Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response. Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue. Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p<0.001, n=20). VCAN accumulation was increased even in the earliest stages of acinar-to-ductal metaplasia in KPC mice. Areas of intense stromal VCAN staining trended toward reduced CD8+ T cell infiltration (4.9 vs 7.3 cells/high power field (hpf), p=0.3). IHC analysis of human PDAC revealed elevated VCAN accumulation across all stages compared to the normal adjacent tissue (n=231, p<0.001). Areas of high VCAN accumulation demonstrated reduced CD8+ T cells compared to areas of low VCAN (0.6 vs 2.9 cells/hpf, p<0.001). To investigate cell types responsible for enhanced VCAN accumulation in the tumor microenvironment, relative expression (RE) of VCAN was compared in vitro to M0 macrophages. Organotypic cancer spheroids demonstrated increased expression of VCAN from KPC mice (RE=49, n=2) and patient-derived PDAC tissue (RE=14, p=0.01, n=3). M1-polarized BMDMs had increased expression of VCAN (RE=24) compared to M2 BMDMs (RE=8) (p<0.001, n=3). Interestingly, M1 BMDMs cultured in PDAC-conditioned media had reduced RE of M1 markers: TNFα (395 vs 37, p=0.03) and iNOS (24723 vs 4813, p=0.003), and increased M2 markers: Arg-1 (127 versus 1049, p=0.02) and YM-1 (0.5 vs 3, p=0.02). PDAC-conditioned media also reduced VCAN expression of M1 BMDMs (24 vs 9, p=0.02). PSCs derived from human PDAC also demonstrated enhanced RE of VCAN compared to negative controls (RE=68, p=0.017) with no significant change in the presence of PDAC conditioned media (p=0.4). Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Chelsie K. Sievers, Connor J. Maloney, Rosabella T. Pitera, Susan N. Payne, Mitchell G. Depke, Cheri A. Pasch, Linda Clipson, Jillian K. Johnson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican production is driven by both epithelial and stromal cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1904.

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Background: Clinical responses to checkpoint inhibitors in gastrointestinal cancers have largely been limited to MSI-high disease. Proteolysis of an immunosuppressive proteoglycan versican (VCAN) by ADAMTS proteases correlates with enhanced CD8+ T cell infiltration in colorectal cancer independent of mismatch repair status. Here we investigate the potential of this biomarker in pancreatic ductal adenocarcinoma (PDAC) as VCAN is abundant VCAN in this disease. Methods: KPC mice were used to generate mouse PDAC tissue and organotypic spheroids. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissues across 131 patients. IHC was used to determine levels of VCAN, an immunostimulatory fragment versikine (Vkine), and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were derived from BALB/c mice. VCAN-specific ADAMTS protease expression profiles were compared between antitumor (M1) and protumor (M2) mouse macrophages, human pancreatic stellate cells (PSCs) and patient-derived PDAC-organotypic spheroids. Results: IHC analysis of normal murine pancreas demonstrated low VCAN and Vkine staining. VCAN is abundant in metaplastic/neoplastic KPC mouse pancreatic tissues. Rare VCAN proteolytic predominant areas (samples with both low VCAN and high Vkine) in KPC tumors had increased tumor-infiltrating CD8+ T cells/ high-powered field compared to weak areas (17 vs 4, p<0.001). Human PDACs had abundant VCAN expression compared to normal pancreatic tissues (p<0.001, n=118). Vkine was absent from normal tissues and present only at low levels in tumor cores (p<0.001, n=117). No human PDAC VCAN proteolytic-predominant samples were identified. M1 BMDMs demonstrated high relative expression (RE) of VCAN-specific ADAMTS proteases relative to unpolarized M0 BMDMs; however, these levels decreased under KPC-conditioned media (ADAMTS4, 101 vs 5, p=0.003; ADAMTS9, 80 vs 1, p=0.02; ADAMTS15, 103 versus 1.6, p<0.001; ADAMTS20, 881 vs 3.2, p<0.001). Similarly, M2 BMDMs had elevated ADAMTS-levels that were reduced under KPC-conditioned media, (ADAMTS4, 42 vs 0.4, p=0.0029; ADAMTS9, 40 vs 1, p=0.002; ADAMTS15, 23 vs 3, p=0.027; ADAMTS20, 310 vs 0.8, p<0.001; n=3). RE levels of ADAMTS proteases in KPC spheroids was consistently lower than reported above in macrophages. RE of ADAMTS proteases in human PSCs were between 18 and 304. No differences in ADAMTS expression were identified under PDAC-conditioned media in the human PSCs (p=0.3). Conclusions: The VCAN proteolysis predominant phenotype correlates with increased CD8+ T cell infiltration in PDAC though is rarely present in the human disease. PDAC epithelial cells influence the stromal cells to reduce VCAN proteolysis through decreased ADAMTS expression by macrophages. Future studies examining measures to enhance VCAN proteolysis could result in a more immune permissive microenvironment. Citation Format: Philip B. Emmerich, Chelsie Sievers, Hanna Rainiero, Rosabella Pitera, Connor Maloney, Susan Payne, Mitchell Depke, Cheri Pasch, Linda Clipson, Jillian K. Johnson, Kristina Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1909.

Background: Immune therapies have shown great promise for some cancers. To date, pancreatic ductal adenocarcinomas (PDAC) have been largely resistant to immunotherapeutic approaches in part due to their exclusion of tumor infiltrating lymphocytes. The mechanisms by which this exclusion occurs are not well-characterized but are thought to involve both innate and adaptive immune responses. Methods: The primary objective of this study was to investigate how dual anti-PD-1/anti-CSF1R therapy changes the tumor immune cell niche and tumor growth in an allograft version of the KPC mouse model of PDAC (Pdx-1-Cre; LSL-KrasG12D; LSL-Trp53 R172H). Anti-PD1 therapy in the form of monoclonal antibody from BioXCell® was dosed twice weekly via intraperitoneal injections at 200µg/mouse. Anti-CSF1R in the form of the small-molecule inhibitor BLZ945 from Selleckchem® was dosed daily via oral gavage at 200 mg/kg. Human THP-1 cells differentiated and polarized to macrophages were used for viability and gene expression analyses. Results: In B6 recipient mice, the combination anti-PD-1 plus BLZ945 resulted in reduced percent tumor volume change (11% ±17) versus vehicle treated controls (121% ±174) (p=0.02). Flow cytometry of the spleens of tumor-bearing mice demonstrated an increase in conventional dendritic cells with combination treatment compared to controls (15.3 ±2.3% vs 8.3 ±0.6%, respectively; p=0.02). Mice lacking conventional dendritic cells (B6.BATF3-/-) had an increased percent tumor volume change (84% ±78) compared to B6 wild type mice (11% ±16) (p=0.01) when receiving combination treatment. IHC staining of allografted tumors revealed a trend towards increased infiltrating CD8+ T cells/hpf in B6 wild type mice treated with anti-PD-1 or combination therapy (21±24/hpf) compared to control treated mice (11±18/hpf). A similar trend was observed in B6.BATF3-/- mice (2.9±4.4/hpf vs 0.5±0.50/hpf); however, the magnitude was greatly reduced compared to B6 wild type mice. Interestingly, BLZ945 treated tumors did not show evidence of statistically significant reduced macrophage recruitment in vivo (268 vs 241, p=0.16). Macrophage viability in vitro was also unaffected by BLZ945 treatment (fold change in viability 0.03, p=0.6). Preferential changes in macrophage polarization in vitro were observed, with significant reductions in M2 polarization markers Arg-1, YM-1, and CD206 (p= 0.0005, 0.0029, and 0.012, respectively). Conclusions: The combination of an anti-PD-1 agent in combination with inhibition of the macrophage-predominant cytokine, anti-CSF1R, slows PDAC tumor growth in a conventional dendritic cell-dependent manner. Further characterization of the interplay between innate and adaptive immune cells upon immunotherapy treatment should be investigated to increase the efficacy of these therapeutics. Citation Format: Chelsie K. Sievers, Philip B. Emmerich, Hanna R. Rainiero, Connor Maloney, Rosabella Pitera, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. BLZ945 and anti-PD-1 combination immunotherapy modulates the immune landscape in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3209.

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Background: Infiltration of CD8+ T cells has shown important prognostic implications in colorectal cancers (CRC). Our group has recently correlated proteolysis of an extracellular matrix proteoglycan, versican (VCAN), with infiltration of CRCs by CD8+ T cells. Cleavage by ADAMTS proteases generates a bioactive fragment of versican, versikine (VKINE). VKINE stimulates Batf3-driven dendritic cell activation and maturation, which are critical for immunotherapy efficacy. Methods: CT26 murine CRC cells were transfected to constitutively express VKINE. BALB/c mice between 50 and 70 days of age were orthotopically injected with either 105 empty vector (CT26-EV) or VKINE-expressing (CT26-VKINE) cells. Tumor growth was tracked weekly with colonoscopy and growth rate assessed using percent lumen occlusion. Mice were euthanized and dissected once moribund. Tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemical (IHC) staining was performed using standard procedures. Additionally, FFPE samples from human CRC liver metastases (n=9) were obtained under the University of Wisconsin Translational Science Biocore Institutional Review Board-approved protocol and IHC performed for VCAN, VKINE and CD8. The number of CD8+ tumor-infiltrating lymphocytes (TILs) per high-powered field (HPF) within the malignant epithelium was calculated using four separate 400x magnification fields of view. Results: CT26-EV and CT26-VKINE injected mice both had a ~70% tumor initiation rate and these tumors averaged ~50% lumen occlusion by 14 days post injection. Survival data for each group were compared using a log-rank test. A trend towards improved survival was observed in those mice injected with CT26-VKINE (average survival of 27 days for CT26-VKINE compared to 22 days for CT26-EV, p=0.41, n=13). The average number of CD8+ TILs for each tumor were compared. For mice with CT26-VKINE, the average number of CD8+ TILs per HPF was 7.6, compared to 4.7 for mice with CT26-EV (p=0.39). Human CRC liver metastases were analyzed for VCAN, VKINE, and CD8+ T cells. Several lesions had little to no VCAN staining, whereas others had intense pockets or peripheral staining. VKINE staining was more abundant in metastatic CRC lesions than in primary lesions. CD8+ T cells were excluded in the setting of increased VCAN staining and infiltration was noted specifically in areas of VCAN proteolysis (low VCAN and high VKINE staining). These areas of proteolysis were more often present at the tumor margin and correlated with ADAMTS1 expression. Conclusions: Immunotherapies offer a unique treatment option for cancer patients, but their use is limited in CRC. VCAN proteolysis and the generation of bioactive VKINE has potential to influence the ability of CD8+ T cells to infiltrate the tumor in both primary and metastatic CRC, indicating the potential to utilize VCAN and VKINE as immune biomarkers or therapeutic targets. Citation Format: Philip B. Emmerich, Susan N. Payne, Connor J. Maloney, Rosabella T. Pitera, Hanna Rainiero, Gioia Sha, Athanasios T. Papadas, Adam C. Pagenkopf, Michael F. Bassetti, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3146.

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