Fig 1.
Type I collagen- β1 integrin signaling enhances the clonogenic growth of PDAC cells.
(a) Colony formation by BxPC-3, Capan-1 and MIA PaCa-2 cells following growth on type I collagen, fibronectin, or laminin for 96 hours. Data represent the mean ± SD (n = 4) compared to control cell growth on plastic; **P < 0.001; ***P < 0.0001 by ANOVA. (b) Colony formation assay by cells from 2 distinct patient derived xenografts cells cultured on plastic or type I collagen for 96 hours. (c) Secondary colony formation by MIA PaCa-2, and Capan-1 cells. Data represent the mean ± SD (n = 4) compared to control; *P < 0.05, **P < 0.001. (d) Primary and secondary colony formation by MIA PaCa2 cells expressing a scrambled control (shCtrl) or β1 integrin (shBeta1) shRNA after culture on type I collagen for 96 hours. Data represent mean ± SD (n = 4). **P < 0.001.
Fig 2.
FAK overexpression increases PDAC clonogenic growth and tumor initiating capacity.
(a) Ratio of phospho-FAK to total FAK expression by MIA PaCa2 cells expressing a scrambled control (shCtrl) or β1 integrin (shBeta1) shRNA and cultured on plastic or type I collagen for 96 hours. (b) Colony formation by MIA PaCa-2 and Capan-1 cells overexpressing FAK-FL following growth on plastic or type I collagen for 96 hours. Data represent mean ± SD (n = 4) of control versus FAK-FL; *P < 0.05; **P < 0.001; ***P < 0.0001. (c) Tumor growth of MIA PaCa2 cells overexpressing FAK-FL following subcutaneously injection in to NSG mice. *P = 0.04 compared to control (vector).
Table 1.
FAK regulates tumor initiating cell (TIC) frequency in PDAC.
Fig 3.
Type I collagen-β1 integrin-FAK signaling impacts ALDH+ PDAC CSCs.
(a) Frequency of ALDH+ MIA PaCa-2, Capan-1, and BxPC-3 cells following culture on plastic or type I collagen for 96 hours. Data represent mean ± SD (n = 3) compared to cell growth on plastic; *P < 0.05, **P < 0.001, ***P < 0.0001. (b) Frequency of α2β1+ cells within ALDH+ or all tumor cells from 3 distinct patient derived xenografts. (c) Frequency of phospho-FAK+ cells within ALDH+ or all tumor cells from 2 distinct patient derived xenografts as determined by flow cytometry.
Fig 4.
FAK kinase-inhibition decrease clonogenic PDAC growth in vitro and inhibits tumor growth in vivo.
(a) In vitro colony formation by MIA PaCa-2 and Capan-1 cells following treatment with vehicle control (DMSO) or VS-4718 on type I collagen for 96 hours. Data represent mean ± SD (n = 3) of DMSO versus VS-4718; *P < 0.05. (b) Colony formation by 2 distinct patient derived xenograft cells following treatment with vehicle control (DMSO) or VS-4718 on type I collagen for 5 days. (c) Subcutaneous tumor growth of a patient derived xenograft (JH102) following treatment with vehicle control, VS-4718, gemcitabine plus nab-paclitaxel (Gem-Pac), or all three drugs together (Gem-Pac-VS). Five mice were included in each group. **P = 0.0076, *P = 0.03 by ANOVA. (d) Colony formation by MIA PaCa-2 and Capan-1 cells overexpressing FAK-Y397F following growth on type I collagen for 96 hours. Data represent mean ± SD (n = 4) of control versus FAK-Y397F; *P < 0.05, **P < 0.001.
Fig 5.
The loss of FAK inhibits self-renewal.
(a) Frequency of ALDH+ MIA PaCa-2 and Capan-1 cells expressing scrambled control (shCtrl) or FAK shRNA (shFAK) following treatment with doxycycline for 96 hours. Data represent mean ± SD (n = 3) of shCtrl versus shFAK; *P < 0.05. (b) In vitro colony formation by MIA PaCa-2 and Capan-1 cells expressing scrambled control (shCtrl) or FAK shRNA (shFAK) following treatment with doxycycline for 96 hours on type I collagen. Data represents ± SD (n = 4) of control versus hairpin; **P < 0.001. (c) In vivo subcutaneous tumor growth by MIA PaCa-2 cells expressing scrambled control (shCtrl, n = 4) or FAK shRNA (shFAK, n = 7) following treatment with doxycycline. Error bar represents SD; *P = 0.02.
Fig 6.
The loss of FAK inhibits PDAC cell migration in vitro and metastasis in vivo.
(a) In vitro migration by MIA PaCa-2 and Capan-1 cells expressing scrambled control (shCtrl) or FAK shRNA following treatment with doxycycline on type I collagen for 96 hours. Data represent mean ± SD (n = 4) of control versus hairpin; **P < 0.001. (b) In vivo metastases by orthotopic MIAPaCa-2 tumors expressing scrambled control (shCtrl) or FAK shRNA (shFAK) following treatment with doxycycline. Seven mice were used in each group. Control versus hairpin; *P = 0.008.