A CCL8 gradient drives breast cancer cell dissemination

Abstract

The migration of cancer cells towards gradients of chemoattractive factors represents a potential, yet elusive, mechanism that may contribute to cancer cell dissemination. Here we provide evidence for the maintenance of a gradient of increasing CCL8 concentration between the epithelium, the stroma and the periphery that is instrumental for breast cancer cells' dissemination. In response to signals elicited by the neoplastic epithelium, CCL8 production is enhanced in stromal fibroblasts at the tumor margins and in tissues at which breast cancer cells tend to metastasize such as the lungs and the brain. Manipulation of CCL8 activity influences the histology of the tumors and promotes major steps of the metastatic process such as invasion to adjacent stroma, intravasation and ultimately extravasation and seeding. These findings exemplify how gradients of chemoattractive factors such as CCL8, drive metastasis and suggest that interference with their operation may provide means for breast cancer management.

Figure 1

Establishment of a self-sustained CCL8 gradient between breast cancer and stromal cells. (a) Genomic organization of the cytokine cluster that harbors Ccl8 in mouse and humans. Data were compiled from Ensembl Genome Browser (www.ensembl.org). (b) Cytokine expression in human HFFF2 and mouse 3T3 fibroblasts following culture in MDA231 and MDA468 breast cancer cell conditioned media. Ccl8 is the only cytokine in this cluster that specifically responds to breast cancer cells’ media in both mouse and human fibroblasts. (c) Expression of CCL8 in human invasive breast cancer stroma. Data were obtained by Oncomine, https://www.oncomine.org. (d) Breast cancer patients’ survival in relation to CCL8 expression. High CCL8 expression is associated with worse prognosis. Data were obtained from Kaplan-Meier-plotter [Breast Cancer], www.kmplot.com/. High expression is indicated in red, low expression in blue, cutoffs were set automatically by the software. (e) Ccl8 expression in mouse and human fibroblasts following exposure to breast cancer cells’ conditioned media. *, p<0.01, Student’s t-test.

Figure 2

CCL8-induced chemoattraction. (a) Chemoattraction of various breast cancer cells and fibroblasts by increasing amounts of CCL8 in transwells in vitro. (b) MDA231 cells’ migration in response to CCL8 added in the upper or lower chamber of the transwells or distributed evenly between the upper and the lower chamber. (c) Receptors’ expression measured by quantitative PCR in cells migrated through the transwells relative to receptors expression in regular wells (c) Diagrammatic depiction of the CCL8 gradient (red) that originates from the fibroblasts in response to signals from the cancer cells. *, p=0.05, **, p<0.05, ***, p<0.001 Student’s t-test.

Figure 3

Ccl8 expression in tumors, stroma and peripheral tissues. (a) Ccl8 levels of EO771 tumors developed in wt (n=6) and Ccl8KO (n=4) mice. Tumor volumes in all cases analyzed ranged between 200mm³–300mm³. (b) Expression of Ccl8 in the stroma of EO771 tumors growing in wt mice. Right panel shows in higher magnification of the area marked in the left panel by a blue square. (c) Serum Ccl8 levels in relation to tumor volume in different EO771 breast cancer – bearing mice. Ep, epithelium, St, fibroblastic stroma. p<0.05, Pearson’s correlation (d) Tumoral Ccl8 (n=6) and Ccl8 in various organs from tumor free (n=4) and mice bearing EO771 tumors (n=5) (200mm³–300mm³). *, p<0.05 Student’s t-test

Figure 4

Effect of Ccl8 inhibition in EO771 tumors in vivo. (a) EO771 tumor onset in wt mice following administration of a neutralizing antibody for CCL8. (b) Inhibition of mCcl8-induced chemoattraction by a neutralizing antibody for CCL8 in RAW 264.7 macrophages in transwells. (c) EO771 tumor onset in wt and Ccl8KO mice (24). (d) Histology of EO771 tumors in wt and Ccl8KO mice. Genotypes are shown in white insets. The upper panel corresponds to images from the core of the tumor while the lower panel from the margins. Lower right graph indicates cellularity expressed as epithelial cells per high power optic field. (n=3 per genotype). Ep, epithelium, FS, fibroblastic stroma, AC, antipocytic stroma. *, (p<0.05,Student’s t-test). (e) Van Gieson staining of EO771 tumors in control and Ccl8KO mice. (f) Vimentin expression (brown staining) close to tumor margins in EO771 tumors that developed in wt and Ccl8KO mice. Right panel shows in higher magnification of the area marked in the left panel by a black square.

Figure 5

Modulation of CCL8 levels in mice affects the metastatic potential of MDA231 cells. (a) Ccl8 in various organs from tumor free (n=3) and mice bearing MDA231 tumors (n=3) (200mm³–300mm³). (b) Diagrammatic illustration of the experimental design. (c) Expression of CCL8 in matrigel-enclosed fibroblasts inoculated adjacent (about 5mm) to MDA231 tumors or in tumor-free mice (control) (n=3). (d) Inhibition of CCL8-induced chemoattraction by a neutralizing antibody for hCCL8 in MDA231 cells in transwells. (e) Invasion of MDA231-β-Gal cells (blue) in matrigel-enclosed HFFF2 fibroblasts inoculated in nude mice and treated daily for 3 days with neutralizing antibody for CCL8 (aCCL8) (n=5) or IgG (n=4). Sections were counterstained with eosin. Samples from Ab-treated mice exhibited consistently minimal invasion of MDA231 cells while controls had variable invasion of MDA231 cells. Quantification of fibroblasts in control and Ab-treated mice is shown in (f). (g) CTCs’ abundance, in arbitrary units, 24h after they received CCL8 (n=2) or saline (n=2). (h) Diagrammatic illustration of the experimental process followed to reveal the effects of CCL8 inhibition in the seeding of mammary glands from MDA231-β-Gal cells. (i) Representative images of MDA231-β-Gal cells colonizing the mammary glands of mice that received IgG (n=4) or aCCL8 (n=4). Right panel shows the area indicated in dashed square in high magnification. Quantification of the MDA231-β-Gal foci in control and Ab-treated mice is shown in (j).

Figure 6

Diagrammatic illustration of how a gradient of CCL8 is self-sustained to promote dissemination of breast cancer cells. Fibroblasts and probably other stromal cells are activated in response to secreted factors produced by the breast cancer cells (a) and activate CCL8 expression which results in the establishment of a CCL8-gradient (b). This results in the chemoattraction of the cancer cells towards the CCL8 gradient casing progressively their movement along with the shift of the gradient towards the tumor margins at which stromal cells are more abundant (c). Eventually cancer cells intravasate in response to elevated levels of CCL8 in circulation establishing CTC populations (d). Ultimately, niches of increased CCL8 production in peripheral tissues promote the extravasation of cancer cells and the establishment of populations of secondary tumor growth (e).