Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013;9(9):e1003789.
doi: 10.1371/journal.pgen.1003789. Epub 2013 Sep 19.

System-wide Analysis Reveals a Complex Network of Tumor-Fibroblast Interactions Involved in Tumorigenicity

Affiliations
Free PMC article

System-wide Analysis Reveals a Complex Network of Tumor-Fibroblast Interactions Involved in Tumorigenicity

Megha Rajaram et al. PLoS Genet. .
Free PMC article

Abstract

Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Comparative genomic analysis of expression changes induced by breast cancer cells in tumor-supportive fibroblasts, patient-derived carcinoma-associated fibroblasts, and microdissected breast stroma.
(A) Top ten pathways identified by GSEA analysis of exposure of tumor-supportive fibroblasts to breast cancer cells; blue indicates overlap with the top ten pathways activated in patient-derived breast cancer fibroblasts; green indicates overlap with other pathways significantly activated in patient-derived breast cancer fibroblasts; grey indicates no overlap, (B) Top ten pathways identified by GSEA analysis of patient-derived breast cancer fibroblasts relative to their normal counterparts; blue indicates overlap with the top ten pathways activated by exposure of tumor-supportive fibroblasts to breast cancer cells; green indicates overlap with other pathways significantly activated by exposure of tumor-supportive fibroblasts to breast cancer cells; grey indicates no overlap, (C) Top ten pathways identified by GSEA analysis of microdissected breast cancer stroma relative to normal breast stroma, green indicates overlap with pathways significantly activated by exposure of tumor-supportive fibroblasts to breast cancer cells, but not in the top ten; grey indicates no overlap. (D) A tumor-supportive fibroblast gene signature was used in unsupervised clustering to classify normal (green) and tumor (red) microdissected breast stroma samples. (E) The same signature was used in principal component analysis to observe the separation of normal (green) and tumor (red) microdissected breast stroma samples.
Figure 2
Figure 2. Tumor-supportive fibroblasts have profound effects on the composition of the tumor microenvironment.
(A) Tumor-supportive fibroblasts HFFF2 increase Cal51 tumor cell proliferation and increase stromal components (bottom panel) as compared to tumors formed using Cal51 alone (top panel). Immunohistochemical analysis using antibodies to Ki-67 (proliferation), antigen #7/4 (neutrophils and monocytes), CD31 (blood vessels), and α-SMA (mesenchymal cells). Scale bars represent 50 µm for Ki-67, #7/4 and CD31 and 100 µm for α-SMA. (B) Quantification of Ki-67 positive (proliferating) tumor cells. Five different fields of three different tumors per group were scored. Asterisk indicates a significant difference between cell line only (Cal51shNT) and co-injection (Cal51shNT+HFFF2) groups (p<0.01). Data are expressed as the mean ± SEM. (C–E) Similarly performed quantification of monocytes and neutrophils, endothelial cells, and activated mesenchymal cells (p<0.01).
Figure 3
Figure 3. shRNA silencing of CCL2, CCL7 or CCL8 in tumor-supportive fibroblasts reduces their ability to promote tumorigenicity.
(A) Reduction of CCL2 protein in HFFF2 fibroblasts by expressing shRNAs targeting CCL2 as indicated compared to non-targeting (N.T.) control determined by immunoblotting. Beta-actin was used as a loading control. (B) Reduction of CCL7 protein levels in HFFF2 fibroblasts expressing shRNAs targeting CCL7. (C) Reduction in CCL8 RNA levels in HFFF2 fibroblasts expressing shRNAs targeting CCL8 compared to HFFF2 fibroblasts expressing non-targeting (N.T.) control. Asterisk indicates a significant difference (p<0.01) between the experimental (shCCL8) and control (shN.T.) group. (D) Tumorigenicity of Cal51 cells alone or co-injected with HFFF2 cells expressing either no shRNA, control shRNA or shRNAs targeting CCL2. Tumor-take rate for the total injections for each group is indicated. Asterisks indicate significant differences (p<0.01) in tumor volumes for shCCL2-1 and shCCL2-2 compared to control. Hashtag indicates a significant difference (p<0.05) between cell line only (Cal51) and Cal51 co-injected with HFFF2 fibroblasts. Error bars represent SEM. (E) Effects of shRNAs targeting CCL7 (p<0.01). There was no significant difference in the tumor volumes between the two knockdown groups (p = 0.3, 0.3, 0.2 and 0.5 at weeks 3, 4, 5 and 6 post injections respectively). (F) Effects of shRNAs targeting CCL8 (p<0.01). (G) Tumorigenicity of MDAMB231 cells alone or co-injected with HFFF2 cells expressing either no shRNA, control shRNA or shRNAs targeting CCL2. Tumor-take rate for the total injections for each group is indicated. Asterisks indicate significant differences in tumor volumes for shCCL2-1 and shCCL2-2 compared to control (p<0.01). Hashtag indicates a significant difference between cell line only (MDAMB231) and MDAMB231 coinjected with HFFF2 fibroblasts (p<0.05 and n = 10 per group). Errors represent SEM. (H) Effects of shRNAs targeting CCL7 (p<0.01). (I) Effects of shRNAs targeting CCL8 (p<0.01). The lack of effects of the CCL-targeting shRNAs on fibroblast proliferation are shown in Figure S4.
Figure 4
Figure 4. Diverse effects on the tumor microenvironment mediated by fibroblast secretion of the related chemokines CCL2, CCL7 and CCL8.
(A) Immunohistochemical analysis of the effects of suppressing CCL2, CCL7 or CCL8 in tumor-supportive fibroblasts on cancer cell proliferation, immune cell recruitment, blood vessel recruitment and mesenchymal cell activation in Cal51 tumors, using antibodies to Ki-67 (proliferation), antigen #7/4 (immune cells; neutrophils and monocytes), CD31 (endothelial cells of blood vessels) and α-SMA (mesenchymal cell activation). Scale bars represent 50 µm for Ki-67, antigen #7/4 and CD31 panels and 100 µm for α-SMA. (B–E) Quantification of tumor cell proliferation, monocytes and neutrophils, blood vessel endothelial cells, and mesenchymal cell activation. For each property, five different fields of three different tumors per group were scored. Data are expressed as the mean ± SEM. Asterisks indicates a significant difference between the experimental shRNAs and control non-targeting shRNA (p<0.01). # indicates a significant difference in proliferation in Cal51 only group compared to Cal51 coinjected with HFFF2 fibroblasts (p<0.01). Data are expressed as the mean ± SEM.
Figure 5
Figure 5. Suppressing amphiregulin expression in tumor-supportive fibroblasts reduces tumorigenicity and amount of mesenchymal cells.
(A) Reduced amphiregulin protein levels in HFFF2 fibroblasts expressing two distinct shRNAs directed against AREG compared to cells expressing non-targeting shRNA (immunoblotting). β-actin was used as a loading control. shAREG-2 was more potent at suppression both by immunoblotting and quantitative RT-PCR (see also Figure S3). (B) Reduced tumorigenicity of Cal51 cells co-injected with HFFF2 cells expressing either shRNAs targeting AREG as compared to control shRNA. Tumor-take rates for each group are indicated. Asterisks denote significant differences in tumor volumes for shAREG-1 or shAREG-2 compared to control (p<0.05) Data are expressed as the mean ± SEM. (C) As with (B) but for MDA-MB-231 breast cancer cells. p<0.05. (D) Immunohistochemical analysis of the effects of suppressing amphiregulin expression in tumor-supportive fibroblasts HFFF2 on Cal51 tumor cell proliferation and the tumor microenvironment (bottom panel) compared to tumors formed using Cal51 co-injected with control HFFF2 (top panel) as described in the legend to Figure 2. (E) Quantification of Ki-67 positive tumor cells in Cal51 tumors injected with control HFFF2 fibroblasts or shAREG-2 HFFF2 fibroblasts. Five different fields of three different tumors per group were scored. Error bars represent SEM. There was a small decrease in proliferation in the shAREG-2 HFFF2 fibroblast group (not statistical significance, p = 0.06). (F) Similarly performed quantification of monocytes and neutrophils, with a slight decrease observed in the shAREG-2 HFFF2 fibroblast group (not, significant, p = 0.14). (G) Similarly performed quantification of blood vessel endothelial cells, with no significant difference (p = 0.24). (H) Similarly performed quantification of activated mesenchymal cells, asterisk indicates a significant difference (p = 0.002). (I) Pericyte coverage of tumor associated blood vessels in tumors derived from injection of Cal51 cells with HFFF2 fibroblasts expressing shRNA-targeting AREG compared to control. Fifteen vessels from three tumors were examined for each group were analyzed for the ratio of pericytes (α-SMA+) to endothelial cells (CD31+). Data are expressed as the mean ± SEM. (J) The effect of amphiregulin on the replicative rate of wild-type mouse embryonic fibroblasts (WT-MEFs) measured by quantifying BrdU incorporation. Data representative of three independent experiments are shown. Concentration of amphiregulin (AREG ng/ml) is indicated on the x-axis. Asterisk indicates significant differences (p<0.05) between the control (0 ng/ml AREG) and experimental groups (10, 50, 100 and 200 ng/ml AREG). (K) As in J, but assaying the replicative rate of human fibroblasts, HFFF2.
Figure 6
Figure 6. Amphiregulin is a chemoattractant for fibroblasts and helps prevent necrosis and tumor cell death.
(A) Photomicrographs of mouse fibroblasts that have transversed a Boyden chamber in response to different concentrations of amphiregulin in the opposing chamber. Migration was measured 5 hours post plating. Scale bars represent 100 µm. (B) Amphiregulin promotes migration of mouse embryonic fibroblasts (MEFs) in a scratch wound-healing assay (photomicrographs at indicated time after initiation of assay). (C) Quantification of the Boyden migration assay. Data are expressed as the mean ± SEM. Asterisks indicate a significant difference (p<0.05) between experimental and control groups. (D) Quantification of migration in scratch wound healing assay (cells that had moved into the scratched area as percent of control). Asterisk indicates a significant difference between the control and experimental group (p<0.05). (E) Quantitative RT-PCR analysis of α-SMA expression (relative to GAPDH) in HFFF2 fibroblasts (gray bars) and Wi38 fibroblasts (red bars) upon treatment with amphiregulin. Asterisk indicates a significant difference between untreated HFFF2 fibroblasts and those treated at 50 and 100 ng/ml (p value 0.01 and 0.001, respectively). All data are expressed as the mean ± SEM. (F) Necrosis in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting AREG as visualized by H&E staining. Dashed lines indicate necrotic areas. Scale bars represent 1 mm. (G) Quantification of necrosis in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting AREG. Necrotic area was calculated from five different tumors per group. Data are expressed as the mean ± SEM. Asterisks indicate a significant difference (p = 3e-4) between experimental and control groups. (H) EGFR activation (phosphorylation) in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting AREG detected by immunostaining using an antibody to phospho-EGFR (Tyr1068). Scale bars represent 50 µm. (I) Quantification of phospho-EGFR positive tumor cells in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting AREG. Areas positive for pEGFR were calculated from five different fields of five different tumors per group. Asterisk indicates that the experimental group is significantly different than the control (p = 0.007). Data are expressed as the mean ± SEM. (J) Effects of amphiregulin on the viability of Cal51 cells plated on non-adhesive plates and cultured for 24 hours (anoikis assay). Viability was determined by measuring calcein AM uptake. Asterisk indicates that the experimental group is significantly different than the control (p<0.05). Data are expressed as the percentage of viable cells normalized to control with the error bars representing SEM.
Figure 7
Figure 7. Combined inhibition of chemokine and amphiregulin signaling is more effective at blocking the effects of tumor supportive fibroblasts.
(A) Paracrine upregulation of the expression of ligand-receptor pairs upon co-culture of tumor supportive fibroblasts with basal breast cancer cells. The fold-change in ligand expression in tumor-supportive fibroblasts (x-axis) is plotted along with the fold-change in receptor expression in breast cancer cells (y-axis). (B) Tumorigenicity of Cal51 cells expressing either control shRNA or shRNAs targeting CCR1 co-injected with HFFF2 fibroblasts. Tumor take rate for each group is indicated. Asterisks indicate significant differences in tumor volumes for shCCR1-1 and shCCR1-2 co-injection groups compared to control (p<0.01) (C) Tumorigenicity of Cal51 and HFFF2 fibroblasts coinjected in the following combinations: Cal51 cells expressing shRNAs targeting CCR1 co-injected with HFFF2 fibroblasts expressing control shRNA; Cal51 cells expressing control shRNA coinjected with HFFF2 cells expressing shRNA targeting AREG; Cal51 cells expressing shRNAs targeting CCR1 co-injected with HFFF2 fibroblasts expressing shRNA targeting AREG. Tumor take rate for each group is indicated. Asterisks indicate significant differences (p<0.05). (D) Histological and Immunohistochemical analysis of the effects of suppressing CCR1 in Cal51 cells and amphiregulin in tumor-supportive fibroblasts HFFF2 as described in the legend to Figure 2 but also tumor necrosis which was evaluated on hematoxylin and eosin stained sections. Scale bars represent 50 µm for Ki67 and 7/4, 100 µm for α-SMA and 500 µm for hematoxylin and eosin staining. (E) Quantification of microenvironment effects as described in the legend to Figure 2. Five different fields of three different tumors per group were scored. Error bars represent SEM. Hashtag indicates a significant difference in proliferation in the CCR1 silenced group compared to the control where p = 8.9E-04. Asterisk indicates a significant difference in proliferation in the CCR1 and AREG silenced group compared to the control where p = 0.01. (F) Similarly performed quantification of neutrophils and monocytes where Hashtag indicates a significant difference in the CCR1 silenced group compared to the control where p = 4.6E-04. Asterisk indicates a significant difference in the CCR1 and AREG silenced group compared to the control where p = 2.4E-05. (G) Similarly performed quantification of activated mesenchymal cells. Asterisk indicates a significant difference in the CCR1 and AREG silenced group compared to the control where p = 0.003. (H) Similarly performed quantification of necrotic area. Asterisk indicates a significant difference in the CCR1 and AREG silenced group compared to the control where p = 0.02.

Comment in

Similar articles

See all similar articles

Cited by 35 articles

See all "Cited by" articles

References

    1. Egeblad M, Nakasone ES, Werb Z (2010) Tumors as organs: complex tissues that interface with the entire organism. Dev Cell 18: 884–901. - PMC - PubMed
    1. Casey T, Bond J, Tighe S, Hunter T, Lintault L, et al. (2009) Molecular signatures suggest a major role for stromal cells in development of invasive breast cancer. Breast Cancer Res Treat 114: 47–62. - PubMed
    1. Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, et al. (2008) Stromal gene expression predicts clinical outcome in breast cancer. Nat Med 14: 518–527. - PubMed
    1. Ma XJ, Dahiya S, Richardson E, Erlander M, Sgroi DC (2009) Gene expression profiling of the tumor microenvironment during breast cancer progression. Breast Cancer Res 11: R7. - PMC - PubMed
    1. Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, et al. (2004) Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 6: 17–32. - PubMed

Publication types

MeSH terms

Associated data

Feedback