Direct Interactions With Cancer-Associated Fibroblasts Lead to Enhanced Pancreatic Cancer Stem Cell Function

Abstract

Objective: Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs).

Methods: Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against β1 integrin and FAK as well as a small-molecule FAK inhibitor.

Results: Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of β1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth.

Conclusion: Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.

FIGURE 1.

Cancer-associated fibroblasts enhance the clonogenic growth potential of PDAC. A, Colony formation by PDAC cell lines (Capan-1, and BxPC-3) and B, a PDX (JH102) cells following co-culture with CAFs or nHLFs for 7 days. Data represent the mean and SD of 4 experiments. *P < 0.05, **P < 0.005. C, Primary and secondary colony formation by Capan-1 cells cultured with or without CAFs for seven days. Data represent the mean and SD of 4 experiments. *P < 0.05.

FIGURE 2.

Cancer-associated fibroblasts induce EMT and facilitate PDAC migration. A, In vitro migration of Capan-1 cells following culture with CAF-conditioned or control media for 7 days. Data represent the mean and SD of 4 experiments. *P < 0.05, **P < 0.001, ***P < 0.0001. B, E-cadherin staining (red) of Capan-1 cells (GFP) following co-culture with CAFs for 7 days. Arrows indicate E-cadherin negative Capan-1 cells. C, The frequency of E-cadherin negative Capan-1 cells following culture with or without CAFs detected by flow cytometry. D, Relative mRNA expression of EMT associated genes in sorted Capan-1 cells following co-culture with CAFs. Data represent the mean and SD of 3 experiments.

FIGURE 3.

Cancer-associated fibroblasts enhance the frequency of ALDH⁺ PDAC CSCs. A, ALDH expression by Capan-1 cells following 7 days of co-culture with CAFs. Cells treated with DEAB were used as negative control for ALDH staining. Data represent the mean and SD of 3 experiments. *P < 0.05, *** P < 0.0001. B, The frequency of ALDH⁺ cells was analyzed in patient-derived low passage PDX (102) following co-culture with CAF35.

FIGURE 4.

Cancer-associated fibroblasts express type I collagen and enhance integrin-FAK signaling in PDAC. A, αSMA expression of CAFs following co-culture with or without Capan-1 cells for 7 days. Left: immunostaining of αSMA (green) and nucleus-Hoechst 33342 (blue). Right: αSMA expression by CAF35 cells before and after co-cultured with Capan-1 cells. B, Relative mRNA expression of type I collagen by CAF35 cells following co-culture with Capan-1 cells. C, The frequency of pY397-FAK⁺ Capan-1 cells following co-culture with CAFs. D, Colony formation by Capan-1 cells expressing a scrambled control (Ctrl) or β1 integrin (shBeta1) shRNA vectors or treated with PF573228 following co-culture with CAFs. Data represent the mean and SD of 3 experiments. **P < 0.001. E, Colony formation by Capan-1 cells expressing a scrambled control (Ctrl) or FAK (shFAK) shRNA following co-culture with CAFs. Data represent the mean and SD of 3 experiments. *P < 0.05; **P < 0.001, ***P < 0.0001.