Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to its resistance to effective therapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma including cancer associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Gemcitabine (gem) and nab-paclitaxel (nab-p) is standard of care for PDAC with modest efficacy. Immunotherapies, effective in other cancers, are often combined with chemotherapy in an attempt to augment tumor response. We investigated the impact of gem and nab-p on the TME and antigen presentation of PDAC. Methods: A cancer organoid line from KPC (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) mice, was used as a murine model of PDAC. The organoids were treated in vitro with 50uM gem and 5.85uM nab-p for 12-24 hrs and subsequently tested for MHC class I expression via flow cytometry. A co-culture in Matrigel was created using KPC organoids, pro-tumor M2-polarized bone-marrow derived macrophages (BMDMs), and pancreatic stellate cells (PSCs). After 48 hrs the co-culture was treated with gem and nab-p for 24 hrs and collected to assess macrophage and fibroblast polarization markers via flow cytometry. KPC organoids were used to generate allografts in wild-type B6 mice randomized to receive 100 mg/kg gem (i.p.) and 30 mg/kg nab-p (i.v.) or control. After 96 hrs, tumors were processed for flow cytometry analyses. Results: The percentage of MHC I expressing KPC cells increased from baseline following 12 hrs of gem/nab-p treatment (32.7% vs 50.4%, p= 0.12 and declined at 24, 48 and 72 hour post-treatment time points (22.5%, p=0.3, 23.2%, p=0.31, and 13.1%, p=0.06, respectively). In the co-culture, gem and gem/nab-p significantly enhanced immunoregulatory inflammatory CAF (iCAF) subtype over control (17.5 vs 17.6 vs 10%, respectively, p<0.001). A decrease in myofibroblastic CAFs (myCAFs) was also observed. M1 macrophages in the co-culture decreased significantly with gem and gem/nab-p compared to controls (6.7% vs 6.9% vs 13.8%, respectively, p=0.02/p=0.004). There was also a subsequent increase in M2 macrophages. KPC allografts demonstrated no significant change in MHC I levels following a 96-hour gem/nab-p treatment compared to vehicle. The CAF subtypes showed trends similar to the in vitro co-culture, with more iCAFs (14.9% vs 26.3%, p=0.32) and less myCAFs (83.5% vs 71.3%, p=0.31). We were also able to identify an MHC II class of antigen-presenting CAFs (apCAFs), within both our in vitro and in vivo studies at levels less than 1% of all CAFs. However, no change was observed with treatment. Conclusion: Here we demonstrate reduced MHC class I expression, increased M2 TAM polarization, and increased iCAFs, all associated with an immunoregulatory phenotype. These finding suggest that further studies on alternative combinations of immunotherapies are warranted. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Nathaniel Verhagen, Rebecca Destefanis, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Gemcitabine and nab-paclitaxel increase immunosuppressive environment in PDAC [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 5102.
Background: Endometrial cancer exhibits differential immunogenicity across molecular subtypes. Specifically, mismatch repair (MMR) deficiency in a subset of endometrial cancers increases mutational load, potentially leading to improved detection of tumor neoantigens within this context. The surrounding tumor microenvironment also plays a role in modulating the immune response to tumor neoantigens. The extracellular matrix protein versican (VCAN) has been characterized as an immunosuppressive molecule that is overexpressed in multiple cancer types, while its cleavage product, versikine (Vkine), has immunostimulatory properties. The objective of this study is to examine the relationship between VCAN proteolysis and CD8+ T cell tumor infiltration in endometrial cancer. Methods: An endometrial cancer tissue microarray (TMA) was developed containing tumor cores from 258 patients. TMA slides were stained via immunohistochemistry. VCAN and Vkine stains were scored on a scale of 0 to 3 based on intensity of stromal staining. Tumor-infiltrating lymphocytes (TILs) was quantified as the number of CD8+ T cells touching malignant epithelial cells 400X magnification. MMR proteins were scored as absent or present in each sample by pathology. Samples were classified into three groups based on strength of proteolysis: high proteolysis = VCAN 0 or 1 and Vkine 3, low proteolysis = VCAN 3 and Vkine 0 or 1. All other samples were put into the intermediate proteolysis group. Results: Proteolysis of VCAN correlates with increased CD8+ TILs in endometrial cancer. The CD8 mean in the proteolytic high group was 4-fold higher than that of the proteolytic low group (16.8 vs 3.6; Wilcoxon rank sum test, p=0.059; n=55 and n=8, respectively). High VCAN proteolysis, as well as mismatch repair deficiency, correlate with high CD8+ T cell infiltration of endometrial tumors; 78% of the MMR deficient samples, and 62% of the proteolysis high samples, were above the median CD8 count of 5.3. Furthermore, our results suggest that endometrial cancer recurrence is associated with increased VCAN expression in both type I endometrioid (estrogen-dependent) and type II non-endometrioid (estrogen-independent) cancers. Of the type I endometrioid cancers, 11% of those expressing VCAN recurred whereas 0% of non-VCAN expressing tumors recurred (n=92). Similarly, of the type II non-endometrioid cancers, 47% of VCAN expressing tumors recurred, whereas 20% of the non-VCAN expressing tumors recurred (n=80). Conclusions: VCAN and its proteolysis correlated with CD8+ TILs in endometrial cancer. These data indicate potential for VCAN as both a prognostic and an immune biomarker. Citation Format: Kristen Moriah Bischel, Philip Emmerich, Tonela Qyli, Stephanie McGregor, Mitchell Depke, Nathaniel Verhagen, Dustin Deming, Philip Emmerich, Nathaniel Verhagen, Tonela Qyli, Stephanie McGregor, Mitchell Depke. Versican proteolysis in endometrial cancer [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 403.
Background: Esophageal cancer is a highly lethal cancer with a 20% 5-year overall survival despite standard of care treatment.. Pembrolizumab (anti-PD1, Merck) is FDA-approved for PD-L1 positive esophageal cancer, which comprises roughly 40% of all patients. PD-L1 positive (+) esophageal cancers have previously been shown to demonstrate elevated CD8+ T cell infiltration (CD8+ TILs). Versican (VCAN) is an immunosuppressive extracellular matrix proteoglycan which produces an immunostimulatory fragment, versikine (Vkine), upon proteolytic cleavage by ADAMTS proteases. Previously, VCAN proteolysis has been shown to correlate with enhanced CD8+ TILs in colorectal cancer (CRC), indicating potential for use as an immune biomarker. Methods: A human esophageal cancer tissue microarray (TMA) was developed consisting of multiple samples across 106 representative esophageal cancer patients. Immunohistochemistry (IHC) was used to evaluate VCAN and Vkine expression, and a binning system was used to quantify these stains based on stromal intensity (0: 0-10%, 1: 10-25%, 2:25-50%, 3: 50-100%). IHC was also conducted for CD8, and the number of CD8+ tumor-infiltrating cells per 400x field of view was counted. PD-L1 IHC 22C3 pharmDx was performed and quantified per CPS guidelines for GEJ cancers to quantify PD-L1 status. The VCAN proteolytic predominant (VPP) phenotype was defined as both low VCAN (0 or 1) and high Vkine (2 or 3), and VCAN proteolytic weak phenotype (VPW) was defined as either high VCAN (2 or 3) or low Vkine (0 or 1). Results: Overall, 42.7% of all samples were scored as PD-L1+ (CPS≥1). PD-L1+ samples demonstrated greater CD8+ TILs than PD-L1- samples (44.5±71 vs 14±22; p<0.001). The presence of immunosuppressive VCAN negatively correlated with CD8+ TILs (16.8±27 in VCAN+ samples vs 39.4±67 in VCAN- samples; p<0.01). The VPP phenotype was observed in 47.8% of the cancer samples. VPP samples also demonstrated a trend towards greater CD8+ TILs compared to VPW samples (35.9±63 vs 22.7±46; p=0.09); however, this was not statistically significant. Of all samples, 33.9% were both PD-L1+ and VCAN-. These dual PD-L1+ and VCAN- samples demonstrated even greater CD8+ TILs compared to either PD-L1- or VCAN+ samples (49.5±79 vs 18.1±29; p<0.001). Discussion: Our TMA demonstrates enhanced CD8+ TILs in PD-L1+ samples, as previously reported. Additionally, we discovered the presence of VCAN and its proteolysis are independent predictors of enhanced CD8+ TILs in esophageal cancers. These data indicate the potential importance of the tumor matrix in establishing a permissive tumor microenvironment. These data support the potential for combining VCAN testing with PD-L1 testing to further select appropriate patients for immunotherapeutic trials in esophageal cancer. Citation Format: Phillip B. Emmerich, Jacob Witt, Darya Buehler, Mitchell Depke, Nathaniel Verhagen, Linda Clipson, Kristina Matkowskyj, Nataliya Uboha, Andrew Baschnagel, Dustin Deming. PD-L1 status and versican proteolysis predict CD8+ T cell infiltration in esophageal cancer [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 3294.
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