REVIEW published: 23 October 2020 doi: 10.3389/fonc.2020.582884 Therapy-Induced Modulation of the Tumor Microenvironment: New Opportunities for Cancer Therapies Sergi Benavente 1*, Almudena Sánchez-García 2 , Silvia Naches 3 , Matilde Esther LLeonart 2,4 and Juan Lorente 3 1 Radiation Oncology Department, Vall d’Hebron University Hospital, Barcelona, Spain, 2 Biomedical Research in Cancer Stem Cells Group, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Otorhinolaryngology Department, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Spanish Biomedical Research Network Centre in Oncology, CIBERONC, Barcelona, Spain Advances in immunotherapy have achieved remarkable clinical outcomes in tumors with low curability, but their effects are limited, and increasing evidence has implicated tumoral and non-tumoral components of the tumor microenvironment as critical mediators of cancer progression. At the same time, the clinical successes achieved with minimally invasive and optically-guided surgery and image-guided and ablative radiation strategies have been successfully implemented in clinical care. More effective, localized and safer treatments have fueled strong research interest in radioimmunotherapy, which has shown Edited by: Chris Albanese, the potential immunomodulatory effects of ionizing radiation. However, increasingly Georgetown University, United States more observations suggest that immunosuppressive changes, metabolic remodeling, Reviewed by: and angiogenic responses in the local tumor microenvironment play a central role in Walter J. Storkus, tumor recurrence. In this review, we address challenges to identify responders vs. University of Pittsburgh, United States Enza Lonardo, non-responders to the immune checkpoint blockade, discuss recent developments in Institute of Genetics and Biophysics combinations of immunotherapy and radiotherapy for clinical evaluation, and consider (The National Research Council of Italy), Italy the clinical impact of immunosuppressive changes in the tumor microenvironment in *Correspondence: the context of surgery and radiation. Since the therapy-induced modulation of the Sergi Benavente tumor microenvironment presents a multiplicity of forms, we propose that overcoming sbenavente@vhebron.net microenvironment related resistance can become clinically relevant and represents a novel strategy to optimize treatment immunogenicity and improve patient outcome. Specialty section: This article was submitted to Keywords: radiotherapy, immunotherapy, tumor microenvironment, surgery, cancer Cancer Metabolism, a section of the journal Frontiers in Oncology INTRODUCTION Received: 13 July 2020 Accepted: 16 September 2020 Cancer treatment modalities vary considerably depending on stage and location, however surgical Published: 23 October 2020 excision and radiation therapy are an integral part of treatment for most solid tumors. In an era Citation: of exceptionally dynamic evolution of knowledge, some recently published clinical studies have Benavente S, Sánchez-García A, reshaped the role of surgery such as neoadjuvant immunotherapy combinations leading to less Naches S, LLeonart ME and Lorente J invasive surgery for advanced melanoma, antiangiogenics as an alternative to immediate surgery (2020) Therapy-Induced Modulation of the Tumor Microenvironment: New in renal cell carcinoma or upfront treatments making surgery possible for more patients with Opportunities for Cancer Therapies. pancreatic cancer (1). Most therapeutic combinations in clinical trials are based on knowledge Front. Oncol. 10:582884. of resistance mechanisms and recently immunotherapy, which has revolutionized the clinical doi: 10.3389/fonc.2020.582884 management of multiple tumors, has been included in multiple clinical trials which are mainly Frontiers in Oncology | www.frontiersin.org 1 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance based on T cell and pursue a maintained antitumor immune discuss recent developments in combinations of immunotherapy response. Accumulating evidence suggests that conditioning the and radiotherapy for clinical evaluation, and consider the clinical tumor microenvironment (TME) toward an immunomodulatory impact of immunosuppressive changes in the TME in the context state may have a major impact on cancer outcome (2, 3). of surgery and radiation. Overcoming microenvironment related However, the TME comprises all the non-malignant cellular and resistance may have a fundamental impact on treatment efficacy non-cellular components of the tumor, including the immune and patient outcome. system, blood cells, endothelial cells, fat cells, and the stroma. The tumor stroma is a critical component of the TME with CHARACTERIZING THE IMMUNE cancer-promoting capacity as part of the response to treatments and leads to cancer resistance. For example, immunosuppressive FUNCTION IN THE RESPONSE TO cytokine secretion and metabolic alterations strongly participate CHECKPOINT INHIBITOR in the suppression of host immune responses against tumor IMMUNOTHERAPY cells facilitating tumor proliferation. Extensive work exploring the interactions between cancer cells and the TME has been Combinatorial Approaches to Treat done but the advancements still require a better understanding Differences in the Immune Contexture of of the potential targets before implementation in conceptual the TME antitumor strategies. In this regard, recent advances resulting Immunotherapeutic approaches have transformed treatment and in more effective and localized radiation treatments (stereotactic outcomes for some solid tumors, in particular, melanoma and radiosurgery and stereotactic body radiotherapy, SRS/SBRT) can non-small cell lung cancer (NSCLC), but do not benefit the achieve an effective alteration and ablation of tumor stromal majority of patients with cancer and have failed to induce broadly tissue, which can be a singular advantage against tumoral durable responses. Immunotherapy with ICB uses monoclonal immune evasion [reviewed in (4)]. In addition, technological antibodies that target the inhibitory proteins CTL antigen 4 developments have led to minimally invasive surgery with (CTLA-4) or programmed death-1/programmed death-ligand 1 evident clinical benefits in terms of less invasiveness, excellent (PD-1/PD-L1) on T cells or cancer cells to unleash the immune outcomes, and a shorter hospital stay (5). response. However, response rates vary widely and predictive In this review, we address challenges to identify responders factors of response to ICB remain elusive. It has been suggested vs. non-responders to the immune checkpoint blockade (ICB), that PD-L1 expression, high tumor mutational burden (TMB) which is highly influenced by the epitopes displayed in the human leukocyte antigen (HLA) genes of a tumor, and the presence of Abbreviations: 4-1BB, tumor necrosis factor receptor superfamily member 9, CD8+ T cells are prognostic of clinical response to treatment with CD137; A2AR, adenosine receptor A2; APC, antigen-presenting cells; ATM, ataxia telangiectasia mutated; ATP, adenosine triphosphate; CAFs, cancer-associated ICB (6). fibroblasts; CCL2, CC chemokine receptor 2; CD28, cluster of differentiation 28; The distinction between hot, altered (excluded and CD39, cluster of differentiation 39; CD73, cluster of differentiation 73, ecto-5′ - immunosuppressed) and cold tumors, based on the cytotoxic nucleotidase; CD80, cluster of differentiation 80; CD86, cluster of differentiation T cell landscape within a tumor, establishes the important role 86; cGAS, cyclic GMP-AMP synthase; CHK1, checkpoint kinase 1; CSF-1, colony of the TME but only a thorough profiling of the TME can stimulating factor 1; CSF-1R, colony stimulating factor 1 receptor; CTLA-4, CTL antigen 4; CXCL1, C-X-C motif chemokine ligand 1; CXCL2, C-X-C motif analyze the complexity of the tumors and provide dynamic chemokine ligand 2; CXCL10, C-X-C motif chemokine ligand 10; CXCL16, C- information about the complex networks operating in the X-C motif chemokine ligand 16; CXCR4, C-X-C chemokine receptor type 4; TME to guide clinical decisions (7, 8). Combining immune DAMPs, damage-associated molecular patterns; DC, dendritic cells; DDR, DNA and genomic data has revealed six immune subtypes across damage response; DNA-PK, DNA-dependent protein kinase; ECM, extracellular 33 different cancer types including immune (macrophage matrix; HIF-1α, hypoxia-inducible factor-1α; HLA, human leukocyte antigen; HMGB1, high mobility group box 1; HSP90, heat shock protein 90; ICB, or lymphocyte signatures, Th1:Th2 cell ratio, expression of immune checkpoint blockade; IFN, interferon; IFN-γ, interferon gamma; IL-4, immunomodulatory genes) and non-immune parameters interleukin 4; IL-6, interleukin 6; IL-10, interleukin 10; IL-13, interleukin 13; IL- (intratumoral heterogeneity, aneuploidy, neoantigen load, 35, interleukin 35; irAEs, immune-related adverse events; KPNA2, karyopherin overall cell proliferation, and patients’ prognosis) (9). It has subunit alpha 2; LAG3, lymphocyte-activation gene-3; LUM, LUM imaging been proposed that an integrative view of the multi-omics system (Lumicell Inc.); LUM015, cathepsin activatable fluorescent probe; MDSCs, myeloid-derived suppressor cells; MHC-I, major histocompatibility complex-I; experimental platforms and computational power is required NF-κβ, nuclear factor-kappa beta; NK, natural killer; nMOFs, nanoscale metal- to identify signatures of immune response with improved organic frameworks; NP, nanoparticles; NSCLC, non-small cell lung cancer; predictive power (10). OX-40, tumor necrosis factor receptor superfamily member 4, CD134; PARP, It has been clearly established that CD8+ T cells are the poly(ADP-ribose) polymerase; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species; SBRT, stereotactic body radiotherapy; ultimate effectors of tumor rejection and the strongest predictor SDF-1, stromal cell-derived factor-1; SRS, stereotactic radiosurgery; STING, of ICB response across tumor types. Significantly, the functional stimulator of interferon genes; TAMs, tumor-associated macrophages; TGFβ, variability of tumor-infiltrating T cells can influence their transforming growth factor β; Th1, T helper type 1; Th2, T helper type 2; TIGIT, T cytotoxicity. Subsets with reactivation of dysfunctional CD8+ , cell immunoglobulin and ITIM domain; TIM-3, T cell immunoglobulin and mucin memory-like CD8+ TCF7+ , CD103+ tumor-resident CD8+ , and domain-3; TLR, toll-like receptors; TLR3, toll-like receptor 3; TLR7/8, toll-like receptors 7/8; TLR9, toll-like receptor 9; TMB, tumor mutational burden; TME, Tcf1+ PD-1+ CD8+ with stem-like properties T cells have shown tumor microenvironment; TNFα, tumor necrosis factor α; Treg, regulatory T cells; durable responses. CD4+ T cell subpopulations that play a critical Trex1, three-prime repair exonuclease 1; VEGF, vascular endothelial growth factor. role in immunotherapy include CD4+ Th1 cells that generate Frontiers in Oncology | www.frontiersin.org 2 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance functional CD8+ T cell responses, CD4+ FoxP3+ regulatory T been established although the potential of analyzing dynamics cells (Treg ) generally associated with suppression of antitumor and plasticity of TME networks will offer more powerful immune responses in several cancers although responses to stratification systems between responders and non-responders. CTLA-4 blockade have been shown, and CD4+ FoxP3− PD-1Hi (4PD-1Hi ) T cells can indicate a negative prognosis when there is Interactions Within the TME persistence after PD-1 blockade (6). Emerging factors associated Interactions between malignant and non-malignant cells create with ICB response include B cells and tertiary lymphoid the TME (Figure 1). Non-malignant cells are usually highly structures (11, 12). As for innate immune populations, BDCA- dynamic and display tumor-promoting capabilities. Major 3+ CLEC9A+ dendritic cells (DC) and XCL1-producing NK cells non-malignant cell types found in the TME are immune are linked to ICB response (13). cells, vasculature and lymphatic vessels, and fibroblasts. Cell ICB is most efficacious in tumors with a high degree communication is accomplished by a network of cytokines, of T cell infiltration (hot tumors), such as melanomas and chemokines, and diverse metabolites that reacts to changes in the NSCLC. Alternative combinations include other checkpoint physical and chemical characteristics of the tissue (23). Cancer molecules, such as T cell immunoglobulin and mucin domain- treatment effects induce a variety of mechanisms which lead to T 3 (TIM-3), lymphocyte-activation gene-3 (LAG3), and T cell cell exclusion and avoidance of their cytotoxic function (24) that immunoglobulin and ITIM domain (TIGIT) in the case of T ultimately shift the balance of stromal cell phenotypes in the TME cell exhaustion; or co-stimulatory checkpoint proteins, including toward an immunosuppressive state. These pro-tumorigenic OX-40, CD28, and 4-1BB ligand receptor to enhance T cell responses to therapy can induce local and/or systemic changes expansion or effector functions. Preliminary results also suggest that underlie tumor recurrence and treatment resistance. a potential role of microbiome modulation. On the other hand, In a broad sense, the mechanisms leading to a pro- immune cold tumors, including pancreatic and prostate cancers, tumorigenic microenvironment can be grouped into three are not well-infiltrated by immune cells. Therefore, research categories: immune cell regulation, metabolic reprogramming, efforts have focused on making cold tumors hot by increasing and hypoxia (4). The biochemical and physical properties of the immune infiltration and activity, such as vascular normalization, TME undergo substantial changes during tumor evolution and increasing the neoantigen burden, oncolytic therapy, vaccines, treatment determined by the increased demand for blood vessels adoptive T cell therapy, T cell immunomodulators, and to endure tumor growth, which requires an adequate supply of radiotherapy. Clinical strategies in immune-altered tumors have oxygen and nutrients delivered through the blood vasculature. an impact on T cell trafficking, inhibition of hypoxia-associated The resulting abnormal vessels are leaky and compressed which pathways, and the immune suppressive microenvironment (14). can induce a dense stromal reaction and reduction of blood flow As more combinations of immunotherapeutic strategies reach that promotes hypoperfusion. The TME then becomes hypoxic the clinical arena, two clinical challenges become more relevant. with enhanced potential for tumor progression in multiple ways. Checkpoint disruption leads to a wide range of inflammatory In this situation, hypoxia reduces immune cell activity and the toxicities grouped as immune-related adverse events (irAEs). The TME acquires an immunosuppressive phenotype (25). Hence, majority occur in barrier tissues (gastrointestinal or pulmonary better understanding and reprogramming of these components mucosa, skin) or in endocrine glands. Although many are may greatly influence cancer outcome. mild, they can carry considerable morbidity, lead to reduced Clinically, this may significantly limit cancer treatment treatment dosage and/or duration, and on occasions may be efficacy and represent a shift in our understanding of tumor fatal (e.g., in patients with pre-existing autoimmunity) (15, 16). progression and resistance. Major emphasis has been placed on On the other hand, it has been suggested that irAEs could help advancing clinical applications that strengthen the effectiveness select responders to ICB in bladder cancer (17). Secondly, some of immunotherapies, leading to rapid regulatory approval of ICB patients experience an acceleration of tumor growth kinetics with combined with targeted therapies and/or chemotherapy in large poor survival called hyperprogression which, at present, remains numbers of patients with cancer, facilitating their incorporation difficult to characterize (18, 19). into clinical practice. However, in spite of the extensive use of The composition of the TME is dynamic and evolves during surgery and radiation strategies in cancer, as a definitive strategy ICB treatment. It has been suggested that the TME evolves in early or moderately-advanced stages of cancer, as part of a differently between responders and non-responders. Of interest, multimodal strategy in advanced loco-regional disease and, more stronger differences were found early on-treatment than before recently in selected cases of oligometastatic disease, there is very the ICB based on the differences in the densities of CD4+ limited understanding of the biological changes in the TME or CD8+ T cells and the expression of PD-1/PD-L1 after two induced by local treatments. or three anti-PD-1 doses than at baseline (20, 21). Another interesting feature is that PD-1 blockade can induce clonal replacement preferentially of exhausted CD8+ T cells, meaning RADIOIMMUNOTHERAPY COMBINATIONS that T cells present at baseline may show reduced proliferation and that the response to ICB could be due to T cell clones that Radioimmunotherapy is an area of extensive research due to the enter the tumor during the course of treatment (22). potential immunomodulatory effects of ionizing radiation and Clinical relevance of distinctions in the immune contexture has established a new paradigm in which radiation is as efficient mainly based on the cytotoxic landscape of T cells in tumors has as its capacity to elicit tumor-targeting immune responses (2). Frontiers in Oncology | www.frontiersin.org 3 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance FIGURE 1 | A schematic representation of the immunosuppressive TME. In a tumor, cancer cells coexist with immune cells, fibroblasts, and blood vessels to form the TME. Cancer cells can alter the microenvironment and promote cancer growth and dissemination. Ionizing radiation is able to induce immunogenic cell transcription of the IFN-stimulated genes. The cytoplasmic death, a form of cell death that promotes a T cell-based three-prime repair exonuclease 1 (Trex1), induced by immune response against antigens derived from dying cells, radiation, is a negative regulator of this pathway. The enhances antigen presentation, and activates cytotoxic T cells. release of IFN type I from APC supports antigen uptake Cytosolic DNA from dying cells function as neoantigens by Batf3+ DC and cross-presentation of tumor antigens that are highly immunogenic. Radiation induces the release to CD8+ T cells. Activated CD8+ T cells are recruited of danger signals, including calreticulin, high mobility to the irradiated tumor site by cytokines upregulated by group box 1 (HMGB1), and adenosine triphosphate radiation (CCL2, CXCL1, CXCL10, and CXCL16). In addition, (ATP), which are collectively known as damage-associated radiation enhances the expression of major histocompatibility molecular patterns (DAMPs), and support the recruitment complex-I (MHC-I) antigens on cancer cells that favor antigen and maturation of antigen-presenting cells (APC), migrate presentation (4). to lymph nodes, and prime a cytotoxic T cell-dependent immune response. Critical to the immunogenicity of radiotherapy is the DNA Damage Response Following fragmentation of nuclear DNA from the DNA damage Radiation and Exposure of Neoantigens response (DDR) of radiation, shuttled to the cytoplasm Tumor cell-intrinsic events driven by DNA damage are where it activates cyclic GMP-AMP synthase/stimulator central to the immunomodulatory effects of radiotherapy. of interferon genes (cGAS/STING) pathways and induces Radiation-induced DNA damage alters gene transcription and Frontiers in Oncology | www.frontiersin.org 4 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance modulates the expression of tumor neoantigens, resulting TLR9 in combination with radiotherapy can enhance antitumor in activation of innate and/or adaptive antitumor immune immunity with long-term tumor control. Mechanistically, TLR response (6, 26, 27). The finding that a patient with metastatic, can enhance DC-mediated cross-presentation and activation of treatment refractory NSCLC who responded to ipilimumab T cells. Novel formulations of TLR agonists with reduced toxicity plus radiotherapy was carrying a mutation in a KPNA2 and precise and image-guided radiation techniques are favorable gene, upregulated in expression by radiation; tumor-specific aspects for this strategy (30, 31). T cell clones were developed in peripheral blood shortly after completion of radiotherapy and the first dose of ipilimumab to a metastatic site and remained elevated while the patient achieved a complete response in all of the non- Addressing the Evasive Objective of irradiated lesions supports the hypothesis of in situ tumor Durable Responses of vaccination (28). Radiation-Immunotherapy Combinations Identification of genetic determinants of radiotherapeutic Studies on resistance to ICB reveal a complex and rapidly efficacy has remained elusive but a recent report identifies evolving network of mechanisms of immune resistance specific genetic ATM inactivation to be strongly associated with to each host and tumor (32). The absence of biomarkers that clinical benefit from radiotherapy. The identification of identify the different types of resistance obliges the use of a radiosensitive phenotype across multiple cancer types empirical approaches to target them. inaugurates the possibility of further testing in prospective The immunogenicity of radiation has been approached with clinical trials and progress in personalized radiation strategies. two different strategies, one that emphasizes the local interaction For example, patients with metastatic tumors harboring a of radiotherapy and the immune system where the majority of somatic ATM mutation may receive a reduced dose of radiation clinical knowledge has been accumulated, and a second strategy with the goal of reducing toxicity and maintaining tumor where focal radiation elicits systemic disease control (abscopal control (Pitter et al., accepted). effect) known as in situ tumor vaccination that has attracted a Defects in DDR have been exploited for drug development as lot of attention. The basis for combining ICB with radiotherapy radiosensitizers including poly(ADP-ribose) polymerase (PARP), stems from the fact that radiation upregulates PD-L1, which leads checkpoint kinase 1 (CHK1), DNA-dependent protein kinase to CD8+ T cell exhaustion. In addition, many tumors devoid (DNA-PK), or the chaperone HSP90 inhibitors. Radiation of T cells at baseline (and secondary lack of PD-L1 expression damage in the context of defective DDR pathways generates on effector T cells) could benefit from the radiation-induced micronuclei in cancer cells that activate cGAS/STING pathways increase in PD-L1 and the combination (33). In the case of and propagate an inflammatory response that can enhance CTLA-4, upon radiation, it is recruited to the membrane of radiation effects. Adding ICB to the immunomodulation activated T cells and binds to the ligands CD80 and CD86, induced by DDR inhibitors plus radiotherapy is a new area expressed on DC and other APC, thereby attenuating T cell of clinical research that can provide additional insights into activation (34). the immunomodulatory effects of radiation given that DDR Tumor burden has been regarded as a surrogate for inhibitors can enhance the immunostimulatory effects of ICB effectivity based on clinical observations that adjuvant radiation while ICB can target the immunosuppressive radiation ipilimumab in resected stage III melanomas obtains major effects (27). benefits in recurrence-free survival and overall survival (48.3 and 65.4% at 5 years, respectively) (35), and locally advanced NSCLC treated with definitive chemoradiation followed by Central Role of Dendritic Cell Maturation in adjuvant durvalumab in the PACIFIC trial with an impressive Radiation-Induced Immunological prolongation of time to death or distant metastasis from 16.2 Response to 28.3 months and a favorable toxicity profile (36). Moreover, DC are a sparsely distributed immunological component of in patients that do respond to ICB, failure frequently occurs the TME with high biological heterogeneity that play a in sites of previous disease, with 60% of failures in anti-PD- central role in linking innate and adaptive immune responses. 1/PD-L1 treated NSCLC and 39% of failures in anti-PD-1 treated Therefore, DC are a key element in the immunostimulatory melanoma (37, 38). Although it is not specific criteria, the best effect of radiotherapy. It has been recently reported that poorly outcome with ablative radiation in oligometastatic clinical trials radioimmunogenic murine tumors fail to activate DC following has been obtained in patients with low tumor burden and as local treatment, and that it could be successfully reverted with an consolidation (39, 40). exogenous adjuvant, resulting in tumor cures (29). Therefore, While the majority of clinical studies have targeted a single it could be hypothesized that in patients with a poor TME, metastatic site, abscopal responses are relatively rare, and the combination of radiation with adjuvants that promote DC mainly in melanoma and NSCLC (41). Improved outcomes maturation or target the immunosuppressive TME can improve have been obtained in several phase 2 clinical trials using tumor control. local consolidation with ablative doses of radiation in the Toll-like receptors (TLR) signaling pathways activate innate oligometastatic setting (39, 40, 42) while ongoing phase immunity and regulate adaptive immune responses. Preclinical 3 trials are investigating whether this approach may lead evidence suggests that TLR-agonists targeting TLR3, TLR 7/8 or to improve overall survival in a subset of patients with Frontiers in Oncology | www.frontiersin.org 5 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance limited metastatic disease (NCT02417662, NCT03137771, CLINICAL IMPLICATIONS OF NCT02364557, NCT03862911, and NCT03721341). TECHNOLOGICAL DEVELOPMENTS It has been recently reported that tumor-resident CD8+ T cells play a significant role in mediating the immune effects Surgery and radiation remain strong curative modalities for of radiotherapy. Even if proliferation decreases after radiation, treatment of established solid tumors but treatment failure their functionality, measured as production of IFN-γ, augments, continues to be a significant problem. The best established role of and mediates the early antitumoral effect of local SBRT doses. surgical oncology is the complete removal of the tumor, with an Nonetheless, as newly infiltrating CD8+ T cells play a key role in additional strong foundation to question the elective treatment antitumor immunity, that may also be the case with radiation- of uninvolved regional lymph nodes in a large variety of tumor induced immunogenicity (43). If radiation could increase the types and resection of metastatic disease which is increasingly population of pro-immunogenic T cell subtypes within the local offered to selected patients with indolent oligometastatic disease TME, it would enhance the response to ICB. This hypothesis (52). Critical to all of them is securing negative surgical margins. raises the possibility that targeting multiple metastatic sites with Less invasive technologies and advances in imaging SBRT to achieve complete cytoreduction in the metastatic setting leading to minimally invasive and robot-assisted surgeries may become clinically relevant (44). Moreover, the irradiation of are revolutionizing surgical care (5). Likewise, advanced each visible metastasis addresses the challenge of heterogeneity image guidance and motion management strategies are by attempting to convert each target into an in situ vaccine shaping new therapeutic radiation strategies enabling the safe (45). Clinical support data comes from a phase 2 clinical trial in administration of ablative doses of radiation (2). Advanced NSCLC with up to 4 metastatic sites (93% had <2 metastases), imaging is fundamental and uniquely placed to serve both which underwent locally ablative treatment with metastasectomy margin negativity rates and future radiation strategies. or multi-site SBRT followed by pembrolizumab, with median Surgical margin positivity rate (cancer cells at the edge of survival of 19.1 months (vs. 6.6 months in historical controls) and tumor resection) has not significantly improved in recent decades favorable toxicity profile (46). and when it does occur prognosis is significantly affected in many Research has been very controversial with variations in dose tumor types. Margin positivity rates across all types of cancer or fraction for radiation delivery in the metastatic setting, range from 15 to 60% (53). A recent report on positive surgical where the role of treatment parameters such as duration (more margins in the ten most common solid cancers has identified oral or <7 days), fraction size (1.8–3 to 8–30 Gy) and scheduling cavity cancer with the highest rate with up to 25% of cases, no (single or multiple fractions) are largely unknown. While a change over time, with significant effects on tumor relapse and short course (1–5 fractions) of high dose radiation can be safely overall survival. For advanced disease, the rates ranged between administered and is able to elicit an immunogenic response 20.9% (breast) and 65.5% (prostate) with related worse outcome that can benefit from the addition of ICB, the predominance in seven tumor types (54). Although not a true resistance type, of the immunosuppressive effects of radiation may limit the we propose the term “margin-missing” effect to characterize this effectiveness of ablative doses of radiation, especially if single situation which leads to treatment failure and resistance. fractions are used (47, 48). Nonetheless, the immune context Fluorescence-guided surgery, which allows intraoperative of the tumor type or even the metastatic organ may require visualization of tumors, is an evolving image-guided surgical a different dose and/or fractionation to elicit an immunogenic strategy to help differentiate tumor cells from normal response. This possibility offers the potential to reduce the surrounding tissues in real time. Near-infrared fluorescence dose and volumes of radiation and still prove efficacious. imaging has a higher tumor to background ratio, high tissue In the PACIFIC trial (36), immunotherapy was administered penetration (5–10 mm), and little interference from intrinsic sequentially (i.e., following chemoradiation) with a good toxicity fluorescence. Indocyanine green is the most widely used probe in profile but data on toxicity of concomitant radiation and fluorescence-guided surgery although tumor detectability is not immunotherapy in the clinical setting is scarce. Yet the biological very good and optical technology is still evolving (55). context remains to be proven that would favor multiple rounds More than 50% of patients with cancer receive radiotherapy, of high-end ablative dose schedules in oligometastatic patient as which defines its leading role in cancer management, in advocated by some groups (49). Another concept of potential particular for several locally advanced solid tumors. The latest clinical relevance that has been put forward is the possibility that developments in radiotherapy have swiftly enabled local dose the immunomulatory effect of low-dose radiation for stromal escalation making it possible to deliver high doses of radiation modulation could favor T cell infiltration and enhance the with incredibly high anatomical precision and reduced risk of immune response (47, 50). long-term adverse effects. As a consequence, relevant clinical The next generation of clinical trials addressing benefit has been achieved in a variety of cancer types such as radiotherapy-immunotherapy combinations will have to prostate, gynecologic, breast, head and neck cancers, and brain include immunological read-outs with proper endpoints for and lung metastases (2). However, no significant advance has immune monitoring as well as the identification of immune occurred in the past 30 years in the development of strategies biomarkers that optimize the selection of treatment strategies that enhance radiation effects. On the other hand, due to the (31, 51). recognition that the immune system can strongly contribute Frontiers in Oncology | www.frontiersin.org 6 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance to therapeutic responses to radiation, radioimmunotherapy has Reprogramming the immunosuppressive TME also converts become an intensive area of research. tumors not responding to ICB to responding ones (62). This The current challenge in near-infrared fluorescence-guided platform may serve to reshape immunosuppressive TME and surgery is to design probes with high selectivity for tumors and synergize with other therapies. clear visualization, referred to as smart probes, which are only Recent clinical data in melanoma and NSCLC have shown activated at the tumor site (turn-on probes). There was a recent that response to ICB in individual patients with metastasis report about the design, synthesis, and characterization of three vary depending on the anatomical location of the metastasis, novel polymeric turn-on nanoprobes that are activated at the untangling the importance of the local TME in antitumor tumor site by cysteine cathepsins (highly expressed in multiple immunity. Of interest, tissue specific response to immune tumor types) showing a stable and well-defined signal from the checkpoint inhibition depends on the cancer type, which implies tumor during the whole surgical procedure in orthotopic breast that responsive and non-responsive sites are different among cancer and melanoma models resulting in less tumor recurrence patients with NSCLC or melanoma (63, 64). These heterogeneous and prolonged survival compared with standard commercial responses are an evident clinical problem, since patients with probes (56). This is a significant lead toward real-time image- responses to ICB in all lesions survive longer than those with guided tumor margin assessment during surgical oncology. response in some of the lesions (65). Potential mechanisms Emerging approaches seek to integrate analytical tools with include myeloid cell exclusion and alteration of T cell activation optical technology to help improve the decision-making of in response to tumor growth and local factors, but this will fluorescence-guided surgery to reduce margin positivity rates. require unraveling a very complex network of interactions For example, combinations of fluorescence-guided surgery have for differential responsiveness across different tissue sites of been made with mass spectrometry (57), Raman spectroscopy tumor deposits. (58), and hyper spectral imaging (59). The most clinically advanced nanoprobe is LUM015 (a Regulatory T Cells pegylated cathepsin-activatable probe) which is undergoing eight Treg cells are a small subset of circulating CD4+ T cells clinical trials, including a pivotal phase 3 study (60). The phase 3 with potent suppressive functions with a central role in trial is a multicenter study with the primary objective of assessing regulating immune responses and maintaining self-tolerance the ability of LUM015 and LUM fluorescence-guided surgery although they also impede antitumor immunity. In contrast system to detect residual tumors in 250 breast cancer patients with circulating Treg cells, intratumoral Treg cells maintain an undergoing lumpectomy (NCT03686215). active configuration, suggesting that antigen stimulation may play an important role in the activation and accumulation of Treg cells in the TME. The immunosuppression mediated by CLINICAL IMPLICATIONS OF THE Treg cells is mainly mediated by the release of anti-inflammatory IMMUNOSUPPRESSIVE ENVIRONMENT cytokines including IL-10 and transforming growth factor β (TGFβ), facilitating proliferation of CD4+ T cells to Treg cells, It has been traditionally assumed that recurrent tumors arise while suppressing proliferation to CD8+ T cells and NK cells. from transformed neoplastic clones that are more resistant to In addition, Treg cells can also reprogram macrophages to the oncological therapies, however, an early experience challenged M2 phenotype (via IL-4, IL-10, and IL-13) and favor MDSCs this view and hypothesized that primary and recurrent tumors infiltration (via IL-10 and IL-35) (66). of equal size did have different microenvironments that Immunological cell death induced by radiation upregulates or explained their response to therapies. The study found that releases DAMPs, including ATP, with further recruitment and while small primary tumors had a healthy population of activation of DC to initiate the antitumor immune response but antitumor effector CD8+ T lymphocytes, recurrent tumors ATP is rapidly catabolized in the TME into adenosine by the had an immunosuppressive condition consisting in expanded enzymes CD39 and CD73. Local accumulation of extracellular populations of tumor-associated macrophages (TAMs), Treg cells, adenosine suppresses DC and CD8+ T cells and promotes and pro-tumoral cytokines that inhibited cytotoxic CD8+ T proliferation of Treg cells, increases the expression of CTLA-4 lymphocytes. These changes were also identified in regional and adenosine receptor A2 (A2AR) on Treg cells, and enhances draining lymph nodes. Disruption of these immunosuppressive the polarization of macrophages to the M2 phenotype. Radiation pathways restored the efficacy of the tumor vaccine in recurrent can also induce conversion of ATP to adenosine through the tumors, as if they were primary tumors (61). induction of reactive oxygen species (ROS) and TGFβ. Thus, Research in preclinical models has shown that a syringeable targeting of A2AR, CD73, and TGFβ may reduce resistance immunomodulatory multidomain nanogel (iGel) containing to immunotherapy in the radiotherapy setting (33). Blockade gemcitabine, imiquimod, and clodronate locally applied as a of CD73 plus radiotherapy restored radiation-induced DC postsurgical treatment is able to deplete immunosuppressive infiltration of tumors in a poor immunogenic setting, and the cells from the TME (myeloid-derived suppressor cells (MDSCs), addition of CTLA-4 blockade improved control of non-irradiated M2 macrophages, and Treg cells), increase immunogenicity, lung metastases in murine models. These findings set the stage for and induce immunogenic cell death. Indeed, it generates clinical testing CD73 in patients who carry cGAS/STING tumors systemic antitumor immunity and a memory T cell that or show upregulation of soluble CD73 following radiotherapy to significantly inhibits tumor recurrence and lung metastases. determine if CD73 blockade can enhance responses to ICB (67). Frontiers in Oncology | www.frontiersin.org 7 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance Treg cells also express PD-1 at a low level in the blood and shift M2 cells to M1 by targeting secreted immunosuppressive at a high level in tumors, promoting the suppressive activity factors released by cancer cells and cells in the TME (Figure 2). of PD-1-expressing Treg cells upon antibody-mediated PD-1 Preclinical studies suggest that macrophage manipulation blockade (68). Recently reported, ∼10% of cancer patients treated to avoid recruitment or prevent M2 polarization produce a with anti-PD-1 antibody develop hyperprogressive disease, significant enhancement of the radiation effect irregardless characterized by rapid cancer progression. Treg -specific depletion of the tumor model [reviewed in (81)]. The increase in prior to, or combined with, an anti-PD-1 antibody may prevent radiosensitivity with this strategy has been attributed to blockade hyperprogressive disease and enhance the effectiveness of anti- of vasculogenesis. If angiogenesis supports the formation of PD-1 therapy (69). tumor blood vessels from the sprouting of local vessels, tumors can also develop or repair blood vessels from circulating proangiogenic cells mainly from the bone marrow, which is Macrophages known as vasculogenesis (82). This effect could be exploited TAMs account for the largest fraction of the myeloid infiltrate in in radiation treatments, namely if the increase in hypoxia that the majority of solid tumors. The tumor-associated macrophage occurs at the end of radiation through recruitment of circulating compartment is highly dynamic in time (during tumor proangiogenic cells to rescue damaged tumor vasculature and progression and response to treatment) and space (at different promote tumor recurrence can be reversed. A first-in-human tumor sites) through an extensive remodeling of energy clinical trial of glioblastoma examined the effects of CXCR4 metabolism. In addition, the tumor-associated macrophage blockade through a 4-week continuous infusion of plerixafor, compartment is highly heterogeneous both within and across a small molecule CXCR4 inhibitor, at the end of irradiation tumors in response to environmental changes ranging from in newly diagnosed glioblastoma and showed high efficacy and a pro-inflammatory (M1) to an anti-inflammatory (M2) state. local control with an excellent median survival time of 21.3 However, the M1/M2 phenotypes represent the extremes of a months. Unexpectedly, a high proportion of patients had out-of- continuum and the plasticity of these cells makes therapeutic field recurrences with local tumor control which deserves further targeting challenging. Solid experimental evidence informs that evaluation (83). the crosstalk between TAMs and the immune cells facilitates A relevant aspect that remains unanswered is whether the an immunosuppressive environment by supporting angiogenesis effect of blocking the CXCR4 pathway could be more pronounced and extracellular matrix (ECM) remodeling, promoting active with ablative doses of radiation which seems likely since greater recruitment of Treg cells, and expression of PD-L1, paving the vascular damage would be expected. Furthermore, it is of interest way for metastatic development (70). M2 polarization is mostly to know if CXCR4 blockade can enhance tumor immunity. Very mediated by growth factors and cytokines secreted by cancer cells limited information suggests that T cell exclusion from cancer that reach M2 cells via exosomes (71). Intriguingly, ontogeny cell deposits secondary to SDF-1 could be overcome by inhibiting can influence the functional profile of TAMs, i.e., tissue-resident the CXCR4 axis, improving the effect of checkpoint inhibitors or vs. circulating macrophages, such that they can have opposing stroma normalizing strategies in pancreatic cancer (84, 85) and functions depending on the tumor type (72). Based on these triple-negative breast cancer (86) models. findings, it has been speculated that macrophage origins may be important in determining the permissiveness of an organ to Pre-metastatic Niche and Exosomes metastatic growth. In addition to TAMs, radiation also recruits MDSCs in the Preliminary studies have evaluated the influence of irradiated tumors by tumor-secreted factors like SDF-1. MDSCs radiation in macrophage polarization. Macrophages are encompass a heterogeneous population of polymorphonuclear highly radioresistant due to high production of anti-oxidative MDSCs and monocytic MDSCs which inhibit the activity of molecules such as manganese superoxide dismutase by a CD8+ T cells. Moreover, MDSCs play a prominent role in the mechanism depending on tumor necrosis factor α (TNFα) establishment of the pre-metastatic niche, promote angiogenesis signaling and nuclear factor-κβ (NFκβ) activation (73). Early and facilitate the development of metastasis (87). studies established that radiation exposure recruited bone Tumors induce the formation of microenvironments in marrow-derived CD11b+ monocytes/macrophages to irradiated distant sites that support future metastatic tumor growth sites (74, 75) and related it to the transcription factor hypoxia- before their arrival at these sites, known as pre-metastatic inducible factor-1α (HIF-1α) and effectors stromal cell-derived niches. Tumor-secreted factors and tumor-shed extracellular factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) vesicles promote a sequence of events that start with vascular (76, 77). Therapy can polarize macrophages to the M2 phenotype leakiness, and are followed by alteration of local cells in with very high levels of proangiogenic molecules through the the TME, recruitment of MDSCs, and finally attraction of treatment-induced expression of colony stimulating factor 1 circulating tumor cells (88). Following seeding in a secondary (CSF-1), the ligand for the colony stimulating factor 1 receptor organ, cancer cells interact with their environment to create (CSF-1R) on macrophages, which can be prevented by CSF-1R the metastatic niche. The microenvironment in pre-metastatic antagonists and enhance radiation effects (78, 79). niches is immunosuppressive and MDSCs are the main cellular Ongoing research efforts are directed toward the alteration of component, however, migration of MDSCs into pre-metastatic the macrophage phenotype to attenuate immunosuppression and niches and subsequent activation is not well-characterized. More improve antitumor immunity (80). Current approaches aim to than 100 different immunosuppressive tumor-secreted proteins Frontiers in Oncology | www.frontiersin.org 8 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance FIGURE 2 | Macrophage targeting in cancer. Macrophages are primarily recruited to tumors to acquire a pro-tumorigenic phenotype (M2 state). Several strategies target TAMs aiming to reprogram them into a pro-inflammatory phenotype (M1 state). Most macrophage-targeted therapies are focused on CSF-1R inhibitors. Another approach is via CXCR4 blockade, which acts on vasculogenesis and has been tested in the clinical setting after radiotherapy in glioblastoma. have been identified (89). Fibronectin accumulates and anchors resected tumor was the main source of exosomal release (95). to collagen in the ECM to facilitate the adherence of circulating However, in animal models of abdominal cancer surgery can tumor cells through high affinity of membrane integrins (90). The induce increased levels of ROS, that may downregulate tight vascular changes allow for uptake of tumor-secreted exosomes junctions in the endothelium and peritoneum, form intercellular by cancer-associated fibroblasts (CAFs) in the local stroma, gaps and expose the underlying ECM; which can promote which contributes to the formation of a tumor-associated integrin-binding of circulating tumor cells during surgery, and desmoplastic stroma, characteristic of many carcinomas (91) result in an excess of liver metastases in a colorectal cancer (Figure 3). Exosomes are extracellular vesicles released by model (96). exocytosis and essential to intercellular communication. They Research in animal models of breast cancer known to produce can contain genetic material, proteins, and lipids; they can immunosuppressive MDSCs in the spleen and lungs, has shown be found in all body fluids and are considered to be major that surgical resection of the primary tumor decreased levels drivers of pre-metastatic niche formation (92). Measurement of of MDSCs in the spleen but persisted in the lungs for 2 weeks exosomal microRNA has been shown to accurately reflect tumor after resection, indicative of a pro-metastatic environment. Post- progression in several cancer types (93, 94) as well as dropping surgical treatment with gemcitabine depleted lung MDSCs and levels of exosomal microRNA after surgery indicate that the decreased posterior metastatic disease (97). Frontiers in Oncology | www.frontiersin.org 9 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance FIGURE 3 | Role of the pre-metastatic niche in cancer metastasis. Primary tumor cells produce soluble factors and exosomes (A) to trigger the formation of an immature pre-metastatic niche in the target organ (B). Primary tumor conditions (hypoxia, acidity, and interstitial pressure) promote tumor cell migration into the blood vessels. Tumor-secreted factors and exosomes mobilize bone marrow-derived cells (such as CD11b+ myeloid cells) and suppressive immune cells (such as MDSCs, Treg , and TAMs) to target organs (C). Interactions with local stroma, hypoxia and active ECM remodeling may create a niche with suitable microenvironment conditions for tumor cell colonization (D). Surgery, inflammation, and immunosuppression may increase the number and survival of circulating tumor cells and favor the development of metastasis. Accumulating evidence indicates that exposure to radiation cetuximab, radiation, and ipilimumab; exosomes were isolated induces the release of exosomes (98–100) that could contribute from plasma and the molecular cargo contents (derived from to radioresistance but additional mechanistic understanding Treg cells) could separate patients who remained free 2 years after to define potential interventions is lacking. The potential treatment from those who did not (101). role of exosomes has also been explored as biomarkers of An important aspect required to characterize extracellular disease outcome in head and neck cancer patients treated with vesicles is the development of highly specific detection Frontiers in Oncology | www.frontiersin.org 10 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance techniques. Since the distribution of extracellular vesicles in the into a quiescent state) to ICB in pancreatic cancer, and through TME depends on the cellular function, it is highly necessary to TGFβ blockade combined with immune checkpoint inhibition in visualize them in freshly resected tissues. There was a recent multiple tumor types (111). report about an intraoperative optical imaging system that was able to provide rich details and molecular contrast thanks to a label-free multimodal nonlinear optical technology in human CLINICAL IMPLICATIONS OF METABOLIC breast cancer showing good correlation with stained histological REMODELING slides. The enriched areas with extracellular vesicles in the microenvironment correlated with macroscopic tissue deposits Metabolic crosstalk across all cellular compartments is as well as increasing distance from tumor to margin (102). responsible for homeostasis and evolution of the TME. All A recent publication has shown that after surgical removal of cells of the TME, both malignant and non-malignant, compete resected primary lung, breast, and esophageal cancer, low-dose for nutrients and oxygen, which are generally limited, especially adjuvant therapy with epigenetic therapy can disable the pre- in a stiffened and poorly vascularized TME, or secondary to metastatic niche and inhibit the formation of lung metastases the accumulation of the excessive production of metabolites by by avoiding the trafficking of MDSCs and promoting their cancer cells. Additional aspects that influence how the TME differentiation into a macrophage-like phenotype (103). These reacts include immune-related substances released by cancer preclinical findings represent a novel paradigm to be tested in and/or immune cells, mechanical forces in the ECM, and clinical trials. reactions to treatment (112). Although the metabolic pathways are shared between cellular Cancer-Associated Fibroblasts compartments of the TME, the singularity of the reaction of Fibroblasts, the major cell type in the TME, are critical stromal cells to energy demands is crucial. TAMs and CAFs are determinants of cellular crosstalk (104). CAFs, a subpopulation recruited to the tumor bed and activated in response to different of activated fibroblasts, are difficult to identify and in practice, stressful situations, such as limited nutrient disposal, hypoxia, are described as any mesenchymal cell that lacks lineage markers and oxidative stress, attracted by cytokines such as TGFβ and for epithelial cells, endothelial cells, and leukocytes. CAFs are CXCL2 or ROS from cancer cells [reviewed in (113)]. In such proliferative, migratory, and highly secretory cells that promote complex interactions, metabolites can serve different roles such extensive tissue remodeling which influences the physical and as being a source of energy or communicate signals between chemical properties of the tumor and increases the ECM stiffness, different cellular compartments, and metabolism byproducts which promotes malignancy in experimental models. An can favor an immunosuppressive phenotype. CAFs can rapidly extensive range of functions have also been attributed to CAFs, adapt to these poor conditions through glycolysis and fatty including secretion of growth factors, cytokines, and exosomes acid oxidation in mitochondria. This increased consumption that promote tumor growth and alter treatment responses. The of glucose is coupled with extensive lactate secretion, which principal effect of CAFs is considered to be immunosuppressive acidifies the TME and facilitates the activation of TAMs (114). with IL-6, SDF-1, and TGFβ as well-established mediators (105). The result of this swift metabolic adaptation of CAFs is the These CAFs contribute to a rigid matrix that creates a physical secretion of ECM-remodeling enzymes that promote fibrosis and barrier that leads to vessel compression and reduces diffusion of further limit the availability of nutrients and oxygen, establishing therapeutic agents to cancer cells which are particularly relevant a dynamic circuit in which lactate accumulation, glucose for colorectal and pancreatic cancer (106, 107). CAFs are also deprivation, and hypoxic conditions stimulate the recruitment effective in the remodeling of the tumor vasculature through the and activation of additional stromal cells (113). Hypoxia secretion of vascular endothelial growth factor (VEGF), fibroblast supports the stabilization of the transcription factor HIF-1α to growth factor, and IL-6 to enhance angiogenesis (108, 109) foster glycolysis. In this setting, HIF-1α also mediates CAF- (Figure 4). secretion of proangiogenic factors such as VEGF, and hypoxia Emerging evidence implicates CAFs in immune escape and contributes to tumor progression by stimulating CAFs to secrete resistance to immunotherapy but not all subpopulations seem immunomodulatory molecules, growth factors, antioxidants, and to have the same functions. A comprehensive identification ECM-remodeling enzymes. Taken together, the response of CAFs of specific subsets of CAFs and their function is needed to under poor nutritional conditions promotes tumor progression become a viable targeting option (110). Currently, several through engagement of endothelial cells. In addition, altered preclinical strategies that target specific subsets of CAFs are metabolism of cancer cells can create a gradient of metabolites under development (109). around the tumor that can signal the distance to blood vessels Two promising strategies are normalization of activated and tailor the secretion of VEGF to match the tumor spatial CAFs, which intends to revert the activated state into a organization and optimize the angiogenic response (115), and the quiescent state or to induce them to acquire tumor-suppressor metabolic switch in the TME may add to the disrupted immune phenotypes (111), and targeting CAF-derived ECM proteins, cell metabolism (80). either their production or degradation to alleviate the ECM Amino acids synthesize nucleotides and are also intermediate stiffness (109). Reprogramming of CAFs to enhance immune metabolites that contribute to other bioenergetic pathways. responses, normalizing their ECM, is being investigated through Glutamine is an abundant nutrient that provides carbon and the addition of vitamin D analogs (known to convert them nitrogen for pathways that contribute to energy formation, redox, Frontiers in Oncology | www.frontiersin.org 11 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance FIGURE 4 | Cancer-associated fibroblasts remodel the tumor stroma. The pro-tumorigenic functions of CAFs are generally associated with their highly secretory activity. Secretory functions and matrix remodeling contribute to tumor invasion and angiogenesis. In addition, secreted soluble factors also contribute to immune reprogramming and tumor growth. Metabolic remodeling by CAFs supports an immunosuppressive microenvironment and promotes tumor growth. homeostasis, macromolecular synthesis and signaling for cancer- A link has been recently proposed between ECM stiffness and cell growth of particular relevance in hypoxic conditions (116). metabolic transformation that facilitates tumor progression. It Endothelial cells form the lining of blood vessels and was found that through metabolic crosstalk between CAFs and lymphatics and require glycolysis for proliferation and migration cancer cells, aspartate secreted by CAFs maintains cancer cell during angiogenesis. As the tumor grows, new vessels are proliferation while glutamate secreted by cancer cells balances required to supply the tumor with nutrients and oxygen and the redox state of CAFs to promote ECM remodeling. This the endothelial cells increase their synthetic and energetic amino acid exchange among glutamate and aspartate offers new demands. Sprouting, the formation of new vessels, is a well- targeting options for both stromal and cancer cells (120). known mechanism in the angiogenic process and an area of intensive research. Although endothelial metabolism has been mainly described as glycolytic, recent findings suggest that CLINICAL IMPLICATIONS OF ANGIOGENIC mitochondrial oxidative phosphorylation is also required for RESPONSES endothelial cell proliferation during angiogenesis (117). The interplay between metabolic remodeling and immune An abnormal vasculature is a paramount characteristic of solid regulation in cancer is an active area of investigation. Preclinical tumors, with suboptimal function resulting from a leaky and models in glioblastoma have identified that tryptophan and immature vessel network (via overexpression of proangiogenic adenosine metabolism result in accumulation of Treg cells molecules such as VEGF), and compression of these anomalous and M2 macrophages, contributing to an immuno-suppressive vessels by physical forces (via TME cells and the ECM phenotype. Future studies will need to define the role of the molecules they produce) (121). The resulting hypoxia enforces intermediary metabolites of these pathways to determine their the stimulation of immune checkpoints and infiltration of therapeutic function (118). immunosuppressive cells in the TME (122). Specifically, hypoxia Preclinical results with the prodrug JHU083, a glutamine up-regulates immune checkpoints, reprograms TAMs to an M2 antagonist that targets glutaminase and a broad range of state, may influence the efficacy of antigen presentation by DC, glutamine-requiring enzymes, provide a strong and differentiated and affects the function of T cells, while hypoperfusion stiffens metabolic response in which cancer cells stop growth, through the TME that becomes a physical barrier to T cell infiltration into depletion of glutamine pathways and impairment of glucose the tumor (123). uptake, and in addition stimulates T cell functionality, even An emerging field of interest investigates the synergy with persistent antitumor memory (119). Disengaging the of immune-vascular interactions to promote an antitumor metabolism of cancer cells and that of T cells is an evolving effect (124). The objective of this strategy is to induce therapeutic concept. vascular normalization that needs to be coupled to vessel Frontiers in Oncology | www.frontiersin.org 12 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance decompression (to avoid vessel collapse). Restoring vessel normalization should improve perfusion in tumors with leaky function by normalizing tumor stroma has been evaluated in vessels (131), and the combination when both co-exist. However, preclinical models through targeting angiotensin signaling with addressing the cause of hypoperfusion and identifying the anti-hypertensive agents (125) or inhibiting SDF-1/CXCR4 (86) normalization window for each tumor is challenging (126). which can target CAFs and collagen/hyaluronan to decompress Since tumor perfusion is key for the efficacy of tumor vessels and improve perfusion and effect of the ICB. immunotherapy, perfusion markers could be used as markers for Vascular normalization can be achieved with antiangiogenic immunotherapy prediction (132). agents to improve tumor perfusion and treatment delivery, but it is dose- and time-dependent, making outcome predictions FUTURE PERSPECTIVES for combinations of antiangiogenics, stroma normalization and immune therapies difficult to optimize (126) (Figure 5). While recent studies have improved our understanding of Successful clinical evidence that the combination of ICB with mechanisms supporting immune resistance, we still have an antiangiogenic drugs has been recently reported in lung (127), incomplete view of how the TME works as a whole. We propose renal (128, 129), and endometrial (130) cancer. However, the that advancements in cancer metabolism and nanotechnology potential to improve the treatment outcome of this approach is represent promising areas of research that have the potential under evaluation in an ongoing clinical trial, which tests the role to significantly improve our understanding of immune escape of adding losartan (an antihypertensive angiotensin inhibitor) in nutrient- and oxygen-poor environments which may lead to to chemo-radiation (delivered via SBRT) and nivolumab in opportunities for therapeutic intervention. pancreatic cancer patients (NCT03563248). A comprehensive understanding of the metabolic needs Apparently, any method that improves tumor perfusion is of cancer cells has been achieved during this past decade. likely to enhance immunotherapy. It has been proposed that Significantly, metabolic signatures and hypoxia within the TME strategies that normalize the stroma would be more beneficial impact the immune function. The fact that these findings have in tumors with abundant compressed vessels, while vascular been translated into actionable anticancer targets provides the FIGURE 5 | Strategies to improve tumor perfusion increase tumor immunogenicity. Angiogenesis, desmoplasia, and inflammation promote leaky and compressed tumor vessels. Vascular normalization strengthens the vessel wall reducing intercellular gaps and improving perfusion. Blood vessel decompression by depletion of CAFs or ECM reperfuses the vessel and augments perfusion. As a result, reprogramming of the TME to an immunomodulatory state enhances antitumor immunity. Frontiers in Oncology | www.frontiersin.org 13 October 2020 | Volume 10 | Article 582884 Benavente et al. Therapy-Induced Resistance basis for a metabolic characterization of the TME to identify (NBTXR3, a high-Z nanomaterial with high-level electron novel targets and signatures in the future. Indeed, better density that increases energy dose deposit within cells) plus technologies to investigate cancer metabolism at the single-cell radiation vs. radiation alone has recently demonstrated level without disrupting the tissue will be required to achieve a meaningful clinical benefit in locally advanced soft tissue deeper understanding of the role of metabolism in cancer. sarcoma by doubling pathologic response rates (16 vs. 8%) Advancements in nanotechnology have been effectively (138). Significantly, recent research has reported that radiation- developed in cancer therapy. Innovative nanomedicines can activated hafnium oxide NP can augment tumor infiltrates of use the conditions and characteristics of the TME to deliver CD8+ T cells and generate an antitumor immune response, with therapeutics with increased precision, while providing for systemic effect on untreated tumors on the same animals in a signal outputs that allow to follow their effects in real time. murine model of colon cancer (139). Likewise, recent advances in nanotechnology have broadened Newly designed hafnium-based nanoscale metal- opportunities for the development of radiosensitizers in synergy organic frameworks (nMOFs) have demonstrated effective with other treatment modalities. We highlight recent progress of radioenhancement for low-dose radiation in preclinical models. nanotechnology between radiotherapy and immunotherapy. The combination of nMOF-mediated radiotherapy and PD-L1 blockade extended the local therapeutic effects of radiation to Metabolic Rewiring of the TME distant tumors via systemic antitumor immunity. This powerful The complex interplay between cellular crosstalk, interactions platform can minimize toxic effects by lowering the administered in the ECM and the biochemical environment within a tumor dose of radiation; it can be redesigned for rational tuning and has an impact on the metabolic phenotype and polarization of can significantly strengthen the effect of immunotherapy for immune cells. Thus, the concerted actions of different immune treatment of non-immunogenic tumors (140, 141). subsets suppress or promote growth. Solid tumors have a dynamic oxygen supply with hypoxic regions where interactions CONCLUSION among immune cells are not well understood. Untangling these interactions might offer new potential for response prediction. Evolution in the technological delivery of radiation and precision Tumor infiltrating lymphocytes are at a metabolic disadvantage surgery parallels the rapid progress in immune biology that within the TME since tumor cells impede their access to nutrients identifies novel strategies to enhance the antitumor immune needed for activation and acidify the TME through lactate response. In this setting, alterations in the TME could become accumulation, favoring a Treg phenotype (133). especially relevant to optimize treatment immunogenicity and Targeting specific metabolic alterations shared by tumor enhance patient outcome. cells and tumor promoting immune populations in the TME Defining the individual response of tumors to surgery and is a new strategy under evaluation. Preclinical research has radiation offers the possibility to design innovative treatment focused on fatty acid metabolism as a source of metabolic strategies and re-adapt treatment to new emerging targets. This plasticity in cancer cells (134), carbon metabolism to stimulate could have a major impact since it potentially represents a novel antitumor activity of macrophages (135), or targeting metabolism way to enhance local and systemic treatments. of ferroptosis (a form of death that relies on ROS) in tumors (136), among others. Strategies that reduce immunomodulatory AUTHOR CONTRIBUTIONS metabolites are also under evaluation, which include altering the acidic microenvironment, blocking the thryptophan metabolism The structure of the manuscript was compiled by all authors. pathway, inhibition of adenosine within the TME (33), or SB and ML wrote the first draft. AS-G and SB assembled the avoiding lactate accumulation in the TME (137). figures. All authors reviewed and approved the final version of A coordinated approach, which takes into account tumor the manuscript. types and tumor biology with detailed molecular links between cancer genotypes and metabolic dependency in a longitudinal fashion, will be best suited to detect the patient populations that FUNDING are most likely to benefit from metabolism-targeted therapies. This work was supported by grants from the Instituto de Salud Carlos III (ISCIII): PI15/01262 (ML) and CP03/00101 Nanoparticle-Mediated Immunogenic Cell (ML), co-financed by the European Regional Fund (ERDF) Death and AECC (Spanish Association of Cancer Research) Nanoparticles (NP) have been increasingly studied for Founding Ref. GC16173720CARR (ML). AS-G was granted radiosensitization. The combination of hafnium oxide NP by VHIR fellowships. REFERENCES Oncology. J Clin Oncol. (2020) 38:1081. doi: 10.1200/JCO.19. 03141 1. Markham MJ, Wachter K, Agarwal N, Bertagnolli MM, Chang 2. 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