Esha Mathew

Myeloid cells are required for PD-1/PD-L1 checkpoint activation and the establishment of an immunosuppressive environment in pancreatic cancer

Background Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity. Objective The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. Methods Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells (mostly myeloid cell population) were depleted by diphtheria toxin treatment during tumour initiation or in established tumours. Results Depletion of myeloid cells prevented KrasG12D-driven pancreatic cancer initiation. In pre-established tumours, myeloid cell depletion arrested tumour growth and in some cases, induced tumour regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T-cell anti-tumour activity by inducing the expression of programmed cell death-ligand 1 (PD-L1) in tumour cells in an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent manner. Conclusion Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer.

CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice.

Zhang and colleagues show that Kras-expressing epithelial cells recruit CD4+ T cells that repress the activity of CD8+ T cells to establish the immunosuppressive microenvironment in the iKras* mouse model of pancreatic cancer; elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells. Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as pancreatic intraepithelial neoplasias (PanIN). PanIN formation is accompanied by the establishment of an immunotolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we genetically eliminate CD4+ T cells in the iKras* mouse model of pancreatic cancer, in the context of pancreatitis, to determine the functional role of CD4+ T cells during mutant Kras-driven pancreatic carcinogenesis. We show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. Furthermore, we show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation and provide a rationale to explore immunomodulatory approaches in pancreatic cancer. Cancer Immunol Res; 2(5); 423–35. ©2014 AACR.

Mesenchymal Stem Cells Promote Pancreatic Tumor Growth by Inducing Alternative Polarization of Macrophages

Pancreatic cancer is characterized by an extensive desmoplastic stroma, the functional relevance of which is poorly understood. Activated fibroblasts are a prevalent component of the stroma, and traditionally, these cells have been considered as a homogenous population derived from pancreatic stellate cells. In this study, we highlight a previously unappreciated heterogeneity of the fibroblast population within the stroma. In particular, a subset of stromal fibroblasts has characteristics of mesenchymal stem cells (MSCs). MSCs are present in the normal pancreas as well as in the carcinomatous pancreas (CA-MSCs). Here, we determine that CA-MSCs have increased tumor-promoting function compared with MSCs in normal pancreas. This ability to promote tumor growth is associated with CA-MSCs’ unique ability to promote alternative macrophage polarization. Thus, our study identifies a previously uncharacterized cell population within the stroma and sheds light on tumor-promoting interactions between different components of the stroma. Significance Targeting the stroma is emerging as a new paradigm in pancreatic cancer; however, efforts to that effect are hampered by our limited understanding of the nature and function of stromal components. Here, we uncover previously unappreciated heterogeneity within the stroma and identify interactions among stromal components that promote tumor growth and could be targeted therapeutically.

The transcription factor GLI1 modulates the inflammatory response during pancreatic tissue remodeling

Background: Gli1 is induced during pancreatic injury; its function remains unknown. Results: Loss of Gli1 in the stromal compartment impairs cytokine production, myeloid cell recruitment, and tissue repair. Conclusion: Gli1 regulates remodeling during pancreatic tissue recovery. Significance: This study defines a novel role for GLI1 in pancreas remodeling and expands the repertoire of target genes mediating GLI1 cellular functions. Pancreatic cancer, one of the deadliest human malignancies, is almost uniformly associated with a mutant, constitutively active form of the oncogene Kras. Studies in genetically engineered mouse models have defined a requirement for oncogenic KRAS in both the formation of pancreatic intraepithelial neoplasias, the most common precursor lesions to pancreatic cancer, and in the maintenance and progression of these lesions. Previous work using an inducible model allowing tissue-specific and reversible expression of oncogenic Kras in the pancreas indicates that inactivation of this GTPase at the pancreatic intraepithelial neoplasia stage promotes pancreatic tissue repair. Here, we extend these findings to identify GLI1, a transcriptional effector of the Hedgehog pathway, as a central player in pancreatic tissue repair upon Kras inactivation. Deletion of a single allele of Gli1 results in improper stromal remodeling and perdurance of the inflammatory infiltrate characteristic of pancreatic tumorigenesis. Strikingly, this partial loss of Gli1 affects activated fibroblasts in the pancreas and the recruitment of immune cells that are vital for tissue recovery. Analysis of the mechanism using expression and chromatin immunoprecipitation assays identified a subset of cytokines, including IL-6, mIL-8, Mcp-1, and M-csf (Csf1), as direct GLI1 target genes potentially mediating this phenomenon. Finally, we demonstrate that canonical Hedgehog signaling, a known regulator of Gli1 activity, is required for pancreas recovery. Collectively, these data delineate a new pathway controlling tissue repair and highlight the importance of GLI1 in regulation of the pancreatic microenvironment during this cellular process.

Epithelial Notch signaling is a limiting step for pancreatic carcinogenesis

BackgroundPancreatic cancer is one of the deadliest human malignancies, with few therapeutic options. Re-activation of embryonic signaling pathways is commonly in human pancreatic cancer and provided rationale to explore inhibition of these pathways therapeutically. Notch signaling is important during pancreatic development, and it is re-activated in pancreatic cancer. The functional role of Notch signaling during pancreatic carcinogenesis has been previously characterized using both genetic and drug-based approaches. However, contrasting findings were reported based on the study design. In fact, Notch signaling has been proposed to act as tumor-promoter or tumor-suppressor. Given the availability of Notch inhibitors in the clinic, understanding how this signaling pathway contributes to pancreatic carcinogenesis has important therapeutic implications. Here, we interrogated the role of Notch signaling specifically in the epithelial compartment of the pancreas, in the context of a genetically engineered mouse model of pancreatic cancer.MethodsTo inhibit Notch signaling in the pancreas epithelium, we crossed a mouse model of pancreatic cancer based on pancreas-specific expression of mutant Kras with a transgenic mouse that conditionally expresses a dominant negative form of the Mastermind-like 1 gene. MAML is an essential co-activator of the canonical Notch signaling-mediated transcription. DNMAML encodes a truncated MAML protein that represses all canonical Notch mediated transcription in a cell autonomous manner, independent of which Notch receptor is activated. As a result, in mice co-expressing mutant Kras and DNMAML, Notch signaling is inhibited specifically in the epithelium upon Cre-mediated recombination. We explored the effect of epithelial-specific DNMAML expression on Kras-driven carcinogenesis both during normal aging and following the induction of acute pancreatitis.ResultsWe find that DNMAML expression efficiently inhibits epithelial Notch signaling and delays PanIN formation. However, over time, loss of Notch inhibition allows PanIN formation and progression.ConclusionsEpithelial-specific Notch signaling is important for PanIN initiation. Our findings indicate that PanIN formation can only occur upon loss of epithelial Notch inhibition, thus supporting an essential role of this signaling pathway during pancreatic carcinogenesis.

Epithelial-Myeloid cell crosstalk regulates acinar cell plasticity and pancreatic remodeling in mice

Dedifferentiation of acini to duct-like cells occurs during the physiologic damage response in the pancreas, but this process can be co-opted by oncogenic Kras to drive carcinogenesis. Myeloid cells infiltrate the pancreas during the onset of pancreatic cancer, and promote carcinogenesis. Here, we show that the function of infiltrating myeloid cells is regulated by oncogenic Kras expressed in epithelial cells. In the presence of oncogenic Kras, myeloid cells promote acinar dedifferentiation and carcinogenesis. Upon inactivation of oncogenic Kras, myeloid cells promote re-differentiation of acinar cells, remodeling of the fibrotic stroma and tissue repair. Intriguingly, both aspects of myeloid cell activity depend, at least in part, on activation of EGFR/MAPK signaling, with different subsets of ligands and receptors in different target cells promoting carcinogenesis or repair, respectively. Thus, the cross-talk between epithelial cells and infiltrating myeloid cells determines the balance between tissue repair and carcinogenesis in the pancreas.

Abstract A096: Myeloid cells are required for pancreatic carcinogenesis and PD-1/PD-L1 checkpoint activation

Myeloid cells, including both macrophages and immature myeloid cells/myeloid derived suppressor cells (MDSCs), accumulate during the progression of pancreatic cancer. The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer, and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. Primary mouse pancreatic cancer cells were transplanted into CD11b-DTR mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells were depleted by Diphtheria Toxin treatment during tumor initiation or in established tumors. Depletion of myeloid cells prevented KrasG12D driven pancreatic cancer initiation. Myeloid cells are required for sustained MAPK signaling in pancreatic epithelial cells during the onset of carcinogenesis, notwithstanding the expression of oncogenic Kras. In pre-established tumors, myeloid cell depletion arrested tumor growth and in some cases, induced tumor regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T cell anti-tumor activity by inducing the expression of Programmed cell death-ligand 1 (PD-L1) in tumor cells in an EGFR/MAPK dependent manner. Treatment with MEK inhibitors lowers the intratumoral expression of PD-L1 and renders the tumor susceptible to PD-1 blockade. Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK dependent regulation of PD-L1 expression on tumor cells. Derailing this cross-talk between myeloid cells and tumor cells is sufficient to restore anti-tumor immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer. Note: This abstract was not presented at the conference. Citation Format: Yaqing Zhang, Ashley Velez-Delgado, Esha Mathew, Dongjun Li, Flor M. Mendez, Kevin Flannagan, Andrew D. Rhim, Diane M. Simeone, Gregory L. Beatty, Marina Pasca di Magliano. Myeloid cells are required for pancreatic carcinogenesis and PD-1/PD-L1 checkpoint activation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A096.

Abstract A48: CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice

This abstract is also being presented as a short talk in Session 6: Immunology. A full abstract is printed in the Proffered Abstracts section (PR11) of the Conference Proceedings. Citation Format: Yaqing Zhang, Wei Yan, Esha Mathew, Filip Bednar, Shanshan Wan, Meredith A. Collins, Rebecca A. Evans, Theodore H. Welling, Robert H. Vonderheide, Marina Pasca di Magliano. CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A48.

Abstract A51: Novel Hedgehog co-receptors in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. The Hedgehog (Hh) signaling pathway is upregulated in PDAC. 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is active in early PanIN lesions and persists as the cancer progresses. Interestingly, Hh ligands are secreted by the tumor cells and function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor growth. PDAC is characterized by hypovascularity and a highly desmoplastic stroma. The downstream effects of Hh activation in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdon), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Gas1, Boc, and Cdon are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. During cancer progression, expression of all three co-receptors is prevalent throughout the desmoplastic stroma. Functional studies on Gas1-/-;Boc-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wildtype MEFs, indicating that these co-receptors are important for Hh signal transduction. Despite a reduced Hh-response, we found that Gas1-/-;Boc-/- MEFs promoted the growth of significantly larger, more vascularized tumors than their wildtype counterparts. A more physiologically relevant experiment with pancreatic fibroblasts and primary pancreatic tumor cells yielded the same counterintuitive result: removal of Gas1 and Boc from fibroblasts decreases Hh-response, and yet tumor-promoting potential was significantly increased. Moreover, chick chorioallantoic membrane (CAM) assays with tumor cells and fibroblasts revealed that Gas1-/-;Boc-/- MEFs stimulated blood vessel growth. We repeated these experiments with MEFs in which all three co-receptors were removed. Gas1-/-;Boc-/-;Cdon-/- MEFs displayed a more significantly abrogated Hh-response than both their Gas1-/-;Boc-/- and wildtype counterparts. However subcutaneous co-injection and CAM experiments revealed that these MEFs did not promote tumor growth, behaving similarly to Smo-/- MEFs in previously published findings. These results indicate the importance of Hh dosage in pancreatic cancer, which has important clinical implications. In particular, we found that intermediate levels of Hh signaling, such as that resulting from pharmaceutical inhibition, resulted in a marked angiogenesis response. This finding may partially account for the recent failure of Hh inhibition in a pancreatic cancer clinical trial. Citation Format: Esha Mathew, Yaqing Zhang, Alexander M. Holtz, Kevin T. Kane, Jane Song, Benjamin L. Allen, Marina Pasca di Magliano. Novel Hedgehog co-receptors in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A51.

Abstract IA24: Oncogenic Kras and the pancreatic cancer microenvironment

Oncogenic mutations in the KRAS gene are almost invariably associated to pancreatic cancer in humans. Genetic engineered mice that express oncogenic Kras in the pancreas develop pancreatic cancer in a step-wise manner that resembles the progression of the human disease. Invasive cancer is preceded by Pancreatic Intraepithelial Neoplasia (PanIN) formation. In humans and in mice, pancreatic cancer and PanINs are associated to extensive accumulation of fibro-inflammatory stroma. The interactions between epithelial cells and individual components of the stroma, as well as the interaction among components of the stroma are not fully understood. We have recently identified mesenchymal stromal cells (MSCs) as one of the components of the PanIN stroma. MSCs are also present, albeit at lower frequency, in the normal pancreas. Interestingly, PanIN-derived MSCs have increased tumor-promoting ability compared to normal-pancreas-derived MSCs. MSCs have been shown to regulate the recruitment of macrophages in other tumor types. Accordingly, we find that MSCs promote macrophage recruitment in vivo. Moreover, both normal-pancreas derived MSCs and PanIN-derived MSCs promote differentiation of macrophages from bone marrow progenitors in vitro. PanIN-derived MSCs have the unique ability to promote polarization to M2- macrophages. Finally, depletion of macrophages blocks the tumor-promoting effect of MSCs in co-transplantation experiments with pancreatic cancer cells. These preliminary data our data, and previous studies in the literature, provided the rationale for an investigation of the role of monocytes-macrophages in the pancreatic cancer microenvironment. Therefore, we crossed the iKras* mouse model of pancreatic cancer with CD11b-DTR mice that allow depletion of all CD11b+ lineages (monocytes and macrophages) upon administration of Diphtheria Toxin (DT). The iKras* mouse allows inducible and reversible expression of oncogenic Kras. Expression of oncogenic Kras and induction of acute pancreatitis leads to PanIN formation and accumulation of a stroma rich in macrophages. The polarization status of these macrophages is largely M2. Inactivation of oncogenic Kras reverses the accumulation of macrophages, indicating that macrophage infiltration requires signals derived from oncogenic Kras-expressing epithelial cells (directly, or mediated by other cell types within the stroma such as MSCs or fibroblasts). Depletion of macrophages prevents PanIN formation; depletion of macrophages after PanINs have formed leads to their regression. Moreover, macrophage depletion prevents tumor establishment and causes arrest or tumor growth or regression in pre-established transplanted tumors. Thus macrophages emerge as key cell components within the pancreatic cancer stroma. We are currently investigating the mechanism underlying the requirement for macrophages during pancreatic carcinogenesis. Citation Format: Esha Mathew, Yaqing Zhang, Flor Mendez, Fil Bednar, Marina Pasca di Magliano. Oncogenic Kras and the pancreatic cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA24.