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Supplementary Table S1 shows patient information, Supplementary Table S2 shows cell numbers analyzed from each human PDAC sample, Supplementary Table S3 shows statistics of the clustering analysis of the human PDAC single cell RNA sequencing, Supplementary Table S5 shows statistics of the human PDAC ductal cell analysis, Supplementary Table S7 shows statistics of the human PDAC myeloid cell analysis, Supplementary Table S9 shows statistics of the human PDAC T & NK cell analysis, Supplementary Table S11 shows cell numbers analyzed for fibroblast subtypes from each human PDAC sample, Supplementary Table S12 shows statistics of the human PDAC fibroblast analysis, Supplementary Table S17 shows statistics of all viable KPC mouse cell analysis, Supplementary Table S19 shows statistics of the KPC mouse fibroblast-enriched fraction analysis, Supplementary Table S21 shows statistics of the fibroblasts from KPC mouse fibroblast-enriched population

Supplementary Table S16 shows protein activity of transcription factors in human CAFs, as analyzed by VIPER

PDF file - 99K, Protein expression of DR5, FasL, and CD24 compared between naive low avidity T cells, low avidity T cell activated in vivo, and low avidity T cells activated in vitro.

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population “antigen-presenting CAFs” and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression.Significance:Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II–expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors.See related commentary by Belle and DeNardo, p. 1001.This article is highlighted in the In This Issue feature, p. 983

(A-B) Tumor-bearing FVB/N and neu/N mice were treated with 1 dose of ADU-S100, and 24 hours later (A) necrotic tumor cells were evaluated by hematoxylin and eosin staining. (B) The surface area of necrosis in ADU-S100- and HBSS-treated FVB/N and neu/N mice was scored. At 24 hours post IT injection of ADU-S100 or HBSS numbers of activated caspase 3+ cells were evaluated by hematoxylin and eosin in FVB/N and neu/N mice. (C) A representative stain for active caspase 3 is shown for an ADU-S100 tumor from FVB/N mouse. (D) Relative active caspase 3 scored from treated FVB/N and neu/N mice are shown. Data is representative of 3 independent experiments of 5 mice/group. * p < 0.05, ** p

The supplemental tables include: antibodies used for flow cytometry; gene expression of genes involved in innate and adaptive immunity from whole tumors isolated from neu-N mice.

(A) 162 differentially expressed genes (Affymetrix Mouse Gene 2.0 ST array, 3 independent experiments, ANOVA p

Neu/N mice were received 5 x 104 NT2.5 cells in the right cranial mammary fat pad (proximal injected) and left dorsal gluteal subcutaneous tissue (distal un-injected). When tumor sizes reached 62.5-mm3 (5 x 5 mm) neu/N mice were administered IT injections of ADU-S100 or HBSS with or without αOX40 receptor agonistic and αPD-L1 antagonistic antibodies as previously described in the material and methods section. Tumor growth in the injected NT2.5 tumor (proximal injected) and dorsal gluteal (distal un-injected) were evaluated over the course of 50 days. This data is cumulative of two independent experiments of 5 mice/group.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.

Tumor bearing FVB/N (A) and neu/N (B) mice were evaluated for lymphocytic infiltrate on hematoxylin and eosin stained, paraffin-fixed tumors. CD4+ and CD8+ T cell infiltrates within tumors isolated from FVB (C) and neu/N (D) mice. Data is representative of 2 experiments of 4 - 5 mice/group. * p < 0.05, ** p

This file contains supplemental figures that includes: FACS gating strategies; QC plots for NanoString cell typing; survival and tumor growth of neu-N mice receiving ENT and ICIs without ï�¡-HER2; circulating MDSCs and absolute numbers of G-MDSCs infiltrating neu-N and Panc02 tumors; G-MDSCs quantified using flow cytometry from neu-N tumors or from hepatic Panc02 metastases; MDSC functional markers by flow cytometry and expression of PD-L1 on G-MDSCs; patterns of myeloid cell functional markers on gene expression profiling; qPCR validation of NanoString, and T cell proliferation and IFNï�§ï€ production within the TME; western blots depicting protein expression levels of STAT3 and phospho-STAT3 and representative proteins from NanoString pathways; the exhaustion state of CD8+ T cells.

At 24 hours post IT injection of ADU-S100 or HBSS, cytokine and chemokine production within the TME of non-tumor bearing FVB/N mice were evaluated by ELISA. (A) Significant amounts of TNFα and IL-6 were produced in the TME in response to ADU-S100. (B) Significant amounts of CCL2, CCL5, and CCL11 were produced in the TME in response to ADU-S100. (C) Significant amounts of CXCL1 were produced in the TME in response to ADU-S100, whereas CXCL9 and CXCL10 were already highly expressed independent of ADU-S100 injection.

R script of TCGA computational analysis

PDF file - 71K, Percent of adoptively transferred low avidity T cells present in the mice compared between untreated mice and mice treated with Cy+vaccine and Cy+vaccine+OX40.

Stimulator of interferon genes (STING) signaling induces IFNβ production by intratumoral dendritic cells (DC), driving T-cell priming and recruitment into the tumor microenvironment (TME). We examined to what extent preexisting antigen-specific tolerance influenced the efficacy of in situ delivery of a potent STING-activating cyclic dinucleotide (CDN), ADU S-100, against established HER-2+ breast tumors. ADU S-100 induced HER-2–specific CD8+ T-cell priming and durable tumor clearance in 100% of nontolerant parental FVB/N mice. In contrast, ADU S-100 did not sufficiently prime HER-2–specific CD8+ T cells in tolerant neu/N mice, resulting in only delayed tumor growth and tumor clearance in 10% of the mice. No differences in IFNβ production, DC priming, or HER-2–specific CD8+ T-cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2–specific CD8+ T cells were defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high numbers of PD1 and OX40 receptors on their CD8+ T cells, and PD-L1 was highly expressed on both myeloid and tumor cells. Modulating PD-L1 and OX40 receptor signaling combined with intratumoral ADU S-100 administration enhanced HER-2–specific CD8+ T-cell activity, clearing tumors in 40% of neu/N mice. Thus, intratumoral STING agonists could potently prime tumor antigen–specific CD8+ T-cell responses, and adding PD-L1 blockade and OX40 receptor activation can overcome antigen-enforced immune tolerance to induce tumor regression. Cancer Immunol Res; 5(6); 468–79. ©2017 AACR.

PDF file - 134K, Demonstration of the ability of alphaOX40 and alpha41BB agonists to temporarily eliminate established tumors.

Supplementary text

PDF file - 202K, Microarray data determining differences in gene expression between high and low avidity T cells.

(A) Expression of immune checkpoint receptors on CD4+ T cells in the TME. (B) Expression of PD-L1 on myeloid and tumor cells in the spleen and TME. (C-D) Increased expression of PD-1 on CD8+ T cells (C) and PD-L1 on myeloid cells (D) was noted 24 hours after IT ADU-S100 injection (gray bar) vs. HBSS (black bar). Statistical significance is determined by student''s t-test. * p < 0.05, ** p

PDF file - 84K

In untreated, tumor bearing FVB/N and neu/N mice intratumoral chemokine gradients were evaluated by multiplex ELISA. FVB/N express increased amounts of CXCL9, CCL3, CCL4, and CCL5, whereas neu/N mice express increased amounts of CCL2. Data is representative of 3 independent experiments involving 5 mice/group. Statistical significance was determined by multiple students'' t-test and a Bonferroni-Dunn posttest was performed to correct for increasing type I error. * p < 0.05, ** p

Supplementary Figure S1 shows the data quality and clustering metrics of the human PDAC single cell transcriptomes, Supplementary Figure S2 shows the distribution of sub-clusters of cells throughout the human dataset, Supplementary Figure S3 shows pathway analysis in human PDAC iCAFs and myCAFs, Supplementary Figure S4 shows the data quality and clustering metrics of the KPC mouse PDAC single cell transcriptomes, Supplementary Figure S5 shows pathway analysis in different KPC CAFs, Supplementary Figure S6 shows that Podoplanin is a pan-fibroblast marker in murine PDAC, Supplementary Figure S7 shows the validation of apCAFs in human PDAC, Supplementary Figure S8 shows the characterization of apCAFs in KPC and orthotopic transplantation models

Supplementary Figures and Tables

Immune-checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TMEs such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs), whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti–PD-1, anti–CTLA-4, or both significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene-expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B–producing CD8+ T effector cells in mice treated with combination therapy. Gene-expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

A major barrier to vaccines in cancer treatment is their failure to activate and maintain a complete cancer-specific CD8+ effector T-cell repertoire. Low-avidity T cells are more likely to escape clonal deletion in the thymus when compared with high-avidity T cells, and therefore comprise the major population of effector T cells available for activation in patients with cancer. However, low-avidity T cells fail to traffic into the tumor microenvironment and function in eradicating tumor under optimal vaccination conditions as opposed to high-avidity T cells that escape clonal deletion and function in tumor killing. We used high- and low-avidity T-cell receptor transgenic CD8+ T cells specific for the immunodominant epitope HER2/neu (RNEU420–429) to identify signaling pathways responsible for the inferior activity of the low-avidity T cells. Adoptive transfer of these cells into tumor-bearing vaccinated mice identified the members of apoptosis pathways that are upregulated in low-avidity T cells. The increased expression of proapoptotic proteins by low-avidity T cells promoted their own cell death and also that of other tumor-specific CD8+ T cells within their local environment. Importantly, we show that this proapoptotic effect can be overcome by using a strong costimulatory signal that prevents the activation-induced cell death and enables the low-avidity T cells to traffic into the tumor and assist in tumor clearance. These findings identify new therapeutic opportunities for activating the most potent anticancer T-cell responses. Cancer Immunol Res; 2(4); 307–19. ©2014 AACR.

(A) Tumor bearing neu/N mice were treated with isotype control (gray line) αCD4 (dashed gray line), αCD8 (dashed black line), or αCD4 + αCD8 (dotted black line) depleting antibodies. Depletion was initiated a week prior to NT2.5 tumor implantation and carried out with biweekly IP injections for the duration of the study. Depletion efficacy of 90% was confirmed before NT2.5 tumor implantation and upon completion of the study. As previously described mice were treated with IT ADU-S100 in sequence with αOX40 receptor agonistic and αPD-L1 antagonistic antibodies. Untreated tumor-bearing neu/N mice were included as control (black line). Tumor growth was followed for 35 days. (B) Serum from tumor-bearing neu/N mice receiving mock or ADU-S100 IT injection sequenced with either αOX40 receptor agonistic and αPD-L1 antagonistic antibodies or isotype matched controls was harvested 7 days post IT injection and HER-2 specific IgG antibody was measured. (C) HER-2-specific IgG antibody was measured in the cohorts of mice from figure (A) 7 days after IT-ADU-S100 injections. Statistical significance was determined through one-way Anova with significant differences in means re-evaluated using Bonferroni post-test. All data is cumulative of 2 - 3 experiments of 5 mice/group. * p < 0.05, ** p

Supplementary figure legends (S1-S11) clean AND supplementary table legends (1-2) clean.

Supplemental Data: Figure S1: Pathologic representation of hemispleen liver metastases; Figure S2: Treatment schema; Figure S3: Supplemental survival; Figure S4: FACS PD1/PDL1 expression; Figure S5: Myeloid cell FACS; Figure S6: Multiplex IHC of myeloid cell and HA high/low; Figure S7: CXCR4 survival; Figure S8: Multiplex and RNAseq PD1/PDL1

Purpose:Myeloid cells are a prominent immunosuppressive component within the stroma of pancreatic ductal adenocarcinoma (PDAC). Previously, targeting myeloid cells has had limited success. Here, we sought to target the myeloid cells through modifying a specific stromal component.Experimental Design:A murine model of metastatic PDAC treated with an irradiated whole-cell PDAC vaccine and PDAC specimens from patients treated with the same type of vaccine were used to assess the immune-modulating effect of stromal hyaluronan (HA) degradation by PEGPH20.Results:Targeting stroma by degrading HA with PEGPH20 in combination with vaccine decreases CXCL12/CXCR4/CCR7 immunosuppressive signaling axis expression in cancer-associated fibroblasts, myeloid, and CD8+ T cells, respectively. This corresponds with increased CCR7− effector memory T-cell infiltration, an increase in tumor-specific IFNγ, and improved survival. In the stroma of human PDACs treated with the same vaccine, decreased stromal CXCR4 expression significantly correlated with decreased HA and increased cytotoxic activities, suggesting CXCR4 is an important therapeutic target.Conclusions:This study represents the first to dissect signaling cascades following PDAC stroma remodeling via HA depletion, suggesting this not only overcomes a physical barrier for immune cell trafficking, but alters myeloid function leading to downstream selective increases in effector memory T-cell infiltration and antitumor activity.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden and therefore few neoantigen targets that can be recognized by cytotoxic T cells. Most PDACs are thus insensitive to either single or dual immune checkpoint inhibitor (ICI) therapy. Personalized neoantigen vaccines can expand the number and repertoire of anti-tumor T cells that infiltrate the tumor and mediate cytotoxicity. To model a personalized neoantigen vaccine treatment strategy in PDAC, we previously developed PancVAX, a peptide-based vaccine targeting 12 neoantigens expressed in the murine pancreatic cell line Panc02 (Kinkead et al, JCI Insight 2018). Although we observed increased T cell infiltration present in the tumor post-vaccination, these cells expressed high levels of exhaustion markers. We therefore hypothesized that sequential administration of anti-CTLA-4 and anti-PD-1 would enhance the pool of T cells primed by the neoantigen vaccine and maintain activation of antigen-experienced T cells, respectively, to yield optimal and durable neoantigen-specific anti-tumor immunity in PDAC. To address this, mice bearing subcutaneous Panc02 tumors were vaccinated with two rounds of the PancVAX neoantigen vaccine followed by anti-CTLA-4 and anti-PD-1 3 days later. Anti-PD-1 maintenance was given twice weekly beginning at the first vaccine dose. Twelve days after the last peptide vaccine dose, tumors were harvested and dissociated into single-cell suspensions for paired single-cell RNA-sequencing and TCR-sequencing. Mice that were untreated or given ICIs without PancVAX had the highest proportions of CD8+ T cells expressing exhaustion markers. PancVAX-treated mice had more intratumoral cycling CD8 T cells and effector CD8+ T cells with high cytotoxic gene expression. Among mice treated with PancVAX, tumors from mice treated with PancVAX + anti-PD1 or PancVAX + anti-PD1 + anti-CTLA-4 had the highest proportions of effector CD8+ T cells. Ongoing analyses include differential gene expression and pathway analysis between treatment conditions in the T cell compartment in mice treated with combination ICI and PancVAX. Additionally, we will assess changes in T cell clonality and diversity within the tumors when mice are treated with single or combination therapy. These results will define a transcriptional signature associated with the generation of a productive anti-tumor immune response when neoantigen vaccines and ICI are used in combination. This work demonstrates how the addition of ICIs to personalized neo-epitope vaccines for PDAC can further enhance the quality of vaccine-induced T cell effector function in an otherwise immunologically cold tumor type and supports their inclusion in neoantigen vaccination strategies for patients with PDAC. Citation Format: Jacob T. Mitchell, Amanda Huff, Emily Davis-Marcisak, Fangluo Chen, Todd D. Armstrong, Luciane T. Kagohara, James Leatherman, Rulin Wang, Srinivasan Yegnasubramanian, Elizabeth M. Jaffee, Elana J. Fertig, Neeha Zaidi. Combination PancVAX neo-epitope vaccine with anti-CTLA-4 and anti-PD-1 antibodies enhances infiltration of cytotoxic T cells and mitigates T cell exhaustion in a murine model of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5076.

Pancreatic ductal adenocarcinoma (PDAC) is most often diagnosed at an advanced stage. Newly diagnosed patients therefore have a dismal five-year survival rate of 11%. However, PDAC progresses from pre-invasive pancreatic intraepithelial neoplasia (PanIN) over at least a decade. Throughout this transition, the tissue microenvironment becomes increasingly immunosuppressive. Early PanINs may therefore be more amenable to immune-based interception strategies; however, little is known about the pre-malignant lesion immune microenvironment in PDAC. We hypothesized that the identification of the immune landscape of PanINs will elucidate the immuno-dynamic changes that occur during PanIN-to-PDAC progression and identify novel strategies for intercepting PDAC. Here, we use an inducible mouse model to study the evolution of immunosuppression from PanINs to PDAC. We pair spatial molecular profiling of our mouse model with profiling of human tissue in a cohort of patients with normal tissue, chronic pancreatitis, PanIN, and PDAC. To examine the evolution of the immune microenvironment throughout PanIN-to-PDAC progression, we first optimized a tamoxifen-inducible Pdx1-CreERT2 mouse that controls KRASG12D expression and knocks out p53. The impact of KRASG12D expression on the immune cell landscape in PanIN and PDAC lesions was examined by immunohistochemistry (IHC) and RNA in situ hybridization on mouse pancreas. We used imaging mass cytometry (IMC) of 35 immune markers to better classify and quantify the immune cell subtypes. Our analyses thus far reveal increased Tregs as PanINs progress to PDAC. Furthermore, although CD3+ T cells are recruited to tumors, these immune cells are strictly restricted to the immediate edge of the tumor and predominantly consist of Tregs. For our human analyses, FFPE pancreas sections from treatment naïve patients who had undergone surgical resection without neoadjuvant chemotherapy were evaluated. Each section contained regions of normal tissue, chronic pancreatitis, PanIN and PDAC. These were evaluated using IHC, IMC, and spatial transcriptomics to allow us to spatially evaluate the immune populations associated with lesions and PDAC. The relative density and localization of myeloid and lymphoid cell types in both PanIN and PDAC regions revealed an initial influx of CD8+ and CD4+ T cells to PanINs and a progressively immunosuppressive microenvironment in subsequent stages. While PanINs and PDAC both recruited immune cells, the phenotypes of the immune infiltrates were distinct and revealed unique immune pathways that could contribute to immunosuppression as PanINs develop into PDACs. Our proteomic and transcriptomic data from mouse and human pancreas show that mutant KRAS driven premalignant lesions recruit an evolving immune response that readily becomes immunosuppressive as progression to PDAC occurs. Citation Format: Melissa R. Lyman, Jacob T. Mitchell, Luciane T. Kagohara, Benjamin Barrett, Amanda Huff, Sarah Shin, Gabriella Longway, Anuj Gupta, Lalitya Andaloori, Todd D. Armstrong, Daniel Haldar, Robert Anders, Elizabeth Thompson, Nilo Azad, Won Jin Ho, Elizabeth Jaffee, Elana J. Fertig, Neeha Zaidi. Evolution of immune cell composition and functionality as pancreatic intraepithelial neoplasia progresses to pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2873.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here, we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8(+) T cells and macrophage subpopulations toward MHC-II–expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD-1/PD-L1 axis expression in the setting of PTPN22 inhibition could be further leveraged with PD-1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

Purpose: Myeloid cells are a prominent immunosuppressive component within the stroma of pancreatic ductal adenocarcinoma (PDAC). Previously, targeting myeloid cells has had limited success. Here, we sought to target the myeloid cells through modifying a specific stromal component. Experimental Design: A murine model of metastatic PDAC treated with an irradiated whole-cell PDAC vaccine and PDAC specimens from patients treated with the same type of vaccine were used to assess the immune-modulating effect of stromal hyaluronan (HA) degradation by PEGPH20. Results: Targeting stroma by degrading HA with PEGPH20 in combination with vaccine decreases CXCL12/CXCR4/CCR7 immunosuppressive signaling axis expression in cancer-associated fibroblasts, myeloid, and CD8+ T cells, respectively. This corresponds with increased CCR7− effector memory T-cell infiltration, an increase in tumor-specific IFNγ, and improved survival. In the stroma of human PDACs treated with the same vaccine, decreased stromal CXCR4 expression significantly correlated with decreased HA and increased cytotoxic activities, suggesting CXCR4 is an important therapeutic target. Conclusions: This study represents the first to dissect signaling cascades following PDAC stroma remodeling via HA depletion, suggesting this not only overcomes a physical barrier for immune cell trafficking, but alters myeloid function leading to downstream selective increases in effector memory T-cell infiltration and antitumor activity.

Immune checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TME's such as breast cancer present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs) whose functioning prohibits both T cell activation and infiltration. To date, most studies focus on use of ICI in triple negative disease. Our work aims to uncover the efficacy of ICI in both early and advanced HER2 positive (HER2+) disease and to advance our understanding of how to improve response rates to these new promising therapies. We are using a HER-2/neu transgenic mouse model with tumor challenge of syngeneic cell lines to test the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti-PD-1 and anti- CTLA-4, on primary and metastatic disease. We are examining treatment effects on primary tumor growth, metastatic burden, and survival. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated using fluorescence-activated cell sorting, gene expression profiling, and ex vivo suppression assays. Western blots, qPCR and other in vitro assays will be performed on MDSCs to investigate mechanisms behind response. In the HER2+ mouse model of early stage disease, we show that combining ENT, with ICis significantly improves survival and delays tumor growth. Preliminary data in models of advanced disease, show only ENT + a-PD-1 improves survival and metastatic burden. Conversely, in the metastatic model, ENT + a-CTLA-4 negatively effects survival and metastatic burden. In primary tumors, ENT + ICIs leads to significantly decreased suppression by granulocytic-MDSCs. However, MDSC infiltration and function is not affected in lungs containing macrometastatic disease. Interestingly, we found an increase in activated granzyme-B-producing CD8+ T effector cells in mice treated with combination therapy in both primary and metastatic tumors. Finally, gene expression profiling of MDSCs from primary tumors identified significant changes in immune-related pathways, and identified a common downstream regulator –STAT3. Studies are ongoing to evaluate the mechanistic role of STAT3 in the response observed in primary tumors and to determine if STAT3 is involved in response in the metastatic setting. In summary, addition of ENT to ICIs significantly affects overall survival in early stage models of HER2+ breast cancer however, only addition of a-PD-1 to ENT is beneficial in models of advanced disease. Additionally, the mechanism of action in early stage disease involves altered infiltration and function of MDSCs, allowing for a more robust adaptive immune response. However, a different mechanism of action is likely responsible for the responses seen in advanced stage disease. These novel findings provide a rationale for combination therapy in patients with HER2+ breast cancer and suggest responses to this combination therapy are linked to stage of disease likely due to different mechanisms of action. Citation Format: Roussos Torres ET, Rafie C, Armstrong T, Jaffee EM. Epigenetic modulation—unlocking the potential of checkpoint inhibition in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-10.

Immune-checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TMEs such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs), whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti–PD-1, anti–CTLA-4, or both significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene-expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B–producing CD8+ T effector cells in mice treated with combination therapy. Gene-expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

BackgroundThe majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed at the metastatic stage, and standard therapies have limited activity with a dismal 5-year survival rate of only 8%. The liver and lung are the most common sites of PDAC metastasis, and each have been differentially associated with prognoses and responses to systemic therapies. A deeper understanding of the molecular and cellular landscape within the tumor microenvironment (TME) metastasis at these different sites is critical to informing future therapeutic strategies against metastatic PDAC.ResultsBy leveraging combined mass cytometry, immunohistochemistry, and RNA sequencing, we identify key regulatory pathways that distinguish the liver and lung TMEs in a preclinical mouse model of metastatic PDAC. We demonstrate that the lung TME generally exhibits higher levels of immune infiltration, immune activation, and pro-immune signaling pathways, whereas multiple immune-suppressive pathways are emphasized in the liver TME. We then perform further validation of these preclinical findings in paired human lung and liver metastatic samples using immunohistochemistry from PDAC rapid autopsy specimens. Finally, in silico validation with transfer learning between our mouse model and TCGA datasets further demonstrates that many of the site-associated features are detectable even in the context of different primary tumors.ConclusionsDetermining the distinctive immune-suppressive features in multiple liver and lung TME datasets provides further insight into the tissue specificity of molecular and cellular pathways, suggesting a potential mechanism underlying the discordant clinical responses that are often observed in metastatic diseases.

In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines., Structural parameters, including the solvent exposed surface area of the neoepitope and the position and spatial configuration of the mutated residue can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.

Almost all pancreatic ductal adenocarcinomas (PDA) develop following KRAS activation, which triggers epithelial transformation and recruitment of desmoplastic stroma through additional transcriptional and epigenetic regulation, but only a few of these regulatory mechanisms have been described. We profiled dysregulated miRNAs starting with the earliest premalignant pancreatic intraepithelial neoplasias (PanIN) in genetically engineered mutated KRAS and P53 (KPC) mice programmed to recapitulate human PDA tumorigenesis. We identified miR-21 and miR-224 as cell-specific and compartment-specific regulators in PanINs and PDA. miR-21 is overexpressed in tumor epithelial cells of premalignant ducts, while miR-224 is overexpressed in cancer-associated fibroblasts in PDA stroma. Inhibition of miR-21 reverted protumorigenic functionalities to baseline levels. Overexpression of miR-224 induced activated phenotypes in normal fibroblasts. In vivo miR-21 inhibition improved survival in established PDA. Importantly, early systemic miR-21 inhibition completely intercepted premalignant progression. Finally, an evaluation of miR-21 expression in the PDA cohort of The Cancer Genome Atlas identified a correlation between tumor epithelial cell content and miR-21 expression in human tumors providing further rationale for conducting human studies. Thus, miR-21 may be useful for early PanIN detection, and for intercepting developing premalignant pancreatic lesions and other KRAS-driven premalignancies.

Immunotherapies that modulate T cell function have been firmly established as a pillar of cancer therapy, whereas the potential for B cells in the antitumor immune response is less established. B cell–activating factor (BAFF) is a B cell–activating cytokine belonging to the TNF ligand family that has been associated with autoimmunity, but little is known about its effects on cancer immunity. We find that BAFF upregulates multiple B cell costimulatory molecules; augments IL-12a expression, consistent with Be-1 lineage commitment; and enhances B cell antigen-presentation to CD4(+) Th cells in vitro. In a syngeneic mouse model of melanoma, BAFF upregulates B cell CD40 and PD-L1 expression; it also modulates T cell function through increased T cell activation and TH1 polarization, enhanced expression of the proinflammatory leukocyte trafficking chemokine CCR6, and promotion of a memory phenotype, leading to enhanced antitumor immunity. Similarly, adjuvant BAFF promotes a memory phenotype of T cells in vaccine-draining lymph nodes and augments the antitumor efficacy of whole cell vaccines. BAFF also has distinct immunoregulatory functions, promoting the expansion of CD4(+)Foxp3(+) Tregs in the spleen and tumor microenvironment (TME). Human melanoma data from The Cancer Genome Atlas (TCGA) demonstrate that BAFF expression is positively associated with overall survival and a TH1/IFN-γ gene signature. These data support a potential role for BAFF signaling as a cancer immunotherapy.

Immunotherapy has shown limited success in prostate cancer; this may be partially explained by its immunosuppressive tumor microenvironment (TME). Although androgen-deprivation therapy (ADT), the most common treatment for prostate cancer, initially promotes a robust T cell infiltrate, T cell responses are later attenuated. Based on the castration-sensitive Myc-CaP model, we developed an antigen-specific system to study CD8 T cell tolerance to prostate tumors. This model is unique in that CD8 T cells recognize a bona-fide tumor antigen (Her-2/neu), rather than an overexpressed xenogenic antigen like chicken ovalbumin or influenza hemagglutinin. Using this novel model, we demonstrate robust tolerance that is not alleviated by TLR agonists or ADT. This model may serve as a novel and useful tool to further interrogate methods by which to augment anti-tumor cancer immune responses to prostate cancer. Significance Prostate cancer is a leading cause of cancer-related death in men worldwide, with an estimated 33,000 deaths projected in the U.S. in 2020. Although primary (localized) tumors can be cured by surgery or radiation, approximately 40% of patients eventually develop recurrent disease. While initially responsive to androgen-deprivation, many patients with recurrent prostate cancer eventually progress to a more advanced disease state known as metastatic castration-resistant prostate cancer (mCRPC); this is the lethal phenotype. These studies describe a novel androgen-responsive murine cell line that expresses a bona-fide tumor antigen (Her-2/neu). Pre-clinical work with this model shows robust and antigen-specific CD8 T cell tolerance, providing a novel preclinical model to study CD8 T cell tolerance to prostate tumors.

Background: Checkpoint inhibition is a very successful treatment strategy in cancers that are naturally immunogenic by attracting T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers are not highly immunogenic likely due to an immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME is to use epigenetic modulation to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and sensitize tumors to checkpoint modulation. We hypothesize that combinatorial therapy primes the TME by altering infiltration and function of MDSCs leading to a more robust T cell response. Methods: We are using a HER-2/neu transgenic mouse model with tumor challenge of syngeneic cell lines to test the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti -programmed cell death protein (a-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (a-CTLA-4) antibodies, with and without anti-HER2 antibodies. We will examine treatment effects on tumor growth, and hope to identify co-stimulatory and inhibitory factors regulating T cell and MDSC responses. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated in primary tumors using fluorescence-activated cell sorting, nanostring gene expression profiling, and immunohistochemistry. Results: We found significant improvement in survival and delay in tumor growth in mice treated with ENT in combination with a-PD-1 and/or a-CTLA-4. Addition of anti-HER2 therapy to ENT and a-CTLA4 or a-PD1 also significantly improves survival and delay in tumor growth. We also found addition of ENT to checkpoint inhibition leads to significantly increased infiltration of granulocytic-MDSCs into the TME. We demonstrate an increase in CD8+ T effector cells in mice treated with combination therapy. Flow cytometric evaluation of markers of T cell activation, exhaustion, and MDSC function demonstrate significantly increased T cell activation, exhaustion, and myeloid function however it is unclear how this directly effects the phenotype we have observed in these mice. Gene expression profiling of both MDSCs and lymphocytes infiltrating tumors is underway to help determine significant changes in immune related pathways that lead to our observed outcomes. Conclusions: Addition of ENT to checkpoint inhibition significantly increases infiltration of innate and adaptive immune cells into the highly tolerant neu-N breast tumors and leads to improved survival and decreased tumor burden. Functional assays are underway and future studies will further delineate changes in immune infiltration as well as genetic alterations responsible for these observations. It is our hope that these novel findings will provide further rationale for combination therapy and improve the response rate of these immune therapies in patients with breast cancer. Citation Format: Roussos Torres ET, Ma H, Christmas B, Armstrong T, Jaffee EM. Combination checkpoint inhibition and epigenetic modulation promotes tumor suppression and improves survival in Her2+ models of breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-08-01.

Epigenetic control of gene expression is a major determinant of tumor phenotype and has been found to influence sensitivity to individual chemotherapeutic agents. Glutathione peroxidase 3 (GPX3, plasma glutathione peroxidase) is a key component of cellular antioxidant regulation and its gene has been reported to be methylated in specific tumor types. GPX3 role in oxidative damage has been associated with sensitivity to platinums in other tumors but its importance in colorectal cancer (CRC) has not been determined. We examined the role of GPX3 methylation in colorectal carcinoma in determining sensitivity to platinum drugs using primary tumor specimens, cell lines, knockdown cell lines, and tumor cell line xenografts. We find GPX3 promoter region methylation in approximately one third of CRC samples and GPX3 methylation leads to reduced GPX3 expression and increased oxaliplatin and cisplatin sensitivity. In contrast, in cell lines with high baseline levels of GPX3 expression or with the ability to increase GPX3 expression, platinum resistance is increased. The cisplatin IC50 in GPX3-methylated cell lines is approximately 6-fold lower than that in GPX3-unmethylated lines. Additionally, knockdown cell lines with essentially no GPX3 expression require N-acetylcysteine to survive in culture underscoring the importance of GPX3 in redox biology. In vivo, GPX3 methylation predicts tumor xenograft sensitivity to platinum with regression of GPX3 knockdown xenografts with platinum treatment but continued growth of GPX3 wild type xenografts in the presence of platinum. These studies demonstrate the importance of GPX3 for CRC cells resistance to platinums and the potential utility of GPX3 methylation status as a predictive biomarker for platinum sensitivity in CRC.

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population “antigen-presenting CAFs” and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. Significance: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II–expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors. See related commentary by Belle and DeNardo, p. 1001. This article is highlighted in the In This Issue feature, p. 983

Checkpoint inhibition is a very successful treatment strategy in cancers that are naturally immunogenic by attracting T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers are not highly immunogenic likely due to an immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME into one that is immune responsive, is to use epigenetic modulation to expose tumor antigens, promote cytokine production, thus, attracting inflammatory cells into the tumor. Epigenetic modulation also has the potential to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and thus, sensitizing tumors to checkpoint modulation. We hypothesize that combinatorial therapy primes the TME by increasing immunogenicity via alteration of T cells and MDSCs. We also hypothesize that combination therapy will significantly affect immune modulatory pathways. Alterations in these could serve as a functional read out of combinatorial therapy. We are using a Her-2/neu (neu-N) transgenic mouse model which through the use of various syngeneic cell lines has the capability to inform the response of Her2 and triple negative subtypes of breast cancer to immunotherapy. This model enables us to study the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat, and checkpoint inhibitors anti -programmed cell death protein (PD1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, on tumor growth and metastatic progression, and to help identify co-stimulatory and inhibitory factors regulating T cell and MDSC responses. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated in primary tumors and metastases using fluorescence-activated cell sorting and immunohistochemistry. Thus far, there is a significant improvement in survival and delay in tumor growth in mice inoculated with neu expressing tumor cells treated with combination anti-PD1, anti-CTLA4 and entinostat as well as with anti-PD1 in combination with Entinostat. Preliminary results also suggest that epigenetic modulation with entinostat in combination with checkpoint inhibition improves the immune response within these tumors as evidenced by increased CD8+/T-regulatory cell infiltration, and increased monocytic-MDSCs into the TME. There are ongoing survival studies testing this combination therapy in mice inoculated with triple negative breast tumor cells. Future studies will further delineate the mechanisms by which epigenetic therapy alters the function of the inflammatory response to immunotherapy. Citation Format: Evanthia T. Roussos Torres, Hayley Ma, Brian Christmas, Todd Armstrong, Elizabeth M. Jaffee. Promotion of immunogenicity using epigenetic modulation and immune checkpoint inhibition in mouse models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3667. doi:10.1158/1538-7445.AM2017-3667