Reduction In Myeloid-derived Suppressor Cells Research Articles

TLR2 agonist promotes myeloid-derived suppressor cell polarization via Runx1 in hepatocellular carcinoma

Myeloid-derived suppressor cells (MDSCs) play a critical role in maintaining the tumor immune microenvironment; thus, the promotion of MDSC polarization will improve immunotherapies for cancers. However, the mechanisms involved in controlling MDSC polarization in hepatocellular carcinoma remain largely unclear. In this study, we found that injection of Pam3CSK4 attenuated the process of tumor growth, along with reduction of MDSC and recovery of T cell function. Moreover, Pam3CSK4 promoted MDSC polarization by targeting Runx1. Runx1 inhibitor reversed the therapeutic effect of Pam3CSK4 by increasing tumor size and weight and decreasing the survival rate of tumor mice. In addition, targeting Runx1 reduced the expression of CD11c, F4/80, CD80/CD86 and MHC-II in MDSC after Pam3CSK4 stimulation in vivo and in vitro. MDSC also exhibited consistent changes with increasing reactive oxygen species (ROS) production after Pam3CSK4 and Ro5-3335 treatment. RNA sequence data revealed that tfrc, steap3, and gclm were up-regulated in the Pam3CSK4/Ro5-3335 group compared with Pam3CSK4 treatment alone, suggesting that the regulatory effect of TLR2 and Runx1 on MDSC might act through the ferroptosis pathway. Overall, our study has identified a critical role for TLR2 and Runx1 in regulating the differentiation and function of MDSCs and has provided a new mechanism of controlling MDSC polarization during HCC immunotherapy.

Abstract 1345: p300/CBP bromodomain inhibitor CCS1477 enhances the efficacy of immune checkpoint blockade therapy in cancer treatment

Abstract Background: Blockade of programmed death-ligand 1 (PD-L1) by therapeutic antibodies has shown to be a promising strategy in cancer therapy, yet clinical response in many types of cancer is limited. Tumor cells secrete PD-L1 through exosomes or splice variants, which has been described as a new mechanism for the resistance to PD-L1 blockade therapy in multiple cancers. Our previous findings showed that the histone acetyltransferases p300/CBP could be recruited to the promoter of CD274 (encoding PD-L1) to regulate the expression of PD-L1. P300/CBP inhibition abrogated this process and reduced the secretion of exosomal PD-L1 by blocking the transcription of CD274, which combined with the anti-PD-L1 antibody to reactivate T cells function for tumor attack. In this study we have tested the outcome of combined immune checkpoint inhibitors with CCS1477, a novel p300/CBP bromodomain inhibitor, in pre-clinical models of prostate cancer and melanoma. Results: CCS1477 significantly decreased human prostate (DU145/PC-3) and melanoma (A375/SK-MEL-28) cancer cell line PD-L1 expression, as well as exosome secreted PD-L1 after 24 hours treatment. Moreover, consistent with these findings in vitro, CCS1477 (2.5mg//kg) also reduced PD-L1 expression in a syngeneic mouse prostate model (TRAMP-C2) and syngeneic mouse melanoma model (B16-F10). Meanwhile, CCS1477 treatment significantly increased tumor-infiltrating CD8+ and CD4+ T cells in syngeneic mouse models. Monotherapy treatment with CCS1477 or immune checkpoint inhibitors (anti-PD-L1 antibody for prostate cancer or anti-PD-L1 and anti-CTLA4 antibody for melanoma) resulted in significant but modest efficacy. Strikingly, the combination of immune checkpoint inhibitors with CCS1477 showed robust synergistic responses resulting in tumor regression. Mechanistically, CCS1477 treatment induced significant loss of H3K27 acetylation from super-enhancers of CD274, which subsequently induced a significant reduction of PD-L1 expression and exosome secreted-PD-L1. Also, we found that CCS1477 treatment significantly decreased the abundance of circulating and tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which are known to play important roles in tumor immune evasion by inhibiting T cell’s activation. Reduction of MDSCs and exosome secreted-PD-L1 preserved T-cell function and elicited robust immunotherapy response combined with immune checkpoint blockade agents. Conclusions: These observations illuminate a clinically relevant hypothesis for combining CCS1477 with immune checkpoint blockade in the treatment of cancer. Citation Format: Jinghui Liu, Xinyi Wang, Katelyn Jones, Nigel Brooks, Neil Pegg, Xiaoqi Liu. p300/CBP bromodomain inhibitor CCS1477 enhances the efficacy of immune checkpoint blockade therapy in cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1345.

Abstract LB007: A novel approach to target tumor immune microenvironment and improve checkpoint immunotherapies

Abstract Background: Pancreatic cancer remains 3rd deadliest disease, with less than 7-10% survival rate. Little progress has been seen in patient’s outcome due to high desmoplasia and chemo-resistance. Immunotherapy has shown promising results in cancers, except pancreatic cancer due to their characteristic fibrotic tumor microenvironment. The therapies are unable to penetrate fibrotic tumor leading to insufficient availability of therapeutic drugs at the tumor site. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas, that makes it an excellent protein tumor target. This study is unique as it utilizes MUC13Ab for targeting the pancreatic tumor site and SPION nanoparticle system for delivering the stroma depleting drug (curcumin), which would help in improving immunotherapy response. Methods: The inhouse generated MUC13Ab have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS). Conjugation efficiency of the SPION-Anti-MUC13 particles was seen through cell uptake studies, by measuring fluorescence intensity, Prussian blue staining. Invasion assay and migration assay was carried out on KPC cells. We have used female C57BL/6J black mice, orthotopic mice model for investigating targeting efficacy of MUC13-SPION-CUR. Immune checkpoint therapy (PDL-1 and CTLA-4) was administrated along with MUC13-SPION-CUR and conjugated with fluorescent indocyanine green (ICG) dye for monitoring the tumor growth. Further, immunostimulatory effect of the nano formulation was done using flow cytometry. Results: Our results showed that MUC13Ab conjugated SPIONS can efficiently internalize the PDAC cells. SPION-MUC13 using Indocyanine dye (ICG) specifically reached to the tumor site in an orthotopic syngeneic mouse model of PDAC as indicated by ICG fluorescence. Additionally, the combination formulation inhibited the tumor growth and showed more survival rate with CTLA-4. The combined treatment with CTLA-4 increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 43% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 23.8% (FoxP3+CD25+CD45+CD3+) in KrasG12D; LSL-Trp53R172H syngeneic mouse model of PDAC. Similar results were observed in SP-CUR-M13+PDL-1 group, which showed reduction in MDSCs (by 26.6%) and Tregs (by 0.1%) as compared with PDL-1 alone. Conclusion: The formulation softens up the tumors for therapies that resulted in improved response to checkpoint immunotherapies in a pancreatic orthotopic mice model. Therefore, this study indicates high significance of MUC13-SPIONS-CUR for achieving pancreatic tumor specific delivery of drugs. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients. Citation Format: Poornima Shaji, Ana Martinez, Melida Flores Cantu, Anupam Dhasmana, Neeraj Chauhan, Fnu Shabnam, Vincent Diego, Meena Jaggi, Stephen Behrman, Murali M. Yallapu, Subhash C. Chauhan, Sheema Khan. A novel approach to target tumor immune microenvironment and improve checkpoint immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB007.

Abstract 1283: Single intra-tumoral injection of gaseous nitric oxide induces an adaptive immune response in a mouse CT-26 solid tumor model

Abstract Background: Metastases are responsible for a significant portion of the morbidity and mortality in cancer patients. In situ destruction of tumor mass has been reported to provide the immune system with an antigen source for the induction of antitumor immunity, which can then attack distant metastases. Nitric oxide (NO) acts as a signaling molecule in multiple disease states, including cancer. NO has been shown to activate innate and adaptive responses of the immune system against tumors. In a previous Beyond Air study, 89% of CT26 tumor bearing mice treated with 50,000 parts per million (ppm) gaseous NO (gNO), rejected a secondary tumor induction. Here, we assessed the effects of a single intra-tumoral injection of gNO at concentrations of 20,000-50,000 ppm on immune cell recruitment in the tumor, spleen, and blood. Methods: Mouse colon carcinoma (CT26) tumor bearing Balb/c mice underwent a single intra-tumoral treatment with 20,000 (n=3) or 50,000 (n=4) ppm gNO for 5 minutes. Mice treated with N2 (n=3), under the same conditions, served as a control. Tumors were removed 14 days post treatment and were histologically analyzed to test for immune cell penetration, inflammation, and necrosis. Immune cells from the spleen and blood were harvested 21 days post treatment and assessed via flow cytometry. Results: 1. Treatment with 50,000 ppm gNO resulted in higher levels of inflammation, lymphocytes and T-cell infiltration, and necrosis within the tumor compared to both N2 and 20,000 ppm NO, as viewed histologically. 2. Flow cytometry analysis of immune cells from the spleen revealed a 1.3-fold increase of both B cells and IFN-γ expressing T cells with 50,000 NO treatment, and a 1.3-fold increase of B cells and 1.1-fold increase of IFN-γ expressing T cells with 20,000 NO treatment, compared to N2. 3. A 29% reduction of myeloid-derived suppressor cells (MDSCs) was detected in spleens of mice treated with 50,000 ppm gNO, and 40% for 20,000 ppm, compared to mice treated with N2. Conclusion: Intra-tumoral injection of ultra-high doses of NO at 20,000 and 50,000 ppm gNO led to an increased recruitment and concentration of T and B cells. Additionally, 50,000 ppm NO led to an increase of lymphocyte infiltration to the tumor. Taken together, the data suggest that the increase in lymphocyte infiltration, B and T cell population, and the reduction of MDSCs, are indicative of an adaptive immune response that results in rejection of secondary tumors in gNO-treated mice. Citation Format: Hila Confino, Matan Goldshtein, Yakir Levi, Lidor Malka, Amir Avniel, Steve Lisi, Ido Wolf. Single intra-tumoral injection of gaseous nitric oxide induces an adaptive immune response in a mouse CT-26 solid tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1283.

Regional infusion of a class C TLR9 agonist enhances liver tumor microenvironment reprogramming and MDSC reduction to improve responsiveness to systemic checkpoint inhibition

Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.

Phase 2 trial of bavituximab with chemoradiation and adjuvant temozolomide in newly diagnosed glioblastoma.

2030 Background: Glioblastoma (GBM) and tumor endothelial cells express phosphatidylserine (PS), a highly immunosuppressive membrane phospholipid. PS receptors engage with immune cells, leading to expansion of myeloid-derived suppressor cells (MDSCs) which promote an immunosuppressive and pro-angiogenic tumor microenvironment. Bavituximab (BAV) – a chimeric monoclonal antibody – binds to β2-glycoprotein 1 (β2-GP1) to form a complex of β2-GP1 with PS, resulting in immune activation against tumor cells and anti-angiogenic effects. Pre-clinical data in GBM models suggest synergistic effects of PS blockade, radiation (RT), and temozolomide (TMZ). Here, we present results from a phase II trial (NCT03139916) of BAV, RT and TMZ in GBM patients. Methods: 33 adults with newly diagnosed IDH-wild-type GBM were enrolled and received 6 weeks of RT+TMZ, followed by 6 cycles of TMZ. BAV (3 mg/kg) was given weekly, starting at week 1 of RT+TMZ, for 18 weeks with the option to continue if tolerated. The primary endpoint was the proportion of patients alive at 12 months (OS-12). The null hypothesis would be rejected if OS-12 was ≥ 72%. As an exploratory endpoint, the immune profile in tumor tissue and peripheral blood mononuclear cells (PBMCs) was assessed using nanoString and multispectral immunofluorescence, with the goal to assess on-target effects of BAV in longer vs. shorter surviving patients (split based on median survival). Relative cerebral blood flow (rCBF) from dynamic susceptibility contrast MRI was also obtained. Results: 24 patients were alive at 12 months and OS-12 was 73% (95% CI 59-90%) so the study met its primary endpoint. Median OS was 15.4 months. As best response, 79% of patients had stable disease, 12% had a partial response and 9% had progressive disease. Eight grade 3 or 4 adverse events were seen (no grade 5 AEs). Ten pre-treatment and 7 post-treatment tissue samples were available. Analysis of RNA from pre-treatment tumor specimens showed a significantly positive shift in myeloid-related gene expression in patients with longer survival, with enrichment of 116 and 120 transcripts as well as downregulation of 2 and 1 gene for PFS and OS, respectively. There was no differential expression in PBMCs. Including all tissue samples, there was a marked reduction of MDSCs after BAV compared to time of diagnosis (p = 0.011). Decreased rCBF post-RT/pre-cycle 1 TMZ was associated with improved OS (HR 4.63, p = 0.029). Conclusions: OS-12 was 73%, meeting the primary endpoint and suggesting potential activity of BAV in newly diagnosed GBM. BAV leads to on-target depletion of intratumoral immunosuppressive MDSCs and anti-angiogenic effects. As expected, based on the mechanism of action of BAV, there was no difference in PBMC gene expression profile in patients with long and short survival. Combining BAV with immune checkpoint inhibitors in the future may augment tumor immune response. Clinical trial information: NCT03139916.

Radiation dose and schedule influence the abscopal effect in a bilateral murine CT26 tumor model

Radiotherapy (RT) can induce immune-mediated responses in local irradiated tumors, and non-irradiated distant metastasis is termed the abscopal effect. Here, we aimed to evaluate the impact of different RT doses and fractions on anti-tumor responses within local irradiated and distance non-irradiated tumor microenvironments. In mice bearing CT26 tumors, the primary tumor was irradiated with three different RT doses (16 Gy × 1F, 10 Gy × 2F, and 3 Gy × 10F) with the same biologically effective dose. Tumor volumes and immune cells changes were assessed in irradiated and non-irradiated tumors. Survival times were evaluated over 90 days. Only 16 Gy × 1F radiation increased CD8 + T cells number in the irradiated (p = 0.043) and non-irradiated (p = 0.047) tumors compared to the untreated group. A high frequency of tumor-associated macrophages-1 (TAM-1) and low TAM-2 was found in 16 Gy × 1F irradiated mice.Moreover, 16 Gy × 1F significantly induced interferon gamma (IFNγ)-producing CD8 + cells in the spleen compared to controls (p = 0.021). Hypofraction regimens (16 Gy × 1F, 10 Gy × 2F) caused a reduction in myeloid-derived suppressor cells in the irradiated tumors. We detected A modest growth delay in both flank tumors and long-term survival after hypofraction treatments (16 Gy × 1F, 10 Gy × 2F). A single high RT dose increased CD8 + cells number in irradiated (p = 0.000) and non-irradiated (p = 0.002) tumors approximal up to 2 points along with significant induction of IFN-γ production by CD8 + cells in the spleen when combined with anti- programmed death ligand-1 (PDL-1) (p = 0.000). Combination therapy was also associated with bilateral tumor growth control and increased life span in mice. Hypofractionated RT schedules, especially single high dose, seem the most effective regimen for inducing an abscopal effect. Immune checkpoint inhibitors could promote RT-induced systemic effects.

Bedside formulation of a personalized multi-neoantigen vaccine against mammary carcinoma

BackgroundHarnessing the immune system to purposely recognize and destroy tumors represents a significant breakthrough in clinical oncology. Non-synonymous mutations (neoantigenic peptides) were identified as powerful cancer targets. This knowledge can...

Intra-arterial infusion chemotherapy utilizing cisplatin inhibits bladder cancer by decreasing the fibrocytic myeloid-derived suppressor cells in an m6A-dependent manner

Intra-arterial infusion chemotherapy (IAIC), using immunomodulatory cisplatin, is a novel treatment for bladder cancer (BC) that allows the delivery of specific drugs to the local malignant lesion. To explore the immunomodulatory effect of cisplatin during IAIC, we detected the proportion of immunosuppressed cells in BC tissue from eight BC patients, with the reduction of myeloid-derived suppressor cells (MDSCs), more specifically fibrocytic-MDSCs (f-MDSCs). Further, we demonstrated that cisplatin inhibits their proliferation and immunosuppressive activity. f-MDSCs promote tumor proliferation and metastasis in the BC immune environment. Then, we analyzed the genetic differences detected in samples before and after chemotherapy and found that granulocyte colony-stimulating factors (G-CSF) decreased after IAIC. Furthermore, G-CSF methylation decreased following treatment with cisplatin. Specifically, treatment with cisplatin decreased the methylase (METTL3) levels in BC cells, which is important for G-CSF production. Collectively, cisplatin decreased the number of f-MDSCs during IAIC, by blocking G-CSF methylation via targeting METTL3.

Immunodepletion of MDSC By AMV564, a Novel Tetravalent Bispecific CD33/CD3 T Cell Engager Restores Immune Homeostasis in MDS in Vitro

Myelodysplastic syndromes (MDS) are age-dependent and genetically diverse hematopoietic neoplasms associated with inflammation, cytopenias, myeloid dysplasia and risk for acute myeloid leukemia (AML) progression. Mounting evidence indicates that emergence of somatic gene mutations arising in hematopoietic stem and progenitor cells (HSPC) with age plays an important role in MDS pathogenesis. Our investigations indicate that sustained activation of innate immune response directs inflammation in the bone marrow (BM) microenvironment that contributes to hematopoietic and immune impairment through local generation of inflammatory cytokines, expansion of regulatory T- cells, and up-regulation and activation of pattern recognition receptors. Critical cellular effectors are the inflammation-associated immature myeloid cells termed myeloid-derived suppressor cells (MDSCs). Human MDSCs, distinguished phenotypically by CD33high and HLA-DR−Lin−, expand and are activated in cancers. We previously reported that MDSCs are profoundly expanded in the local BM microenvironment of MDS where they contribute to MDS hematopoietic BM failure. MDSCs suppress T-cell responses and impair CD4+/CD8+ function, suggesting that this may be a key mechanism fostering clonal escape from anti-tumor immune response. Indeed, the MDSC population size limits the benefit of immune checkpoint antagonists in the treatment of solid tumors and effective strategies to deplete MDSCs are lacking. Therefore, we hypothesized the high CD33 surface density in MDSCs can be exploited for selective immune-depletion by the T-cell engager AMV564, a novel CD33/CD3 tetravalent bispecific antibody that recognizes both CD33 and CD3, currently in clinical development for AML (NCT03144245). Depletion of MDSCs may restore immune function, hematopoiesis and improve sensitivity to checkpoint inhibitors. Primary BM mononuclear cells (BMMNC) from 15 MDS patients were treated with AMV564 or isotype IgG control for 5-7 days in vitro . AMV564 treatment of MDS BMMNC eliminated CD33+ MDSCs in a dose-dependent manner and expanded CD4+ and CD8+ T-cells compared to controls (P ≤0.001). Proliferation of CD4+ and CD8+ T-cells, measured by Brdu incorporation, more than doubled with AMV564 treatment (P ≤0.001). IFNγ, a T-cell activation biomarker, dramatically increased in AMV564 treated cells indicating T-cells are fully activated by AMV564 at concentrations as low as 0.7 ng/ml (P ≤0.01). Moreover, depletion of MDSCs and immunological restoration was accompanied by significant improvement in colony-forming capacity after AMV564 treatment (P ≤0.01). Checkpoint inhibition alone with 10 µg/ml of anti-PD1 antibody did not result in reduction of MDSCs and T-cell activation in MDS BMMNCs, demonstrating that anti-PD1 alone is insufficient to appropriately activate T-cells or modulate MDSC cell number. Interestingly, combined treatment of MDS BMMNC with anti-PD1 and AMV564 yielded more than additive T-cell activation and increased colony forming capacity (P ≤0.01), indicating the potential to augment MDS-specific immune responses. Our findings demonstrate that AMV564 treatment selectively depletes MDSCs to reactivate T lymphocytes by improving both quantity and quality of immune responses leading to the improvement of hematopoiesis. These preclinical data provide a strong rationale for clinical investigation of this innovative approach in patients with MDS. DisclosuresGuenot:Amphivena Therapeutics, Inc.: Employment. Fox:Amphivena Therapeutics, Inc.: Consultancy.

PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma.

New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

Abstract LB-391: Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression

Abstract Triple-negative breast cancer (TNBC) affects only about 10-15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC is urgently needed. Insulin-like growth factor-2 (IGF2) is highly expressed in TNBCs in African-American and Latina patients. Since hormone receptors, like insulin-like growth factor-1/insulin (IGF1R/IR, are known to be enriched and to cross-communicate in subtypes of TNBC, we assessed effects of IGF1R/IR antagonists on proliferation of human and murine TNBC cells in vitro. Combination treatment of BMS-754807 (IGF1R/IR) with NVP-AEW541 (IGF1R inhibitor) reduced proliferation of human MDA-MB-231, BT549, HCC1937 and murine 4T1 TNBC cells in vitro (P<0.001). Moreover, combination treatment using BMS-754807 and NVP-AEW541 significantly reduced MDA-MB-231 tumor xenograft progression in vivo (P<0.001). IHC study of tumor tissues from in vivo experiments confirmed reduced Ki-67 expression with combination treatments. Among breast cancer subtypes, TNBC is characterized by significant numbers of tumor infiltrating lymphocytes (TILs), a predictive marker for response to immunotherapy. Further, recent reports from clinical trials using immune checkpoint inhibitors demonstrate that a subset of TNBC patients respond to treatment with immune checkpoint inhibitors (ICIs). It is also notable that increased expression of IGF1Rs are identified on immune cells responsible for mounting adaptive and humoral immune responses. Using murine 4T1 TNBC implants in syngeneic, immune-competent BALB/c mice, we find that combination treatment with BMS-754807 and anti-PD-L1 antibody significantly blocks tumor progression as compared to both controls and with either agent used alone (P<0.0001). In addition, single cell analysis of the tumor microenvironment after treatment with BMS-754807 alone or combined with nti-PD-L1 antibody combination therapy shows a significant reduction in myeloid-derived suppressor cells (MDSCs). Moreover, BMS-754807 combined with anti-PD-L1 antibody increased infiltrating effector T cells and significantly enhanced the T central memory population (Tcm) (P<0.01). Functionally, it is reported that Tcm subsets of immune cells produce effector cytokines in response to antigens and can readily proliferate and differentiate into effector cells. Use of immunotherapy is a promising management option for a subset of TNBC patients, and combination treatments using IGF1R antagonists with immune checkpoint inhibitor may constitute a new treatment strategy to combat this deadly disease. [Funding by NCI U54 CA1433930; California Breast Cancer Research Program; UCLA Jonsson Citation Format: Nalo M. Hamilton, Diana C. Marquez-Garban, Lorena P. Burton, Begonya Comin-Anduix, Alejandro J. Garcia, Jaydutt V. Vadgama. Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-391.

Evaluation of antitumor immunity by a combination treatment of high-dose irradiation, anti-PDL1, and anti-angiogenic therapy in murine lung tumors.

C57BL/6 mice implanted in the flank with murine Lewis lung carcinoma cells were randomized into control, anti-angiogenic, anti-PD-L1, radiotherapy (RT), RT + anti-angiogenic, RT + anti-PD-L1, and RT + anti-PD-L1 + anti-angiogenic therapy groups. Immune response and immunophenotyping were determined by flow cytometry. Vasculature analysis after RT and anti-angiogenic therapy was assessed by quantified power Doppler sonography. Antitumor response, survival, and rechallenged tumor growth were evaluated. RT increased PD-L1 expression on CD8+ T, CD4+ T, dendritic, myeloid-derived suppressor cells (MDSCs), and tumor cells and increased PD-1 expression on CD8+ and CD4+ T cells. Anti-angiogenic therapy insignificantly decreased the RT-induced PD-1 expression on CD8+ and CD4+ T cells, implying a weak reversal of the immune-suppressive environment. Transient vessel collapse was observed within days after RT, and blood flow recovered at 1week after RT. RT + anti-PD-L1 suppressed the tumor growth, improved survival, and prolonged immune memory capable of protecting against tumor recurrence, evidenced by local accumulation of CD8+ T cells and reduction in MDSCs in microenvironment. Similar and more prominent effects were observed when anti-VEGF was added to RT + anti-PDL1 therapies, implying an additive, rather than synergistic, antitumor immunity. Phenotypic analyses revealed that anti-cancer treatments increased the proportion of effector memory T cells in TILs and splenocytes, and RT, alone or in combination with other treatments, further increased the proportion of central memory T cells in splenocytes. These results provide evidence on operating the immunosuppressive tumor environment and offer insights into the design of the new combination treatment.

Hypofractionated Irradiation Suppressed the Off-Target Mouse Hepatocarcinoma Growth by Inhibiting Myeloid-Derived Suppressor Cell-Mediated Immune Suppression.

Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear.Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry.Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an “in situ tumor vaccine” and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells.Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

A35 Dendritic Cell in Situ Vaccination Potentiates Anti-PD-1 Efficacy and Induces Immunoediting in a Murine Model of NSCLC

Studies reveal that responses to checkpoint blockade in non-small cell lung cancer (NSCLC) are associated with high tumor mutational burden (TMB), preexisting CD8+ T-cell infiltration, and high baseline PD-L1 expression within the tumor microenvironment (TME). In contrast, co-occurring KRAS/LKB1 mutation is associated with primary resistance to PD-1 blockade and decreased overall survival. In preclinical studies as well as a phase I clinical trial, we have discovered that intratumoral (IT) vaccination with gene-modified dendritic cells expressing CCL21 (CCL21-DC) promotes tumor effector T-lymphocyte infiltration, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could restore tumor antigen presentation and promote T-cell priming and activation, thereby sensitizing nonresponsive NSCLC tumors to checkpoint blockade. Although genetically engineered murine models (GEMMs) of NSCLC bear driver mutations of the disease, recent studies reveal that these GEMMs possess low mutational burden. We established novel GEMMs of NSCLC [KrasG12D (K), KrasG12DP53-/- (KP), KrasG12DP53+/-Lkb1-/- (KPL)] bearing common driver mutations and varying mutational loads by in vitro exposure of tumor cell lines to the carcinogen N-methyl-N-nitrosourea (MNU). Our preclinical KPL model with high TMB recapitulates the immunologic phenotype of human disease, and contains a predominance of myeloid-derived suppressor cells (MDSC), low tumor-infiltrating lymphocytes (TILs), and low PD-L1 expression within the TME. As anticipated, the KPL tumors are resistant to anti-PD-1 therapy, even with increased mutational load. We evaluated IT CCL21-DC combined with anti-PD-1 therapy in immunocompetent mice bearing KPL tumors with high TMB, and observed that IT CCL21-DC vaccination induces infiltration of autologous T lymphocytes and conventional type I DCs (cDC1s) into the TME and sensitizes the tumors to anti-PD-1 therapy. Combination therapy also reprogrammed the myeloid compartment, resulting in a significant reduction of MDSCs and a concurrent increase in CD11b+Ly6GhiLy6Clo monocyte/myeloid population. Whole-exome sequencing (WES) of tumors revealed immunoediting and selective depletion of tumor subclones post IT CCL21-DC and anti-PD1 combination therapy. Future studies will evaluate the evolution of the T-cell receptor (TCR) repertoire in response to the combination treatment and define functional responses to neoepitopes. These studies will enhance our understanding of the molecular mechanisms of tumor vaccination and facilitate the development of rational combination strategies.

Exercise reduces immune suppression and breast cancer progression in a preclinical model.

Exercise is associated with favorable changes in circulating immune cells and improved survival in early-stage breast cancer patients, but the mechansims remain to be fully elucidated. Preclinical studies indicate that physical activity started before tumor injection reduces tumor incidence and progression. Here we tested whether exercise has anti-tumor effects in mice with established 4T1 mammary carcinoma, a mouse model of triple negative breast cancer. Exercise slowed tumor progression and reduced the tumor-induced accumulation of myeloid-derived suppressor cells (MDSCs). The reduction in MDSCs was accompanied by a relative increase in natural killer and CD8 T cell activation, suggesting that exercise restores a favorable immune environment. Consistently, exercise improved responses to a combination of programmed cell death protein 1 (PD-1) blockade and focal radiotherapy. These data support further investigations of exercise in breast cancer patients treated with combinations of immunotherapy and cytotoxic agents to improve cancer outcomes.

CD19-targeting fusion protein combined with PD1 antibody enhances anti-tumor immunity in mouse models

ABSTRACT In our previous studies, using a B cell vaccine (scFv-Her2), the targeting of tumor-associated antigen Her2 (human epidermal growth factor receptor-2) to B cells via the anti-CD19 single chain variable fragment (scFv) was shown to augment tumor-specific immunity, which enhanced tumor control in the prophylactic and therapeutic setting. However, the fusion protein displayed limited activity against established tumors, and local relapses often occurred following scFv-Her2 treatment, indicating that scFv-Her2-induced responses are inadequate to maintain anti-tumor immunity. In this study, targeting the IV region (D4) of the extracellular region of Her2 to B cells via CD19 molecules (scFv-Her2D4) was found to enhance IFN-γ-producing-CD8+ T cell infiltration in tumor tissues and reduced the number of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). However, negative co-stimulatory molecules such as programmed cell death protein-1 (PD-1), CD160, and LAG-3 on T cells and programmed death protein ligand-1 (PD-L1) on tumor cells were upregulated in the tumor microenvironment after scFv-Her2D4 treatment. Further, anti-PD1 administration enhanced the efficacy of scFv-Her2D4 and anti-tumor immunity, as evidenced by the reversal of tumor-infiltrating CD8+ T cell exhaustion and the reduction of MDSCs and Treg cells, which suppress T cells and alter the tumor immune microenvironment. Moreover, combining this with anti-PD1 antibodies promoted complete tumor rejection. Our data provide evidence of a close interaction among tumor vaccines, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and tumor vaccine therapy.

Sunitinib Treatment-elicited Distinct Tumor Microenvironment Dramatically Compensated the Reduction of Myeloid-derived Suppressor Cells.

The clinical response rate of prostate cancer to tyrosine kinase inhibitor (TKI) monotherapy is low. The mechanisms of resistance to TKI are unclear. This study aimed to examine if the tumor microenvironment (TME) is involved in the resistance. The anti-vascular effect of Sutent was examined by immunofluorescent staining in TRAMP-C1 tumor. The percentage of CD11b+ population were analyzed by flow cytometry. The level of cytokines and chemokines were measured by multiplex immunoassay. The Sutent monotherapy caused 1.5 days of tumor growth delay, chronic hypoxia, and more mature vasculature. Sutent monotherapy increased the percentage of polymorphonuclear myeloid-derived suppressor cells (MDSCs) in peripheral blood. The evolved TME triggered the re-distribution of myeloid cells in chronically hypoxic areas. The multiplex immunoassay indicated higher levels of several cytokines and chemokines both in tumors and the blood. Sunitinib treatment induced a distinct tumor microenvironment that impaired the efficient reduction of MDSCs by TKI.

Co-delivery of Bee Venom Melittin and a Photosensitizer with an Organic-Inorganic Hybrid Nanocarrier for Photodynamic Therapy and Immunotherapy.

Photodynamic therapy (PDT) is a clinical cancer treatment modality based on the induction of therapeutic reactive oxygen species (ROS), which can trigger immunogenic cell death (ICD). With the aim of simultaneously improving both PDT-mediated intracellular ROS production and ICD levels, we designed a serum albumin (SA)-coated boehmite ("B"; aluminum hydroxide oxide) organic-inorganic scaffold that could be loaded with chlorin e6 (Ce6), a photosensitizer, and a honey bee venom melittin (MLT) peptide, denoted Ce6/MLT@SAB. Ce6/MLT@SAB was anchored by a boehmite nanorod structure and exhibited particle size of approximately 180 nm. Ce6/MLT@SAB could significantly reduce hemolysis relative to that of free MLT, while providing MLT-enhanced PDT antitumor effects in vitro. Compared with Ce6@SAB, Ce6/MLT@SAB improved Ce6 penetration of cancer cells both in vitro and in vivo, thereby providing enhanced intracellular ROS generation with 660 nm light treatment. Following phototreatment, Ce6/MLT@SAB-treated cells displayed significantly improved levels of ICD and abilities to activate dendritic cells. In the absence of laser irradiation, multidose injection of Ce6/MLT@SAB could delay the growth of subcutaneous murine tumors by more than 60%, compared to controls. When combined with laser irradiation, a single injection and phototreatment with Ce6/MLT@SAB eradicated one-third of subcutaneous tumors in treated mice. The addition of an immune checkpoint blockade to Ce6/MLT@SAB phototreatment further augmented antitumor effects, generating increased numbers of CD4+ and CD8+ T cells in tumors with concomitant reduction of myeloid-derived suppressor cells.

Repurposing of the anti-helminthic drug niclosamide to treat melanoma and pulmonary metastasis via the STAT3 signaling pathway

The incidence of melanoma is increasing rapidly worldwide. Additionally, new and effective candidates for treating melanoma are needed because of the increase in drug resistance and the high metastatic potential of this cancer. The STAT3 signaling pathway plays a pivotal role in pathogenesis of melanoma, making STAT3 a promising anticancer target for melanoma therapy. Niclosamide, an FDA-approved anti-helminthic drug, has been identified as a potent STAT3 inhibitor that suppresses STAT3 phosphorylation at Tyr705 and its transcript activity. In this study, we evaluated the biological activities of niclosamide in melanoma in vitro and in vivo. Niclosamide potently inhibited the growth of four melanoma cell lines and induced the apoptosis of melanoma cells via the mitochondrial apoptotic pathway. Further, western blot analysis indicated that cell apoptosis was correlated with activation of Bax and cleaved caspase-3 and decreased expression of Bcl-2. Moreover, niclosamide markedly impaired melanoma cell migration and invasion, reduced phosphorylated STAT3Tyr705 levels, and inhibited matrix metalloproteinase-2 and −9 expression. Additionally, in a xenograft model of A375, intraperitoneal administration of niclosamide inhibited tumor growth and tumor weight in a dose-dependent manner without obvious side effects. Histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells and p-STAT3Try705-positive cells and increase in cleaved caspase-3-positive cells. Notably, niclosamide significantly inhibited pulmonary metastasis in a B16-F10 melanoma lung metastasis model, including the number of lung metastatic nodules and lung/body coefficient. Importantly, a marked reduction in myeloid-derived suppressor cells (Gr1+CD11b+) infiltration in the pulmonary metastasis tissue was observed. Taken together, these results demonstrate that niclosamide is a promising candidate for treating melanoma.