Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE); even combining IR with immune checkpoint inhibitors has shown only anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug, disulfiram (DSF), complexed with copper (DSF/Cu) to induce tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anticancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs relative to spontaneous lung metastasis. In addition, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anticancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral (i.t.) injection of DSF/Cu and IR(12Gy) demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8+ and CD4+ cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, dendritic cells (DC), and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8+ and CD4+ cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anticancer immune response that results in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

Reprogramming the Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD-1 Therapy.

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Adding blockade of the anti-programmed cell death protein (PD)-1 pathway to gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers but with low response rates. Here, we studied the effects of anti-cytotoxic T lymphocyte antigen (CTLA)-4 when combined with anti-PD-1 and gemcitabine/cisplatin in orthotopic murine models of ICC. This combination therapy led to substantial survival benefits and reduction of morbidity in two aggressive ICC models that were resistant to immunotherapy alone. Gemcitabine/cisplatin treatment increased tumor-infiltrating lymphocytes and normalized the ICC vessels and, when combined with dual CTLA-4/PD-1 blockade, increased the number of activated CD8+Cxcr3+IFNγ+ T cells. CD8+ T cells were necessary for the therapeutic benefit because the efficacy was compromised when CD8+ T cells were depleted. Expression of Cxcr3 on CD8+ T cells is necessary and sufficient because CD8+ T cells from Cxcr3+/+ but not Cxcr3-/- mice rescued efficacy in T cell‒deficient mice. Finally, rational scheduling of anti-CTLA-4 "priming" with chemotherapy followed by anti-PD-1 therapy achieved equivalent efficacy with reduced overall drug exposure. These data suggest that this combination approach should be clinically tested to overcome resistance to current therapies in ICC patients.

Preventing NK cell activation in the damaged liver induced by cabozantinib/PD-1 blockade increases survival in hepatocellular carcinoma models.

The addition of anti-VEGF antibody treatment to immune checkpoint blockade (ICB) has increased the efficacy of immunotherapy in advanced hepatocellular carcinoma (HCC). Despite an initial promise, adding multitargeted kinase inhibitors of VEGFR with ICB has failed to increase survival in HCC. To reveal the mechanisms underlying treatment failure, we studied the effects of cabozantinib/ICB using orthotopic murine HCC models with or without liver damage. We monitored tumor growth and liver function, recorded survival outcomes, and performed immune profiling studies for intra-tumoral and surrounding liver. Cabozantinib/ICB treatment led to tumor regression and significantly improved survival in mice with normal livers. However, consistent with the clinical findings, combination therapy failed to show survival benefits despite similar tumor control when tested in the same models but in mice with liver fibrosis. Moreover, preclinical and clinical data converged, showing that activating immune responses by cabozantinib/ICB treatment induced hepatoxicity. Immune profiling revealed that combination therapy effectively reprogrammed the tumor immune microenvironment and increased NK cell infiltration and activation in the damaged liver tissue. Surprisingly, systemic depletion of NK reduced hepatotoxicity elicited by the combination therapy without compromising its anti-cancer effect, and significantly enhanced the survival benefit even in mice with HCC and underlying liver fibrosis. These findings demonstrate that preventing NK activation allowed for maintaining a favorable therapeutic ratio when combining ICB with cabozantinib in advanced HCC models.

Stressed target cancer cells drive nongenetic reprogramming of CAR T cells and solid tumor microenvironment.

The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumors is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquire early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogram and reverse the immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells of healthy donors or metastatic female breast cancer patients, induce robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a promising therapy for solid tumors.

Turning anecdotal irradiation-induced anti-cancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells.

Irradiation (IR) induces immunogenic cell death (ICD) in tumors, but it rarely leads to the abscopal effect (AE). However, combining IR with immune checkpoint inhibitors has shown anecdotal success in inducing AEs. In this study, we aimed to enhance the IR-induced immune response and generate reproducible AEs using the anti-alcoholism drug disulfiram (DSF) and copper complex (DSF/Cu) via induction of tumor ICD. We measured ICD in vitro and in vivo. In mouse tumor models, DSF/Cu was injected intratumorally followed by localized tumor IR, creating an in situ cancer vaccine. We determined the anti-cancer response by primary tumor rejection and assessed systemic immune responses by tumor rechallenge and the occurrence of AEs, i.e., spontaneous lung metastasis. Additionally, we analyzed immune cell subsets and quantified proinflammatory and immunosuppressive chemokines/cytokines in the tumor microenvironment (TME) and blood of the vaccinated mice. Immune cell depletion was investigated for its effects on the vaccine-induced anti-cancer response. The results showed that DSF/Cu and IR induced more potent ICD under hypoxia than normoxia in vitro. Low-dose intratumoral injection of DSF/Cu and IR demonstrated strong anti-primary and -rechallenged tumor effects and robust AEs in mouse models. These vaccinations also increased CD8 + and CD4 + cell numbers while decreasing Tregs and myeloid-derived suppressor cells in the 4T1 model, and increased CD8+, DC, and decreased Treg cell numbers in the MCa-M3C model. Depleting both CD8 + and CD4 + cells abolished the vaccine's anticancer response. Moreover, vaccinated tumor-bearing mice exhibited increased TNFα levels and reduced levels of immunosuppressive chemokines/cytokines. In conclusion, our novel approach generated an anti-cancer immune response, resulting in a lack of or low tumor incidence post-rechallenge and robust AEs, i.e., the absence of or decreased spontaneous lung metastasis in tumor-bearing mice. This approach is readily translatable to clinical settings and may increase IR-induced AEs in cancer patients.

"Isolation and characterization of a novel hormone receptor positive mammary adenocarcinoma MCa-P1362 with stromal drivers of tumor growth, metastasis, and drug resistance".

Preclinical models that display spontaneous metastasis are necessary to improve therapeutic options for hormone receptor positive breast cancers. In this study, we conducted a detailed cellular and molecular characterization of MCa-P1362, a novel syngeneic Balb/c mouse model of metastatic breast cancer. MCa-P1362 cancer cells expressed estrogen receptors (ER), progesterone receptors (PR), and HER-2 receptors. MCa-P1362 cells proliferate in vitro and in vivo in response to estrogen, yet do not depend on steroid hormones for tumor progression. Further characterization of MCa-P1362 tumor explants shows that they contain a mixture of epithelial cancer cells and stromal cells. Based on transcriptomic and functional analyses of cancer and stromal cells, stem cells are present in both populations. Functional studies demonstrate that crosstalk between cancer and stromal cells promotes tumor growth, metastasis, and drug resistance. MCa-P1362 may serve as a useful preclinical model to investigate the cellular and molecular basis of hormone receptor positive tumor progression and therapeutic resistance.

Cancer cell plasticity and MHC-II-mediated immune tolerance promote breast cancer metastasis to lymph nodes.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen-specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

Absence of Tissue-Sparing Effects in Partial Proton FLASH Irradiation in Murine Intestine.

Ultra-high dose rate irradiation has been reported to protect normal tissues more than conventional dose rate irradiation. This tissue sparing has been termed the FLASH effect. We investigated the FLASH effect of proton irradiation on the intestine as well as the hypothesis that lymphocyte depletion is a cause of the FLASH effect. A 16 × 12 mm2 elliptical field with a dose rate of ~120 Gy/s was provided by a 228 MeV proton pencil beam. Partial abdominal irradiation was delivered to C57BL/6j and immunodeficient Rag1-/-/C57 mice. Proliferating crypt cells were counted at 2 days post exposure, and the thickness of the muscularis externa was measured at 280 days following irradiation. FLASH irradiation did not reduce the morbidity or mortality of conventional irradiation in either strain of mice; in fact, a tendency for worse survival in FLASH-irradiated mice was observed. There were no significant differences in lymphocyte numbers between FLASH and conventional-dose-rate mice. A similar number of proliferating crypt cells and a similar thickness of the muscularis externa following FLASH and conventional dose rate irradiation were observed. Partial abdominal FLASH proton irradiation at 120 Gy/s did not spare normal intestinal tissue, and no difference in lymphocyte depletion was observed. This study suggests that the effect of FLASH irradiation may depend on multiple factors, and in some cases dose rates of over 100 Gy/s do not induce a FLASH effect and can even result in worse outcomes.

Indole-3-Propionic Acid, a Gut Microbiota Metabolite, Protects Against the Development of Postoperative Delirium.

OBJECTIVE: The aim was to determine preoperative gut microbiota metabolites that may be associated with postoperative delirium (POD) development in patients and further study in rodents. SUMMARY BACKGROUND DATA: POD occurs in 9% to 50% of older patients undergoing anesthesia/surgery but lacks effective treatments or prevention. High-throughput metabolomics using liquid chromatography with tandem mass spectrometry has accelerated disease-related biomarkers discovery. We performed metabolomic studies in humans to identify potential metabolite biomarkers linked to POD and examined potential mechanisms in rodents. METHODS: We performed a prospective observational cohort study to examine the metabolomic changes that were associated with the development of POD. Then the gut microbiota-related metabolomic changes were recapitulated by gut microbiota perturbation in rodents. POD was assessed in mice using a battery of behavioral tests including novel objective test, Y-maze test, open-field test, and buried food test. The mechanisms through which gut microbiota-related metabolomic changes influenced POD were examined using chemogenetics. RESULTS: Indole-3-propionic acid (IPA) is a gut microbiota metabolite that belongs to the indole family. Baseline plasma levels of IPA were significantly inversely correlated with the onset of POD in 103 (17 cases) human individuals. This relationship was validated in preclinical mouse models for POD: reducing IPA levels through gut microbiota perturbation promoted POD-like behavior. More importantly, IPA administration deterred POD-like behavior. Colonization of germ-free mice with mutant Clostridium sporogenes that did not produce IPA-promoted POD-like behavior. Chemogenetic studies revealed that the protective effect of IPA in mice was mediated, in part, by peroxisome proliferator-activated receptor gamma coactivator 1-alpha in hippocampal interneurons. CONCLUSIONS: Gut microbiota-derived IPA is an important molecule implicated in the pathogenesis of POD, which could potentially be harnessed for POD prevention.

Stressed target cancer cells drive nongenetic reprogramming of CAR T cells and tumor microenvironment, overcoming multiple obstacles of CAR T therapy for solid tumors.

The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumor is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach massively reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquired early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogrammed and reversed immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells (PBMC) of healthy or metastatic breast cancer patients, induced robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a novel therapy for solid tumor.

Inhibition of CXCR4 Enhances the Efficacy of Radiotherapy in Metastatic Prostate Cancer Models.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

Proton FLASH effects on mouse skin at different oxygen tensions.

Objective. Irradiation at FLASH dose rates (>40 Gy s-1) has received great attention due to its reported normal tissue sparing effect. The FLASH effect was originally observed in electron irradiations but has since been shown to also occur with both photon and proton beams. Several mechanisms have been proposed to explain the tissue sparing at high dose rates, including effects involving oxygen, such as depletion of oxygen within the irradiated cells. In this study, we investigated the protective role of FLASH proton irradiation on the skin when varying the oxygen concentration.Approach. Our double scattering proton system provided a 1.2 × 1.6 cm2elliptical field at a dose rate of ∼130 Gy s-1. The conventional dose rate was ∼0.4 Gy s-1. The legs of the FVB/N mice were marked with two tattooed dots and fixed in a holder for exposure. To alter the skin oxygen concentration, the mice were breathing pure oxygen or had their legs tied to restrict blood flow. The distance between the two dots was measured to analyze skin contraction over time.Main results. FLASH irradiation mitigated skin contraction by 15% compared to conventional dose rate irradiation. The epidermis thickness and collagen deposition at 75 d following 25 to 30 Gy exposure suggested a long-term protective function in the skin from FLASH irradiation. Providing the mice with oxygen or reducing the skin oxygen concentration removed the dose-rate-dependent difference in response.Significance. FLASH proton irradiation decreased skin contraction, epidermis thickness and collagen deposition compared to standard dose rate irradiations. The observed oxygen-dependence of the FLASH effect is consistent with, but not conclusive of, fast oxygen depletion during the exposure.

Reprogramming Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD1 Therapy.

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Anti-PD-L1 immunotherapy combined with gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers, but responses are seen only in a minority of patients. Here, we studied the roles of anti-PD1 and anti-CTLA-4 immune checkpoint blockade (ICB) therapies when combined with gemcitabine/cisplatin and the mechanisms of treatment benefit in orthotopic murine ICC models. We evaluated the effects of the combined treatments on ICC vasculature and immune microenvironment using flow cytometry analysis, immunofluorescence, imaging mass cytometry, RNA-sequencing, qPCR, and in vivo T-cell depletion and CD8+ T-cell transfer using orthotopic ICC models and transgenic mice. Combining gemcitabine/cisplatin with anti-PD1 and anti-CTLA-4 antibodies led to substantial survival benefits and reduction of morbidity in two aggressive ICC models, which were ICB-resistant. Gemcitabine/cisplatin treatment increased the frequency of tumor-infiltrating lymphocytes and normalized the ICC vessels, and when combined with dual CTLA-4/PD1 blockade, increased the number of activated CD8+Cxcr3+IFN-γ+ T-cells. Depletion of CD8+ but not CD4+ T-cells compromised efficacy. Conversely, CD8+ T-cell transfer from Cxcr3-/- versus Cxcr3+/+ mice into Rag1-/- immunodeficient mice restored the anti-tumor effect of gemcitabine/cisplatin/ICB combination therapy. Finally, rational scheduling of the ICBs (anti-CTLA-4 "priming") with chemotherapy and anti-PD1 therapy achieved equivalent efficacy with continuous dosing while reducing overall drug exposure. In summary, gemcitabine/cisplatin chemotherapy normalizes vessel structure, increases activated T-cell infiltration, and enhances anti-PD1/CTLA-4 immunotherapy efficacy in aggressive murine ICC. This combination approach should be clinically tested to overcome resistance to current therapies in ICC patients.

Wnt inhibition alleviates resistance to immune checkpoint blockade in glioblastoma.

Wnt signaling plays a critical role in the progression and treatment outcome of glioblastoma (GBM). Here, we identified WNT7b as a heretofore unknown mechanism of resistance to immune checkpoint inhibition (αPD1) in GBM patients and murine models. Acquired resistance to αPD1 was found to be associated with the upregulation of Wnt7b and ß-catenin protein levels in GBM in patients and in a clinically relevant, stem-rich GBM model. Combining the porcupine inhibitor WNT974 with αPD1 prolonged the survival of GBM-bearing mice. However, this combination had a dichotomous response, with a subset of tumors showing refractoriness. WNT974 and αPD1 expanded a subset of DC3-like dendritic cells (DCs) and decreased the granulocytic myeloid-derived suppressor cells (gMDSCs) in the tumor microenvironment (TME). By contrast, monocytic MDSCs (mMDSCs) increased, while T-cell infiltration remained unchanged, suggesting potential TME-mediated resistance. Our preclinical findings warrant the testing of Wnt7b/ß-catenin combined with αPD1 in GBM patients with elevated Wnt7b/ß-catenin signaling.

Increased CD8+ T-cell Infiltration and Efficacy for Multikinase Inhibitors After PD-1 Blockade in Hepatocellular Carcinoma.

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.

Glycocalyx mechanotransduction mechanisms are involved in renal cancer metastasis.

Mammalian cells, including cancer cells, are covered by a surface layer containing cell bound proteoglycans, glycoproteins, associated glycosaminoglycans and bound proteins that is commonly referred to as the glycocalyx. Solid tumors also have a dynamic fluid microenvironment with elevated interstitial flow. In the present work we further investigate the hypothesis that interstitial flow is sensed by the tumor glycocalyx leading to activation of cell motility and metastasis. Using a highly metastatic renal carcinoma cell line (SN12L1) and its low metastatic counterpart (SN12C) we demonstrate in vitro that the small molecule Suberoylanilide Hydroxamic Acid (SAHA) inhibits the heparan sulfate synthesis enzyme N-deacetylase-N-sulfotransferase-1, reduces heparan sulfate in the glycocalyx and suppresses SN12L1 motility in response to interstitial flow. SN12L1 cells implanted in the kidney capsule of SCID mice formed large primary tumors and metastasized to distant organs, but when treated with SAHA metastases were not detected. In another set of experiments, the role of hyaluronic acid was investigated. Hyaluronan synthase 1, a critical enzyme in the synthetic pathway for hyaluronic acid, was knocked down in SN12L1 cells and in vitro experiments revealed inhibition of interstitial flow induced migration. Subsequently these cells were implanted in mouse kidneys and no distant metastases were detected. These findings suggest new therapeutic approaches to the treatment of kidney carcinoma metastasis.

Dendritic cell paucity in mismatch repair-proficient colorectal cancer liver metastases limits immune checkpoint blockade efficacy.

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.

Solid stress impairs lymphocyte infiltration into lymph-node metastases.

Strong and durable anticancer immune responses are associated with the generation of activated cancer-specific T cells in the draining lymph nodes. However, cancer cells can colonize lymph nodes and drive tumour progression. Here, we show that lymphocytes fail to penetrate metastatic lesions in lymph nodes. In tissue from patients with breast, colon, and head and neck cancers, as well as in mice with spontaneously developing breast-cancer lymph-node metastases, we found that lymphocyte exclusion from nodal lesions is associated with the presence of solid stress caused by lesion growth, that solid stress induces reductions in the number of functional high endothelial venules in the nodes, and that relieving solid stress in the mice increased the presence of lymphocytes in lymph-node lesions by about 15-fold. Solid-stress-mediated impairment of lymphocyte infiltration into lymph-node metastases suggests a therapeutic route for overcoming T-cell exclusion during immunotherapy.

Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas.

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.

Combining losartan with radiotherapy increases tumor control and inhibits lung metastases from a HER2/neu-positive orthotopic breast cancer model.

BACKGROUND: Patients with metastatic HER2/neu-positive (HER2/neu +) breast cancer (BC) often experience treatment resistance, disease recurrences and metastases. Thus, new approaches for improving the treatment of HER2/neu + BC to prevent metastatic dissemination are urgently needed. Our previous studies have shown that losartan, an angiotensin receptor blocker, increases tumor perfusion and decreases hypoxia in a number of tumor models. Hypoxia reduces the efficacy of radiation and increases metastases. We therefore hypothesized that by modifying tumor stroma and increasing oxygenation, losartan will improve the outcome of radiotherapy and inhibit disease progression in a highly metastatic HER2/neu + murine BC model. METHODS: We established a metastatic HER2/neu + murine BC line (MCa-M3C) and used it to generate mammary fat pad isografts in syngeneic female FVB/N mice. Starting on day 3 after orthotopic tumor implantation, we administered a 7-day losartan treatment (40 mg/kg BW, gavage daily); or a 7-day losartan treatment followed by 20 Gy single dose local irradiation (S-IR) on day 10 (tumor size ~ 100 mm3), or 20 Gy local fractionated (5 × 4 Gy daily) irradiation (F-IR) on days 10-14. We analyzed tumor-growth delay (TGD), development of spontaneous lung metastases, animal survival, tumor vascular density, and tumor hypoxia. RESULTS: Treatments with S-IR, F-IR, Losartan + S-IR, or Losartan + F-IR resulted in a significantly increased TGD (8-16 days) in MCa-M3C tumors versus controls. However, the combination of Losartan + S-IR and Losartan + F-IR further enhanced tumor response to radiation alone by increasing TGD an additional 5 to 8 days for both single and fractionated dose irradiation (P < 0.01), decreasing lung metastasis (Losartan + IR vs. Control, P < 0.025), and increasing animal survival (Losartan + IR vs. Control, P = 0.0303). In addition, losartan treatment significantly increased tumor vascularity (P = 0.0314) and decreased pimonidazole positive (hypoxic) area (P = 0.0002). CONCLUSIONS: Combining losartan with local irradiation significantly enhanced tumor response, at least in part via reduced tumor hypoxia presumably due to increased tumor perfusion. Our findings suggest that combining losartan with radiotherapy is a potential new treatment strategy for local control and inhibiting metastasis in HER2 + BC.