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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.

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.