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. 2013 Mar;15(3):249-62.
doi: 10.1593/neo.121950.

Breast fibroblasts modulate early dissemination, tumorigenesis, and metastasis through alteration of extracellular matrix characteristics

Nancy Dumont  1 , Bob Liu, Rosa Anna Defilippis, Hang Chang, Joseph T Rabban, Anthony N Karnezis, Judy A Tjoe, James Marx, Bahram Parvin, Thea D Tlsty
Affiliations

VSports注册入口 - Breast fibroblasts modulate early dissemination, tumorigenesis, and metastasis through alteration of extracellular matrix characteristics

Nancy Dumont et al. Neoplasia. 2013 Mar.

Abstract

A wealth of evidence has now demonstrated that the microenvironment in which a tumorigenic cell evolves is as critical to its evolution as the genetic mutations it accrues. However, there is still relatively little known about how signals from the microenvironment contribute to the early events in the progression to malignancy. To address this question, we used a premalignant mammary model to examine how fibroblasts, and the extracellular matrix (ECM) proteins they secrete, influence progression to malignancy. Their effect on metastatic malignant cells was also assessed for comparison. We found that carcinoma-associated fibroblasts, and the distinct aligned ECM they deposit, can cause both premalignant and malignant mammary epithelial cells to assume a mesenchymal morphology that is associated with increased dissemination and metastasis, while benign reduction mammoplasty fibroblasts favor the maintenance of an epithelial morphology and constrain early dissemination, tumor growth, and metastasis. Our results suggest that normalizing the organization of the ECM could be effective in limiting systemic dissemination and tumor growth VSports手机版. .

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Figures

Figure 1
Figure 1
Premalignant mammary epithelial cells acquire a mesenchymal morphology when co-cultured with CAFs but not RMFs. (A) Representative 10x images of epithelial cells co-cultured with RMFs or CAFs for 3 days. (B) Immunoblot analysis of Fn, N-cadherin (N-cad), and E-cadherin (E-cad) in lysates of vHMEC-ras0.5 cells isolated by FACS after they had been cultured either alone, with RMFs, or with CAFs for 3 days. Representative immunoblots are shown. (C) Flow cytometric analysis of Thy-1 and EpCAM expression in vHMEC-ras0.5 cells cultured as above in B. Bar graphs represent the averages ± SEM of multiple experiments combined. For Thy-1: alone (n = 12), RMF146 (n = 6), CAF1363 (n = 11). For EpCAM: alone (n = 3), RMF146 (n = 3), CAF1363 (n = 2). Student's t test for Thy-1: alone versus RMF146 (P < .001), alone versus CAF1363 (P < .001); for EpCAM: alone versus RMF146 (P = .17), alone versus CAF1363 (P = .02). Statistically significant differences in expression between cells in co-culture and the cells alone are denoted with asterisk.
Figure 2
Figure 2
The CAF-induced mesenchymal phenotype is due to differences in the organization of the ECM deposited by CAFs. (A) Representative 10x images of ECM stained with picrosirius 4 days after seeding and removal of fibroblasts (top panels) and of vHMEC-ras0.5 cells overlaid on top of ECM (bottom panels). (B) Flow cytometric analysis of Thy-1 expression in vHMEC-ras0.5 cells overlaid on control ECM (no fibroblasts seeded) or ECM deposited by RMFs or CAFs for 2 days. Analysis of one representative experiment is shown. Inset shows percentage of Thy-1+ cells based on no antibody control for histogram shown. (C) Representative 10x images of vHMEC-ras0.5 cells co-cultured with RMFs or CAFs for 3 days in the absence or presence of collagenase. (D) Flow cytometric analysis of Thy-1 expression in the CMFDA-labeled vHMEC-ras0.5 cells of the experiment shown in C; top row, no collagenase; bottom row, + collagenase. Analysis of one representative experiment is shown.
Figure 3
Figure 3
The CAF-induced mesenchymal phenotype is dependent on the activation of the Smad, Erk, Jun, and Rho signaling pathways. (A) Representative immunoblots of signaling proteins expressed in vHMEC-ras0.5 cells after they were cultured either alone (lane 1) or with RMFs (lane 2) or CAFs (lane 3) for 3 days. (B) Representative 10x images of vHMEC-ras0.5 cells co-cultured with CAFs in the absence or presence of different pathway kinase inhibitors, as indicated: TβRKi (LY364947, 10 µM), MEK inhibitor (U0126, 10 µM), JNK inhibitor II (10 µM), p38MAPKi (SB203580, 10 µM), PI3K inhibitor (LY294002, 10 µM), mTORi (rapamycin, 10 nM), and ROCK inhibitor (Y27632, 40 µM).
Figure 4
Figure 4
CAFs deposit aligned matrix and promote the dissemination of premalignant cells in vivo. (A) Bioluminescence imaging, hematoxylin and eosin (H&E), lamin A/C, and picrosirius staining of mammary glands harvested 3 weeks following orthotopic injection of 1 x 106 ras0.5-GFPLux alone or in combination with 3 x 106 fibroblasts. All images were captured at 10x (higher 40x magnification images are available in Figure W7). The location of ras0.5-GFPLux cells is indicated by arrows in the H&E and outlined with a white dashed line in the picrosirius image. (B) Bar graphs represent the averages ± SEM of mammary ECM abundance and alignment obtained through automated image analysis (alone, n = 18; RMF163, n = 14; CAF559, n = 18). Student's t test comparing ECM abundance: alone versus RMF163 (P = .4), alone versus CAF559 (P < .001), RMF163 versus CAF559 (P = .03); comparing ECM alignment: alone versus RMF163 (P = .4), alone versus CAF559 (P < .001), RMF163 versus CAF559 (P < .001). (C and D) qPCR analysis of EpCAM levels in genomic DNA extracted from blood collected 3 weeks following: (C) injection of 1 x 106 ras0.5-GFPLux cells alone (n = 4), or in combination with 3 x 106 RMF163 (n = 4), or3 x 106 CAF559 (n = 8). Student's t test comparing qPCR replicates from alone versus RMF163 (P = .04), alone versus CAF559 (P = .4), RMF163 versus CAF559 (P = .003); and (D) injection of 2.5 x 106 ras0.5-GFPLux cells into RMF163 (n = 4) or CAF559 (n = 4) pre-injected fat pads. Student's t test comparing qPCR replicate values of nanogram EpCAM per milliliter of blood collected from mice in the RMF163 group versus CAF559 group (P = .03). Statistically significant differences between groups are denoted with asterisk.
Figure 5
Figure 5
RMFs and CAFs differentially modulate primary tumor growth. (A) Tumor growth curves of 231-Lux cells alone (green) or in combination with RMFs (blue) or CAFs (red). Representative experiments performed with RMFs and CAFs isolated from three different individuals each are shown. P values correspond to Student's t test comparing growth curves in the presence of each fibroblast relative to 231-Lux cells alone. Experiments with each RMF and CAF were performed at least twice. (B) Graph of fold change in final tumor volume and weight in the presence of RMFs isolated from four different individuals (in blue from left to right: 163, 156, 9, 146) or CAFs isolated from three different individuals (in red from left to right: 1366, 559, 1363) relative to the 231-Lux cells alone for all experiments combined. The number of mice in each group and the P values comparing the effects of each RMF and CAF on tumor volume and weight relative to the 231-Lux cells alone are shown in Table 2.
Figure 6
Figure 6
RMFs suppress, while CAFs enhance, the metastatic potential of malignant cells in vivo. Graphs of fold change in (A) the number of metastatic foci per lung or (B) lung luminescence as a measure of total pulmonary metastatic burden in the presence of RMFs (in blue from left to right: 163, 156, 9, 146) or CAFs (in red from left to right: 1366, 559, 1363) relative to the 231-Lux cells alone for all experiments combined. The number of mice in each group and the P values comparing the effects of each RMF and CAF on metastatic burden relative to the 231-Lux cells alone are shown in Table 3.
Figure 7
Figure 7
CAFs deposit aligned matrix and promote dissemination of malignant cells in vivo. Representative 20x images of tumors stained with (A) Ki67, (B) CC3, or (C) picrosirius, grown in the absence (alone) or presence of RMF146 or CAF1363, as indicated. Staining was quantified using three to four randomly chosen fields from each tumor section. Alone (n = 34, 15, 10), RMF146 (n = 12, 8, 8), and CAF1363 (n = 22, 12, 7) for stains shown in A, B, and C, respectively. Quantification of stains is shown in bar graphs as average ± SEM for each group. P values correspond to Student's t test comparing scores of tumors grown in the presence or absence of fibroblasts. For Ki67: alone versus RMF146 (P = .11), alone versus CAF1363 (P = .72); for CC3: alone versus RMF146 (P <.001), alone versus CAF1363 (P = .8); for ECM abundance: alone versus RMF146 (P = .09), alone versus CAF1363 (P < .001); for ECM alignment: alone versus RMF146 (P = .04), alone versus CAF1363 (P < .001). (D) qPCR analysis of EpCAM levels in genomic DNA extracted from blood collected 8 weeks following injection of 5 x 105 231-Lux cells alone (n = 9) or in combination with 1.5 x 106 RMF146 (n = 4), or 1.5 x 106 CAF1363 (n = 7). Student's t test comparing qPCR replicates from alone versus RMF146 (P = .15), alone versus CAF1363 (P = .04), and RMF146 versus CAF1363 (P = .01). (E) Representative immunoblots of Fn, TNC, biglycan, and LOX expression in MDA-MB-231 cells (231), RMFs (163, 156, 009, 146, Mes), and CAFs (1366, 559, 1363). “Mes” denotes an RMF that induced the aligned mesenchymal phenotype. Statistically significant differences between endpoints measured in tumors grown alone versus with fibroblasts are denoted with asterisk.
Figure 8
Figure 8
Induction of the aligned mesenchymal phenotype by an RMF enhances dissemination but is not sufficient to promote metastasis. (A) Representative 10x images of ras0.5-GFPLux and 231-Lux cells cultured in the absence (alone) or presence of RMF-Mes (+RMF-Mes) for 3 days. (B) qPCR analysis of EpCAM levels in genomic DNA extracted from blood shown as average ± SEM. (C) Graph depicting the average number of surface metastases per lung ± SEM. (D) Graph depicting the quantification of lung luminescence ± SEM as a measure of total metastatic burden. All measurements shown in B to D were taken 8 weeks following injection of 5 x 105 231-Lux cells alone (n =17) or in combination with 1.5 x 106 RMF-Mes (n = 12). Student's t test comparing results obtained in the presence of RMF-Mes relative to the 231-Lux cells alone (CTCs, P = .007; surface metastases, P = .8; total metastatic burden, P = .9). Statistically significant difference is denoted with asterisk.
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