Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy

Annals of Oncology Advance Access published April 10, 2016

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Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy

J.M. Pitt1,2,3, A. Marabelle1,2,4, A. Eggermont1, J-C. Soria1,3,4,5, G. Kroemer6,7,8,9,10,11,12, L. Zitvogel1,2,3,13,14

Institut de Cancérologie Gustave Roussy Cancer Campus (GRCC), 94800, Villejuif, France

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INSERM Unit U1015, 94800, Villejuif, France

3

Université Paris Sud, Université Paris-Saclay, Faculté de Médecine, 94276, Le Kremlin Bicêtre,

France 4

INSERM Unit U981, 94800, Villejuif, France

5

Drug Development Department (DITEP), 94800, Villejuif, France

6

INSERM U848, 94800, Villejuif, France

7

Metabolomics Platform, GRCC, 94800, Villejuif, France

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Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM U

1138, 75006, Paris, France 9

Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, 75015, Paris, France

10

Université Paris Descartes, Sorbonne Paris Cité, 75006, Paris, France

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Université Pierre et Marie Curie, 75005, Paris, France

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Karolinska Institute, Department of Women's and Children's Health, Karolinska University

Hospital, 17176, Stockholm, Sweden 13

INSERM Unit U932, Institut Curie, Paris Cedex 05, France

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Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 507, 94800, Villejuif,

France

© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].

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2 Corresponding author: Pr Laurence ZITVOGEL, Gustave Roussy Cancer Campus, 114 rue Edouard Vaillant, 94805 VILLEJUIF Cedex, France, E-mail : [email protected], Phone : +33 1 42 11 50 41, Fax : +33 1 42 11 60 94

Abstract The tumor microenvironment (TME) is an integral part of cancer. Recognition of the essential nature of the TME in cancer evolution has led to a shift from a tumor cell-

that supports tumor growth and metastatic dissemination. Accordingly, novel targets within the TME have been uncovered that can help direct and improve the actions of various cancer therapies, notably immunotherapies that work by potentiating host antitumor immune responses. Here, we review the composition of the TME, how this attenuates immunosurveillance, and discuss existing and potential strategies aimed at targeting cellular and molecular TME components.

Key Message The tumor microenvironment (TME) is an integral part of cancer, acting as a complex ecosystem supporting tumor growth and metastatic dissemination while attenuating immunosurveillance. In this review we describe the composition of the TME and how this attenuates anti-tumor immune responses, and discuss the existing and upcoming strategies aimed at targeting cellular and molecular TME components.

Key Words: Tumor microenvironment, immunotherapy, cancer, angiogenesis, anticancer therapy, immunosuppression

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centered view of cancer development to the concept of a complex tumor ecosystem

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Introduction Although much focus in cancer biology has been directed on cancer cell genetic and epigenetic alterations that drive malignancy, a wealth of new information has emerged revealing how the functionality of the tumor microenvironment (TME) determines its integral and indispensible role in tumor anatomy and physiology. It is now increasingly accepted that, rather than working alone, cancer cells interact closely with the extracellular matrix (ECM) and stromal cells, which together form the major construct of the TME [1]. Within the TME infrastructure, a variety of

secrete, these drive a chronic inflammatory, immunosuppressive, and pro-angiogenic intratumoral environment. Cancer cells are able to adapt and grow in such environments with significantly less likelihood of detection and eradication by host immunosurveillance. As our knowledge of the TME increases, so does the number of biological molecules and mechanistic pathways potentially targetable for cancer treatment. Here we review the input of the TME in existing development along with current and future TME-targeting treatment strategies.

The composition of the tumor microenvironment and its interaction with the host immune system A variety of cell types are found in the TME, which accumulate at different stages of tumor development. Some of the principal cell types to arrive in tumors during their early development are infiltrating inflammatory cells, bone marrow-derived hematopoietic and endothelial progenitor cells, and carcinoma-associated fibroblasts [2]. Early infiltration of tumors by immune cells such as macrophages, lymphocytes, natural killer (NK) cells, and dendritic cells (DC) is crucial for tumor control [3]. The

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immune and non-immune cell types are found and, with the many factors that they

4 anti-cancer immune response generated by these cells is however inhibited by the action of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSC) regulatory T cells (Tregs), and type 2-polarized macrophages (M2), which are intrinsically associated with the developing TME (Figure 1) [3, 4]. Cancer cells are able to communicate with other cells and components of the TME through two principal pathways, the first being contact-dependent mechanisms between the particular cancer cell and another cell or with the extracellular matrix (ECM), the second being contact-independent mechanisms via soluble molecules such as

constitutive part of the TME are originally recruited from either the surrounding tissues or from the bone marrow, these making up the cellular components such as endothelial cells, mesenchymal cells, fibroblasts, and myeloid and lymphoid inflammatory cells [1, 5]. Within the TME, stromal cells can become ‘educated’ to become a variety of other cell types that facilitate and sustain cancer cells. This is able to occur due to the phenomenal nature of the TME, which is one of chronic inflammation [6].

Chronic inflammation in the tumor microenvironment benefits cancer cells The health of tissues in homeostatic conditions is largely influenced by leukocytes, which defend against threats posed by pathogens and foreign agents. At the first line of this defense are the innate immune cells, such as granulocytes (e.g., neutrophils), macrophages, DCs, NK cells, and innate lymphocytes. When tissue homeostasis is perturbed by a potential threat such as infection, these cells sense the danger via pattern recognition receptors that are specific for pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), and respond by

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cytokines, lipid mediators, and growth factors. The stromal cells that form a

5 producing cytokines and chemokines that activate and recruit other immune cell types, in doing so initiating an immune response against the danger [7, 8]. As part of this response, tissue-resident macrophages and mast cells additionally secrete matrixremodeling proteins and cytokines that locally activate surrounding stromal cells to help defend against the threat [6]. The relatively low populations of DCs present in a perturbed tissue have the key responsibility of linking the innate immune response to the antigen-specific lymphocytes of the adaptive immune response [9]. Through antigen presentation in the context of cytokine signaling and upregulation of co-

and are subsequently able to undergo clonal expansion on recognition of their specific foreign antigen in the perturbed environment. This mounted T lymphocyte population performs the elimination of the perceived threat, specifically where it is located (given that the cognate foreign antigen is adequately displayed by major histocompatibility [MHC] molecules on the surface of infected or transformed cells), and once eliminated, inflammation resolves and tissue homeostasis is reestablished.

In tumors, this inflammation fails to resolve, giving rise to a chronic inflammatory microenvironment [6]. This is of significant benefit to cancer cells both directly and indirectly, since they, and the stromal cells that support them, are potentially able to feed from the continuous supply of mitogenic growth factors and cytokines – transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and fibroblast growth factors being only a handful of examples. Also at play in this chronic inflammatory environment are the diverse classes of proteolytic enzymes (metallo, serine, and cysteine proteases) that modify the structure and functions of the tumoral ECM; these predominantly secreted by intratumoral macrophages, monocytes and

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stimulatory molecules on the part of the DC, naïve CD4+ and CD8+ T cells are primed

6 granulocytes [6, 10]. The persistent presence of these factors, which normally facilitate tissue repair, provide an additional survival advantage to cancer cells since they activate and maintain angiogenesis, impair anti-cancer lymphocyte functions, facilitate metastasis, and blunt cell death following ECM detachment [1].

Suppression of anti-tumoral immune responses in the tumor microenvironment The TME is profoundly immunosuppressive, which is a key reason for why most cancer therapies that operate (or in part operate) via stimulation of immune cell

often have the means to evade detection and destruction by immune cells at almost every conceivable immune mechanistic level. Developing tumors are able to recruit bone marrow-derived cells and promote their differentiation toward phenotypes that facilitate tumor survival [3]. Macrophages, which may be considered the prototypical bone marrow-derived cell, can promote tumor progression through facilitating angiogenesis, invasion, and metastasis in vivo – this being dependent on their functional state that is usually instilled by the make-up of the inflammatory milieu of the TME [11]. It is believed that cancer cells exploit the plastic nature of macrophages to elicit distinct functions at various stages of tumor progression, such as the production of EGF to increase invasive potential of cancer cells expressing EGF receptor [11]. The populations of tumor-associated macrophages (TAMs) may also change temporally in line with the development of the tumor.

MDSCs, another bone marrow derived cell potentially sharing a common progenitor with TAMs, have a remarkable ability to suppress immune responses [4]. MDSCs are best considered as a heterogeneous population of immature myeloid cells, which

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actions against cancer continue to display limited clinical efficacy. Indeed, tumors

7 likely arise due to the chronic inflammatory environment associated with tumors [12, 13]. Indeed, the S100 protein family [14, 15] and prostaglandin E2 [16], which are found in such inflammatory environments, have each been shown to stimulate the intratumoral induction and accumulation of MDSCs. MDSCs are substantially increased in cancer patients, in various compartments, with this correlating with cancer progression [11]. MDSCs primarily suppress antitumoral immune responses by inhibiting the actions and activation of T cells and NK cells within the TME. They block immune responses in this way through the production of arginase and nitric

The most crucial and direct control of tumor cells is performed by CD4+ T helper and CD8+ cytotoxic T lymphocytes (CTL), as confirmed by the many correlations between the intratumoral presence of these cells and positive clinical prognoses [17]. CD4+ T helper (Th) lymphocytes act as crucial regulators of inflammation specific to the threat encountered, with a large (and expanding) list of Th subsets defined thus far (including Th1, Th2, Th17, Th9, and Th22). Th1 responses, characterized by T cell production of interferon (IFN)-γ, TNF-α, and interleukin (IL)-2, are considered to be the essential subset for tumor rejection. IFN-γ generated from Th1 responses may also synergize with the IL-17 produced from Th17 cells, together promoting the secretion of the chemokines CXCL9 and CXCL10, which can recruit tumor cell-targeting CTLs into the TME [6]. However, evidence also suggests Th1 responses contribute to tumor escape via IFN-γ-mediated expression of the inhibitory checkpoint molecule programmed cell death ligand 1 (PD-L1) [18], or via the selection of resistant clones through tumor immunoediting [19, 20]. In addition, alongside the chronic inflammatory TME, long-term exposure to tumor antigen induces Th1 cells and other

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oxide, and by expanding local Treg populations [4].

8 T cells that lack the typical polyfunctional phenotype (i.e. the ability to secrete high levels of several cytokines), and that express inhibitory receptors such as PD-L1, lymphocyte activation gene 3 protein (LAG-3), and T cell immunoglobulin domain and mucin domain protein 3 (TIM-3) [21]. These so-called ‘exhausted’ T cells have a much-limited anti-tumoral efficiency. Intriguingly, however, there remains a positive correlation between the number of CD8+ tumor-infiltrating lymphocytes (TILs) and favorable clinical prognoses [17, 21], suggesting these cells must retain or be able to retrieve some form of anti-tumor efficacy. Indeed, blocking the interaction between

(as well as anti-viral [22]) immunity [23-25]; this forming an important target in the TME, which will be described in more detail below.

Other subsets of CD4+ T cells however inhibit antitumor immune responses. A significant source of immunosuppression in the TME arises from CD4+ Tregs, which become significantly enriched in the tumors of cancer patients [26]. Tregs inhibit the antitumor activity of CTLs and NK cells either directly, or indirectly via APCs [27, 28]. This is mediated through the direct interaction of Tregs with these immune cells and/or Treg production of immunoregulatory cytokines such as IL-10 and TGF-β [29]. As with other T cells, Tregs require both priming in the tumor-draining lymph node plus exposure to antigen in the TME to be able to suppress CTLs, which results in CTL acquisition of an ‘exhausted’ PD-1+TIM-3+ phenotype [28]. Additionally to Tregs, Th2 cells can also block T cell-induced tumor rejection. In contrast with Th1 cells, Th2 cells can instead induce T cell anergy, loss of T cell-mediated cytotoxicity, and enhance humoral immunity [30, 31]. Th2 cells block T cell-induced tumor rejection through their production of Th2 cytokines, such as IL-4, IL-5, and IL-13,

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PD-L1 and its receptor, programmed cell death protein 1 (PD-1), restores anti-tumor

9 and their expression of B7-H3, which act to regulate Th1-mediated responses while inducing the formation of immunosuppressive type 2-polarized macrophages [6, 27]. Breast and pancreatic cancer can be heavily infiltrated with Th2 cells, which accelerate tumor growth [32].

Evidence suggests the TME can also directly impair intratumoral T cell proliferation, acting as another mechanism for tumor evasion of immunosurveillance. An example of this has been shown with the production of indole 2,3-dioxygenase (IDO) within

generate kynurenine, which together promotes the conversion of naïve T cells to Tregs [33], inhibition of Treg reprogramming to potentially anti-tumoral Th cells [34], and increases MDSC functions through upregulation of IL-6 expression [33, 35].

B cells, defined by their receptor (BCR), can functionally exist in various stages of differentiation in tumor beds, such as immunoglobulin (Ig)-producing plasma cells and immunosuppressive regulatory B cells (Breg). Evidence suggests B cells can either participate in or inhibit antitumor immune responses dependent upon their subtype [36]. Ig produced by plasma cells can activate the complement cascade, regulating the clotting cascade, and favor metastases [37]. B cells may also inhibit the efficacy of certain cancer therapies. In three different mouse prostate cancer models, stimulation of immune responses following oxaliplatin-mediated immunogenic cell death was found to be inhibited by the recruitment of a population of B cells that expressed IgA, PD-L1, and IL-10; the appearance of these cells dependent on TGF-β receptor signaling [38].

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the TME. IDO produced by myeloid cells and cancer cells catabolizes tryptophan to

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Immunotherapeutic targeting of the tumor microenvironment Given the multiple mechanisms used by tumors to promote their development, there exist many potential angles of attack for cancer therapy, these including the targeting of excessive immunoregulation, angiogenesis, inflammation, and tumor cell communication with the ECM.

Targeting tumor immunoregulation via immune checkpoint blockade Immunotherapy has revolutionized the treatment of cancer, the forefront of this

checkpoint’ regulators, notably cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and the PD-1/PD-L1 axis [39].

Efficient activation of naïve T cells against tumor antigens requires additional costimulatory signals, provided by the engagement of CD28 on the T cell surface membrane with B7 molecules (CD80 and CD86) present on the surface of the antigen-presenting cell (typically a DC). Provision of tumor antigens in the context of MHC molecules occurs here only if the antigen-presenting cell has phagocytosed tumor cell material released following non-apoptotic tumor cell death. T cell activation is strictly regulated at the level of costimulation, since this activation also upregulates the surface expression of CTLA-4, which has a high homology to CD28 and binds B7 molecules with much higher affinity, to the point where CTLA-4 eventually blocks costimulation and thus inhibits the T cell response. Tregs also have a strong expression level of CTLA-4, thus anti-CTLA-4 antibody therapy can also deplete these cells from the tumor TME, resulting in a subsequent release in suppression of anti-tumoral CTL activity [40]. The preclinical success of anti-CTLA-

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recently being the therapeutic success attributed to antibody blockade of ‘immune

11 4 antibodies in achieving tumor rejection in animal models led to the development of ipilimumab, an antibody against human CTLA-4 [39]. Ipilimumab induced a considerable improvement in overall survival of patients with metastatic melanoma [41, 42] (and has shown clinical responses in several other cancers [39]), leading to its approval by the FDA in 2011.

Like ipilimumab, antibodies toward human components of the PD-1/PD-L1 pathway have also been subject to successful clinical testing. The PD-1/PD-L1 axis is believed

antigen recognition by the T cell receptor [23-25, 39]. PD-L1 can be expressed on many cell types, including epithelial cells, endothelial cells, and tumor cells, following their exposure to IFN-γ produced by Th1 responses [43, 44]. When PD-1expressing activated T cells engage PD-L1 on such cells, the response of the T cell is attenuated, this providing an efficient means for tumor cells to resist T cell attack [22, 39]. Antibodies that block the PD-1/PD-L1 immune checkpoint have demonstrated excellent therapeutic efficacy in a variety of tumor types. Tumor regressions have been shown following anti-PD-L1 antibody trials in melanoma, non-small cell lung cancer (NSCLC), bladder cancer, and renal cell carcinoma [45, 46]. The end of 2014 saw the FDA approval of two anti-PD-1 antibodies, pembrolizumab [47] and nivolumab [48]. Pembrolizumab treatment induced strong response rates in a metastatic melanoma cohort [47], which was supported in a subsequent study demonstrating an overall response rate of 26% in patients with progressive disease post ipilimumab treatment [49]. Nivolumab, in parallel, was seen to increase the objective response and overall survival of metastatic melanoma patients as compared with dacarbazine chemotherapy in a phase III study [48]. Nivolumab has since

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to be less involved in T cell costimulation, but rather in the signaling mediated by

12 received FDA approval for patients with previously treated advanced or metastatic NSCLC following improved overall survival over docetaxel chemotherapy [50, 51], and has more recently been approved for metastatic renal cell carcinoma following demonstration of antitumor activity [52] and improved overall survival and adverse event profiles versus everolimus treatment [53].

The phenomenal success of immune checkpoint blockade has been one of the greatest achievements in cancer immunotherapy thus far, and has generated much research

blockade of LAG-3 and TIM-3 [54]. These therapies are however often associated with immune-related adverse events that require clinical management [55], and fail to have therapeutic efficacy in the majority of patients. Given the non-overlapping mechanisms of action of CTLA-4 and PD-1, combination therapy targeting both pathways simultaneously has been suggested as potentially more efficacious than either therapy alone [39], particularly following encouraging in vivo data from murine tumor models [56, 57]. In a phase I study combining nivolumab with ipilimumab, the objective response rate for all patients treated by the concurrent-regimen was 40%, with evidence of clinical activity observed in 65% of patients. Moreover, at the maximum doses associated with an acceptable level of adverse events, 53% of patients had objective responses (these patients having tumor regressions of 80% or more). Immune-related adverse events from the dual treatment were manageable [58]. Following this study, a phase 3 trial investigated progression-free survival in previously untreated patients with metastatic melanoma receiving this same combination, beside nivolumab and ipilimumab monotherapies. The median progression-free survival was 11.5 months in the combination group, compared with

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and development of other potential checkpoint inhibition strategies, for example

13 2.9 months with ipilimumab and 6.9 months with nivolumab. Notably, patients with PD-L1-positive tumors had similar median progression-free survival, of 14 months, when treated with either the combination or nivolumab alone, whereas the combination was by far the most effective regimen in patients with PD-L1-negative tumors [59]. Additional studies that aim to show whether such combination therapies are safe and effective are in progress [39].

Targeting immunoregulatory cells and their secreted factors

Tregs in the TME, the targeting of this immunoregulatory cell population poses an additional favorable approach to better immune tumor control. Interestingly, it has been shown that several long-standing chemotherapies modulate Tregs and their functions, which may contribute toward their efficacy. Low continuous (‘metronomic’) doses of cyclophosphamide (CTX), as an example, promote tumor immunity by selectively targeting and eliminating Tregs (albeit higher CTX doses cause increased immune cell cytotoxicity and thus immunosuppression) [60-62]. CTX has also been observed to support the host anti-tumor immune response by favoring Th17 differentiation [63], through stimulating DC function and presentation via type I interferon (IFN) induction [64, 65], and by restoring T cell and NK cell effector functions in advanced cancer patients [66]. Reduction in the number and function of Treg cells in the TME, and thereby an increased CTL to Treg ratio, has also been observed on treatment with other anti-cancer agents, including paclitaxel, arsenic trioxide, the vascular endothelial growth factor (VEGF) receptor inhibitor sunitinib [67], and gemcitabine [68]. Gemcitabine has been demonstrated to induce additional immunogenic effects, such as the induction of class I human leukocyte antigen (HLA)

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Given the potent suppression of immune-mediated tumor regression resulting from

14 [69], enhancing DC cross presentation for CTL responses [70], and the ability to selectively kill MDSCs that contribute to immunosuppression within the TME [71, 72]. Accordingly, gemcitabine in combination with DC-based vaccines has shown some promising results in patients with pancreatic cancer [73, 74].

Targeting Tregs directly in the TME has been proposed as another method to reestablish the anti-tumoral immune response. Tregs within tumors are known to be enriched for the cell surface markers CTLA-4 and OX-40, and one study has shown

them from the TME [75]. By directly injecting mouse tumors with anti-CTLA-4 and anti-OX-40 antibodies to deplete Tregs, along with the TLR9-activating agonist CpG to trigger the innate immune response, the authors showed the establishment of a systemic antitumor immune response capable of eradicating disseminated disease in mice. Furthermore, this treatment modality was effective against established lymphoma in the central nervous system, which is traditionally considered to be a sanctuary for tumor cells in the face of systemic therapies. This study suggested antibody therapy could be used to target TILs locally, thereby inducing an effective systemic immune response [75].

Targeting the immunosuppressive factors released by Tregs into the TME is an additional strategy, which although supported by various lines of pre-clinical evidence has not as yet been tested sufficiently in the clinical setting [76, 77]. This strategy would require the use of cytokine-specific antibodies or receptors able to neutralize the suppressive mediator in question. Notable Treg-produced immunoregulatory factors targetable by this approach might include TGF-β, IL-10, and IL-35 [78]. Of

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that Tregs may be targeted by using antibodies against these molecules to deplete

15 these, TGF-β would seem a particularly desirable target given its roles in promoting metastasis and tumor stroma formation, beside its potent inhibitory effects upon T cells and NK cells while promoting the generation and expansion of Tregs [79].

Intratumoral delivery of immunomodulatory cytokines (or genes encoding cytokines) is an alternative strategy that has been trialed in the clinic. This requires a high delivered concentration of the cytokine inside the tumor, along with its persistence at sufficient quantities to elicit a therapeutic response and a non-toxic concentration of

cytokines IL-2, IL-12, TNF-α, type I IFNs, and granulocyte macrophage colonystimulating factor (GM-CSF), with varying results [76]. Worthy of mention here is the intratumoral delivery of GM-CSF, a growth factor that recruits DCs and facilitates their maturation and expansion necessary to stimulate T cell responses, and that has been used in many clinical trials to stimulate immune activity (notably in trials investigating cancer antigen vaccination) [80-84]. GM-CSF has been delivered by oncolytic viruses to the lesions of patients with metastatic melanoma [85, 86], where it was found to increase numbers of MART-1-specific T cells and decrease populations of Tregs and MDSCs [85]. A 26% objective response rate and one-year overall survival of 58% were also observed [86], which promoted the development of this approach [84]. A subsequent phase III trial examining intratumoral delivery of talimogene laherparepvec (T-VEC), the lead oncolytic viral GM-CSF delivery construct, revealed a therapeutic benefit against melanoma with a higher durable response rate and a trend for longer median overall survival (p = 0.051) compared with subcutaneous GM-CSF [87]. T-VEC has since been approved by the FDA for treatment of unresestable cutaneous, subcutaneous, and nodal lesions in patients with

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the cytokine outside of the TME. Intratumoral delivery has been trialed for the

16 recurrent melanoma after initial surgery [88]. Nonetheless, in the majority of patients, there is so far little evidence to suggest significant systemic antitumor activity and abscopal effects on distant tumors – this being a critical aspect to the success of such intratumoral therapies. Again, combination therapies may prove more promise here, encouraged by clinical observations such as that advanced melanoma patients benefit from the combination of GM-CSF and ipilimumab, with longer survival and reduced toxicity with this combination compared to ipilimumab monotherapy [89]. A phase 3 study is currently exploring the combination of T-VEC with pembrolizumab for

Targeting the tumor structure to promote immune-mediated tumor regressions

Cancer cells continually create a TME that supports their growth and survival as the tumor increases in size. Essential parts of this process include the stimulation of angiogenesis to gain continued access to oxygen and nutrients, and a level of control over the entry of the cells and soluble factors that could enter tumors through this new vasculature [91].

Targeting angiogenesis while maintaining adequate immune cell infiltration More than 1000 clinical trials involving anti-angiogenic therapeutics have been conducted worldwide, with several agents now approved including the anti-VEGF antibody bevacizumab, and the multi-tyrosine kinase receptor inhibitor sunitinib [92]. However, benefits in overall survival following anti-angiogenic therapy have been disappointing in comparison to the predictions from pre-clinical testing, with the

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unresected melanoma (NCT02263508) [90].

17 majority of cancer patients losing their response to therapy, or not responding at all [92]. Questions have also been raised regarding how anti-angiogenic treatment may alter the behavior and progression of tumors, with concerns that this may potentiate invasiveness or metastasis [92]. Although there may be limitations in targeting angiogenesis per se, attention has been focused on the use of anti-angiogenic therapeutics as a means to increase the efficacy of immunotherapy [93]. The targeting of VEGF signaling has been observed to induce tumor vasculature normalization, which in turn can increase extravasation and the number of TILs following adoptive

note, this ‘vascular normalization’ appears to be achievable with lower doses of antiVEGF receptor antibodies, which may also help to prevent the differentiation of TAMs toward an immune inhibitory M2-like phenotype [95] and could block VEGFmediated inhibition of DC maturation [96]. Therefore, aiming toward vascular normalization with anti-angiogenic therapies in combination with immunotherapy may make a more successful strategy than tumor vessel destruction with these therapies. In an example of this strategy, a recent study has shown the combination of bevacizumab and ipilimumab could be safely administered and revealed that VEGF-A blockade modulated inflammation, lymphocyte trafficking, and immune regulation [97].

Overcoming TME-mediated restriction of T cell access to cancer cells Following their priming in the tumor draining lymph nodes, T cells must then travel via the circulatory system to reach tumors. In order to perform their anti-tumoral functions within the tumor, they must extravasate, replicate, and gain proximity to tumor cells. The TME can influence all of these requisites so to evade the exposure of

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cell transfer [94] and cancer cell vaccine [95] therapies in experimental settings. Of

18 tumor cells to the adaptive immune response [98]. Hamzah et al. were the first to identify regulator of G-protein signaling 5 (Rgs5) as a master gene responsible for aberrant tumor vasculature [99]. Here, Rgs5 deficiency resulted in pericyte maturation, vascular normalization, and reduced tumor hypoxia and vessel leakiness; this culminating in a brisk influx of immune effector cells into tumor beds and enhanced mouse survival.

Extravasation and accumulation of T cells in tumors is largely influenced by T cell

production of reactive nitrogen species within the TME can posttranslationally modify chemokines, resulting in a profound impact on TIL accumulation, with tumorspecific T cells instead being trapped in the stroma surrounding cancer cells [100]. Moreover, a recent study has shown that DNA methylation is a mechanism used by tumors to repress production of the Th1-type chemokines CXCL9 and CXCL10, this epigenetic silencing resulting in decreased T cell trafficking to the TME [101]. This study also found that treatment with epigenetic modulators could remove this repression to reinstate effector T cell infiltration of tumors in vivo, culminating in greater control of tumor progression and improvement in the efficacy of PD-L1 checkpoint blockade [101]. If such epigenetic reprogramming of tumors can be translated to the clinical setting, this could be an approach to boost TILs as a preconditioning for immunotherapy.

In addition, there is evidence to suggest that in several different tumor types, expression of the apoptosis inducer Fas ligand (FasL) upon the endothelium of the tumor vasculature can be induced by the TME, this effectively establishing a selective

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chemokine gradients, which can be blocked by tumor cells. For example, the

19 immune barrier that promotes tolerance in tumors [102]. FasL expression enabled endothelial cells to kill CTLs, but not Tregs, via Fas-mediated apoptosis, the latter resistant to FasL exposure due to their increased surface expression of c-FLIP. Accordingly, FasL expression was associated with a scarce tumoral infiltration of CTLs and a predominance of Tregs. Downregulation of endothelial FasL may make a future target for enhancement of cancer immunotherapy, this potentially achievable through inhibition of either VEGF-A or cyclooxygenase (COX) [102].

additional hurdles posed by the TME to counter their presence and function. First, cancers can impose metabolic restriction on T cells. A recent study has shown that glucose restriction imposed by ovarian cancers can upregulate the expression of miR101 and miR-26a microRNAs in T cells, which act to constrain expression of the methyltransferase EZH2, this resulting in reduced T cell polyfunctional cytokine expression [103]. Glucose consumption by mouse sarcoma cells has also been shown to suppress mTOR activity, glycolytic capacity, and IFN-γ production within T cells, this allowing progression of tumors [104]. Interestingly, this was found to be reversed with blockade of the PD-1/PD-L1 axis or blockade of CTLA-4, predicting that immune checkpoint blockade might be potentially most therapeutically effective in patient tumors with higher glycolytic rates.

Second, since the TME is in most cases the predominant site of T cell clonal expansion, a strong and active presence of intratumoral DCs is required. Over the past decade, a wealth of studies from our group and others have identified strategies for increasing intratumoral DCs and their functionality in instigating T cell-mediated

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Even if T cells manage to reach the proximity of cancer cells, they must resist

20 immune responses [64, 105-112]. This can be achieved with the informed selection of existing anticancer chemotherapies, targeted therapies, and radiotherapy [113], notably those that induce immunogenic cell death within tumors (described in detail elsewhere [114]). This form of cancer cell death exposes a variety of immunostimulatory molecules (e.g., ATP, HMGB1, calreticulin) alongside tumor cell antigens, the former activating intratumoral DCs via their cognate surface membrane receptors or inflammasomes to present the latter to antigen-specific T cells within tumors (Figure 2) [105, 106, 108, 109, 111]. Activation of the stimulator of interferon

shown as another effective means to generate adaptive immune responses against tumors. This pathway is activated by cytosolic DNA (e.g., DNA derived from tumor cells following their death), which is converted by cyclic GMP-AMP synthase (cGAS) to cyclic GMP-AMP (cGAMP), which in turn binds and activates STING leading to type I IFN production [115]. In vivo studies have demonstrated a crucial role for STING pathway activation, type I IFN production and signaling on the BATF3 lineage of DCs for T cell recruitment to tumors and spontaneous antitumor T cell responses [115-117], suggesting a potential application for STING agonists as cancer therapeutics [115]. Also worthy of mention is the recent finding that oncogenic WNT/Beta-catenin signaling in melanoma cells correlates with an absence of intratumoral CD103+ DCs and T cells, this occurring via Beta-catenin-mediated suppression of the chemokine CCL4. This immune evasion mechanism of melanoma cells facilitated their resistance to anti-PD-L1/anti-CTLA-4 mAb-based therapies in experimental murine tumor models [118].

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genes (STING) innate immune sensing mechanism within intratumoral DCs has been

21 Finally, the DC activation status in tumor beds is also regulated by another compartment of the body, the mutualistic community of gut commensals. Vétizou et al. [119] and Sivan et al. [120] recently unraveled the role of Bacteroides fragilis and Bifidobacteria, respectively, in dictating DC antigen processing and maturation functions during tumor progression; this contributing to effective treatment with immune checkpoint blockade (Figure 3).

Beyond successful priming and activation by intratumoral DCs, T cells must

immunosuppression in the TME (an example being IDO activity), and must resist threats to their viability such as upregulated death molecules issued by myelomonocytic cells (e.g., FasL and TNF-related apoptosis inducing ligand [TRAIL]). The development of hypoxic regions within rapidly growing solid tumors can also contribute to T cell suppression, via the increased production of adenosine in such areas. Hypoxia-inducible factors (HIF) respond to hypoxic conditions by activating transcriptional programs that influence cellular metabolism and stimulate angiogenesis; this includes upregulation of the ectonucleotidases CD39 and CD73. The action of these two enzymes on ATP released in hypoxic tumor environments results in the creation of adenosine, which in turn disables cytotoxic effector functions of both NK and CD8+ T cells, and inhibits Th1 responses, predominantly via A2A adenosine receptor signaling [121].

Correcting these aspects of the TME via interventions targeting TAMs and/or MDSCs, with IDO inhibitors, or with adenosine receptor antagonists may be a step to overcoming such direct inhibition of TILs [98, 121]. For example, a combinatorial

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additionally overcome direct inhibition of their replication posed by drivers of

22 approach of IDO inhibitors with immune checkpoint blockade has been shown to increase TILs and their functional capacities in the TME, this mediating rejection of both IDO-positive and IDO-negative tumors of low immunogenicity in experimental settings [122, 123]. Clinical studies are currently in progress to assess the safety and efficacy of such combinatorial strategies (clinical trials: NCT02073123, NCT01604889, and NCT02327078).

Conclusions

clinical breakthroughs over the last 5 years. In order to eradicate cancer cells, effector immune cells must first be relieved from the multiple suppressive networks and activation barriers that constitute the TME. This can be, and has been, achievable through the targeted inhibition of pivotal factors at the heart of such networks, for example inhibitors of IDO, angiogenesis, and antibody-mediated blockade of immune checkpoints and neutralization of cytokines. The greatest future advances will perhaps come with combination therapies of these and other treatments.

The optimizing of our approach to treating cancers will progress hand-in-hand with the improved understanding of interactions between stromal cells, and between stromal cells and immune cells. A significant hurdle to overcome here, which may be solvable as these interactions are deciphered, is the TME-regulated spatial distribution of T cells leading to their physical exclusion from the vicinity of cancer cells. Other important considerations include the stages of tumorigenesis at which a given therapy would be most effective (particularly with respect to combinatorial regimes), and the potentially different constitution of the TME at metastatic sites; the latter likely to

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The shift in our understanding of the host-tumor relationship has allowed significant

23 require cancer treatments that target endothelial cells [11]. For this, it will be important to continue developing reliable biomarkers that indicate the type of TME present in a specific tumor [17]. Following from the outstanding clinical efficacy already observed with cancer immunotherapy, personalized modulation of the TME to a status favorable to antitumor immune responses will likely bring even greater benefit to cancer patients receiving these and other oncotherapies.

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24 ACKNOWLEDGEMENTS We thank the Association de recherche contre le Cancer (ARC) for supporting JMP.

Funding JMP is supported by ARC. GK and LZ were supported by the Ligue Nationale contre le Cancer (Equipes labellisées), Agence Nationale pour la Recherche (ANR AUTOPH, ANR Emergence), European Commission (ArtForce), ISREC, Swiss Bridge foundation, European Research Council Advanced Investigator Grant (to GK),

Fondation de France, Cancéropôle Ile-de-France, Fondation Bettencourt-Schueller, the LabEx Immuno-Oncology, the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); the SIRIC Cancer Research and Personalized Medicine (CARPEM), and the Paris Alliance of Cancer Research Institutes (PACRI). No grant numbers apply.

Disclosure The authors have declared no conflicts of interest.

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Fondation pour la Recherche Médicale (FRM), Institut National du Cancer (INCa),

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2011; 208: 2005-2016.

38 FIGURE LEGENDS

Figure 1: Good guys versus bad guys in the tumor microenvironment: a balance. The balance between a large number of different immune cells, immune factors, and signaling molecules determines the outcome of the anti-tumor immune response. CTL, cytotoxic T lymphocyte; Th, T helper; pTh17, pathogenic T helper 17; NK, natural killer; DC, dendritic cell; IFN, interferon; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor; TGF, transforming growth factor;

suppressor cell; TAM, tumor-associated macrophage.

Figure 2: Immunogenic chemotherapy and radiotherapy to restore the tumor microenvironment. Exposure of tumor cells to certain types of chemotherapy or to γ-ray irradiation induces a pattern of tumor cell death that stimulates anti-cancer host immune responses. This immunogenic cell death (ICD) is characterized by an ER stress response that results in exposure of calreticulin (CRT) on the cell surface membrane of the dying cell, the release from dying cells of HMGB1, and the induction of autophagy, which results in release of ATP. These molecules interact with CD91, TLR4, and P2RX7 receptors, respectively, on dendritic cells (DC), which can derive from myeloid precursors recruited by Ccl2 and ATP signaling post ICD. This results in DC maturation and DC secretion of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β, which in turn can enable an early recruitment of IL-17-producing γδ T cells. The ensuing inflammatory environment, alongside DC uptake of tumor antigens, facilitates generation and activation of anti-tumor CTL, Th1, and

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IDO, indole 2,3-dioxygenase; PGE, prostaglandin E; MDSC, myeloid-derived

39 “pathogenic” (p)Th17 cell responses (the latter characteristically producing both IL17 and IFN-γ). These T cells can be attracted into tumor beds via tumor cell secretion of the chemokine Cxcl10, which results from autocrine and paracrine type I IFN signaling among tumor cells post chemotherapy. CTL, cytotoxic T lymphocyte; Th, T helper; DC, dendritic cell; IFN, interferon; IL, interleukin.

Figure 3: Microbiota can augment DC function and thus contribute to anticancer immunity.

antibiotics, pro-biotics) can impact host anti-cancer immunity. Anti-cancer medications have also been found to modulate the microbiota composition and function/integrity of the intestinal mucosal barrier, which may facilitate their immunemediated therapeutic efficacy. For example, greater uptake of distinct bacterial species (such as Bacteroides fragilis and Bifidobacteria) by DCs of the lamina propria in the context of immune checkpoint blockade therapies can significantly enhance DC antigen processing and presentation functions (e.g., upregulation of costimulatory molecules and antigen presentation molecules such as CD40 and MHC class II, respectively), and ensure DC production of cytokines such as IL-12, which together increase the generation of anti-tumor T cell responses. Increased translocation of select bacterial species (e.g., Lactobacillus johnsonii) to secondary lymphoid organs following cyclophosphamide (CTX) chemotherapy-mediated intestinal barrier disruption amounts to similar DC-orientated effects. ICB, immune checkpoint blockade; CTL, cytotoxic T lymphocyte; Th, T helper; pTh17, pathogenic T helper 17; DC, dendritic cell; LN, lymph node; IFN, interferon; IL, interleukin.

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Alterations in the composition of the gut microbiota (e.g., following administration of

40 ABBREVIATIONS Aex – ascites-derived exosome CEA – carcinoembryonic antigen CTL – cytotoxic T lymphocyte CTX – cyclophosphamide Dex – DC-derived exosome DTH – delayed-type hypersensitivity IFN – interferon

MM – malignant melanoma MVB – multivesicular body NSCLC – non-small cell lung cancer TAA – tumor-associated antigen TLR – Toll-like receptor

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MAGE – melanoma-associated antigen

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