Abstract The microenvironment where a tumor originates plays an important role in its initiation, growth, progression and metastatic capability. Since in most cancers the microenvironment is not derived from the malignant clone and does not contain oncogenic mutations, it is likely that the tumor microenvironment is reprogrammed epigenetically to support the growth of the tumor. To test this hypothesis, Fibroblasts associated with primary pancreatic adenocarcinomas (N=7) & healthy fibroblast controls (N=4) were analyzed for genome wide alterations in DNA cytosine methylation by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR). Methylome profiling revealed widespread aberrant cytosine methylation in Pancreatic cancer associated fibroblasts (CAFs) with demethylation of many important gene promoters as the predominant epigenetic event. In addition to loss of methylation, aberrant hypermethylation of GALNTS, JAZF1, MTCH1, SP8, GRB14 was also seen in CAFs. In addition to epigenetic differences, pancreatic CAFs were also found to have widespread transcriptmoic differences as seen by parallel gene expression profiling. Next, we analyzed tumor mediated reprogramming of microenvironment at a higher resolution by a Massive parallel sequencing based methylation analysis (HELP-Tagging) on CAFs differentiated from mesenchymal stem cells (MSCs) in the presence of pancreatic cancer cell conditioned medium. HELP-tagging showed widespread epigenetic reprogramming with 11,100 hypomethylated and 1709 hypermethylated loci. Comparison of in-vitro reprogramed loci with the aberrantly methylated loci from primary CAFs showed a core set of 140 loci that were commonly differentially methylated in both samples. The chemokine receptor CXCR4 was observed to overexpressed & demethylated in both cohorts & was found to be expressed on the surface of primary pancreatic CAFs by immunohistochemistry. Functional studies demonstrated that co-culture of pancreatic cancer cells with CAFs (from MSCs) led to significant increase in malignant cell invasion when compared to co-culture with naïve MSCs. This increased invasion was abrogated by blockade of CXCR4 by AMD-3100 and by knockdown of CXCR4 by siRNAs in orthotopically derived CAFs; demonstrating a critical role for this receptor in regulating tumor promoting abilities of the microenvironment. Thus our results reveal for the first time that pancreatic CAFs are characterized by widespread epigenomic reprogramming that includes loss of methylation at many important loci. Validation of an aberrantly demethylated target, CXCR4, shows that inhibition of this receptor can abrogate the ability of CAFs in promoting cancer cell invasion. These results also provide a preclinical rationale for the use of clinically relevant CXCR4 antagonist AMD-3100 (plerixafor) in pancreatic cancer. Citation Format: Tushar D. Bhagat, Yanique Rattigan, Brijesh Patel, Strepell Mirte, Yiting Yu, Davendra Sohal, Matthias Bartenstein, Orsolya Giricz, Shanisha AK Gordon, Nishanth Vallumsetla, Rahul Polineni, Meher Walia, Paraic Kenny, John Greally, Debabrata Banerjee, Anirban Maitra, Amit Verma. Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2013-1504