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Hypoxia is a unique characteristic of the solid tumor microenvironment. Hypoxia contributes to multi-drug resistance, metastasis and cancer relapse through numerous molecular pathways, but at the same time provides an opportunity for the development of novel drugs or modalities specifically targeting hypoxic tumor regions. Given the high significance of tumor hypoxia in therapeutic results, we here discuss a variety of hypoxia-adopted strategies, and their potential and utility in the treatment of deep-seated hypoxic tumor cells. We discuss the merits and demerits of these approaches, as well as their combination with other approaches such as photodynamic therapy. We also survey the currently available 3D hypoxia modeling systems, in particular organoid-based microfluidics. Finally, we discuss the potential and the current status of preclinical tumor hypoxia approaches in clinical trials for advanced cancer. We believe that multi-modal imaging and therapeutic hypoxia adopted drug delivery platforms could provide better efficacy and safety profiles, and more importantly personalized therapy. Determining the hypoxia status of tumors could offer a second chance for the clinical translation of hypoxia-based agents, such as hypoxia activated prodrugs (HAPs) from bench to bedside.

Supplemental Figures 1-11 and Supplementary table.

The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer, we examined here the causal role of CD8+ T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C breast cancer cells orthotopically in wild-type or Cxcr3−/− female mice and initiated intensity-controlled ExTr sessions when tumors reached approximately 100 mm3. We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8+ T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti–PD-1 alone or in combination with anti–CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion and by decreased hypoxia. ExTr boosted CD8+ T-cell infiltration, with enhanced effector function. CD8+ T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3−/− mice, supporting the causal role of the CXCL9/CXCL11–CXCR3 pathway. ExTr also sensitized ICB-refractory breast cancers to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8+ T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of breast cancer.

The lack of second-line treatment for relapsed ovarian cancer necessitates the development of improved combination therapies. Targeted therapy and immunotherapy each confer clinical benefit, albeit limited as monotherapies. Ovarian cancer is not particularly responsive to immune checkpoint blockade, so combination with a complementary therapy may be beneficial. Recent studies have revealed that a DNA methyl transferase inhibitor, azacytidine, alters expression of immunoregulatory genes in ovarian cancer. In this study, the antitumor effects of a related DNA methyl transferase inhibitor, decitabine (DAC), were demonstrated in a syngeneic murine ovarian cancer model. Low-dose DAC treatment increases the expression of chemokines that recruit NK cells and CD8+ T cells, promotes their production of IFNγ and TNFα, and extends the survival of mice bearing subcutaneous or orthotopic tumors. While neither DAC nor immune checkpoint blockade confers durable responses as a monotherapy in this model, the efficacy of anti–CTLA-4 was potentiated by combination with DAC. This combination promotes differentiation of naïve T cells into effector T cells and prolongs cytotoxic lymphocyte responses as well as mouse survival. These results suggest that this combination therapy may be worthy of further consideration for improved treatment of drug-resistant ovarian cancer. Cancer Immunol Res; 3(9); 1030–41. ©2015 AACR.

Supplementary Figure 1: Decitabine upregulates major histocompatibility complex I on BR5FVB1-Akt cells in a dose-dependent manner.

Supplementary Figures 1-8

Figure legends for Supplementary Figures 1-8

Supplementary Table 1: A list of genes regulated by decitabine treatment in BR5FVB1-Akt cells.

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

Immune checkpoint blockade combined with antiangiogenic therapy induces vascular normalization and antitumor immunity and is efficacious in hepatocellular carcinoma (HCC); but whether and how initial immunotherapy affects the efficacy of subsequent antiangiogenic therapy are unknown. We evaluated a cohort of HCC patients (n = 25) who received the pan–vascular endothelial growth factor receptor multikinase inhibitor sorafenib after initial therapy with an antiprogrammed cell death protein (PD)–1 antibody and found superior outcomes in these patients (12% overall response rate to sorafenib and a median overall survival of 12.1 months). To prove this potential benefit, we examined the impact of an anti–PD-1 antibody on response to subsequent sorafenib treatment in orthotopic models of murine HCC. Prior anti–PD-1 antibody treatment amplified HCC response to sorafenib therapy and increased survival (n = 8-9 mice per group, hazard ratio = 0.28, 95% confidence interval = 0.09 to 0.91; 2-sided P = .04). Anti–PD-1 therapy showed angioprotective effects on HCC vessels to subsequent sorafenib treatment, which enhanced the benefit of this therapy sequence in a CD8+ T-cell–dependent manner. This priming approach using immunotherapy provides an immediately translatable strategy for effective HCC treatment while reducing drug exposure.

SummaryTumor-draining lymph nodes are critical sites for generating tumor antigen-specific T cells and are associated with durable immune responses. However, lymph nodes are often the first site of metastasis and lymph node metastases portend worse outcomes. Through cross-species single cell gene expression analysis of breast cancer progression and metastasis to lymph nodes, we uncovered features that define the heterogeneity, plasticity, and immune evasion of cancer cells. Notably, a subpopulation of metastatic cancer cells in the lymph node were marked by high levels of MHC class II (MHC-II) gene expression both in mice and humans. Mechanistically, the IFN-γ and JAK/STAT signaling pathways mediate MHC-II expression in cancer cells. Ablation of IFNGR1/2 or CIITA, the transactivator of MHC-II, in cancer cells prevented tumor progression. Interestingly, MHC-II+ cancer cells lacked co-stimulatory molecule expression, engendered the expansion of regulatory T cells and blunted CD4+ effector T cells in the tumor-draining lymph nodes and favor tumor progression. Overall, our data suggests that cancer cell plasticity during breast cancer progression and metastasis to lymph nodes endows metastatic cells with the ability to avoid immune surveillance. These data provide the basis for new opportunities to therapeutically stimulate anti-cancer immune responses against local and systemic metastases.

Glioblastoma (GBM) remains a highly aggressive and uniformly fatal primary tumor, which resists cytotoxic, targeted, antiangiogenic, and immune therapies, even when used in combination. Here we report that tumor endothelial cell dysfunction confers resistance to immunotherapy in preclinical GBM models. Anti-VEGF-therapy-induced vascular normalization is insufficient to fully restore the endothelial cell function. Strikingly, concomitant blockade of Ang2, VEGF, and PD1 reprograms dysfunctional endothelial cells to quasi-antigen presenting cells and upregulates receptors required for cytotoxic T lymphocyte entry into the tumor. Blocking VEGF, Ang2, and PD1 induces durable anti-tumor T cell responses. Upregulation of the transcription factor T-bet is both necessary and sufficient for generating resident memory T cells elicited by this combination therapy. In summary, our study reveals the role of Ang2 in resistance to PD1-blockade and provides a compelling rationale for clinical evaluation of blocking Ang2 along with VEGF and PD1 in GBM patients.Statement of SignificanceOur study is the first to demonstrate Ang2 as a resistance pathway for both αVEGF and αPD1 in GBM. Concomitant blockade of Ang2 reprograms endothelial cells to recruit, activate and retain CD8 T cells, overcomes resistance to αVEGF and αPD1, and imparts T cell memory formation via T-bet in GBM.

The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer, we examined here the causal role of CD8+ T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C breast cancer cells orthotopically in wild-type or Cxcr3−/− female mice and initiated intensity-controlled ExTr sessions when tumors reached approximately 100 mm3. We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8+ T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti–PD-1 alone or in combination with anti–CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion and by decreased hypoxia. ExTr boosted CD8+ T-cell infiltration, with enhanced effector function. CD8+ T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3−/− mice, supporting the causal role of the CXCL9/CXCL11–CXCR3 pathway. ExTr also sensitized ICB-refractory breast cancers to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8+ T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of breast cancer.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Tumor-draining lymph nodes are critical sites for generating tumor antigen-specific T cells and are associated with durable immune responses. However, lymph nodes are often the first site of metastasis and lymph node metastases portend worse outcomes. Through cross species single cell gene expression analysis of breast cancer progression and metastasis to lymph nodes, we uncovered features that define the heterogeneity, plasticity, and immune evasion of cancer cells. Notably, a subpopulation of metastatic cancer cells in the lymph node were marked by high levels of MHC class II (MHC-II) gene expression both in mice and humans. Mechanistically, the IFN-γ and JAK/STAT signaling pathways mediate MHC-II expression in cancer cells. Ablation of IFNGR1/2 or CIITA, the transactivator of MHC-II, in cancer cells prevented tumor progression. Interestingly, MHC-II+ cancer cells lacked co-stimulatory molecule expression, engendered the expansion of regulatory T cells and blunted CD4+ effector T cells in the tumor draining lymph nodes and favor tumor progression. Overall, our data suggests that cancer cell plasticity during breast cancer progression and metastasis to lymph nodes endows metastatic cells with the ability to avoid immune surveillance. These data provide the basis for new opportunities to therapeutically stimulate anti-cancer immune responses against local and systemic metastases. Citation Format: Pin-Ji Lei, Ethel Pereira, Patrik Andersson, Zohreh Amoozgar, Jan Willem Van Wijnbergen, Meghan J. O'Melia, Hengbo Zhou, Sampurna Chatterjee, William Wee Ho, Jessica M. Posada, Ashwin Srinivasan Kumar, Satoru Morita, Charlie Chuang, Ilgin Ergin, Dennis Jones, Peigen Huang, Semir Beyaz, Timothy P. Padera. Single cell analysis of breast cancer progression and metastasis to lymph nodes reveals cancer cell plasticity and MHC class II-mediated immune regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1298.

Ependymoma is a common type of brain tumor in children, often resistant to available cytotoxics. Molecular profiling studies have led to a better understanding of ependymoma pathology and revealed a critical role of EPHB2. However, the immune system's role in tumor progression and treatment response remains poorly understood. Here, we show that the multikinase inhibitor dasatinib potently inhibits EPHB2 and ABL1 and the growth of ependymoma both in vitro and in vivo. Using an immunocompetent mouse model of ependymoma that faithfully recapitulates its immune microenvironment, we found an increased frequency of M2-like tumor-associated macrophages (TAMs), which proportionally increased with tumor size. Treatment with dasatinib reprogrammed the immune microenvironment of ependymoma by polarizing the TAMs toward an M1-like phenotype and increasing CD8 T cell activation. Dasatinib treatment induced complete regression of established mEPN-Ephb2 tumors in 78% of the animals and protected survivors against tumor recurrence. These studies indicate that dasatinib may be an effective therapy for this molecular subgroup of pediatric brain tumors and should be tested in clinical trials. Citation Format: Jun Ren, Zohreh Amoozgar, Yuhui Zhao, Pinji Lei, William W. Ho, Sylvie Robergea, Peigen Huang, Dan G. Duda, Lei Xu, Rakesh K. Jain. Targeting EPHB2/ABL1 restores anti-tumor immunity in a preclinical model of ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2343.

Chimeric antigen receptor (CAR)-T cells have revolutionized the treatment of multiple types of hematological malignancies, but have shown limited efficacy in patients with glioblastoma (GBM) or other solid tumors. This may be largely due to the immunosuppressive tumor microenvironment (TME) that compromises CAR-T cells’ delivery and antitumor activity. We previously showed that blocking vascular endothelial growth factor (VEGF) signaling can normalize tumor vessels in murine and human tumors, including GBM, breast, liver, and rectal carcinomas. Moreover, we demonstrated that vascular normalization can improve the delivery of CD8+ T cells and the efficacy of immunotherapy in breast cancer models in mice. In fact, the US FDA (Food and drug administration) has approved seven different combinations of anti-VEGF drugs and immune checkpoint blockers for liver, kidney, lung and endometrial cancers in the past 3 years. Here, we tested the hypothesis that anti-VEGF therapy can improve the delivery and efficacy of CAR-T cells in immunocompetent mice bearing orthotopic GBM tumors. We engineered two syngeneic mouse GBM cell lines (CT2A and GSC005) to express EGFRvIII—one of the most common neoantigens in human GBM—and CAR T cells to recognize EGFRvIII. We found that treatment with the anti-mouse VEGF antibody (B20) improved CAR-T cell infiltration and distribution throughout the GBM TME, delayed tumor growth, and prolonged survival of GBM-bearing mice compared with EGFRvIII-CAR-T cell therapy alone. Our findings provide compelling data and a rationale for clinical evaluation of anti-VEGF agents with CAR T cells for GBM patients.

Metastasis is the major cause of cancer-related deaths. Invasive primary cancers often metastasize after circulating tumor cells (CTCs) enter the bloodstream or lymph node to colonize adjacent tissue or distant anatomical locations. CTCs interact with immune cells and metastatic microenvironments, survival signaling, and chemotherapeutic resistance. Among immune cells, natural killer (NK) cells can, directly and indirectly, interact with CTCs to control cancer metastasis. Understanding the molecular mechanisms that drive NK cells mediated recognition and elimination of CTCs may pave the way for a new generation of anti-CTC molecularly targeted immunotherapies. In this review, we will discuss i) the role of CTCs in metastases, ii) CTCs in the context of the tumor microenvironment, iii) CTCs immune escape, and finally, iv) the potentials of NK cell-based therapies alone, or in combination with nanomedicine for targeted-immunotherapies of metastatic diseases.

Significance Immune checkpoint blockade (ICB) has been efficacious in several cancer types. However, mismatch repair–proficient (pMMR) metastatic colorectal cancer (CRC), ∼95% of total metastatic CRC cases, typically does not respond to ICB. Here, we show that orthotopic liver metastasis mouse models of pMMR CRC cell lines are unresponsive to ICB and recapitulate the resistance of human disease, unlike subcutaneous tumors of the same cell lines. We also show that just like the human disease, orthotopic pMMR CRC liver metastases have a paucity of T cells and dendritic cells, and that treatment with Flt3L sensitizes the liver metastases to ICB. Our findings highlight that orthotopic tumor models, and not subcutaneous models, should be used for preclinical studies of cancer immunotherapy., Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair–proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.

Glioblastoma (GBM) is the most common form of malignant primary brain tumor and despite optimal treatment, long-term survival remains incredibly rare. Radiation therapy (RT) leads to successful initial tumor regression but recurrence is inevitable. Previous studies have shown that ionizing radiation changes the composition of the tumor microenvironment and alters the expression of immune-related markers on tumor cells. Extracellular vesicles (EVs) are vesicular bodies of cellular origin and secreted by nearly every cell. They have been shown to carry a variety of cargo (DNA, proteins, RNAs, lipids) which can be taken up by other cells. Notably, previous studies highlight that EVs drive GBM progression and immune evasion by acting as multifunctional signaling complexes. Here we show that ionizing radiation of GBM cells results in an altered EV secretome and further facilitates the uptake of EVs in recipient cells. In addition, EVs secreted by GBM cells following radiation modulate the tumor microenvironment by manipulating the innate as well as the adaptive immune response. Macrophages exposed to EVs from irradiated GBM cells switched to the tumor supportive M2 phenotype and exhibited decreased phagocytotic ability.We dissected a novel mechanism by which GBM evades the immune system with the help of EVs following RT, pointing towards novel therapeutic strategies to prevent GB recurrence.

This single-arm phase II study investigated the efficacy and safety of cabozantinib combined with nivolumab in metastatic triple-negative breast cancer (mTNBC). The primary endpoint was objective response rate (ORR) by RECIST 1.1. Biopsies at baseline and after cycle 1 were analyzed for tumor-infiltrating lymphocytes (TILs), PD-L1, and whole-exome and transcriptome sequencing. Only 1/18 patients achieved a partial response (ORR 6%), and the trial was stopped early. Toxicity led to cabozantinib dose reduction in 50% of patients. One patient had a PD-L1-positive tumor, and three patients had TILs > 10%. The responding patient had a PD-L1-negative tumor with low tumor mutational burden but high TILs and enriched immune gene expression. High pretreatment levels of plasma immunosuppressive cytokines, chemokines, and immune checkpoint molecules were associated with rapid progression. Although this study did not meet its primary endpoint, immunostaining, genomic, and proteomic studies indicated a high degree of tumor immunosuppression in this mTNBC cohort.

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

Immunotherapy with immune checkpoint blockade (ICB) has shown limited benefits in hepatocellular carcinoma (HCC) and other cancers, mediated in part by the immunosuppressive tumor microenvironment (TME). As p53 loss of function may play a role in immunosuppression, we herein examine the effects of restoring p53 expression on the immune TME and ICB efficacy. We develop and optimize a CXCR4-targeted mRNA nanoparticle platform to effectively induce p53 expression in HCC models. Using p53-null orthotopic and ectopic models of murine HCC, we find that combining CXCR4-targeted p53 mRNA nanoparticles with anti-PD-1 therapy effectively induces global reprogramming of cellular and molecular components of the immune TME. This effect results in improved anti-tumor effects compared to anti-PD-1 therapy or therapeutic p53 expression alone. Thus, our findings demonstrate the reversal of immunosuppression in HCC by a p53 mRNA nanomedicine when combined with ICB and support the implementation of this strategy for cancer treatment.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

Coronavirus disease 2019 (Covid-19) is highly contagious with limited treatment options. Early and accurate diagnosis of Covid-19 is crucial in reducing the spread of the disease and its accompanied mortality. Currently, detection by reverse transcriptase-polymerase chain reaction (RT-PCR) is the gold standard of outpatient and inpatient detection of Covid-19. RT-PCR is a rapid method; however, its accuracy in detection is only ~70–75%. Another approved strategy is computed tomography (CT) imaging. CT imaging has a much higher sensitivity of ~80–98%, but similar accuracy of 70%. To enhance the accuracy of CT imaging detection, we developed an open-source framework, CovidCTNet, composed of a set of deep learning algorithms that accurately differentiates Covid-19 from community-acquired pneumonia (CAP) and other lung diseases. CovidCTNet increases the accuracy of CT imaging detection to 95% compared to radiologists (70%). CovidCTNet is designed to work with heterogeneous and small sample sizes independent of the CT imaging hardware. To facilitate the detection of Covid-19 globally and assist radiologists and physicians in the screening process, we are releasing all algorithms and model parameter details as open-source. Open-source sharing of CovidCTNet enables developers to rapidly improve and optimize services while preserving user privacy and data ownership.

The failure of anti-VEGF/R and immune checkpoint therapies to improve overall survival in Phase III clinical trials in glioblastoma (GBM) is considered to be due in part to the prevalent immunosuppression in the GBM tumor microenvironment. Immune suppression is mediated in part by resident microglia and bone-marrow-derived myeloid cells recruited during tumor progression. A paper by Blank et al published in a recent issue of The Journal of Pathology proposes a myeloid cell-mediated mechanism that could contribute to resistance to anti-VEGF/R in GBM patients. A granulocyte-rich GBM tumor microenvironment may push the associated microglia/macrophages to exhibit an activated and immune suppressive phenotype. The identification of pro-angiogenic factors produced by microglia/macrophages and granulocytes in such a tumor microenvironment may offer new targets for improving antiangiogenic therapy of GBM beyond VEGF. Further, consideration of parameters such as IDH status, corticosteroid dosage, tumor mutational burden, gender, vascular function, and pericyte coverage could exploit current immunotherapies to the fullest to reprogram the granulocyte-rich immunosuppressive GBM tumor microenvironment to an immunostimulatory one. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer (BC), we examined here the causal role of CD8(+) T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C BC cells orthotopically in wild-type or Cxcr3(−/−) female mice and initiated intensity-controlled ExTr sessions when tumors reached ~100 mm(3). We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8(+) T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti-PD-1 alone or in combination with anti-CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion, and decreased hypoxia. ExTr boosted CD8(+) T-cell infiltration, with enhanced effector function. CD8(+) T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8(+) T cells and the antitumor effects of ExTr were abrogated in Cxcr3(−/−) mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway. ExTr also sensitized ICB-refractory BCs to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8(+) T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of BC.

Glioblastoma (GBM) shows high level of resistance to currently available treatments including the standard of care and immunotherapy, representing the most fatal cancer type. Our study revealed that immune suppression by regulatory T cells (Treg) secondary to therapy with immune checkpoint blocker (anti-PD1) confers this resistance. In the GBM tumor microenvironment, Treg cells with increased suppressive phenotype were found of which frequency and anergic phenotype increase after ICB therapy, potentially contributing to the resistance. Targeting Treg has a dual-barreled effect on enhancing anti-tumor immunity: GBM is highly infiltrated with Treg while CD8 T cells are excluded. In view of Treg's intrinsic reactivity to self-antigens, mobilizing converted Treg as effector T cells can be an effective strategy to a tumor type that expresses a low level of neoantigens including GBM. Our study revealed that GITR (glucocorticoid induced TNFR family related protein) is a desirable therapeutic target based on its increased expression on GBM Tregs as compared to peripheral Tregs. Engagement of GITR with agonistic antibody led to conversion of Treg to Th1-like effector T cells, which is accompanied with downregulation of Helios and IL-10 expression that are associated with Treg suppressive function. Through combining anti-GITR with anti-PD1 therapy, tumor recognition by converted Treg and CD8 T cells could be enhanced via IFNg induced promotion of MHC class I and II expression by GBM cells, which also resulted in T cell memory formation in the long-term survivors. To obtain clinically relevant information, we established a standard of care regimen consisting of surgery, radiation, and chemotherapy for orthotopic mouse GBM. We found that the anti-GITR +anti-PD1 therapy tailored to the GBM specific TME synergizes with the standard of care, suggesting a translational potential in patients. Citation Format: Zohreh Amoozgar, Jonas Kloepper, Jun Ren, Rong En Tay, Samuel W. Kazer, Evgeny Kiner, Shanmugarajan Krishnan, Jessica M. Posada, Mitrajit Ghosh, Emilie Mamessier, Christina Wong, Gino B. Ferraro, Ana Batista, Nancy Wang, Mark Badeaux, Sylvie Roberge, Lei Xu, Peigen Huang, Alex K. Shalek, Dai Fukumura, Hye-Jung Kim, Rakesh K. Jain. Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P057.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared to their subcutaneous tumor counterparts. Furthermore, treatment with FMS-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC. Citation Format: William W. Ho, Igor L. Gomes-Santos, Shuichi Aoki, Meenal Datta, Kosuke Kawaguchi, Nilesh P Talele, Sylvie Roberge, Jun Ren, Hao Liu, Ivy X Chen, Patrik Andersson, Sampurna Chatterjee, Ashwin S. Kumar, Zohreh Amoozgar, Qixian Zhang, Peigen Huang, Mei Rosa Ng, Vikash P Chauhan, Lei Xu, Dan G. Duda, Jeffrey W. Clark, Mikael J. Pittet, Dai Fukumura, Rakesh K Jain. Dendritic cell paucity in mismatch repair-proficient colorectal cancer liver metastases limits the efficacy of immune checkpoint blockade [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P061.

Immunotherapy has emerged as a major therapeutic modality in oncology. Currently, however, the majority of patients with cancer do not derive benefit from these treatments. Vascular abnormalities are a hallmark of most solid tumours and facilitate immune evasion. These abnormalities stem from elevated levels of proangiogenic factors, such as VEGF and angiopoietin 2 (ANG2); judicious use of drugs targeting these molecules can improve therapeutic responsiveness, partially owing to normalization of the abnormal tumour vasculature that can, in turn, increase the infiltration of immune effector cells into tumours and convert the intrinsically immunosuppressive tumour microenvironment (TME) to an immunosupportive one. Immunotherapy relies on the accumulation and activity of immune effector cells within the TME, and immune responses and vascular normalization seem to be reciprocally regulated. Thus, combining antiangiogenic therapies and immunotherapies might increase the effectiveness of immunotherapy and diminish the risk of immune-related adverse effects. In this Perspective, we outline the roles of VEGF and ANG2 in tumour immune evasion and progression, and discuss the evidence indicating that antiangiogenic agents can normalize the TME. We also suggest ways that antiangiogenic agents can be combined with immune-checkpoint inhibitors to potentially improve patient outcomes, and highlight avenues of future research.