Folfirinox-dependent reprogramming of macrophages by primary pancreatic cancer CAFs
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ABSTRACT: The pancreatic cancer tumor microenvironment is dominated primarily by cancer-associated fibroblasts (CAFs) and tumor associated macrophages (TAMs). The interecellular dialogue between CAFs and TAMs and the resulting impact on chemoresistance, specifically to Folfirinox, remains poorly understood. In this study, we sought to understand the impact of primary PDAC CAFs on macrophage reprogramming in a folfirinox-dependent manner and performed various cellular assays (viability, immunophenotyping, phagocytosis, secretory profile analysis) and transcriptomic analysis.
Project description:Pancreatic ductal adenocarcinoma is an extreme lethal cancer with a poor treatment response to all the current chemotherapies. We generated a library of PDAC organoid lines from resected tumors, five from treatment-naive patients and five from patients who received neoadjuvant FOLFIRINOX (eight cycles). We assessed weather residual cancer cells in the tumor lesions of the patients treated with neoadjuvant FOLFIRINOX developed resistance to the treatment. Further we perfromed transcriptome analysis to investigate: 1- weather organoids recapitualting their corresponding tumor tissue 2- To investigate any modifications in the expression of the genes inflicted by FOLFIRINOX treatment.
Project description:Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer associated fibroblasts (CAFs). CAFs promote tumor growth, metastasis and treatment resistance. We aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAFs) were generated by continuous incubation in 100nM gemcitabine. Gene expression differences between treatment naïve CAFs (N-CAFs) and R-CAFs were compared by array analysis. Immortalized human pancreatic CAFs were grown for 30 days in either control media or media containing 100nM gemcitabine. RNA was then isolated and hybidized on U133 Plus 2.0 Affymetrix arrays.
Project description:Tumor stroma of pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant and heterogeneous cancer-associated fibroblasts (CAFs) that are critically involved in chemoresistance. However, the underlying mechanism of CAFs in chemoresistance is unclear. Here, we show that CAFR, a CAFs subset derived from oxaliplatin-resistant PDAC patients, produces more IL8 than CAFS isolated form oxaliplatin-sensitive PDAC patients. CAFR derived IL8 promotes PDAC oxaliplatin chemoresistance. Base on long non-coding RNA (lncRNA) profiling in tumor cells incubated with CAF, we identify that UPK1A-AS, which is directly induced by IL8/NF-kappa B signaling, functions as a pro-chemoresistant lncRNA and is critical for active IL8-induced oxaliplatin resistance. Impressively, blocking UPK1A-AS1 activation increases sensitivity of tumor cell response to oxaliplatin chemotherapy in vivo. Mechanically, UPK1A-AS1 strengths the interaction of Ku70 and Ku80 to enhance double-strand DNA break (DSB) repair, thereby coordinating the NHEJ pathway. Clinically, UPK1A-AS1 expression is positively correlated with IL8 expression, poor chemotherapeutic response and shorter PFS of PDAC patients. Collectively, our study discovers a lncRNA-mediated mechanism for CAF derived IL8 paracrine-dependent oxaliplatin resistance and highlights UPK1A-AS1 as potential therapeutic targets.
Project description:Purpose: FOLFIRINOX has become standard therapy for patients with advanced stages pancreatic ductal adenocarcinoma (PDAC). However, only a subset of patients benefits from this therapy and biomarkers to guide clinical decisions are lacking. This study aimed to discover circulating microRNAs (miRNAs) as potential stratifying and monitoring biomarkers in patients with PDAC treated with FOLFIRINOX and to investigate their functional roles. Methods: A microarray was used in plasma samples from a first cohort of 11 patients selected based on their long versus short progression-free survival (PFS) after FOLFIRINOX. Nine miRNAs were validated using RT-qPCR in an independent cohort (n=43). The best discriminative miRNA was evaluated in tumor tissue samples and associated with clinicopathological features by Cox regression analyses. In vitro studies explored its role on cell proliferation, key downstream targets, and interaction with 5-fluorouracil, irinotecan, and oxaliplatin. Results: MiR-181a-5p was validated as significantly down-regulated in non-progressive compared to progressive patients after FOLFIRINOX. In multivariate analysis, this down-regulation correlated with improved PFS and overall survival, especially combined with CA19.9 decline (log-rank p<0.001, HR=0.153, 95% C.I. 0.067–0.347 and log-rank p=0.033, HR=0.201, 95% C.I. 0.070–0.576, respectively). Overexpression of miR-181a-5p increased proliferation of PDAC cells and inversely correlated with expression of ATM. Furthermore, inhibition of miR-181a-5p coupled with oxaliplatin exposure increased DNA damage and decreased cell viability. Conclusion: Our findings endorse miR-181a-5p as a biomarker for monitoring response to FOLFIRINOX and prognostication. MiR-181a-5p inhibition can potentially enhance sensitivity to oxaliplatin by amplifying the DNA damage response and cell death.
Project description:Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer associated fibroblasts (CAFs). CAFs promote tumor growth, metastasis and treatment resistance. We aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAFs) were generated by continuous incubation in 100nM gemcitabine. Gene expression differences between treatment naïve CAFs (N-CAFs) and R-CAFs were compared by array analysis.
Project description:CAFs, ascTAMs and M1- and TAM-like-MDMs were treated with 1 μM of the Prostacyclin analoga MRE and DMSO (as solvent control). The aim of this experiment is to see which effect MRE (Prostacyclinanaloga) has on RNA expression (activation of signaling pathways). Background: Prostacyclin receptor expression is elevated in TAMs. Prostacyclinsynthase expression was detected mainly in CAFs. (
Project description:Hepatocellular carcinoma (HCC) is a malignancy that is challenging to treat. Cancer-associated fibroblasts (CAFs) are reported to promote the malignant behavior of HCC cells via cytokines. Conophylline (CnP) has been reported to suppress activated hepatic stellate cells in liver fibrosis. We aimed to determine whether CnP is useful in suppressing tumor growth in HCC. We investigated whether CnP could suppress the HCC-promoting effect of CAFs derived from HCC tissues in vitro and in vivo. CAFs promoted the proliferation and invasion of HCC cells. CnP suppressed activated CAFs expressing α-smooth muscle actin (αSMA) and inhibited the HCC-promoting effects of CAFs. CnP significantly reduced the levels of cancer-promoting cytokines such as interleukin (IL)-6 (IL-6), IL-8, C-C Motif Chemokine Ligand 2 (CCL2), angiogenin, and osteopontin, which are secreted by CAFs. An in vivo study demonstrated that combined therapy with CnP and sorafenib against CAFs and HCC cells showed the strongest inhibition of tumor growth compared with the control and single treatment groups. Transcriptome analysis revealed that GPR68 in CAFs was strongly suppressed by CnP. The cancer-promoting effects of cytokines were eliminated by knockdown of GPR68 in CAFs. CnP inhibited the HCC-promoting effect of CAFs by suppression a number of HCC-promoting cytokines, which are secreted from CAFs expressing GPR68. Combination therapy with CnP and existing anti-cancer agents may be a promising therapeutic strategy in overcoming refractory HCC with activated CAFs.
Project description:We aimed to determine the differences in gene expression levels between SNU668 cells and SNU668 cells co-cultured with CAFs or treated with recombinant human GAS6 protein using microarray analysis. We selected genes that showed a fold change increase of more than 2-fold compared to WT in SNU668 cells. Then, we identified genes involved in chemoresistance.
Project description:To understand the molecular mechanisms that underpin pro-tumourigenic functions of cancer-associated fibroblasts (CAFs) we compared the proteome, acetylome, phosphoproteome of human immortalised breast cancer CAFs with those of the normal mammary fibroblasts that they were generated from. Based on the results obtained, we have also analysed proteome changes when CAFs were treated with the P300/CBP inhibitor c646.