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Table S2. Statistical tests

Supplementary Figures and Table S1

Glioblastoma multiforme (GBM) is a lethal form of primary brain cancer. A new study now implicates beta-secretase 1 (BACE1) as a crucial regulator of pro-tumoral IL-6–STAT3 signaling in GBM-associated macrophages. An effective BACE1 inhibitor, clinically developed for Alzheimer’s disease, may offer new hope for GBM treatment.

Early detection and adjuvant therapies have significantly improved survival of patients with breast cancer over the past three decades. In contrast, management of metastatic disease remains unresolved. Brain metastasis is a late complication frequently observed among patients with metastatic breast cancer, whose poor prognosis calls for novel and more effective therapies. Here, we report that active hypoxia inducible factor-1 (HIF1) signaling and loss of the miRNA let-7d concur to promote brain metastasis in a recently established model of spontaneous breast cancer metastasis from the primary site to the brain (4T1-BM2), and additionally in murine and human experimental models of breast cancer brain metastasis (D2A1-BM2 and MDA231-BrM2). Active HIF1 and let-7d loss upregulated expression of platelet-derived growth factor (PDGF) B/A in murine and human brain metastatic cells, respectively, while either individual silencing of HIF1α and PDGF-A/B or let-7d overexpression suppressed brain metastasis formation in the tested models. Let-7d silencing upregulated HIF1α expression and HIF1 activity, indicating a regulatory hierarchy of the system. The clinical relevance of the identified targets was supported by human gene expression data analyses. Treatment of mice with nilotinib, a kinase inhibitor impinging on PDGF receptor (PDGFR) signaling, prevented formation of spontaneous brain metastases in the 4T1-BM2 model and reduced growth of established brain metastases in mouse and human models. These results identify active HIF1 signaling and let-7d loss as coordinated events promoting breast cancer brain metastasis through increased expression of PDGF-A/B. Moreover, they identify PDGFR inhibition as a potentially actionable therapeutic strategy for patients with brain metastatis. Significance: These findings show that loss of miRNA let-7d and active HIF1 signaling promotes breast cancer brain metastasis via PDGF and that pharmacologic inhibition of PDGFR suppresses brain metastasis, suggesting novel therapeutic opportunities.

microRNAs are short noncoding RNAs that regulate protein expression posttranscriptionally. We previously showed that miR-155 promotes effector CD8+ T-cell responses. However, little is known about the regulation of miR-155 expression. Here, we report that antigen affinity and dose determine miR-155 expression in CD8+ T cells. In B16 tumors expressing a low-affinity antigen ligand, tumor-specific infiltrating CD8+ T cells showed variable miR-155 expression, whereby high miR-155 expression was associated with more cytokine-producing cells and tumor control. Moreover, anti–PD-1 treatment led to both increased miR-155 expression and tumor control by specific CD8+ T cells. In addition, miR-155 overexpression enhanced exhausted CD8+ T-cell persistence in the LCMV cl13 chronic viral infection model. In agreement with these observations in mouse models, miR-155 expression in human effector memory CD8+ T cells positively correlated with their frequencies in tumor-infiltrated lymph nodes of melanoma patients. Low miR-155 target gene signature in tumors was associated with prolonged overall survival in melanoma patients. Altogether, these results raise the possibility that high miR-155 expression in CD8+ tumor-infiltrating T cells may be a surrogate marker of the relative potency of in situ antigen-specific CD8+ T-cell responses.

PD-1/PD-L1 pathway plays a role in inhibiting immune response. Therapeutic antibodies aimed at blocking the PD-1/PD-L1 interaction have entered clinical development and have been approved for a variety of cancers. However, the clinical benefits are reduced to a group of patients. The research in combined therapies, which allow for a greater response, is strongly encouraging. We previously characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) with antitumor activity in both melanoma and breast carcinoma, as well as immunomodulatory activity. We hypothesize that the combined treatment with P2Et and anti-PD-L1 can improve the antitumor response through an additive antitumor effect. We investigated the antitumor and immunomodulatory activity of P2Et and anti-PD-L1 combined therapy in B16-F10 melanoma and 4T1 breast carcinoma. We analyzed tumor growth, hematologic parameters, T cell counts, cytokine expression, and T cell cytotoxicity. In the melanoma model, combined P2Et and anti-PD-L1 therapy has the following effects: decrease in tumor size; increase in the number of activated CD4 + and CD8 + T cells; decrease in the number of suppressor myeloid cells; increase in PD-L1 expression; decrease in the frequency of CD8 + T cell expressing PD-1; improvement in the cytotoxic activity of T cells; and increase in the IFN γ secretion. In the breast cancer model, P2Et and PD-L1 alone or in combination show antitumor effect with no clear additive effect. This study shows that combined therapy of P2Et and anti-PD-L1 can improve antitumor response in a melanoma model by activating the immune response and neutralizing immunosuppressive mechanisms.

Background Immune checkpoint blockade (ICB) with antibodies against PD-1 or PD-L1 may provide therapeutic benefits in patients with non-small cell lung cancer (NSCLC). However, most tumours are resistant and cases of disease hyper-progression have also been reported. Materials and Methods Genetically engineered mouse models of KrasG12Dp53null NSCLC were treated with cisplatin along with antibodies against angiopoietin-2/VEGFA, PD-1 and CSF1R. Tumour growth was monitored by micro-computed tomography and the tumour vasculature and immune cell infiltrates were assessed by immunofluorescence staining and flow cytometry. Results Combined angiopoietin-2/VEGFA blockade by a bispecific antibody (A2V) modulated the vasculature and abated immunosuppressive macrophages while increasing CD8+effector T cells in the tumours, achieving disease stabilization comparable or superior to cisplatin-based chemotherapy. However, these immunological responses were unexpectedly limited by the addition of a PD-1 antibody, which paradoxically enhanced progression of a fraction of the tumours through a mechanism involving regulatory T cells and macrophages. Elimination of tumour-associated macrophages with a CSF1R-blocking antibody induced NSCLC regression in combination with PD-1 blockade and cisplatin. Conclusions The immune cell composition of the tumour determines the outcome of PD-1 blockade. In NSCLC, high infiltration of regulatory T cells and immunosuppressive macrophages may account for tumour hyper-progression upon ICB. Disclosure Information A. Martinez-Usatorre: None. E. Kadioglu: None. C. Cianciaruso: None. B. Torchia: None. J. Faget: None. E. Meylan: None. M. Schmittnaegel: None. I. Keklikoglou: None. M. De Palma: None.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and anti-angiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras (G12D/+);p53 (-/-) NSCLC, including a mismatch repair-deficient variant (Kras (G12D/+);p53 (-/-);Msh2 (-/-)) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, anti-angiogenic therapy, and chemotherapy. Anti-angiogenic blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) markedly slowed progression of autochthonous lung tumors but, contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that anti-angiogenic treatment facilitated tumor infiltration by PD-1(+) regulatory T cells (T(regs)), which were more efficiently targeted by the PD-1 antibody than CD8(+) T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R)-dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-β (TGFβ)-rich tumor microenvironment that supported PD-1(+) T(regs). Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated T(regs), redirected the PD-1 antibody to CD8(+) T cells, and improved the efficacy of anti-angiogenic immunotherapy, achieving regression of most tumors.

Antitumor T cell responses involve CD8+ T cells with high affinity for mutated self-antigen and low affinity for nonmutated tumor-associated Ag. Because of the highly individual nature of nonsynonymous somatic mutations in tumors, however, immunotherapy relies often on an effective engagement of low-affinity T cells. In this study, we studied the role of T cell affinity during peripheral priming with single-peptide vaccines and during the effector phase in the tumor. To that end, we compared the antitumor responses after OVA257–264 (N4) peptide vaccination of CD8+ T cells carrying TCRs with high (OT-1) and low (OT-3) avidity for the N4 peptide in B16.N4 tumor-bearing C57BL/6 mice. Additionally, we assessed the response of OT-1 cells to either high-affinity (B16.N4) or low-affinity (B16.T4) Ag-expressing tumors after high-affinity (N4) or low-affinity (T4) peptide vaccination. We noticed that although low-affinity tumor-specific T cells expand less than high-affinity T cells, they express lower levels of inhibitory receptors and produce more cytokines. Interestingly, tumor-infiltrating CD8+ T cells show similar in vivo re-expansion capacity to their counterparts in secondary lymphoid organs when transferred to tumor-free hosts, suggesting that T cells in tumors may be rekindled upon relief of tumor immunosuppression. Moreover, our results show that αPD-1 treatment enhances tumor control of high- and low-affinity ligand-expressing tumors, suggesting that combination of high-affinity peripheral priming by altered peptide ligands and checkpoint blockade may enable tumor control upon low-affinity Ag recognition in the tumor.

Therapy by adoptive transfer of ex vivo-expanded tumor-infiltrating or genetically modified T cells may lead to impressive clinical responses. However, there is a need to improve in vivo persistence and functionality of the transferred T cells, in particular, to face the highly immunosuppressive environment of solid tumors. Here, we investigate the potential of miR-155, a microRNA known to play an important role in CD8+ T cell fitness. We show that forced expression of miR-155 in tumor antigen-specific T cells improves the tumor control of B16 tumors expressing a low-affinity antigen ligand. Importantly, miR-155-transduced T cells exhibit increased proliferation and effector functions associated with a higher glycolytic activity independent of exogenous glucose. Altogether, these data suggest that miR-155 may optimize the antitumor activity of adoptively transferred low-affinity tumor-infiltrating lymphocytes (TILs), in particular, by rendering them more resistant to the glucose-deprived environment of solid tumors. Thus, transgenic expression of miR-155 may enable therapeutic targeting of self-antigen-specific T cells in addition to neoantigen-specific ones. Keywords: microRNA-155, CD8+ T cells, TCR antigen affinity, therapeutic vaccine, adoptive cell transfer, oncoimmunology

The interaction strength between CD8+ T cells' TCR and cognate peptide-MHC (pMHC) impacts on the CD8+ T cell response against pathogens and tumors ( Martinez-Usatorre, Donda, Zehn, & Romero, 2018 ; Zehn, Lee, & Bevan, 2009 ). CD8+ T cell responses against tumors are characterized by the presence of low affinity CD8+ T cells specific for nonmutated tumor associated self-antigens (TAA) and potentially high affinity tumor specific CD8+ T cells recognizing mutated self-antigens ( Gros et al., 2016 ; Kvistborg et al., 2012 ; McMahan & Slansky, 2007 ). High affinity T cells display enhanced survival, expansion capacity and tumor control ( Martinez-Usatorre et al., 2018 ; Schmid et al., 2010 ). In fact, recent clinical trials using neoantigen tumor vaccines showed prolonged progression free survival in melanoma patients ( Ott et al., 2017 ; Sahin et al., 2017 ), while only modest clinical efficacy was obtained with TAA vaccines ( Romero et al., 2016 ). However, the highly individual nature of neoantigens constitutes a major technical and economical hurdle for routine clinical application. Thus, the characterization of TAA-specific CD8+ T cell responses may reveal new strategies to enhance their anti-tumor properties. In parallel, the identification of high affinity antigens and CD8+ T cells may be essential to design effective tumor vaccines and adoptive cell transfer therapies. Therefore, in this chapter, we describe how to generate tumor cell lines with stable expression of affinity-ranged antigens and methods to assess T-cell affinity.

Therapy by adoptive transfer of ex vivo-expanded tumor-infiltrating or genetically modified T cells may lead to impressive clinical responses. However, there is a need to improve in vivo persistence and functionality of the transferred T cells, in particular, to face the highly immunosuppressive environment of solid tumors. Here, we investigate the potential of miR-155, a microRNA known to play an important role in CD8+ T cell fitness. We show that forced expression of miR-155 in tumor antigen-specific T cells improves the tumor control of B16 tumors expressing a low-affinity antigen ligand. Importantly, miR-155-transduced T cells exhibit increased proliferation and effector functions associated with a higher glycolytic activity independent of exogenous glucose. Altogether, these data suggest that miR-155 may optimize the antitumor activity of adoptively transferred low-affinity tumor-infiltrating lymphocytes (TILs), in particular, by rendering them more resistant to the glucose-deprived environment of solid tumors. Thus, transgenic expression of miR-155 may enable therapeutic targeting of self-antigen-specific T cells in addition to neoantigen-specific ones.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC), often in combination with chemotherapy. However, only a minority of the patients respond and sustained remissions are rare. Anti-angiogenic therapy improves tumor response to ICB in mouse models of cancer and in patients with EGFR-mutant NSCLC, but the contribution of anti-angiogenic therapy to the clinical efficacy of ICB in patients with KRAS-mutant NSCLC remains unclear. We used genetically engineered mouse models of KrasG12D/p53null NSCLC, including a mismatch repair-deficient variant (KrasG12D/p53null/Msh2null) with higher mutational burden, and longitudinal imaging of individual tumors to study tumor response and resistance to combinations of ICB, anti-angiogenic therapy, and cisplatin chemotherapy. We found that anti-angiogenic blockade of VEGFA and angiopoietin-2 (ANGPT2) with the bispecific antibody A2V was superior to single VEGFA inhibition and markedly slowed tumor progression. But, contrary to findings in other tumor models, the addition of a PD1 antibody to A2V did not provide additive benefits and even accelerated growth of a fraction of the tumors. We implicated tumor-associated macrophages (TAMs) and PD-1+regulatory T cells (T-regs) in this growth-promoting response, and identified a pre-clinical strategy involving the differential targeting of two distinct TAM subsets that unleashed the therapeutic potential of anti-angiogenic immunotherapy and achieved regression of more than 80% of the lung tumors. Citation Format: Amaia Martinez-Usatorre, Ece Kadioglu, Chiara Cianciaruso, Alan Guichard, Bruno Torchia, Sina Nassiri, Martina Schmittnaegel, Carola H. Ries, Etienne Meylan, Ioanna Keklikoglou, Michele De Palma. Determinants of tumor resistance to anti-angiogenic immunotherapy in mouse models of Kras-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 75.

Polyphenols have tumoricidal effects via anti-proliferative, anti-angiogenic and cytotoxic mechanisms and have recently been demonstrated to modulate the immune response through their anti- or pro- oxidant activity. Nevertheless, it remains controversial whether antioxidant-rich supplements have real beneficial effects on health, especially in complex diseases such as cancer. We previously identified a polyphenol-rich extract obtained from Caesalpinia spinosa (P2Et) with anti-tumor activity in both breast carcinoma and melanoma. The present work evaluated the ability of P2Et extract to modulate the immune system in either the steady state or following tumor challenge. We found that the prophylactic treatment of healthy mice increased the number of CD4 + and CD8 + activated T, NK, regulatory T, dendritic and myeloid-derived suppressor cells in lymphoid organs together with a significant increase in plasma IL-6. Interestingly, this pre-conditioning of the host immune system with P2Et did not involve a protective effect against the control of tumor growth and metastasis in transplantable models of melanoma (B16) and breast cancer (4T1), but in contrast, a detrimental effect was observed in both models. We further demonstrated that this effect was at least partly due to an increase in regulatory T cells, myeloid-derived suppressor cells, and proinflammatory cytokines, with a concomitant decrease in CD4 + and CD8 + T cells. Taken together, these results suggest that the anti-tumor and immunomodulation properties of the P2Et extract critically depend on the presence of the tumor and might be mediated by the complex interactions between the tumor cells and the other components of the tumor microenvironment.

Group 2 innate lymphoid cells (ILC2s) are involved in human diseases, such as allergy, atopic dermatitis and nasal polyposis, but their function in human cancer remains unclear. Here we show that, in acute promyelocytic leukaemia (APL), ILC2s are increased and hyper-activated through the interaction of CRTH2 and NKp30 with elevated tumour-derived PGD2 and B7H6, respectively. ILC2s, in turn, activate monocytic myeloid-derived suppressor cells (M-MDSCs) via IL-13 secretion. Upon treating APL with all-trans retinoic acid and achieving complete remission, the levels of PGD2, NKp30, ILC2s, IL-13 and M-MDSCs are restored. Similarly, disruption of this tumour immunosuppressive axis by specifically blocking PGD2, IL-13 and NKp30 partially restores ILC2 and M-MDSC levels and results in increased survival. Thus, using APL as a model, we uncover a tolerogenic pathway that may represent a relevant immunosuppressive, therapeutic targetable, mechanism operating in various human tumour types, as supported by our observations in prostate cancer., Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth.

Recent findings suggest that part of the anti-tumor effects of several chemotherapeutic agents require an intact immune system. This is in part due to the induction of immunogenic cell death. We have identified a gallotannin-rich fraction, obtained from Caesalpinia spinosa (P2Et) as an anti-tumor agent in both breast carcinoma and melanoma. Here, we report that P2Et treatment results in activation of caspase 3 and 9, mobilization of cytochrome c and externalization of annexin V in tumor cells, thus suggesting the induction of apoptosis. This was preceded by the onset of autophagy and the expression of immunogenic cell death markers. We further demonstrate that P2Et-treated tumor cells are highly immunogenic in vaccinated mice and induce immune system activation, clearly shown by the generation of interferon gamma (IFN-γ) producing tyrosine-related protein 2 antigen-specific CD8+ T cells. Moreover, the tumor protective effects of P2Et treatment were abolished in immunodeficient mice, and partially lost after CD4 and CD8 depletion, indicating that P2Et’s anti-tumor activity is highly dependent on immune system and at least in part of T cells. Altogether, these results support the hypothesis that the gallotannin-rich fraction P2Et’s anti-tumor effects are mediated to a great extent by the endogenous immune response following to the exposure to immunogenic dying tumor cells.

Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy.

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39– Tim3– Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39– Tim3– Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.

Chemotherapy faces the problem of chemo-resistance in large part of tumors, leading to the appearance of metastasis difficult to eliminate. Over the past decade an improved understanding of the effect of immunogenic chemotherapy on tumor-host interactions has led to the concept of immunogenic cell death (ICD), a type of cell death capable to convert a tumor into an in situ vaccine through the generation of danger signals, dendritic cells activation an finally breaking tolerance, which leads to the destruction of residual tumor cells. Polyphenols are natural compounds from plants, traditionally used for cancer treatment, that exhibit multiple biological activities including anti-oxidation, anti-angiogenesis and pro-apoptosis, which together may exert potent anti-tumor activity. However, little is known about the mechanism of action of polyphenols. We previously obtained a normalized polyphenol rich fraction from Ceasalpina spinosa (P2Et) that exerted cytotoxic activity on several tumor cell lines. Moreover, P2Et showed anti-tumor activity in the 4T1 transplantable model through ICD induction. Vaccination with P2Et-pretreated 4T1 cells yielded long lasting ex vivo IL-2, TNF-α, IL-4, IL-5, and IFN-γ secreting multifunctional T cells after specific 4T1 cell stimulation. In this work we investigate the activation of the immune system after in vivo P2Et treatment of B16 tumor bearing mice. Analysis of spleen, lymph nodes and tumors shows increase numbers of CD44 high, CD4 and CD8 T cells, as well as NK cells in P2Et treated mice compared to non-treated ones. In addition, in vitro, phagocytosis of P2Et treated tumor cells induce activation of DC, and in vivo P2Et induces recruitment and activation of cross-presenting DCs (CD11c+, CD11b+, Ly6C+ or CD11c+ CD8α+) in spleen and tumors, which could lead to the effective activation of CD8 T cells favoring a better tumor control. In fact, vaccinated mice treated with P2Et cells are able to effectively control the growth of transplanted tumor. Accordingly, we observed that P2Et anti-tumor activity is highly dependent on an intact immune system, as P2Et anti-tumor activity is lost in RAG γc KO immunodeficient mice. In summary, P2Et exert its anti-tumor activity through the activation of the endogenous immune system, playing an important role not only in the destruction of the primary tumor but also in controlling metastasis.