Purpose Translation of the PDX model into a method for practical personalized cancer treatment is prevented by the intense resources and time necessary to generate and test each tumorgraft. We aimed to develop a high-throughput ex vivo drug testing approach that can be used for personalized cancer treatment design. Experimental Design We developed a unique ex vivo live tissue sensitivity assay (LTSA), in which precision-cut and uniform small tissue slices derived from pancreatic ductal adenocarcinoma PDX tumors were arrayed in a 96-well plate and screened against clinically relevant regimens within 3-5 days. The correlation between the sensitivities of tissue slices to the regimens and patients’ clinical responses and outcome were statistically analyzed. The results of LTSA assay were further confirmed with biochemical methods in vitro and animal PDX model in vivo. Results The ex vivo tissue slices remain viable for at least 5 days, and the tumor parenchyma, including stroma, vascular structures, and signaling pathways, are all retained. The sensitivities of the ex vivo tissue slices to gemcitabine and irinotecan was consistent with the clinical responses and outcomes of the patients from whom the tumorgrafts were derived (r=0.77; p=0.0002). Retrospective analysis showed that the patients who received LTSA sensitive regimens had remarkably longer progression free survival than patients who received LTSA resistant regimens (16.33 vs 3.8 months, n=18, p=0.011) Conclusions The results from these PDX and LTSA methods reflect clinical patients’ responses and could be used as a personalized strategy for improving systemic therapy effectiveness in pancreatic cancer patients.

Running title: CT-based subtypes of pancreatic ductal adenocarcinoma. Keywords Statement of translational relevanceThere are currently limited methods to stratify patients with pancreatic ductal adenocarcinoma (PDAC) into prognostic groups based on disease biology. We focused on the morphological features of the disease on computed tomography (CT) scans and correlated these features with genomic, pathological, and clinical data. We found that tumors with a distinct interface in relation to the surrounding parenchyma (called high delta tumors) had more aggressive biological features than tumors without a distinct interface (called low delta tumors). Patients with high delta tumors were more likely to develop early distant metastasis and die faster than those with low delta tumors. Mathematical modeling suggested that stromal elements strongly influenced the morphological patterns seen on CT scans. These findings indicate that a universally available diagnostic test can be used to interrogate the biology of this deadly disease, providing a means to stratify patients at diagnosis and aiding the design of future clinical trials.

Introduction Patient-derived xenografts (PDX) are being increasingly utilized in preclinical oncologic research. Maintaining large colonies of early generation tumor-bearing mice is impractical and cost-prohibitive. Optimal methods for efficient long-term cryopreservation and subsequent reanimation of PDX tumors are critical to any viable PDX program. We sought to compare the performance of “Standard” and “Specialized” cryoprotectant media on various cryopreservation and reanimation outcomes in PDX tumors. Standard (10% DMSO media) and Specialized (Cryostor®) media were compared between overall and matched PDX tumors. Primary outcome was reanimation engraftment efficiency (REE). Secondary outcomes included time-to-tumor-formation (TTF), time-to-harvest (TTH), and potential loss of unique PDX lines. Overall 57 unique PDX tumors underwent 484 reanimation engraftment attempts after previous cryopreservation. There were 10 unique PDX tumors cryopreserved with Standard (71 attempts), 40 with Specialized (272 attempts), and 7 with both (141 attempts). Median frozen time of reanimated tumors was 29 weeks (max.177). Tumor pathology, original primary PDX growth rates, frozen storage times, and number of implantations per PDX model were similar between cryoprotectant groups. Specialized media resulted in superior REE (overall: 82% vs. 39%, p<0.0001; matched: 97% vs. 36%, p<0.0001; >52 weeks cryostorage: 59% vs. 9%, p<0.0001), shorter TTF (overall 24 vs. 54 days, p=0.0051; matched 18 vs. 53 days, p=0.0013) and shorter TTH (overall: 64 vs. 89 days, p=0.009; matched: 47 vs. 88 days, p=0.0005) compared to Standard. Specialized media demonstrated improved REE with extended duration cryostorage (p=0.048) compared to Standard. Potential loss of unique PDX lines was lower with Specialized media (9% vs. 35%, p=0.017). In conclusion, cryopreservation with a specialized cryoprotectant appears superior to traditional laboratory-based media and can be performed with reliable reanimation even after extended cryostorage.

Purpose Pancreatic ductal adenocarcinoma (PDAC) is lethal cancer whose primary tumor is characterized by dense composition of cancer cells, stromal cells and extracellular matrix (ECM) composed largely of collagen. Within the PDAC tumor microenvironment, activated pancreatic stellate cells (PSC) are the dominant stromal cell type and responsible for collagen deposition. Lumican is a secreted proteoglycan that regulates collagen fibril assembly. We have previously identified that the presence of lumican in the ECM surrounding PDAC cells is associated with improved patient outcome after multimodal therapy and surgical removal of localized PDAC. Experimental Design Lumican expression in PDAC from 27 patients was determined by IHC and quantitatively analyzed for co-localization with PSC. In vitro studies examined the molecular mechanisms of lumican transcription and secretion from PSC (HPSC, HPaSteC), and cell adhesion and migration assays examined the effect of lumican on PSC in a collagen-rich environment. Results Here we identify PSC as a significant source of extracellular lumican production through quantitative IHC analysis. We demonstrate that the cytokine, transforming growth factor-β (TGF-β), negatively regulates lumican gene transcription within HPSC through its canonical signaling pathway and binding of SMAD4 to novel SBEs identified within the promoter region. Additionally, we found that the ability of HPSC to produce and secrete extracellular lumican significantly enhances HPSC adhesion and mobility on collagen. Conclusion Our results demonstrate that activated pancreatic stellate cells within PDAC secrete lumican under the negative control of TGF-β; once secreted, the extracellular lumican enhances stellate cell adhesion and mobility in a collagen rich environment.

PURPOSE The combination chemotherapy of fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) has provided clinically meaningful improvement for pancreatic ductal adenocarcinoma (PDAC). We previously uncovered a role for the serine hydrolase carboxylesterase 2 (CES2) in mediating intratumoral activation of the prodrug irinotecan, a constituent of FOLFIRINOX. We aimed to further test the predictive value of CES2 for response to irinotecan using patient-derived xenograft (PDX) models and to elucidate the determinants of CES2 expression and response to FOLFIRINOX treatment among patients with PDAC. METHODS PDXs were engrafted subcutaneously into nude mice and treated for 4 weeks with either saline control or irinotecan. CES2 and hepatocyte nuclear factor 4 alpha (HNF4A) expression in PDAC tissues was evaluated by immunohistochemical and Western blot analysis. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and hemoglobin A1C (HbA1C) levels in patients who underwent neoadjuvant FOLFIRINOX treatment. RESULTS High CES2 activity in PDAC PDXs was associated with increased sensitivity to irinotecan. Integrated gene expression, proteomic analyses, and in vitro genetic experiments revealed that nuclear receptor HNF4A, which is upregulated in diabetes, is the upstream transcriptional regulator of CES2 expression. Elevated CES2 protein expression in PDAC tissues was positively associated with a history of type 2 diabetes (odds ratio, 4.84; P = .02). High HbA1C levels were associated with longer overall survival in patients who received neoadjuvant FOLFIRINOX treatment ( P = .04). CONCLUSION To our knowledge, we provide, for the first time, evidence that CES2 expression is associated with a history of type 2 diabetes in PDAC and that elevated HbA1C, by predicting tumor CES2 expression, may represent a novel marker for stratifying patients most likely to respond to FOLFIRINOX therapy.