Whitney L. Gladney

Immune Escape Mechanisms as a Guide for Cancer Immunotherapy

Immunotherapy has demonstrated impressive outcomes for some patients with cancer. However, selecting patients who are most likely to respond to immunotherapy remains a clinical challenge. Here, we discuss immune escape mechanisms exploited by cancer and present strategies for applying this knowledge to improving the efficacy of cancer immunotherapy. Clin Cancer Res; 21(4); 687–92. ©2014 AACR.

Exclusion of T Cells From Pancreatic Carcinomas in Mice Is Regulated by Ly6Clow F4/80+ Extratumoral Macrophages

BACKGROUND & AIMS Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumors immunosuppressive mechanisms, or both. METHODS Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry. RESULTS Gemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells. CONCLUSIONS Ly6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.

Tumor-derived CCL2 mediates resistance to radiotherapy in pancreatic ductal adenocarcinoma

Purpose: Local tumor growth is a major cause of morbidity and mortality in nearly 30% of patients with pancreatic ductal adenocarcinoma (PDAC). Radiotherapy is commonly used for local disease control in PDAC, but its efficacy is limited. We studied the impact of selectively intervening on radiotherapy-induced inflammation as an approach to overcome resistance to radiotherapy in PDAC. Experimental Design: PDAC cell lines derived from primary pancreatic tumors arising spontaneously in KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx-1 Cre mice were implanted into syngeneic mice and tumors were focally irradiated using the Small Animal Radiation Research Platform (SARRP). We determined the impact of depleting T cells and Ly6C+ monocytes as well as inhibiting the chemokine CCL2 on radiotherapy efficacy. Tumors were analyzed by flow cytometry and IHC to detect changes in leukocyte infiltration, tumor viability, and vascularity. Assays were performed on tumor tissues to detect cytokines and gene expression. Results: Ablative radiotherapy alone had minimal impact on PDAC growth but led to a significant increase in CCL2 production by tumor cells and recruitment of Ly6C+CCR2+ monocytes. A neutralizing anti-CCL2 antibody selectively inhibited radiotherapy-dependent recruitment of monocytes/macrophages and delayed tumor growth but only in combination with radiotherapy (P < 0.001). This antitumor effect was associated with decreased tumor proliferation and vascularity. Genetic deletion of CCL2 in PDAC cells also improved radiotherapy efficacy. Conclusions: PDAC responds to radiotherapy by producing CCL2, which recruits Ly6C+CCR2+ monocytes to support tumor proliferation and neovascularization after radiotherapy. Disrupting the CCL2–CCR2 axis in combination with radiotherapy holds promise for improving radiotherapy efficacy in PDAC. Clin Cancer Res; 23(1); 137–48. ©2016 AACR.

IFNγ and CCL2 Cooperate to Redirect Tumor-Infiltrating Monocytes to Degrade Fibrosis and Enhance Chemotherapy Efficacy in Pancreatic Carcinoma

UNLABELLED Dense fibrosis and a robust macrophage infiltrate are key therapeutic barriers in pancreatic ductal adenocarcinoma (PDAC). CD40 activation can circumvent these barriers by inducing macrophages, originating from peripheral blood monocytes, to deplete fibrosis. The precise mechanism and therapeutic implications of this antifibrotic activity, though, remain unclear. Here, we report that IFNγ and CCL2 released systemically in response to a CD40 agonist cooperate to redirect a subset of Ly6C(+)CCR2(+)monocytes/macrophages to infiltrate tumors and deplete fibrosis. Whereas CCL2 is required for Ly6C(+)monocyte/macrophage infiltration, IFNγ is necessary for tumor-infiltrating monocytes/macrophages to shift the profile of matrix metalloproteinases (MMP) in tumors, leading to MMP-dependent fibrosis degradation. In addition, MMP13-dependent loss of extracellular matrix components induced by a CD40 agonist increased PDAC sensitivity to chemotherapy. Our findings demonstrate that fibrosis in PDAC is a bidirectional process that can be rapidly altered by manipulating a subset of tumor-infiltrating monocytes, leading to enhanced chemotherapy efficacy. SIGNIFICANCE We report that CD40 agonists improve chemotherapy efficacy in pancreatic carcinoma by redirecting tumor-infiltrating monocytes/macrophages to induce fibrosis degradation that is dependent on MMPs. These findings provide novel insight into the plasticity of monocytes/macrophages in cancer and their capacity to regulate fibrosis and modulate chemotherapy efficacy in pancreatic carcinoma.

Abstract 2682: IL-6/STAT3 activation in hepatocytes drives the formation of a pro-metastatic niche in the liver during pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer-related deaths in the United States with metastasis to the liver as the major cause of mortality. While the propensity of PDAC to spread to the liver may reflect mechanical trapping of tumor cells that enter the portal circulation, primary tumor cells have also been suggested to secrete factors that may promote recruitment of myeloid cells to establish a pro-metastatic niche. In this study, we used the LSL-Kras G12D/+ ;LSL-Trp53 R127H/+ ;Pdx-1-Cre (KPC) mouse model of PDAC to investigate the impact of PDAC development on the formation of a pro-metastatic niche in the liver. We found that KPC mice (compared to age- and gender-matched control mice) demonstrated an increased susceptibility to tumor seeding in the liver even prior to development of invasive PDAC. Examination of the liver of KPC mice revealed diffuse activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling, particularly in hepatocytes. Although hepatocytes are recognized as important regulators of inflammation, their role in establishing a pro-metastatic niche is unknown. To define changes in the liver associated with development of a pro-metastatic niche, we performed QuantSeq analysis on RNA isolated from the liver of KPC versus control PC mice. Our results showed increased transcriptional levels of myeloid chemoattractants, particularly serum amyloid A proteins that are predominantly produced by hepatocytes. Consistent with this finding, we observed an accumulation of F4/80 + and Ly6G + myeloid cells in the liver of KPC mice by immunofluorescence microscopy. We next determined the role of tumor cells in driving cellular activation seen in the liver by establishing intraperitoneal and orthotopic models of PDAC. Using these models, we found that implantation of pancreatic tumor cells induced STAT3 activation in hepatocytes and stimulated F4/80 + and Ly6G + myeloid cell recruitment to the liver. To determine whether cellular activation in the liver was associated with systemic release of soluble factors, we performed parabiotic joining of tumor-implanted mice and control wild type mice, and we found evidence of STAT3 activation and myeloid recruitment to the liver in parabiotic pairs. As interleukin-6 (IL-6) is a key inflammatory cytokine that can activate STAT3 signaling, we hypothesized a role for IL-6 directed STAT3 activation in hepatocytes for development of a pro-metastatic niche in the liver. Consistent with this hypothesis, we found that IL-6 receptor blocking antibodies administered after tumor implantation reduced STAT3 activation in hepatocytes and decreased transcriptional levels of hepatocyte-derived chemoattractants. Together, our findings support a role for IL-6/STAT3 signaling in hepatocytes in driving a pro-metastatic niche in the liver during PDAC development. Citation Format: Jae W. Lee, Paige M. Porrett, Chad A. Komar, Whitney L. Gladney, Gregory L. Beatty. IL-6/STAT3 activation in hepatocytes drives the formation of a pro-metastatic niche in the liver during pancreatic tumorigenesis [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 2682. doi:10.1158/1538-7445.AM2017-2682

Abstract 3168: A role for tumor-derived CCL2 in directing leukocyte infiltration and stromal heterogeneity in pancreatic ductal adenocarcinoma

Tumor heterogeneity within solid tumors is a recognized barrier to the efficacy of standard therapies. In pancreatic ductal adenocarcinoma (PDAC) the microenvironment is characterized by varying degrees of cytokine/chemokine production, stromal extracellular matrix, and immune cell infiltrates (Hezel et al. 2006). To investigate stromal phenotypical differences in PDAC, we have used the KrasG12D-; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model which recapitulates tumor heterogeneity seen in human PDAC (Hingorani et al. 2005). Gross histological analysis of KPC tumors allowed us to stratify tumors into 2 broad groups: stromal and non-stromal. We found that stromal tumors were comprised of a significant macrophage infiltrate, whereas non-stromal tumors were generally void of macrophages. We modeled this stromal heterogeneity using tumor cell lines derived from KPC tumors. We found that, when implanted under the skin of wild-type mice, some cell lines produced stromal tumors rich in macrophages, while others formed non-stromal tumors with few macrophages within the microenvironment. Supernatant cultured from these KPC tumor cell lines in vitro revealed elevated CCL2 production, specifically in cell lines from stromal tumors. As CCL2 is a known chemo-attractant for monocytes, we hypothesized that tumor-derived CCL2 contributes to monocyte recruitment and stromal heterogeneity within the tumor microenvironment. To test this hypothesis, we have used an in vitro trans-well migration assay. With this assay, we found that more bone marrow derived cells migrate toward supernatant from stromal tumors relative to non-stromal tumors. This finding was lost in the presence of a CCL2 inhibitor. Ongoing studies are examining a role for tumor-derived CCL2 in regulating tumor-associated stroma in PDAC. Our findings suggest the importance of tumor-immune cell interactions in defining stromal heterogeneity and may offer insight into mechanisms that regulate the stromal compartment in human PDAC. Citation Format: Graham M. Tooker, Whitney L. Gladney, Gregory Beatty. A role for tumor-derived CCL2 in directing leukocyte infiltration and stromal heterogeneity in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3168. doi:10.1158/1538-7445.AM2015-3168

Abstract A54: Ly6Chi inflammatory monocytes mediate tumor regression in a mouse model of spontaneously arising pancreatic ductal adenocarcinoma

Macrophage infiltration into solid malignancies most often portends a poor prognosis. In pancreatic ductal adenocarcinoma (PDAC), macrophages dominate the leukocyte infiltrate with effector T lymphocytes remaining scarce. Strategies to restore productive immunosurveillance in PDAC have largely focused on inducing anti-tumor T cell immunity. However, we have previously shown in patients and a mouse model of PDAC that immune modulation with a CD40 agonist can induce macrophages to facilitate tumor regression. Thus, strategies that restore innate immunosurveillance may hold promise in the treatment of PDAC. To further investigate the role of macrophages as targets for anti-tumor immunotherapy, we utilized the KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model of PDAC. We previously reported a role for liposomal targeted macrophages in facilitating tumor regression induced with CD40 therapy. However, unexpectedly we found by immunohistochemistry that macrophages targeted by liposomes do not infiltrate PDAC tumors in response to CD40 therapy. This finding suggested a role for additional leukocyte subsets in mediating the anti-tumor effect. Using immunohistochemistry, we found that systemic CD40 activation induced the infiltration of Ly6C+ myeloid cells into KPC tumors. Based on this finding, we hypothesized that macrophages may regulate the infiltration of Ly6Chi inflammatory monocytes, which are the critical effector myeloid cells mobilized in the peripheral blood by CD40 agonist therapy. In support of this hypothesis, we found that tumor regression induced with a CD40 agonist was abrogated by treatment with the anti-Ly6C/Ly6G RB6-8C5 depleting antibody. Flow cytometric analysis of peripheral blood revealed that RB6-8C5 and liposomes target distinct myeloid subsets. Whereas RB6-8C5 was found to deplete Ly6ChiCCR2+ inflammatory monocytes, CEL depleted Ly6Cneg myeloid cells. Our findings suggest that CD40-induced tumor regression in PDAC is mediated by inflammatory monocytes which infiltrate tumor lesions under the regulation of extra-tumoral macrophages. Citation Format: Whitney L. Gladney, Graham M. Tooker, Gregory L. Beatty. Ly6Chi inflammatory monocytes mediate tumor regression in a mouse model of spontaneously arising pancreatic ductal adenocarcinoma. [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 A54.