D9; Cat. #BE0164, BioXCell), anti-CD137 (LOB12.3; Cat. #BE0169, BioXCell), and anti-CD19 (1D3; Cat. #BE0150, BioXCell) antibodies [18,19,21,23–26] (Table 1). A dose of 5 × 10−3 mg/kg/72 h in 0.2 mL PBS was administered to the treated group of lung cancer mice from day 15 (tumors visible) up until day 30 (Figure 1). The intraperitoneal route was chosen in order to mimic administration of this type of therapies in clinical settings [19]. For ethical reasons we were not allowed to extend the study protocol longer than 30 days. Also for ethical reasons, only non-treated tumor-bearing mice administered with the vehicle PBS were used as the control group in the study. Food and water were supplied ad libitum and mice were kept under pathogen-free conditions with a 12:12 h light:dark cycle in the animal facilities placed in the Barcelona Biomedical Research Park (PRBB) premises. 2.1.2. In Vivo Measurements Conducted on the Animals Food intake and body weight were measured daily in all the study animals. Tumor area was also measured daily using a specific caliper in all the animals. 2.1.3. Sacrifice and Sample Collection The two experimental groups of mice were sacrificed after 30 days of inoculation of LP07 cells. In each mouse, an intraperitoneal injection of 0.1 mL sodium pentobarbital (60 mg/kg) was inoculated prior to sacrifice. In order to verify total anesthesia depth, the pedal and blink reflexes were assessed in all animals. As the histological features of the subcutaneous tumor and those of the lung metastases are identical in this LP07 mouse model of lung cancer, for practical reasons, the subcutaneous tumor was used for the laboratory experiments. As such, the subcutaneous tumor was extracted from all the mice. A fragment of the tumor specimens was immediately frozen in liquid nitrogen and stored at Cancers 2019, 11, 1301 4 of 16 −80 ◦ C, while the other fragment was immersed in an alcohol-formol to be thereafter embedded in paraffin until further use. Cancers 2019, 11, x 4 of 16 FigureFigure 1. Graphical 1. Graphical time-line time-line representation representation of theofnon-treated the non-treated control control lunglung cancer cancer micemice andand the the lung lung group cancer cancerofgroup of animals animals treated treated with the with the combination combination of monoclonal of monoclonal antibodies.This antibodies. was a This was a controlled controlled study designed according to the ethical regulations on animal experimentation of the study designed according to the ethical regulations on animal experimentation of the Spanish Legislation Spanish Legislation (Real Decreto 53/2013, BOE 34/11370–11421), the European Community Directive (Real Decreto 53/2013, BOE 34/11370–11421), the European Community Directive 2010/63/EU, and 2010/63/EU, and the European Convention for the Protection of Vertebrate Animals Used for the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Experimental and Other Scientific Purposes (1986) at PRBB. The Animal Research Committee Scientific approved Purposes (1986) all animal at PRBB. (Animal experiments The Animal Research Welfare Committee Department approvedSpain, in Catalonia, all animal experiments # EBP-15-1704). (Animal Welfare Department in Catalonia, Spain, # EBP-15-1704). 2.1.2. In vivo measurements conducted on the animals. 2.2. Molecular Biology Analyses Food intake and body weight were measured daily in all the study animals. Tumor area was also measured daily using 2.2.1. Histological Analyses ofaTumor specificSamples caliper in all the animals. Immunohistochemical 2.1.3. Sacrifice and sample techniques collection.were applied on tumor sections in order to explore expression of the proliferation marker Ki-67 and T cells, following previous methodologies [8,12,13,16,33,36–38,42,43]. The two experimental groups of mice were sacrificed after 30 days of inoculation of LP07 cells. Briefly,Infor all the each mouse, target antigens, tumor an intraperitoneal cross-sections injection werepentobarbital of 0.1 mL sodium deparaffinized and then (60 mg/kg) wasantigen inoculatedretrieval was carried prior toout by heating sacrifice. slides In order in a water to verify bath in Tris/Ethylenediaminetetraacetic total anesthesia depth, the pedal and blink reflexes acid were (EDTA) assessed buffer, pH 9, for 30animals. in all min (Ki-67 As the and CD3) orfeatures histological in a pressure-cooker of the subcutaneous (CD4, CD8) tumor andin 0.1 of those Mthecitrate lung buffer, pH 6, for metastases 15 min.areSubsequently, identical in thissamples LP07 mouse weremodel of lung blocked cancer, with 3% Hfor2 O practical 2 for 15reasons, min, the and subcutaneous immediately tumor afterwards was used they were blockedfor the laboratory with 1% BSA experiments. and goat As such,for serum thetwo subcutaneous tumorwere hours. Slides was extracted from all the then incubated at room mice. A fragment of the tumor specimens ◦ was immediately frozen in liquid nitrogen and stored at – temperature for 30 min (CD3) or at 4 C overnight (Ki-67, CD4, CD8) with the following specific primary 80 °C, while the other fragment was immersed in an alcohol-formol to be thereafter embedded in antibodies: paraffinanti-Ki67 (Merck-Millipore, until further use. Darmstadt, Germany), anti-CD3 (DAKO, Glostrup, Denmark), anti-CD4 (Abcam, Cambridge, UK), and anti-CD8 (Abcam). Slides were washed and incubated for 30 min2.2.with biotinylated Molecular universal secondary antibody, followed by another 30 min incubation with Biology Analyses horseradish-conjugated streptavidin and diaminobenzidine for five minutes (kit LSAB+HRP Dako 2.2.1. Histological analyses of tumor samples. Cytomation Inc., Carpinteria, CA, USA) as a substrate. TumorImmunohistochemical sections were counterstainedtechniques were with applied on tumor hematoxylin forsections in orderand two minutes to explore expression were then dehydrated of the proliferation marker Ki-67 and T cells, following previous methodologies and mounted for conventional microscopy. Images of the stained tumors were taken under a light [8,12,13,16,33,36– 38,42,43]. Briefly, for all the target antigens, tumor cross-sections were deparaffinized and then microscope (Olympus BX 61, Olympus Corporation, Tokyo, Japan) coupled with a camera (Olympus antigen retrieval was carried out by heating slides in a water bath in Tris/Ethylenediaminetetraacetic DP 71,acidOlympus Corporation). The number of Ki-67, CD3, and CD8 cells was expressed as the (EDTA) buffer, pH 9, for 30 min (Ki-67 and CD3) or in a pressure-cooker (CD4, CD8) in 0.1 M percentage citrateofbuffer, the cells pH 6, that for were 15 min.positively stained Subsequently, for the samples were corresponding blocked with 3% markers H2O2 forof15the total min, andcells in the tumor preparations. The number of CD4 cells was represented as the immediately afterwards they were blocked with 1% BSA and goat serum for two hours. Slides were number of cells that were positively thenstained incubated foratCD4 roomintemperature the measured area for 30 min(expressed (CD3) or at in micrometers). 4 °C overnight (Ki-67, CD4, CD8) with the following Terminal specific primary deoxynucleotidyl antibodies: transferase- anti-Ki67 mediated uridine 50 -triphosphate (UTP) (Merck-Millipore, Darmstadt, nick- Germany), end labellinganti- (TUNEL) assay. In tumor paraffin-embedded sections, apoptotic nuclei were identified using the TUNEL assay Cancers 2019, 11, 1301 5 of 16 (In Situ Cell Death Detection Kit, POD, Roche Applied Science, Mannheim, Germany) for all study groups following the manufacturer’s instructions and previous studies [38,43]. Briefly, this assay is based on the principle that during the apoptosis of nuclei, genomic DNA may yield double-stranded, low molecular weight fragments as well as single-strand breaks (nicks) in high molecular weight DNA. These DNA strand breaks can be identified by labelling 30 -hydroxyl (30 OH) groups with modified nucleotides in an enzymatic reaction. In this assay, deoxynucleotidyl transferase (TdT), which catalyzes the polymerization of nucleotides to free 30 -OH DNA ends, is used to label DNA strand breaks. Briefly, diaphragm and gastrocnemius muscle sections were fixed and permeabilized. Subsequently, they were incubated with the TUNEL reaction mixture that contains terminal TdT and fluorescein-dUTP. During the incubation period, terminal TdT catalyzed the addition of fluorescein-dUTP at free 30 -OH groups in single- and double-stranded DNA. After washing, the label incorporated at the damaged sites of the DNA was marked by anti-fluorescein antibody conjugated with the reporter enzyme peroxidase. After several washes that removed unbound enzyme conjugate, the peroxidase retained in the immune complex was visualized by a substrate reaction. Apoptotic nuclei were brown, while negative nuclei were blue (hematoxylin counterstaining). In each tumor cross-section, the TUNEL-positive nuclei and the total number of nuclei were counted blindly by two independent observers, who were previously trained for that purpose. Results were expressed as the ratio of total TUNEL positively-stained nuclei to the total number of counted nuclei, as also previously reported [38,43]. A minimum amount of 300 nuclei were counted in each tumor preparation. Final results corresponded to the mean value of the counts provided by the two independent observers (concordance rate 95%). Negative control experiments, in which the TUNEL reaction mixture was omitted, were also conducted. Moreover, rat testicles were used as a positive control in these experiments. 2.2.2. Immunoblotting Protein levels of the target markers were determined using 1D electrophoresis and immunoblotting according to previously published methodologies [8,12,13,16,33,36–38,42]. Frozen tumor samples extracted from mice were homogenized in lysis buffer. The following specific primary antibodies were used to identify the different target markers: protein tyrosine nitration (anti-3-nitrotyrosine antibody, Invitrogen, Eugene, OR, USA), malondialdehyde protein adducts (anti-MDA protein adduct antibody, Academic Bio-Medical Company, Inc., Houston, TX, USA), catalase (anti-catalase antibody, Calbiochem, Darmstadt, Germany), Mn-superoxide dismutase (SOD2, anti-SOD2 antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CuZn-superoxide dismutase (SOD1, anti-SOD1 antibody, Santa Cruz), b-cell lymphoma 2 (BCL-2, anti-BCL-2, antibody Santa Cruz), BCL-2 associated X protein (BAX, anti-BAX antibody, Santa Cruz), nucleoporin p62 (anti-p62 antibody, Sigma-Aldrich, St. Louis, MO, USA), beclin-1 (anti-beclin-1 antibody, Santa Cruz), microtubule-associated protein 1 light-chain 3 (LC3B, anti-LC3B antibody, Cell Signaling, MA, USA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 subunit and phosphorylated p65 subunit (anti NF-κB p65 and p-NF-κB p65 antibodies, Santa Cruz), sirtuin-1 (anti-sirtuin-1 antibody, EMD Millipore, Billerica, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, anti-GAPDH antibody, Santa Cruz) as the protein loading control to confirm identical protein loading among different lanes. Horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit (Thermo Scientific, Rockford, IL, USA) were used to detect the antigens from all samples. For the sake of comparisons between the two groups, all study samples (×18) were run together in the same mini-cell electrophoresis and transfer boxes, respectively, and the corresponding membranes were detected using chemiluminescence in the same platform under identical exposure times. Polyvinylidene difluoride (PVDF) membranes were scanned with the Molecular Imager Chemidoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA) using the software Quantity One version 4.6.5 (Bio-Rad Laboratories), and optical densities of target proteins were quantified using the software Image Lab version 2.0.1 (Bio-Rad Laboratories). Final optical densities (arbitrary units) acquired in each group of mice corresponded to the average value of all the samples (lanes). Values of optical Cancers 2019, 11, 1301 6 of 16 densities (arbitrary units) of LC3B-II/LC3B-I were also calculated as the ratio of LC3B-II (14 kDa) to LC3B-I (16 kDa) protein content. Moreover, all values of the different antigens were calculated as the ratio of the optical densities of the given variable to those of the loading control GAPDH in each study sample, as shown in each figure. Standard stripping methodologies were employed to detect p62 protein levels in the same PVDF membranes of beclin-1. The loading control GAPDH was also detected using stripping methodologies in the study markers: protein tyrosine nitration, MDA-protein adducts, SOD1, SOD2, catalase, BAX, BCL-2, beclin-1, p62, LC3B, NF-κB p65, pNF-κB p65, and Sirtuin-1. Briefly, primary and secondary antibodies were stripped off proteins using a stripping solution (25 nM glycine, pH 2.0, and 1% Sodium Dodecyl Sulphate (SDS)) for 30 min. Membranes were subsequently washed two consecutive times (10 min each) with phosphate buffered saline and tween (PBST) at room temperature. Immediately afterwards, membranes were blocked with 1% Bovine Serum Albumin (BSA) and incubated with specific primary and secondary antibodies following the abovementioned procedures. 2.3. Statistical Analysis Using specific software (StudySize 2.0, CreoStat HB, Frolunda, Sweden) and assuming an alpha error of 0.05 and a minimum of 80% of standard power statistics, the sample size (N = 9/group) was sufficiently great to identify a difference of 700 and 0.6 points in both tumor area and weight variables between groups, respectively. The Shapiro–Wilk test was used to check the normality of the study variables. Therefore, data are expressed as mean and standard deviation in both tables and figures. The Statistical Package for the Social Sciences (SPSS, version 22, SPSS Inc., Chicago, IL, USA) was used to compare the study variables between the two study groups using the unpaired Student’s t-test, and statistical significance was established at p ≤ 0.05. 3. Results 3.1. Monoclonal Antibodies Improved Tumor Burden and Body Weight in Mice As illustrated in Table 2 and Figure 2A, by the end day (30) of the study protocol, in the lung cancer mice compared to non-treated control mice, treatment with the cocktail of monoclonal antibodies had significantly improved the following variables: final body weight, body weight gain with and without tumor, tumor weight (34% reduction), and tumor area (64% reduction). Importantly, levels of Ki-67-positive nuclei were significantly lower (27% reduction), while TUNEL-positively stained nuclei were significantly higher (127% increase) in the tumors of mice treated with the monoclonal antibodies compared to those detected in the non-treated control animals (Figure 2B,C). Table 2. Physiological and tumor characteristics in the study groups of mice. Variables Lung Cancer Mice Lung Cancer + Monoclonal Antibodies Mice Initial body weight (g) 20.41 (1.22) 20.34 (0.79) Final body weight (g) 19.35 (2.25) 21.39 (1.57), * Body weight gain (%) −4.27 (10.47) +5.16 (6.33), * Body weight gain without tumor (%) −15.06 (11.28) −2.66 (8.35), * Tumor weight (g) 2.38 (0.75) 1.57 (0.89), * Variables are presented as mean (standard deviation). Statistical significance: * p ≤ 0.05 between the two study groups of mice. Initial body weight (g) 20.41 (1.22) 20.34 (0.79) Final body weight (g) 19.35 (2.25) 21.39 (1.57), * Body weight gain (%) −4.27 (10.47) +5.16 (6.33), * Body weight gain without tumor (%) −15.06 (11.28) −2.66 (8.35), * Tumor weight (g) 2.38 (0.75) 1.57 (0.89), * Cancers 2019, 11, 1301 Variables are presented as mean (standard deviation). Statistical significance: * p ≤ 0.05 between the 7 of 16 two study groups of mice. Figure 2. (A) Figure 2. (A) Mean Mean values values and standard and standard deviation of deviation ofsubcutaneous subcutaneous tumor area (mm tumor area (mm2 ) of non-treated 2) of non-treated control lung cancer group of mice (black color) and lung cancer mice treated with monoclonal control lung cancer group of mice (black color) and lung cancer mice treated with monoclonal antibodies antibodies group (red color) during the study protocol. (B) Representative histological sections (40×) group (redand color) mean during values andthe standard study protocol. (B)Ki-67 deviation of Representative histological in the subcutaneous tumors ofsections (40×) and mean the non-treated values andcontrol standard deviation mice and of lung the treated Ki-67 in the cancer subcutaneous animals. tumors Black arrowheads pointof the non-treated towards control mice Ki-67-positively stainedlung and the treated nuclei (brownanimals. cancer color), while red arrowheads Black arrowheads point towards point Ki-67 negatively towards stained nuclei Ki-67-positively stained nuclei (purple color). (C) Representative histological sections (40×) and mean values and standard deviation (brown color), while red arrowheads point towards Ki-67 negatively stained nuclei (purple color). of TUNEL in the subcutaneous tumors of the non-treated control mice and the treated lung cancer (C) Representative histological animals. Black arrowheadssections (40×) point towards and mean values TUNEL-positively and stained standard nuclei deviation (brown color), of TUNEL in while red the subcutaneous arrowheads tumors of the TUNEL-negatively point towards non-treated control stainedmice nucleiand thecolor). (purple treated lungsignificance: Statistical cancer animals. *: Black p ≤ 0.05 and ***: p ≤ 0.001 between lung cancer mice compared to arrowheads point towards TUNEL-positively stained nuclei (brown color), while red arrowheads the lung cancer-monoclonal antibodies group. point towards TUNEL-negatively stained nuclei (purple color). Statistical significance: *: p ≤ 0.05 and ***: p ≤3.2. 0.001 between Immune lung cancerinmice Microenvironment compared Response to the lung cancer-monoclonal antibodies group. to the Immunomodulators 3.2. Immune Microenvironment in Response to the Immunomodulators In tumors of mice treated with the monoclonal antibodies, the number of T cells (CD3, CD8, and CD4) was significantly greater than that observed in the non-treated animals (Table 3 and Figure 3). Table 3. Immune microenvironment in the study groups of mice. T Cells Lung Cancer Mice Lung Cancer + Monoclonal Antibodies Mice CD3 + cells (%) 8.34 (0.91) 11.30 (0.78), *** CD4 + (cells/µm2 ) 1.99 × 10−6 (0.44 × 10−6 ) 3.37 × 10−6 (1.49 × 10−6 ), * CD8 + cells (%) 5.16 (1.35) 7.86 (1.04), *** Values are expressed as mean (standard deviation). Statistical significance: * p ≤ 0.05 and *** p ≤ 0.001 between the two experimental groups of mice. (Table (Table33and andFigure Figure 3). 3).Table 3. Immune microenvironment in the study groups of mice. Table 3. Immune microenvironment in the study groups of mice. TTCells Cells Lung Lung Cancer Cancer Mice Mice LungCancer Lung Cancer++Monoclonal MonoclonalAntibodies Antibodies Mice Mice CD3 CD3++cells cells(%) (%) 8.34 8.34 (0.91) (0.91) 11.30 (0.78), 11.30 (0.78), *** *** CD4 2 −6 −6 10−6−6(1.49 −6 * CD4++(cells/µm (cells/µm)2) 1.99 1.99 ×× 10 10−6 (0.44 (0.44 ×× 10 10−6)) 3.37××10 3.37 (1.49× ×1010 −6), ), * CD8 + cells (%) CD8 + cells (%) 5.16 (1.35) 5.16 (1.35) 7.86 (1.04), 7.86 (1.04), *** *** Cancers 2019,Values 11, 1301 Values are areexpressed expressedas asmean mean (standard deviation). Statistical (standard deviation). Statisticalsignificance: significance:* *p p≤ ≤0.05 0.05andand****** p ≤p 0.001 ≤ 0.001 8 of 16 between the two experimental groups of mice. between the two experimental groups of mice. 3. Representative Figure Figure examples 3. Representative examplesofofimmunohistochemistry staining immunohistochemistry staining for for CD3,CD3, CD4,CD4, and in and CD8, CD8, in Figure 3. Representative examples of immunohistochemistry staining for CD3, CD4, and CD8, in tumor tumor samples of the samples of different study the different groups study groupsof of mice. Alltypes mice. All typesof of T cells T cells (CD3, (CD3, CD8,CD8, and are and CD4) CD4) are tumor samples of the different study groups of mice. All types of T cells (CD3, CD8, and CD4) are stainedstained in brown in brown colorcolor (black (black arrows), arrows), whilenegative while negative nuclei nucleiare arestained stainedin purple colorcolor in purple (red arrows). (red arrows). stained in brown color (black arrows), while negative nuclei are stained in purple color (red arrows). 3.3. Tumor Oxidative 3.3. Tumor Stress Oxidative in Response Stress to tothe in Response theImmunomodulators Immunomodulators 3.3. Tumor Oxidative Stress in Response to the Immunomodulators Compared Compared to non-treated to non-treated mice, mice, proteintyrosine protein tyrosine nitration nitrationand oxidation and (MDA-protein oxidation adducts) (MDA-protein adducts) andCompared to non-treated cytosolic SOD1 mice, levels were protein tyrosine significantly greaternitration and oxidation in the tumors of the (MDA-protein adducts) mice treated with the and cytosolic and SOD1 levels cytosolic antibodies, SOD1 levels were weresignificantly greater in the tumors of the mice treated with the the monoclonal while nosignificantly greater inwere significant differences the tumors detectedofinthe mice treatedSOD2 mitochondrial with or monoclonal antibodies, monoclonal antibodies,while nonosignificant while differences weredetected detected in in mitochondrial SOD2 or or catalase protein levels between thesignificant differences two study groups were (Figures 4A,B and 5A–C). mitochondrial SOD2 catalase protein catalase levels protein between levels thethe between two study two studygroups groups (Figure (Figures 4A,B and5A–C). 4A,B and Figure 5A–C). Figure 4. (A) Representative immunoblots and mean values and standard deviation of total protein tyrosine nitration levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by optical densities. (B) Representative immunoblots and mean values and standard deviation of total MDA protein adduct levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by optical densities. Representative GAPDH is shown as the loading control. Statistical significance is represented as follows: **: p ≤ 0.01 between non-treated controls (N = 9) in white bars and treated lung cancer (N = 9) mice in black bars. Definition of abbreviations: MDA, malondialdehyde; GAPDH, glyceraldehyde-3-phospate dehydrogenase; OD, optical densities. densities. (B) Representative immunoblots and mean values and standard deviation of total MDA protein adduct levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by optical densities. Representative GAPDH is shown as the loading control. Statistical significance is represented as follows: **: p ≤ 0.01 between non-treated controls (N = 9) in white bars and treated lung cancer (N = 9) mice in black bars. Definition of abbreviations: MDA, malondialdehyde; GAPDH, Cancers 2019, 11, 1301 9 of 16 glyceraldehyde-3-phospate dehydrogenase; OD, optical densities. Figure 5. (A) Representative immunoblots and mean values and standard deviation of SOD1 Figure 5. (A) Representative immunoblots and mean values and standard deviation of SOD1 protein protein levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by optical levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by optical densities. (B) densities. (B) Representative immunoblots and mean values and standard deviation of SOD2 protein Representative immunoblots and mean values and standard deviation of SOD2 protein levels/GAPDH in subcutaneous tumors of lung cancer (LC) mice as measured by optical densities. levels/GAPDH in subcutaneous tumors of lung cancer (LC) mice as measured by optical densities. (C) Mean values and standard deviation of catalase protein levels/GAPDH in subcutaneous tumors of (C)lung Mean values cancer mice and standard deviation as measured by optical of catalaseRepresentative densities. protein levels/GAPDH GAPDH isinshown subcutaneous tumors as the loading of control. lung cancer mice significance Statistical as measured is by optical densities. represented as follows:Representative GAPDH *: p ≤ 0.05 between is shown non-treated as the(N controls loading = 9) control. Statistical significance is represented as follows: *: p ≤ 0.05 between non-treated controls in white bars and treated lung cancer (N = 9) mice in black bars. Definition of abbreviations: SOD1, (N = 9) CuZn-superoxide in white bars anddismutase; treated lung cancer SOD2, (N = 9) mice dismutase; Mn-superoxide in black bars. Definition GAPDH, of abbreviations: SOD1, glyceraldehyde-3-phospate CuZn-superoxide dehydrogenase; OD, dismutase; SOD2, Mn-superoxide dismutase; GAPDH, glyceraldehyde-3-phospate optical densities. dehydrogenase; OD, optical densities. 3.4. Tumor Apoptosis and Autophagy Markers in Response to Immunomodulators 3.4. Tumor Apoptosis Treatment andmice of the Autophagy Markers with the in of cocktail Response to Immunomodulators monoclonal antibodies induced a significant rise in BAX protein levels in the tumors compared to non-treated animals, while no significant differences Treatment of the mice with the cocktail of monoclonal antibodies induced a significant rise in were found in tumor BCL-2 protein levels between the two study groups (Figure 6A,B). Treatment BAX protein levels in the tumors compared to non-treated animals, while no significant differences with monoclonal antibodies did not induce any significant difference in protein expression levels of were found in tumor BCL-2 protein levels between the two study groups (Figure 6A,B). Treatment either beclin-1 or p62, whereas the ratio of LC3-II to LC3-I was significantly increased in the tumors of with monoclonal antibodies did not induce any significant difference in protein expression levels of the treated mice (Figure 7A–C). either beclin-1 or p62, whereas the ratio of LC3-II to LC3-I was significantly increased in the tumors of the treated mice changed (Figure 7A–C). Cancers Cancers 2019, 2019, 11,11, x 1301 10 of 1016 of 16 Cancers 2019, 11, x 10 of 16 Figure Figure Figure (A) 6.6. (A) 6. (A)Representative Representative immunoblots Representativeimmunoblots immunoblots andand and meanmean mean valuesvalues values and standard and standard and standard deviationof deviation deviation ofBAX BAX of BAX protein protein levels/GAPDH protein levels/GAPDH levels/GAPDH in insubcutaneous tumors tumorsofoftumors in subcutaneous subcutaneous lung lung cancer mice cancer of lung cancer as measured as measured miceby by optical asoptical measured densities. densities. by (B)(B) optical Representative Representative densities. immunoblots immunoblots (B) Representative and and mean immunoblots mean values and mean and values and valuesstandard standard deviation deviation and standard ofof BCL-2 deviation BCL-2 of BCL-2 protein proteinprotein levels/GAPDH levels/GAPDH levels/GAPDH in subcutaneous ininsubcutaneous subcutaneous tumorsof tumors tumors ofofLC LCmice mice as mice as measured as measured measuredby bybyoptical opticaldensities. optical densities. densities.Representative Representative Representative GAPDH GAPDH GAPDH is is is shown shown shown as thethe asasthe loading loading loading control.Statistical control. control. Statistical significance Statistical significance is significance isisrepresented represented represented asasas follows: follows: follows:*: *: p ≤p *:0.05 ≤p0.05 ≤ 0.05 between non-treated controls(N(N= =9)9)ininwhite whitebars bars and and treated between between non-treated non-treated controls controls (N = 9) in white bars andtreated treatedlung lung lung cancer cancer(N cancer ==9)9)mice (N(N = 9) mice inin mice black black bars. bars. in black bars. Definition Definition of of abbreviations:BAX, abbreviations: BAX,BCL-2 BCL-2associated associated X X protein; protein; BCL-2, BCL-2, b-cell b-celllymphoma lymphoma 2; 2;GAPDH, GAPDH, Definition of abbreviations: BAX, BCL-2 associated X protein; BCL-2, b-cell lymphoma 2; GAPDH, glyceraldehyde-3-phospate glyceraldehyde-3-phospate dehydrogenase;OD, dehydrogenase; OD, optical densities. densities. glyceraldehyde-3-phospate dehydrogenase; OD, optical optical densities. Figure 7. (A) Representative immunoblots and mean values and standard deviation of Beclin-1 protein levels/GAPDH in subcutaneous tumors of LC mice as measured by optical densities. (B) Figure Figure (A)(A) 7. 7. Representative Representative Representative immunoblots immunoblots immunoblots andand and mean values mean and values mean and values standard standard and deviation deviation standard of p62 of Beclin-1 deviation protein protein of Beclin-1 levels/GAPDH levels/GAPDH protein in subcutaneous tumors of LC mice as measured by optical densities. in subcutaneous tumors of LC mice as measured by optical densities. (C) Representative immunoblots(B) levels/GAPDH in subcutaneous tumors of LC mice as measured by (B) optical Representative densities. immunoblots and meanand Representative mean values andvalues immunoblots andmean and standard standard values deviation deviation of and of p62deviation totalstandard LC3-II/LC3-I protein levels/GAPDH of p62 protein protein/GAPDH in subcutaneous levels/GAPDH in subcutaneous tumors of LCofmice in subcutaneous tumors as measured tumors LC mice by by of LC mice as measured optical densities. asoptical measured (C) by optical densities. Representative densities.GAPDH Representative immunoblots is shown asand (C) Representative the mean immunoblots loadingvalues and and control. standard Statistical deviation mean values significance and of is represented total LC3-II/LC3-I standard as follows: deviation ofprotein/GAPDH *: p total LC3-II/LC3-I≤ 0.05 between non-treated in subcutaneous protein/GAPDH tumorscontrols (N = as of LC mice in subcutaneous 9) in white tumors ofby measured bars LCoptical and treated mice asdensities. lung cancer measuredRepresentative (N = 9) by optical densities. mice GAPDH in black bars. Representative Definition is shown GAPDH of abbreviations: is shown as the loading p62, as the loading control. Statistical control. Statistical significance significance is represented is represented as follows: as follows: *: p ≤ 0.05 between*: pnon-treated ≤ 0.05 between controls (N = 9) non-treated controls (N bars in white = and9) treated in whitelung barscancer (N = 9) and treated lung micecancer (N = bars. in black 9) mice in black bars. Definition Definition of p62, of abbreviations: abbreviations: p62,p62; nucleoporin LC3, microtube-associated protein 1 light chain 3; GAPDH, glyceraldehyde-3-phospate dehydrogenase; OD, optical densities. Cancers 2019, 11, x 11 of 16 Cancers 2019, 11, 1301 11 of 16 nucleoporin p62; LC3, microtube-associated protein 1 light chain 3; GAPDH, glyceraldehyde-3- phospate dehydrogenase; OD, optical densities. 3.5. Effects of the Immunomodulators on Signaling Markers in Tumors 3.5. Effects of the Immunomodulators on Signaling Markers in Tumors Protein levels of the ratio of p-p65 subunit to total p65 of the NF-κB signaling pathway were Protein levels of the ratio of p-p65 subunit to total p65 of the NF-κB signaling pathway were significantly greater significantly in the greater tumors in the of of tumors thethe mice mice treated treatedwith withthe the monoclonal antibodiesthan monoclonal antibodies than in in those of those non-treated rodentsrodents of non-treated (Figure(Figure 8A). Protein levels of 8A). Protein the deacetylase levels sirtuin-1 of the deacetylase were significantly sirtuin-1 increased were significantly in theincreased tumors of in the tumors treatedofmice compared the treated mice to non-treated compared animals (Figure to non-treated 8B). 8B). animals (Figure Figure 8. (A) Figure Representative 8. (A) Representative immunoblots immunoblots and and mean mean values values and standard and standard deviationdeviation of ratio ofof ratio of pNF- κB/NF-κB protein levels/GAPDH in subcutaneous tumors of lung cancer mice as pNF-κB/NF-κB protein levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by measured by optical densities. optical (B)(B) densities. Representative Representative immunoblots and and immunoblots mean values mean and and values standard deviation standard of sirtuin-1 deviation of sirtuin-1 protein levels/GAPDH in subcutaneous tumors of lung cancer mice as measured protein levels/GAPDH in subcutaneous tumors of lung cancer mice as measured by optical by optical densities. densities. Representative GAPDH is shown as the loading control. Statistical significance is represented as Representative GAPDH is shown as the loading control. Statistical significance is represented as follows: and *: p ≤ 0.05 and ** : p ≤ 0.01 between non-treated controls (N = 9) in white bars and treated follows: and *: p ≤ 0.05 and **: p ≤ 0.01 between non-treated controls (N = 9) in white bars and lung cancer (N = 9) mice in black bars. Definition of abbreviations: NF-κB, nuclear factor kappa-light- treated lung cancer (N = 9) mice in black bars. Definition of abbreviations: NF-κB, nuclear factor chain-enhancer of activated B cells; GAPDH, glyceraldehyde-3-phospate dehydrogenase; OD, optical kappa-light-chain-enhancer densities. of activated B cells; GAPDH, glyceraldehyde-3-phospate dehydrogenase; OD, optical densities. 4. Discussion 4. Discussion In the present study, treatment of the tumor-bearing mice with a cocktail of monoclonal In the present antibodies thatstudy, treatment specifically of theimmune targeted tumor-bearing mice with checkpoints a cocktail of and pathways monoclonal elicited antibodies a significant that specifically targeted immune checkpoints and pathways elicited a significant reduction of tumor proliferation rates and sizes through the induction of several biological reduction of tumor mechanisms proliferation ratesthat andare discussed sizes throughbelow. Thus, theofstudy the induction hypothesis several hasmechanisms biological been confirmed thattoare a great discussed below. extent. Thus, the study hypothesis has been confirmed to a great extent. In the In tumors the tumors of the of the mice mice treatedwith treated with the the cocktail cocktail of ofmonoclonal monoclonal antibodies, antibodies,the area was was the area significantly reduced at the end of the study period. These are relevant findings that confirm the significantly reduced at the end of the study period. These are relevant findings that confirm the efficacy of the treatment with the immunomodulators of the tumor cells in this experimental model efficacy of the treatment with the immunomodulators of the tumor cells in this experimental model of lung cancer in mice. These results are in line with those previously reported, in which a complete of lung cancer in regression mice. of the These tumors results are (melanoma andinlung linecancer) with those previously was attained reported, following in which intratumoral a complete treatment regression of the tumors (melanoma and lung cancer) was attained following intratumoral of the mice with the same cocktail of monoclonal antibodies [18,19]. In the present study, the numbers treatment of theofmice with T cell the same subtypes cocktail of monoclonal also significantly increased in antibodies [18,19]. the tumors of the miceIn that the present received study, thewith treatment numbers of T cell the subtypes checkpointalso significantly increased in the tumors of the mice that received treatment with inhibitors. As opposed the checkpoint to previous investigations [18,19], the immunomodulators were injected inhibitors. intraperitoneally As opposed to previous with the aim toinvestigations mimic the treatments applied [18,19], thein patients, in whom drugs are immunomodulators wereusually injected administered systemically. In this investigation, a complete regression intraperitoneally with the aim to mimic the treatments applied in patients, in whom drugs of the tumors was not are achieved, probably as a result of the systemic administration of the drugs compared to previous usually administered systemically. In this investigation, a complete regression of the tumors was not achieved, probably as a result of the systemic administration of the drugs compared to previous reports [18,19], in which the monoclonal antibodies were administered locally. Nonetheless, in the tumor-bearing mice that received the medical treatment with the immunomodulators a substantial reduction in tumor burden as measured by both tumor weight (34%) and area (64%) was observed. Cancers 2019, 11, 1301 12 of 16 This leads to the conclusion that the treatment, the doses, and the route were effectively administered in the current study. Interestingly, the number of tumor proliferating cells was also significantly lower in the tumors of the mice treated with the monoclonal antibodies than in the non-treated tumor-bearing rodents. These results suggest that cell cycle arrest probably due to alterations in cyclin expression levels may account for the reduced levels of Ki-67-positively stained nuclei encountered in the adenocarcinoma cells of the treated tumor-bearing mice. Moreover, these findings are also in agreement with previous investigations, in which expression levels of Ki-67 were significantly reduced (34%) in the tumors of mice treated with several selective inhibitors of cell survival pathways [12], in those from transgenic mice deficient for either poly(Adenosine Diphosphate Ribose (ADP)-ribose)) polymerases (PARP)-1 or -2 enzymes [33], and in those of rodents treated with pharmacological inhibitors of PARP activity [44]. Taken together, these results are also very consistent with the findings reported herein: reduced tumor area and weight in the tumor-bearing mice treated with the immunomodulators at the end of the study period. Oxidative stress was assessed using several indirect markers in the tumor cells of both groups of mice. Importantly, levels of protein tyrosine nitration and total MDA-protein adducts were significantly greater in the tumor cells of the mice that received treatment with the monoclonal antibodies. These results are in agreement with those previously observed in another investigation, in which protein oxidation levels were also increased in the tumors of Parp-1-/- and Parp-2-/- mice [33]. In the present investigation, levels of the antioxidant enzyme SOD1, but not those of SOD2 or catalase, were significantly greater in the tumors of the lung cancer-bearing mice treated with the monoclonal antibodies. These results are in line with those encountered in the tumors of mice treated with the proteasome inhibitor bortezomib [12]. Furthermore, accumulation of reactive oxygen species (ROS) and glutathione depletion were also shown in tumor cells of mice with colorectal cancer [34], and an oxidative stress-associated mechanism of T cell activation was observed in the stroma of ovarian and colon tumor samples in patients as well [35,45,46]. The rise in the expression of cytosolic SOD1 levels may have been a response to counterbalance the deleterious effects of increased oxidative stress in the tumor cells as previously suggested [12]. Altogether, a rise in several oxidative stress markers was observed in the tumors of the mice treated with the cocktail of monoclonal antibodies. These findings may imply that in response to treatment with the immunomodulators, oxidative stress may drive cell cycle arrest and tumor cell death independently of the immune response [47]. Oxidative stress may also trigger several important cellular pathways such as cell death, apoptosis, and autophagy through signaling pathways such as the redox sensitive NF-κB pathway. In this regard, the ratio levels of active p65 (phosphorylated) to total p65 were greater in the tumors of animals treated with the monoclonal antibodies than in the non-treated mice. Importantly, levels of TUNEL-positive nuclei were also significantly increased in tumors of the mice treated with the immunomodulators. Additionally, Bax protein levels also increased in the tumors of mice treated with the monoclonal antibodies, while no differences in Bcl2 levels were seen between the study groups. These results are consistent with those previously reported, in which different therapeutic strategies also elicited a rise in proapoptotic markers [12,33]. The increase in apoptotic markers of cancer cells was also demonstrated in previous investigations in which the animals were treated with selective inhibitors of PARP activity [44,48–50]. A rise in the autophagy marker LC3B was observed in the tumor cells of the mice treated with the immunomodulators compared to non-treated control rodents. These results imply that autophagy may also mediate the reduced tumor burden observed in the mice that received treatment with the cocktail of monoclonal antibodies. In fact, similar results were previously demonstrated in the tumors of mice that were genetically deficient for either PARP-1 or PARP-2 proteins, especially the latter [33]. The deacetylase sirtuin-1 may play a role in autophagy as a result of its upstream regulation of LC3B [51]. In the current study, a significant rise in protein levels of sirtuin-1 was detected in the Cancers 2019, 11, 1301 13 of 16 tumor cells of the mice treated with the monoclonal antibodies compared to non-treated animals. On the other hand, sirtuin-1 may also play a role in the regulation of tumor microenvironment of the immune cells [52]. These results imply that sirtuin-1 probably interfered with immune cells [52], leading to changes in the tumor microenvironment (from Th2 type to Th1 immunity) as previously demonstrated [18,19]. This may further contribute to the reduced tumor burden observed in the mice treated with the monoclonal antibodies. Despite the relevance of this question, it will have to be fully elucidated in future investigations as it was clearly beyond the objectives of the current investigation. Study Limitations Limitations inherent to the use of an animal experimental model may have occurred in the current investigation as compared to clinical studies. This may partly preclude the generalization of the present study results to clinical settings. Moreover, other limitations may be related to the type of tumor cells and the animal background as well as the type of laboratory techniques employed to identify the different immune cells compared to previous investigations [18,19]. Procedures beyond the histology, such as flow cytometry, may be useful to selectively identify the type and number of the cells contained in the tumors. Nonetheless, these experiments would have required a completely different experimental approach at the time of conducting the animal experiments and when collecting the tumor specimens. On the other hand, the histological approach enabled us to identify topographically the presence of the T cells within the cancer specimens, thus confirming that they were, indeed, part of the tumor microenvironment. Another possible limitation in the study would be related to the lack of additional control groups of mice, such as animals administered with isotype-matched antibodies to confirm the selectivity and specificity of the immunotherapy. Nevertheless, as the efficacy and selectivity of the antibodies used in the present investigation had already been demonstrated in previous studies [18,19] and for ethical reasons, no additional control groups were included. Despite all these limitations, the experiments reported herein shed light onto novel mechanisms whereby immunotherapy may exert beneficial effects in lung adenocarcinoma tumors. 5. Conclusions We have demonstrated that immunomodulators with different mechanisms of action elicited a reduction in the tumor burden as measured by tumor size and weight through several biological mechanisms, namely decreased tumor proliferation rates and increased T cell counts, oxidative stress, apoptosis, autophagy, and signaling pathways, which may have interfered with the immune profile of the tumor microenvironment. Future research should be devoted to the elucidation of the specific contribution of each mechanism (reduced tumor proliferation, increased tumor degradation, and stimulation of the immune tumor microenvironment) to the reduced tumor burden seen in this animal model of lung cancer. These findings may have potential therapeutic implications in patients under treatment with immunomodulators for their lung neoplasms. Author Contributions: Study conception and design: E.B., J.T., D.R.-C.; Animal experiments and sample collection: D.R.-C., J.T.; Molecular biology and histological analyses: J.T., D.R.-C., X.W.; Statistical analyses and data interpretation: J.T., D.R.-C., E.B.; manuscript drafting and intellectual input: E.B., J.T., D.R.-C.; manuscript writing final version: E.B. Funding: This study has been supported by CIBERES; FIS 14/00713 (FEDER); FIS 18/00075 (FEDER); SEPAR 2016; SEPAR 2018; FUCAP 2016; Unrestricted grant from Menarini SA 2018 (Spain), and GlaxoSmithKline SA (Spain) 2018. Acknowledgments: The authors are very grateful to Xavier Duran for his contribution to the statistical analyses of the study results and to Anna Salazar-Degracia for her help with part of the animal experiments. Conflicts of Interest: The authors declare no conflict of interest. Cancers 2019, 11, 1301 14 of 16 References 1. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2018. CA Cancer J. 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