Src-mediated morphology transition of lung cancer cells in three-dimensional organotypic culture

Abstract

A fribotic tumor microenvironment promotes progression of cancer. In this study, we utilize a reconstituted basement membrane mimics Matrigel based three-dimensional organotypic culture (rBM 3-D) to investigate the mechanisms that mediate the tumor promoting effects of the fibrogenic mediators TGF-β1 and type I collagen (Col-1) on lung adenocarcinoma cells. Similar to normal alveolar epithelial cells, the well-differentiated lung adenocarcinoma cells in rBM 3-D culture undergo acinar morphogeneis that features polarized epithelial cell spheres with a single central lumen. Either TGF-β1 or Col-1 modestly distorts acinar morphogenesis. On the other hand, TGF-β1 and Col-1 synergistically induce a transition from acinar morphology into stellate morphology that is characteristic of invasive and metastatic cancer cells. Inhibition of the Src kinase activity abrogates induction of stellate morphology, activation of Akt and mTOR, and the expression of tumor promoting genes by TGF-β1 and Col-1. To a similar extent, pharmacological inhibition of mTOR abrogates the cellular responses to TGF-β1 and Col-1. In summary, we demonstrate that TGF-β1 and Col-1 promote stellate morphogenesis of lung cancer cells. Our findings further suggest that the Src-Akt-mTOR axis mediates stellate morphogenesis. These findings also indicate that rBM 3-D culture can serve as an ideal platform for swift and cost-effective screening of therapeutic candidates at the interface of the tumor and its microenvironment.

Figure 1

Distinct growth patterns of lung cancer cell lines in rBM 3-D organotypic culture. Four human and mouse lung cancer cell lines were cultured in rBM 3-D culture. Morphogenesis of lung cancer cells was visualized by filamentous actin staining and confocal fluorescent microscopy (insets). The insets of A-C were captured at 400x and part D was captured at 200x because of the larger diameter of LLC cell cluster. The growth patterns of lung cancer cells in vivo were visualized using H & E staining (main images, 100x). Representative glandular structures are indicated by arrows in A & C.

Figure 2

Induction of stellate morphology by TGF-β1 and Col-1. A549 and its more aggressive derivative A549LC cells were cultured in rBM 3-D culture (Mat) with or without supplementation of TGF-β1 and/or Col-1. A) Morphogenesis of A549 cells in various culture conditions were visualized by filamentous actin staining and confocal fluorescent microscopy. The images were captured at 100x. B) Similar to part A except that the images were captured from A549LC cells at 200x. C) Phase-contrast microscopy was used to capture images of A549 cells cultured in rBM 3-D (Mat) exposed to the indicated combinations of TGF-β1, Col-1, and PP2 (100x). D) The variants of A549LC cells, A549LCvec and A549LCdnSrc were cultured in rBM 3-D (Mat) in the present or absence of TGF-β1+Col-1 and morphogenesis was monitored as described in part C.

Figure 3

Activated expression of the tumor promoting genes by TGF-β1 in rBM 3-D. Total cell RNA was extracted from A549 cells cultured in rBM 3-D (Mat) culture exposed to the indicated combinations of TGF-β1 and Col-1. The expression of the selected tumor-promoting genes was determined using quantitative RT-PCR. Ratios of each mRNA over the housekeeping gene 36B4 were compared across various culture conditions. A-C) The expression of Myc, PAI-1, and LOX was evaluated, respectively. A fold change was obtained by setting the values from the control group to one. D) The expression of Myc, PAI-1, and LOX was compared between A549 cells cultured in Mat+TGF-β1+Col-1 with or without PP2. A fold change was obtained by setting the values from the PP2 minus group to one. E) Similar to part C except that the expression of Myc, PAI-1, and LOX was compared between A549 cells cultured in Mat+TGF-β1 with or without PP2. Mean and standard deviations were obtained from three independent experiments. *, **, and *** indicate a P value < 0.05, 0.01, and 0.001, respectively.

Figure 4

Activation of the Akt-mTOR axis in stellate morphogenesis. A) Total cell protein was extracted from A549 cells in rBM 3-D culture exposed to the indicated combinations of TGF-β1 and Col-1. The protein levels of total and phosphorylated Src, Akt, and mTOR were determined using immunoblots. B) Similar to A except that total cell protein was extracted from A549LCvec and A549LCdnSrc cells in rBM 3-D culture exposed to TGF-β1 and Col-1. The protein levels of total and phosphorylated FAK, Akt, mTOR, and p70 S6K were determined using immunoblots. C) A549LC cells were cultured in rBM 3D culture supplemented with TGF-β1 and Col-1 in the presence or absence of Torin-1 (250 nM). Morphogenesis of A549LC cells was monitored for 12 days. Images were captured using phase contrast microscopy. D) The culture condition was identical to that described in part B. Total cell RNA was extracted from A549 cells. The expression of LOX, PAI-1, and Myc was determined using quantitative RT-PCR. Ratios of each gene transcript over the housekeeping gene 36B4 were compared across various culture conditions. A fold change was obtained by setting the values from the control group to one. Mean and standard deviations were obtained from three independent experiments. * and ** indicate a P value < 0.05 and 0.01, respectively.