Cancer cell invasion driven by extracellular matrix remodeling is dependent on the properties of cancer-associated fibroblasts

Abstract

Purpose: As one form of tumor invasion, cancer cells can invade the extracellular matrix (ECM) through tracks that have been physically remodeled by cancer-associated fibroblasts (CAFs). However, CAFs are a heterogeneous population with diverse matrix-remodeling capacities. The purpose of this study was to investigate how CAFs with various matrix-remodeling capacities influence cancer cell invasion.

Methods: We established single-cell-derived clones from three primary cultures of CAFs from lung adenocarcinoma patients (Case 1, 5 clones; Case 2, 5 clones; and Case 3, 7 clones). Using a co-culture model, we evaluated the correlations between the number of invaded cancer cells and the remodeling areas generated by CAF clones in each case.

Results: When A549 lung adenocarcinoma cells and CAF clones were co-cultured, both the numbers of invaded cancer cells and the remodeling areas generated by the CAF clones varied greatly. The number of invaded cancer cells was moderately and strongly correlated with the remodeling areas generated by each CAF clone originating from Cases 1 and 2 (R(2) value = 0.53 and 0.68, respectively), suggesting that the remodeling areas in the ECM may determine the number of invaded cancer cells. In contrast, the number of invaded cancer cells was not correlated with the remodeling areas generated by CAF clones originating from Case 3, suggesting that factors other than the remodeling areas might determine the number of invading cancer cells.

Conclusions: These findings showed two types of fibroblast-dependent cancer cell invasion that are dependent on and independent of the remodeling areas generated by CAFs.

Keywords: Cancer-associated fibroblasts; Extracellular matrix remodeling; Fibroblast-dependent cancer invasion; Single-cell-derived clones.

Fig. 1

Invasion abilities of three primary cultures of CAFs and co-cultured cancer cells. a Scheme showing the form of invasion in the ECM for CAFs alone and co-cultures of CAFs and cancer cells. Cancer cells invade by following the CAFs, indicating a fibroblast-dependent form of cancer cell invasion. Examples (b) and quantification (c) of the invasion of the three parental CAFs and A549 cancer cells cultured alone for 48 h using the collagen invasion assay model. Examples (d) and quantification (e, f) of the invasion of co-cultures of A549 cancer cells and parental CAFs for 48 h using the collagen invasion assay model. P1, P2, and P3 represent the parental CAFs from Cases 1, 2, and 3, respectively. Values are the mean ± SEM. Bar 300 μm. *P < 0.001

Fig. 2

Establishment of single-cell-derived clones from primarily cultured CAFs infected with hTERT. a Scheme showing the method used to establish single-cell-derived clones from the primary cultures of CAFs (see “Materials and Methods”). b Examples of the parental CAFs from Case 3 and their single-cell-derived clones. hTERT-infected CAFs express Venus in their cytoplasm (green). The images were fusion images of phase contrast and green fluorescence

Fig. 3

Invasion abilities of CAF clones cultured alone in collagen gel. Representative examples (a) and quantification (b) of the invasion of CAF clones cultured alone for 48 h using the collagen invasion assay model. P1, P2, and P3 represent the parental CAFs of Cases 1, 2, and 3, respectively. Values are the mean ± SEM. Bar 300 μm

Fig. 4

Invasion abilities of cancer cells and CAF clones cultured together in collagen gel. Representative examples (a) and quantification (b, c) of the invasion of co-cultures of A549 cancer cells and CAF clones for 48 h using the collagen invasion assay model. P1, P2, and P3 represent the parental CAFs of Cases 1, 2, and 3, respectively. Values are the mean ± SEM. Bar 300 μm

Fig. 5

a Relationship between the invasion of CAFs and the invaded cancer cells in Cases 1, 2, and 3. R ² = coefficient of determination. b Scheme showing two types of cancer cell invasion, one dependent on and one independent of the remodeling areas generated by CAFs