Secretion of fibronectin by human pancreatic stellate cells promotes chemoresistance to gemcitabine in pancreatic cancer cells

Abstract

Background: Gemcitabine remains a cornerstone in chemotherapy of pancreatic ductal adenocarcinoma (PDAC) despite suboptimal clinical effects that are partly due to the development of chemoresistance. Pancreatic stellate cells (PSCs) of the tumor stroma are known to interact with pancreatic cancer cells (PCCs) and influence the progression of PDAC through a complex network of signaling molecules that involve extracellular matrix (ECM) proteins. To understand tumor-stroma interactions regulating chemosensitivity, the role of PSC-secreted fibronectin (FN) in the development of gemcitabine resistance in PDAC was examined.

Methods: PSC cultures obtained from ten different human PDAC tumors were co-cultured with PCC lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1 and SW-1990) either directly, or indirectly via incubation with PSC-conditioned medium (PSC-CM). Gemcitabine dose response cytotoxicity was determined using MTT based cell viability assays. Protein expression was assessed by western blotting and immunofluorescence. PSC-CM secretome analysis was performed by proteomics-based LC-MS/MS, and FN content in PSC-CM was determined with ELISA. Radiolabeled gemcitabine was used to determine the capacity of PCCs to uptake the drug.

Results: In both direct and indirect co-culture, PSCs induced varying degrees of resistance to the cytotoxic effects of gemcitabine among all cancer cell lines examined. A variable degree of increased phosphorylation of ERK1/2 was observed across all PCC lines upon incubation with PSC-CM, while activation of AKT was not detected. Secretome analysis of PSC-CM identified 796 different proteins, including several ECM-related proteins such as FN and collagens. Soluble FN content in PSC-CM was detected in the range 175-350 ng/ml. Neither FN nor PSC-CM showed any effect on PCC uptake capacity of gemcitabine. PCCs grown on FN-coated surface displayed higher resistance to gemcitabine compared to cells grown on non-coated surface. Furthermore, a FN inhibitor, synthetic Arg-Gly-Asp-Ser (RGDS) peptide significantly inhibited PSC-CM-induced chemoresistance in PCCs via downregulation of ERK1/2 phosphorylation.

Conclusions: The findings of this study suggest that FN secreted by PSCs in the ECM plays a key role in the development of resistance to gemcitabine via activation of ERK1/2. FN-blocking agents added to gemcitabine-based chemotherapy might counteract chemoresistance in PDAC and provide better clinical outcomes.

Keywords: Chemoresistance; Extracellular matrix; Fibronectin; Gemcitabine; Pancreatic cancer; Pancreatic stellate cells.

Fig. 1

PSCs are resistant to gemcitabine-induced cytotoxicity while gemcitabine dose response effects differ among various PCC lines. PDAC-derived human PSCs (a) and PCCs (b) seeded on 96-well plates at a density of 3000 cells/well, were incubated with increasing concentrations of gemcitabine for 48 h and evaluated for cell viability using the MTT assay. Data are mean ± SEM of triplicate determinations. PCC, pancreatic cancer cell; PSC, pancreatic stellate cell

Fig. 2

In direct co-culture with PSCs, PCCs develop resistance to gemcitabine. Equal numbers of PSCs and PCCs seeded together on 96-well plates, cultured for 4 days, were incubated with a single concentration of gemcitabine (10 μM) for 48 h and evaluated for cell viability using MTT assay. Data are mean ± SEM of triplicate determinations. *p < 0.05, **p < 0.01 for control vs gemcitabine. PCC, pancreatic cancer cell; PSC, pancreatic stellate cell

Fig. 3

PSC-CM induces resistance to gemcitabine in PCCs but does not affect gemcitabine uptake. PCCs seeded on 96-well plates were incubated with SFM or PSC-CM for 24 h prior to incubation with (a, b) gemcitabine (10 μM) for 48 h or with (c) transport buffer containing [³H]-gemcitabine for 4 h. For (a), cell viability was determined using the MTT assay. *p < 0.05, **p < 0.01 for control vs gemcitabine; #p < 0.05, ##p < 0.01 and $p < 0.05, $$p < 0.01 for SFM vs PSC-CM in control and gemcitabine groups, respectively. b The table indicates gemcitabine-induced cytotoxicity in percentage, and PSC-CM-induced resistance to gemcitabine, calculated by relative reduction in cytotoxicity between SFM and PSC-CM. *p < 0.05, **p < 0.01 comparing SFM with PSC-CM. For c, gemcitabine uptake was determined by scintillation counting. Data are mean ± SEM of triplicate determinations. d The PCCs were lysed and proteins subjected to immunoblotting using anti-pERK1/2, anti-ERK1/2, anti-PI3K, anti-pAKT, and anti-AKT antibodies. GAPDH was used as a loading control. PCC, pancreatic cancer cell; PSC, pancreatic stellate cell; PSC-CM, PSC-conditioned medium; SFM, serum-free DMEM

Fig. 4

Proteome profiling of PSC-CM. Conditioned medium from ten different PSC cultures were subjected to proteomics analysis using LC-MS/MS. a Top five KEGG pathways identified. b The proteins detected by LC-MS/MS were interrogated in terms of functional annotation by DAVID Bioinformatics Resource tool. The representative GO terms cluster groups with top 10 enrichment score are presented. The horizontal axis represents the significance (p value) for each term, while the vertical axis represents the GO categories for biological processes. c STRING network map of proteins involved in ECM remodeling and their categories based on molecular function. ECM, extracellular matrix; ES, enrichment score; FDR, false discovery rate; GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; PSC, pancreatic stellate cell; STRING search tool for the retrieval of interacting genes/proteins

Fig. 5

Measurement of FN content in PSC-CM and its effect on gemcitabine uptake by PCCs. a Amount of soluble FN in conditioned medium from ten different PSC cultures was measured by ELISA. b Both PCCs and PSCs were lysed and proteins subjected to immunoblotting using anti-FN antibody. GAPDH was used as a loading control. For PCCs, ponceau image also indicates loading. c Cancer cells seeded on 96-well plates with- or without FN-coating were incubated for 24 h prior to incubation with transport buffer containing [³H]-gemcitabine for 4 h. Data are mean ± SEM of triplicate determinations. FN, fibronectin; PCC, pancreatic cancer cell; PSC, pancreatic stellate cell

Fig. 6

FN-inhibitor blocks chemoresistance inducing effect of PSC-secreted FN on PCCs via downregulation of ERK1/2 phosphorylation. a PCCs seeded on 96-well plates with- or without FN-coating as indicated. Cell were incubated with SFM or PSC-CM for 24 h and/or RGDS (FN inhibitor; 20 μM) for 4 h prior to incubation with gemcitabine (10 μM) for 48 h. Cell viability was determined using the MTT assay. *p < 0.05, **p < 0.01 for control vs gemcitabine; #p < 0.05, ##p < 0.01 and $p < 0.05, $$p < 0.01 for SFM vs PSC-CM/FN/RGDS in control (black bars) and gemcitabine (white bars), respectively. b The table indicates gemcitabine-induced cytotoxicity in percentage, PSC-CM-induced resistance to gemcitabine, and resistance following subsequent incubation with RGDS. The resistance developed was calculated by relative reduction in cytotoxicity between SFM and PSC-CM or PSC-CM + RGDS. *p < 0.05, **p < 0.01 comparing SFM with PSC-CM or SFM vs PSC-CM + RGDS. c The cells were lysed and proteins subjected to immunoblotting using anti-pERK1/2 and anti-ERK1/2 antibodies. GAPDH was used as a loading control. Data are the mean ± SEM of triplicate determinations. FN, fibronectin; PCC, pancreatic cancer cell; PSC, pancreatic stellate cell; PSC-CM, PSC-conditioned medium; SFM, serum-free DMEM

Fig. 7

PSC-secreted fibronectin promotes chemoresistance to gemcitabine in PCCs. PSC-secreted fibronectin induces resistance to gemcitabine in PCCs via upregulation of ERK1/2 phosphorylation, while no change in gemcitabine uptake. Addition of RGDS (fibronectin receptor blocking agent) or PD98059 (MEK/ERK inhibitor) to PCCs incubated with PSC-CM protect from chemoresistance progression, via inhibition of ERK1/2 phosphorylation. PCC, pancreatic cancer cell; PSC, pancreatic stellate cell; PSC-CM, PSC-conditioned medium