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Supplementary Table 1. Percentage of CA-MSCs (CD44+CD73+CD49α+ CD90+) from primary human pancreatic cancer derived CAF cultures and accompanying clinical data. Supplementary Table 2. Extent of local invasion, lung tumor emboli, and liver metastasis in tumors that were derived from injections of tumor cells alone, tumor cells + CAF, and tumor cells + CA-MSCs (n=8 per group) at six weeks post implantation. Supplementary Table 3. Percentage of CD10 expressing CA-MSCs from primary human pancreatic cancer derived CAF cultures.

Supplementary Figure 2. Pancreatic cancer associated MSCs promote tumor cell growth and invasion. A. GFP-labeled tumor cells were grown alone or co-cultured with CA-MSCs or CAFs. GFP-labeled tumor cells were grown in growth media, in combination with conditioned media (CM) from CA-MSCs and CM from CAFs. Increased tumor cell numbers when co-cultured with CA-MSC (A) or CA-MSC CM (A) demonstrate the ability of CA-MSCs to induce proliferation in the cancer cells. *p

Supplementary Figure 7. A. Sections from orthotopic pancreatic tumors from control shRNA- MSC vs GM-CSF-shRNA-MSC were analyzed for expression of F4/80, and Arg1 by immunohistochemical and immunofluorescence staining. B. Quantitation of F4/80 positively labeled cells was performed by light microscopy at an optical magnification of 400Ã-, (n=5 animals per group *p

Supplementary Figure 5. Representative Western blot demonstrating the expression of GM-CSF receptors CSF2Rα and CSF2Rβ in primary human pancreatic cancer cells.

Supplementary Figure 6. Pancreatic CA-MSCs promote tumor sphere formation. GFP-labeled tumor cells were grown in growth media, in combination with CM from CA-MSCs and CM from CAFs or co-cultured with CA-MSCs or CAFs (red). Enhanced size of tumor spheres when tumor cells were grown with CA-MSCs in sphere forming media (*p

Supplementary Figure 1. A. FACS analysis of freshly digested pancreatic tumor tissue from two different PDA patients (left) and outgrown CAFs from matching patients cultured using the outgrowth method for 2 weeks (right), demonstrating the percentage of CA-MSCs expressing CD44+ CD73+ CD90+ CD49α+ markers. B. ELISA data quantifying amount of GM-CSF secreted by freshly isolated CA-MSCs and cultured CA-MSCs. C. Sorted single CA-MSCs demonstrate multipotent differentiation capacity in differential culture conditions. Specific cell stains used were Alizarin Red for bone, Oil Red O for adipose and Alcian Blue for cartilage.

Supplementary Figure 4. Differential secretion of cytokines by CA-MSC and CAFs from four different patients.

Supplementary Figure 3. Pancreatic CA-MSCs promote tumor growth. A. BXPC-3 (wild type KRAS) pancreatic cancer cells (5 x 104) transduced with a luciferase-expressing lentivirus were orthotopically injected alone or with same number of CAF or CA-MSC into the pancreatic tail of NOD/SCID mice (n=4 per group). Representative bioluminescent images of animals in each group are shown at 4 weeks after injection depicting tumor growth. B. Bioluminescent-based tumor growth (measured by signal intensity p/s/cm2) of luciferase tumors alone or in combination with CAFs or CA-MSCs (MSCs). Pooled results of the average tumor size {plus minus} SEM from each group. *p

Pancreatic ductal adenocarcinoma (PDA) is characterized by a dense stroma consisting of a prevalence of activated fibroblasts whose functional contributions to pancreatic tumorigenesis remain incompletely understood. In this study, we provide the first identification and characterization of mesenchymal stem cells (MSC) within the human PDA microenvironment, highlighting the heterogeneity of the fibroblast population. Primary patient PDA samples and low-passage human pancreatic cancer–associated fibroblast cultures were found to contain a unique population of cancer-associated MSCs (CA-MSC). CA-MSCs markedly enhanced the growth, invasion, and metastatic potential of PDA cancer cells. CA-MSCs secreted the cytokine GM-CSF that was required for tumor cell proliferation, invasion, and transendothelial migration. Depletion of GM-CSF in CA-MSCs inhibited the ability of these cells to promote tumor cell growth and metastasis. Together, these data identify a population of MSCs within the tumor microenvironment that possesses a unique ability, through GM-CSF signaling, to promote PDA survival and metastasis. Significance: The role of stroma in pancreatic cancer is controversial. Here, we provide the first characterization of MSCs within the human PDA microenvironment and demonstrate that CA-MSCs promote tumorigenesis through the production of GM-CSF. These data identify a novel cytokine pathway that mediates mesenchymal–epithelial cross-talk and is amenable to therapeutic intervention. Cancer Discov; 6(8); 886–99. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 803

Purpose of review This review intends to describe recent studies on pancreatic tumor-associated stroma and potential opportunities and limitations to its targeting. Recent findings One of the defining features of pancreatic cancer is extensive desmoplasia, or an inflammatory, fibrotic reaction. Carcinoma cells live in this complex microenvironment which is comprised of extracellular matrix (ECM), diffusible growth factors, cytokines and a variety of nonepithelial cell types including endothelial cells, immune cells, fibroblasts, myofibroblasts and stellate cells. In addition to the heterogeneity noted in the nonneoplastic cells within the tumor microenvironment, it has also been recognized that neoplastic cancer cells themselves are heterogeneous, and include a subpopulation of stem-cell like cells within tumors termed cancer stem cells. Due to the failure of current therapeutics to improve outcomes in patients with pancreatic cancer, new therapeutic avenues targeting different components of the tumor microenvironment are being investigated. In this review article, we will focus on recent studies regarding the function of the tumor stroma in pancreatic cancer and therapeutic treatments that are being advanced to target the stroma as a critical part of tumor management. Summary Recent studies have shed new light on the contribution of the pancreatic cancer fibroinflammatory stroma to pancreatic cancer biology. Additional studies are needed to better define its full contribution to tumor behavior and how to best understand the optimal ways to develop therapies that counteract its pro-neoplastic properties.

Background and Rationale: Pancreatic ductal adenocarcinoma (PDA) is one of the most stroma-rich cancers that exists. It is hypothesized that stroma contributes to the aggressive nature of PDA. There is strong evidence that mesenchymal stem cells (MSC) are recruited as part of the stroma to the tumor microenvironment. Studies in solid cancers of various organs including lung, stomach and ovaries have reported that MSCs are present in stroma within the cancer tissue and impact tumor biology. However, no published reports on role of MSC in PDA exist, therefore an opportunity exists to identify and functionally characterize MSC in pancreatic cancer and understand their contribution to pancreatic cancer biology. Experimental Design: Human pancreatic cancer associated fibroblasts (CAFs) from patient tumors (n=18) were isolated and grown in low passage cultures. The mesenchymal lineage of the CAF cultures was confirmed by KRAS mutational analysis of both the CAF cells and matching neoplastic epithelial cells. The purity of CAF cultures was also confirmed by immunostaining for immune (CD45) and epithelial (CK19) cell markers. An orthotopic model was established in which 105 GFP labeled tumor cells were mixed with either 105 DsRed labeled CAF or CAF-MSC cells and implanted into the pancreata of NOD-SCID mice (n= 6 mice per group). GFP labeled tumor cells and DsRed CAF/CAF-MSC cells injected alone served as the experimental controls. Growth kinetics and tumor metastasis between different groups were studied using bioluminescence animal imaging and weekly tumor monitoring. Once tumors formed, mice were sacrificed and tumor burden quantified. Blood was collected for analysis of circulating GFP+ tumor cells and RFP+ CAF or CAF-MSC cells. Protein cytokine arrays were performed to identify differentially expressed genes by CAF-MSCs. Results: Primary human CAF cultures were heterogeneous and each contained a unique population of MSCs, as defined by expression of MSC markers (CD90, CD49a, CD73 and CD44), the functional capacity to differentiate into bone, cartilage and adipose cells, and the ability to form colonies when plated at low densities. We observed that CAF-MSCs promoted tumor growth and metastasis to a significantly greater degree than bulk CAF cells, and only CAF-MSCs cells were capable of entering into the circulation. Using human protein cytokine arrays to explore the paracrine factors selectively secreted by CAF-MSC, we found that the cytokine GM-CSF was secreted selectively by CAF-MSCs and not CAFs (n=4). GM-CSF knockdown in CAF-MSCs using targeted shRNA compared to control shRNA resulted in a significant decrease in the development of metastasis in co-injection experiments with primary pancreatic cancer cells using an orthotopic model. Conclusions: Here we provide the first characterization of MSCs within the human PDA microenvironment. We find PDAs have a unique population of MSCs which confer specific biologic properties to PDA. These studies will help us to define important target molecules within the tumor microenvironment involved in key tumor-stromal interactions that may impact therapeutic approaches for patients. This abstract is also presented as Poster B2. Citation Format: Meghna Waghray, Guan Grace, Malica Yalamanchili, Lidong Wang, Michele Dziubinski, Mina Zeinali, Sunitha Nagrath, Diane Simeone. Mesenchymal stem cells in pancreatic cancer possess unique properties in promoting tumor growth and metastasis. [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 PR03.

Background Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma. Methods We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing. Results Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue. Conclusions Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.