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Figures S1-4 and Table S1 Figure S1. N-CAFs and R-CAFs were exposed to the indicated concentrations of gemcitabine for 72h, and viability was measured via MTT assay. 3 biologic replicates were included in the experiment. **p{less than or equal to}0.01, **** p{less than or equal to}0.0001 Figure S2. MiaPaCa-2 cells (1 X 105) were subcutaneously injected with N-CAF cells expressing GFP (3 X 105) in NSG mice. At 6 weeks, tumors were harvested and fluorescence microscopy was performed. Representative images of H&E stained sections as well as fluorescence microscopy are displayed. Only occasional GFP+ CAFs were present after 6 weeks. Figure S3. Primary CAFs were treated with 1µM gemcitabine plus DMSO or the above listed small molecule inhibitors for 96h and SASP mediator expression was compared to untreated cells via qRT-PCR. Because of limited primary cell numbers, the experiment was performed once in multiple cell lines rather than being replicated in any single line. Drug concentrations and targets: SB203580 10µM- p38 MAPK, SP600125 20µM- JNK, BEZ235 1µM- ATM/ATR/DNA-PK/PI3K/mTOR, AZD77622 200nM- Chk1/Chk2, VE821 2µM- ATR, KU55933 20µM- ATM. Figure S4. Combined data from Figure S3. The mean normalized expression of each marker from CAF1-3 is displayed. In this combined analysis, none of the changes reached statistical significance.

Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naïve CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF– or R-CAF–conditioned media and assayed for migration, invasion, and viability in vitro. Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo. R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo, TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo. These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens.Implications: Chemotherapy treatment of pancreatic cancer–associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437–47. ©2016 AACR.

Supplementary Figures S1-S5. Figure S1. MTT assays of CBL knockdown versus isogenic parental PDAC cell lines in serum-supplemented media. Figure S2. Immunoblots of L3.6plCBL siRNA cells versus control highlights autoactivation of EGFR and downstream pAKT when treated with chemotherapy. Figure S3. Chemiluminescent images of a phospho-receptor tyrosine kinase array performed in chemotherapy-treated L3.6plCBL siRNA cells versus control. Figure S4. qRT-PCR, immunoblots and photographs of L3.6plCBL shRNA and Panc-1CBL shRNA tumors versus controls verifies stable gene knockdown and gross similarities between tumors prior to the start of chemotherapy. Figure S5. Photographs of L3.6pl subcutaneous xenografts in NOD-scid-IL2Rγnull mice at the time of necropsy reveals qualitatively that chemoresistance to gemcitabine induced by CBL knockdown is abrogated by the addition of erlotinib.

Purpose: Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDAC) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC.Experimental Design: Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, and AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2Rγnull mice were subcutaneously implanted with PDAC or PDACCBL-low cells on opposite flanks and treated with gemcitabine ± erlotinib for ≥4 weeks.Results: There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-fluorouracil (5-FU) by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDACCBL-low cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib+gemcitabine–treated PDACCBL-low cells exhibited greater apoptosis by cleaved PARP, caspase-3, and Annexin V/PI.Conclusions: Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment. Clin Cancer Res; 21(1); 157–65. ©2014 AACR.

Lung resections are associated with a variety of potential postoperative complications. Not surprisingly, pulmonary complications are most frequent after lung surgery. Cardiac and thromboembolic complications are also important. It is essential that surgeons anticipate the possibility of these complications and take preventative measures whenever possible. When complications do occur, prompt recognition and treatment is required to assure optimal patient outcomes.

Importance The utilization of robot-assisted minimally invasive esophagectomy (RAMIE) for esophageal cancer is increasing, despite limited data comparing RAMIE with other surgical approaches. Objective To evaluate the literature for clinical outcomes of RAMIE compared with video-assisted minimally invasive esophagectomy (VAMIE) and open esophagectomy (OE). Data sources A systematic search of PubMed, Cochrane, Ovid Medline, and Embase databases from January 1, 2013, to May 6, 2020, was performed. Study selection Studies that compared RAMIE with VAMIE and/or OE for cancer were included. Data extraction and synthesis Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guideline, data were extracted by independent reviewers. A random-effects meta-analysis of 9 propensity-matched studies was performed for the RAMIE vs VAMIE comparison only. A narrative synthesis of RAMIE vs VAMIE and OE was performed. Main outcomes and measures The outcomes of interest were intraoperative outcomes (ie, estimated blood loss [EBL], operative time, lymph node [LN] harvest), short-term outcomes (anastomotic leak, recurrent laryngeal nerve [RLN] palsy, pulmonary and total complications, and 90-day mortality), and long-term oncologic outcomes. Results Overall, 21 studies (2 randomized clinical trials, 11 propensity-matched studies, and 8 unmatched studies) with 9355 patients were included. A meta-analysis was performed with 9 propensity-matched studies comparing RAMIE with VAMIE. The random-effects pooled estimate found an adjusted risk difference (RD) of -0.06 (95% CI, -0.11 to -0.01) favoring fewer pulmonary complications with RAMIE. There was no evidence of differences between RAMIE and VAMIE in LN harvest (mean difference [MD], -1.1 LN; 95% CI, -2.45 to 0.25 LNs), anastomotic leak (RD, 0.0; 95% CI, -0.03 to 0.03), EBL (MD, -6.25 mL; 95% CI, -18.26 to 5.77 mL), RLN palsy (RD, 0.01; 95% CI, -0.08 to 0.10), total complications (RD, 0.05; 95% CI, -0.01 to 0.11), or 90-day mortality (RD, -0.01; 95% CI, -0.02 to 0.0). There was low certainty of evidence that RAMIE was associated with a longer disease-free survival compared with VAMIE. For OE comparisons (data not pooled), RAMIE was associated with a longer operative time, decreased EBL, and less pulmonary and total complications. Conclusions and relevance In this study, RAMIE had similar outcomes as VAMIE but was associated with fewer pulmonary complications compared with VAMIE and OE. Studies on long-term functional and cancer outcomes are needed.

Historically, the mainstay of treatment for early-stage lung cancer has been anatomic resection via thoracotomy. Over the last two decades, there has been increasing adoption of video-assisted thoracoscopic surgery (VATS) techniques in this group of patients. VATS resection has been shown to reduce perioperative morbidity and hospital length of stay in multiple series (1-8). Perioperative mortality has also been shown to be lower or equivalent (1-7). While data is limited, several small studies have demonstrated similar survival and recurrence rates with VATS compared to open resection for early-stage lung cancer (9-11).

// Andrew H. Nguyen 1 , Irmina A. Elliott 1 , Nanping Wu 1 , Cynthia Matsumura 1 , Maria Vogelauer 2 , Narsis Attar 2 , Amanda Dann 1 , Razmik Ghukasyan 1 , Paul A. Toste 1 , Sanjeet G. Patel 1 , Jennifer L. Williams 3 , Luyi Li 1 , David W. Dawson 4 , Caius Radu 5 , Siavash K. Kurdistani 2 , Timothy R. Donahue 1, 5 1 Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 2 Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 3 Department of Surgery, Harbor-UCLA Medical Center, Torrance, California, USA 4 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 5 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA Correspondence to: Timothy R. Donahue, email: tdonahue@mednet.ucla.edu Keywords: histone deacetylase inhibitor, SAHA, AP-1, pancreatic cancer, cancer-associated fibroblasts Received: October 04, 2016 Accepted: November 07, 2016 Published: November 24, 2016 ABSTRACT Although histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs, thus far, they have been unsuccessful in early phase clinical trials for pancreatic ductal adenocarcinoma (PDAC). One potential reason for their poor efficacy is the tumor stroma, where cancer-associated fibroblasts (CAFs) are a prominent cell type and a source of resistance to cancer therapies. Here, we demonstrate that stromal fibroblasts contribute to the poor efficacy of HDACi’s in PDAC. HDACi-treated fibroblasts show increased biological aggressiveness and are characterized by increased secretion of pro-inflammatory tumor-supportive cytokines and chemokines. We find that HDAC2 binds to the enhancer and promoter regions of pro-inflammatory genes specifically in CAFs and in silico analysis identified AP-1 to be the most frequently associated transcription factor bound in these regions. Pharmacologic inhibition of pathways upstream of AP-1 suppresses the HDACi-induced inflammatory gene expression and tumor-supportive responses in fibroblasts. Our findings demonstrate that the combination of HDACi’s with chemical inhibitors of the AP-1 signaling pathway attenuate the inflammatory phenotype of fibroblasts and may improve the efficacy of HDACi in PDAC and, potentially, in other solid tumors rich in stroma.

BackgroundPatients with early-stage pancreatic neuroendocrine tumors (PNETs) may develop metastatic recurrences despite undergoing potentially curative pancreas resections. We sought to identify factors predictive of metastatic recurrences and develop a prognostication strategy to predict recurrence-free survival (RFS) in resected PNETs.MethodsPatients with localized PNETs undergoing surgical resection between 1989 and 2015 were identified. Univariate and multivariate analysis were used to identify potential predictors of post-resection metastasis. A score-based prognostication system was devised using the identified factors. The bootstrap model validation methodology was utilized to estimate the external validity of the proposed prognostication strategy.ResultsOf the 140 patients with completely resected early-stage PNETs, overall 5- and 10-year RFS were 84.6% and 67.1%, respectively. The median follow-up was 56months. Multivariate analysis identified tumor size > 5cm, Ki-67 index 8-20%, lymph node involvement, and high histologic grade (G3, or Ki-67 > 20%) as independent predictors of post-resection metastatic recurrence. A scoring system based on these factors stratified patients into three prognostic categories with distinct 5-year RFS: 96.9%, 54.8%, and 33.3% (P

BackgroundThe 2012 Sendai Criteria recommend that patients with 3 cm or larger branch duct intraductal papillary mucinous neoplasms (BD-IPMN) without any additional "worrisome features" or "high-risk stigmata" may undergo close observation. Furthermore, endoscopic ultrasound (EUS) is not recommended for BD-IPMN 3 cm were removed based on size alone.Discussion/conclusionsOur results suggest that "larger" size on noninvasive imaging can indicate high-grade/invasive cysts, and EUS-FNA may help identify "smaller" cysts with high-grade/invasive pathology.

Pancreatic cysts are a group of lesions with heterogeneous malignant potential. Currently, there are no reliable biomarkers to aid in cyst diagnosis and classification. The objective of this study was to identify potential microRNA (miR) biomarkers in endoscopically acquired pancreatic cyst fluid that could be used to distinguish between benign, premalignant, and malignant cysts.A list of candidate miRs was developed using a whole-genome expression array analysis of pancreatic cancer (pancreatic ductal adenocarcinoma) and nonmalignant samples overlapped with existing literature and predicted gene targets. Endoscopically acquired pancreatic cyst fluid samples were obtained from a group of 38 patients who underwent cyst fluid aspiration and surgical resection. Selected miR expression levels in cyst fluid samples were assessed by quantitative real-time-PCR. Additionally, in situ hybridization (ISH) on corresponding cyst tissue samples was performed to identify the source and validate the expression level of fluid miRs.Of the six miRs that were profiled in the study, two showed differential expression in malignant cysts. miR-221 was expressed at significantly higher levels in malignant cysts compared with benign or premalignant cysts (P=0.05). miR-21 was also expressed at significantly higher levels in malignant cysts (P0.01). Additionally, the expression of miR-21 was significantly higher in premalignant cysts than benign cysts (P=0.03). The differential expression of miR-21 among cyst categories was confirmed by ISH.In this small single-center study, miRs are potential pancreatic cyst fluid diagnostic biomarkers. In particular, miR-21 is identified as a candidate biomarker to distinguish between benign, premalignant, and malignant cysts. Additionally miR-221 may be of use in the identification of more advanced malignant disease.

Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naive CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF– or R-CAF–conditioned media and assayed for migration, invasion, and viability in vitro . Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo . R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo , TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo . These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens. Implications: Chemotherapy treatment of pancreatic cancer–associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437–47. ©2016 AACR . This article is featured in Highlights of This Issue, [p. 409][1] [1]: /lookup/volpage/14/409?iss=5

BackgroundWe previously identified a correlation between increased expression of the phosphoinositide 3-kinase (PI3K) regulatory subunit p85α and improved survival in human pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the impact of changes in p85α expression on response to chemotherapy and the regulation of p85α by microRNA-21 (miR-21).Materials and methodsPDAC tumor cells overexpressing p85α were generated by viral transduction, and the effect of p85α overexpression on sensitivity to gemcitabine was tested by MTT assay. Primary human PDAC tumors were stained for p85α and miR-21 via immunohistochemistry and in situ hybridization, respectively. Additionally, PDAC cells were treated with miR-21 mimic, and changes in p85α and phospho-AKT were assessed by Western blot. Finally, a luciferase reporter assay system was used to test direct regulation of p85α by miR-21.ResultsHigher p85α expression resulted in increased sensitivity to gemcitabine (P < 0.01), which correlated with decreased PI3K-AKT activation. Human tumors demonstrated an inverse correlation between miR-21 and p85α expression levels (r = -0.353, P < 0.001). In vitro, overexpression of miR-21 resulted in decreased levels of p85α and increased phosphorylation of AKT. Luciferase reporter assays confirmed the direct regulation of p85α by miR-21 (P < 0.01).ConclusionsOur results demonstrate that p85α expression is a determinant of chemosensitivity in PDAC. Additionally, we provide novel evidence that miR-21 can influence PI3K-AKT signaling via its direct regulation of p85α. These data provide insight into potential mechanisms for the known relationship between increased p85α expression and improved survival in PDAC.

Importance Patients with periampullary adenocarcinomas have widely variable survival. These cancers are traditionally categorized by their anatomic location of origin, namely, the duodenum, ampulla, distal common bile duct (CBD), or head of the pancreas. However, they can be alternatively subdivided histopathologically into intestinal or pancreaticobiliary (PB) types, which may more accurately estimate prognosis. Objectives To identify factors associated with survival in patients with periampullary adenocarcinomas and to compare survival between those having intestinal-type or PB-type cancers originating from the duodenum, ampulla, or distal CBD with those having pancreatic ductal adenocarcinoma (PDAC). Design, Setting, and Participants This study was a retrospective analysis of medical records in a prospectively maintained database. Three pathologists separately evaluated histopathologic phenotypes at a university-based tertiary referral center. Study participants were all patients (N = 510) who underwent pancreatoduodenectomy for adenocarcinoma between January 1995 and December 2014. Main Outcome and Measure Overall survival. Results This study identified 510 patients (mean [SD] age, 66.1 [10.9] years; 245 female [48%]) who underwent pancreatoduodenectomy for adenocarcinomas: 13 duodenal, 110 ampullary, 43 distal CBD, and 344 PDAC. The median overall survival was 61.2 (interquartile range [IQR], 22.0-111.0), 70.4 (IQR, 26.7-147.7), 40.6 (IQR, 15.2-59.6), and 31.4 (IQR, 17.3-86.3) months for patients with cancers of the duodenum, ampulla, distal CBD, or pancreas, respectively ( P = .01), indicating a significant difference between the 4 tumor anatomic locations. Most duodenal (61.5% [8 of 13]) and ampullary (51.8% [57 of 110]) cancers were intestinal type, and most distal CBD tumors were PB type (86.0% [37 of 43]). Those with intestinal-type duodenal, ampullary, or distal CBD adenocarcinomas had longer median overall survival than those with PB type (71.7 vs 33.3 months, P = .02) or PDAC (31.4 months, P = .003). There was no survival difference between PB-type cancers and PDAC (33.3 vs 31.4 months, P = .66). On multivariable analysis, histologic grade (hazard ratio [HR], 1.98; 95% CI, 1.56-2.52; P P = .008), and nodal status (HR, 1.45; 95% CI, 1.12-1.87; P = .05) were significantly associated with survival, while anatomic location was not. Conclusions and Relevance Histopathologic phenotype is a better prognosticator of survival in patients with periampullary adenocarcinomas than tumor anatomic location. Those with PB-type duodenal, ampullary, or distal CBD adenocarcinomas have survival similar to those with PDAC.

Purpose: Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDAC) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC. Experimental Design: Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, and AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2Rγnull mice were subcutaneously implanted with PDAC or PDACCBL-low cells on opposite flanks and treated with gemcitabine ± erlotinib for ≥4 weeks. Results: There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-fluorouracil (5-FU) by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDACCBL-low cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib+gemcitabine–treated PDACCBL-low cells exhibited greater apoptosis by cleaved PARP, caspase-3, and Annexin V/PI. Conclusions: Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment. Clin Cancer Res; 21(1); 157–65. ©2014 AACR.

The optimal surgical management of small nonfunctional pancreatic neuroendocrine tumors (NF-PNETs) remains controversial. We sought to identify (1) clinicopathologic factors associated with survival in NF-PNETs and (2) preoperative tumor characteristics that can be used to determine which lesions require resection and lymph node (LN) harvest. The records of all 116 patients who underwent resection for NF-PNETs between 1989 and 2012 were reviewed retrospectively. Preoperative factors, operative data, pathology, surgical morbidity, and survival were analyzed. The overall 5- and 10-year survival rates were 83.9 and 72.8 %, respectively. Negative LNs (p = 0.005), G1 or G2 histology (p = 0.033), and age

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion. METHODS: In-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers. RESULTS: miR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing α-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21. CONCLUSIONS: miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.

Purpose Adaptive growth of the intestinal mucosa in response to massive gut loss is fundamental for autonomy from parenteral nutrition. Although angiogenesis is essential for cellular proliferation in other tissues, its relevance to intestinal adaptation is unknown. We tested the hypothesis that resection-induced adaptation is associated with new blood vessel growth. Methods Male C57Bl/6 mice underwent either a 50% small bowel resection or a sham (transection and reanastomosis) operation. After 1, 3, or 7 days, capillary density within the intestinal villi was measured using confocal microscopy. A messenger RNA reverse-transcriptase polymerase chain reaction (RT-PCR) array was used to determine angiogenic gene expression during adaptation. Results Mice that underwent small bowel resection had a significantly increased capillary density compared to sham-operated mice at postoperative day 7. This morphological alteration was preceded by significant alterations in 5 candidate genes at postoperative day 3. Conclusion New vessel blood growth is observed in the adapting intestine after massive small bowel loss. This response appears to follow rather than initiate the adaptive alterations in mucosal morphology that are characteristic of adaptation. A better understanding of this progress and the signaling factors involved may improve therapeutic options for children with short gut syndrome.

Introduction: The dense tumor-associated stroma (TAS) characteristic of pancreatic ductal adenocarcinoma (PDAC) plays an important role in disease progression and treatment resistance. PDAC tumor-associated fibroblasts (TAFs), the predominant cell type of the TAS, enhance tumor growth, invasion, metastasis, and chemoresistance. However, the impact of cytotoxic chemotherapy on TAF biology has not yet been reported. Methods: Immortalized human TAFs were grown in either standard media or media containing IC50 doses of gemcitabine (GEM) for 4 weeks to generate chemotherapy-naïve and chemotherapy-resistant lines. Conditioned media (CM) was produced from both naïve and resistant TAFs. MTT assay was used to assess tumor cell (TC) viability in naïve versus resistant CM. TC migration and invasion as well as endothelial cell migration were assessed using modified Boyden chambers with naïve or resistant CM. For in vivo studies, TCs were subcutaneously co-implanted with naïve or resistant TAFs in immunodeficient mice. Gene expression microarray analysis was performed to compare naïve and chemoresistant TAFs. Expression of selected targets was confirmed by qRT-PCR and/or antibody-based cytokine array. IL-8 expression was quantified by ELISA. Expression of phospo- and total forms of ATM, AKT, and S6 were assessed by immunofluorescence and/or Western blot. Results:TCs (Miapaca-2 and Panc1) demonstrated increased viability when grown in the presence of resistant as compared to naïve TAF CM (p Conclusions: Chemotherapy treated TAFs support tumorigenesis and angiogenesis in vitro and in vivo via paracrine signaling. After treatment with GEM, TAFs activate a DDR and manifest a pro-inflammatory gene expression signature. Inhibition of PIKK attenuates this inflammatory response to chemotherapy in TAFs. Citation Format: Paul A. Toste, Andrew H. Nguyen, Brian E. Kadera, Mindy Duong, Luyi Li, Timothy R. Donahue. Chemotherapy treated pancreatic cancer tumor-associated fibroblasts are protumorigenic. [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 A53.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor survival despite current medical and surgical therapies and has a propensity towards subclinical metastasis even with resectable disease. The tumor microenvironment consists of a network of supporting cells, including tumor-associated fibroblasts (TAFs), which promote tumor invasion and metastasis, and contribute to PDAC’s extremely poor prognosis. MicroRNAs (miRs) have recently emerged as critical players in post-transcriptional regulation of pathways in cancer biology. In this study, we identified miRs in fibroblasts induced by PDAC tumor cells (TCs). Further, we study the stable suppression of one of these miRs in TAFs and the effects upon fibroblast migration and invasion, as well as tumor cell proliferation. Methods: Primary pancreatic fibroblasts were cultured with MIA PaCa-2 TC conditioned media and RNA was analyzed by the NanoString MicroRNA Expression Assay. Stable miR knockdown (KD) was achieved with a stably expressed shRNA anti-microRNA by lentiviral vector. Migration was determined by an 18 hour scratch assay and 24 hour invasion by modified Boyden chamber. TC proliferation was measured by MTT assay. In vivo tumor growth was evaluated by subcutaneous TC and TAF co-implantation in NSG mice. Results: In primary pancreatic fibroblasts, MIA PaCa-2 tumor cells induced 62 miRs >50% increase or 30% decrease in expression. The most prevalent induced miR with 2-fold increase in expression was miR-21. miR-21 KD in TAFs was achieved by introducing stable expression of anti-miR-21, which resulted in a 75% decrease in expression (P50% decreased ability to migrate (p Conclusions: Pancreatic cancer TCs can induce tumor supportive miRs in TAFs. miR-21 KD in PDAC TAFs results in decreased TAF migration and invasion, as well as reduced ability to support in vitro and in vivo tumor growth. Citation Format: Andrew Hieu Nguyen, Luyi Li, Paul Toste, Nanping Wu, Timothy R. Donahue. MicroRNA-21 is involved in pancreatic cancer fibroblast tumor supportiveness. [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 B18.