Cancer-associated stromal fibroblasts promote pancreatic tumor progression

Abstract

Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer.

Figure 1

Immortalization of HPSCs. Primary HPSCs were exposed to lentiviral vectors carrying hTERT and SV40-TAg. Immunohistochemical staining of HPSCs transduced with hTERT and SV40-TAg for αSMA (A) and vimentin (B). C, growth curve for primary HPSCs, HPSCs transduced with SV40-TAg, and HPSCs transduced with both hTERT and SV40-TAg. HPSCs carrying hTERT and Tag show no evidence of senescence at >100 passages.

Figure 2

Effect of HPSC-CM on pancreatic tumor cell phenotype. CM from HPSCs (μg/μL) was added to BxPC3 or Panc1 pancreatic tumor cells. A, tumor cell proliferation was measured by MTS assay at 48 h. B, tumor cell invasion was evaluated at 48 h using a modified Boyden chamber assay. C, the ability of tumor cells to form colonies in soft agar was determined at 15 d after addition of HPSC-CM. D, tumor cell proliferation was measured in the presence of conditioned media from nonimmortalized HPSCs. Columns, mean of three experiments. *, P < 0.05 and **, P < 0.005 versus serum-free media (SFM) control. #, P < 0.0005 versus HPSC-CM 1.0 μg/μL.

Figure 3

BxPC3 cells were treated with gemcitabine (100 μmol/L) with or without conditioned media from HPSCs (1 μg/μL) for 48 h. A, apoptosis was detected using TUNEL assay and cell nuclei were stained with DAPI. B, percentage of apoptotic cells was determined in 10 random fields for each condition. SFM, serum-free media.

Figure 4

Conditioned media from HPSCs protects pancreatic tumor cells from apoptosis induced by radiation. BxPC3 cells were treated with 100 Gy of radiation in the presence of either media containing 10% FCS or conditioned media from HPSCs (1 μg/μL). After 24 h, cells were analyzed for caspase activation (A) and percentage of cells undergoing apoptosis (B) and total number of nuclei (C) were calculated. Control, DMEM with 10% FCS; XRT, radiation treatment. *, P < 0.01 versus control; **, P < 0.0001 versus control.

Figure 5

Protein lysates from BxPC3 cells treated with HPSC-CM (1 μg/μL) were analyzed for Erk1/2 and Akt activation by Western blotting at various time points (A) and quantifed by densitometry. Control cells were treated with serum-free media alone. B, change in levels of phosphorylated Erk1/2 and Akt was compared with serum-free controls.

Figure 6

Co-injection of HPSCs with BxPC3 tumor cells in an orthotopic nude mouse model of pancreatic cancer results in increased tumor incidence, metastasis, and tumor size. HPSCs were injected with BxPC3 cells (either 0.5 × 10⁶ or 1 × 10⁶) in varying tumor/stroma ratios. At 8 wk, mice were sacrificed and primary tumor incidence (A) and metastasis (B) were quantified. C, pancreatic tumors were weighed and the luciferase signal from a representative mouse in each group is shown. D, H&E staining showed no tumors in the pancreas of mice injected with 0.5 × 10⁶ BxPC3 cells alone (left) but confirmed tumor development in mice co-injected with BxPC3 and HPSCs (pancreas, center, and peritoneal metastases, right). Each experiment was repeated twice and a summary of all the results is shown. *, P < 0.01 versus tumor/stroma ratio 1:0 or 0:1.