Il-6 signaling between ductal carcinoma in situ cells and carcinoma-associated fibroblasts mediates tumor cell growth and migration

Increased levels of pro-inflammatory cytokines, including IL-6, in tumors and serum of breast cancer patients correlate with poor prognosis [26, 36‐38]. Immunohistochemistry on breast tissue from female patients, age 30 to 81 with an average age of 49.5, confirmed IL-6 protein expression in 65 % of patient samples diagnosed with DCIS (Fig. 1a and b, cf. 1c and d).

We utilized a 3D MAME culture model (Additional file 2: Figure S1), to study the role of IL-6 signaling between human breast DCIS cells and human breast CAFs. The MAME model allows for co-culture of multiple cell types in the context of a three dimensional microenvironment. Our model is advantageous over commercially available 3D platforms as it can be utilized for live cell microscopy, and imaging live cell functional assays (such as proteolysis) [32]. MAME cultures are tractable and can be altered to grow multiple cell types at various ratios or at various relative positions within the matrix. Additionally, the reduced density overlay allows for real-time collection and analysis of cytokine secretion without disrupting the longitudinal growth of the culture.

Here we examined the expression of IL-6 and the associated pro-inflammatory genes interleukin 1β (IL1β) and nuclear factor kappa light chain enhancer of activated B cells 1 (NFκB1) in human breast MCF10.DCIS and SUM102 cell lines. Our data show higher IL-6 mRNA levels in both MCF10.DCIS and SUM102 cell lines, as compared to the MCF10A breast epithelial cell line (Fig. 1e). Levels of IL-6 protein in lysates of the three cell lines were below the level of detection. When we measured secreted IL-6 in conditioned media from MCF10.DCIS or SUM102 cultures, we found that both DCIS cell lines secreted 10 to 30-fold more IL-6 than the MCF10A cells (Fig. 1f).

To test whether blocking IL-6 autocrine signaling affected DCIS cell growth, we treated MCF10.DCIS MAME cultures for 8 days with either an IL-6 neutralizing antibody (nAb) or an equivalent concentration of a species and isotype matched antibody (Fig. 2). IL-6 nAb treatment resulted in a reduction in diameter of the multicellular 3D structures (Fig. 2b, cf. 2a, quantified in 2d). This was reversible as replacement of media on day 8, with media lacking IL-6 nAb, resulted in a significant increase in diameter of the MCF10.DCIS structures after an additional 48 h (Fig. 2c, cf. 2b, quantified in 2d).

To determine the effects of IL-6 nAb on MCF10.DCIS structures, we examined the expression of a panel of candidate genes that have been associated with tumor growth and invasion [39‐43]. The expression of IL-6 was upregulated 2-fold in the treated cultures. The expression of TWIST1, vimentin, and Fibroblast Growth Factor 2 was downregulated greater than 2-fold, while minimal changes were observed in the expression of E-cadherin, N-cadherin, and NFκB1 (Fig. 2e). To test whether pharmacological suppression of IL-6 could reproduce IL-6 nAb mediated growth inhibition, we treated cells with oxymatrine, a naturally occurring inhibitor of IL-6 gene expression. Oxymatrine has been shown to prevent nuclear translocation of NFκB-p65 thereby inhibiting transcriptional activation of its target genes, which include IL-6 [44]. Oxymatrine treatment was able to replicate the growth inhibitory effects observed with IL-6 nAb (Additional file 4: Figure S2B, cf. S2A, quantified in S2C). Neither oxymatrine nor IL-6 nAb treatment resulted in marked cell death as cytotoxicity assays showed no difference in cell viability after 48-hour drug treatment (Additional file 4: Figure S2D).

CAFs represent a population or group of populations of stromal cells that can promote tumor cell growth [14, 45‐47]. The mechanism of supported tumor growth is likely through stromal-epithelial paracrine signaling. Therefore, we next evaluated human breast CAFs to determine their contribution of IL-6 in the tumor microenvironment. Additionally, we examined the role that CAFs play in MCF10.DCIS cell proliferation and motility in the 3D MAME model.

We examined the expression of IL-6 mRNA in normal human fibroblasts and CAFs grown in 3D. Here we found that CAFs exhibited elevated expression of IL-6 mRNA compared to normal fibroblasts (Fig. 3a). Protein levels of IL-6 in FB-NF-i normal fibroblast lysates were near the lower limit of detection and undetectable in NAF-FB or NAF98i lysates. IL-6 levels in CAF40TKi lysates were significantly higher than in FB-NF-i lysates (Fig. 3b). Levels of IL-6 in CAF-conditioned media were higher than in normal fibroblast-conditioned media (Fig. 3c).

We next co-cultured MCF10.DCIS cells with CAFs in 3D, at a seeding ratio of five tumor cells to one CAF [32], to evaluate the effect of CAF-secreted cytokines on DCIS cell proliferation and related changes in morphology of multicellular structures. We found that in MCF10.DCIS:CAF40TKi co-cultures there was an increase in the average diameter and volume of the multicellular structures and a prominent formation of branch-like interconnections between the structures (Fig. 3e, cf. 3d, quantified in Additional file 5: Figure S3A). Fluorescent imaging of MCF10.DCIS:CAF40TKi 3D co-cultures revealed that the branch-like multicellular connections between structures were primarily composed of tumor cells, yet contained some CAFs (Additional file 5: Figure S3B and Additional file 6: Video S1).

We also observed that CAFs induced an increased rate of proliferation in MCF10.DCIS cells. Using a thymidine analog to evaluate the rate of DNA synthesis, we observed that co-cultures had a consistently higher rate of DNA synthesis than CAFs alone or DCIS cells alone (Additional file 7: Figure S4A-D). CAF and DCIS co-culture using a slower growing DCIS cell line, i.e., SUM102, also resulted in changes in multicellular structure formation. We observed the presence of multiple invasive processes in SUM102:CAF co-cultures that were completely absent in cultures of the SUM102 cells alone (Additional file 8: Figure S5C and D, arrow, cf. S5A and B).

CAFs express and secrete a number of proteases, which enhance their ability to migrate and remodel extracellular matrices [48‐51]. In CAF:MCF10.DCIS co-cultures, we evaluated cell:cell interactions and motility. Here we found that MCF10.DCIS spheroids formed attachments to CAFs and remained associated with them throughout an 8-day culture. On day 1 (24 h after seeding) of a co-culture we observed many single DCIS cells and a few CSFE labeled CAFs (Fig. 4a). After 3 days in culture DCIS cells had formed small spheroids that were in contact with one or more CAFs (Fig. 4b). On day 5 we observed CAFs at the invasive edges of DCIS tumor spheroids. A representative image shows a CAF (Fig. 4c, arrow) in contact with a DCIS spheroid. A time-lapse video shows the CAF leading the DCIS spheroid (Additional file 9: Video S2). High magnification imaging shows heterocellular contact between a tumor structure and a single CAF (Additional file 10: Figure S6). We did not observe these invasive characteristics in DCIS cell grown alone, as the tumor structures tended to roll in the extracellular matrix (Additional file 11: Video S3). By day 7 CAFs were seen at the invasive edge of most tumor structures (Fig. 4d).

Using confocal microscopy we examined the 3D spatial organization of the DCIS cells in relation to CAFs. A top view of a 3D reconstruction shows an MCF10.DCIS structure (only nuclei labeled) branching out toward CFSE labeled CAFs (pseudo-colored white) (Fig. 4e, Additional file 12: Video S4). The core of this structure (dashed circle) formed shortly after seeding and grew in size before the two branching outgrowths formed. These outgrowths are reminiscent of the “strand” multicellular migration pattern previously described [52]. A side view of the same structure shows that DCIS cells migrated downward from the core structure toward the CAFs (Fig. 4f).

IL-6 protein expression in these co-cultures was detected using immunofluorescent staining (Additional file 13: Figure S7). A CFSE-labeled CAF showed high expression of IL-6, whereas the leading edge of the DCIS multicellular structure showed a gradient of IL-6 that was strongest near the attachment to the CAF (Additional file 13: Figure S7A, arrow). We also showed that MCF10.DCIS cells that do not migrate to serum-free media (Additional file 13: Figure S7D) migrated toward CAF-conditioned serum-free media (Additional file 13: Figure S7E, S7F).

IL-6 has previously been shown to upregulate cathepsin B [21], a protease associated with breast cancer progression. Therefore, we examined the expression and localization of cathepsin B in relation to active proteolysis in our 3D MAME co-cultures. Our active proteolysis assays were performed during live cell imaging [33]. Fluorescent imaging of live cells in MAME co-culture revealed regions of proteolytic activity (green) around several multicellular structures (Fig. 5a). The cells were then fixed for cathepsin B immunofluorescent staining. Visualizing the same structures revealed cathepsin B expression at the interface between the multicellular structures and the matrix (Fig. 5b). Fluorescent overlay shows co-localization between DQ-collagen IV degradation and areas with high cathepsin B expression (Fig. 5c); however, matrix degradation also occurred in areas that did not stain for cathepsin B (arrow). Upon visualization using light microscopy, the cells in these areas had morphological features consistent with CAFs, which would be consistent with current knowledge of fibroblast migration and their secretion of many proteases in addition to cathepsin B [53, 54].

We then used protease inhibitors to determine the contribution of all cysteine proteases and/or cathepsin B and L, to the overall proteolysis in the MAME culture. We treated cultures with E64d, a cell permeable pan-cysteine protease inhibitor, and found a 30 – 40 % reduction of matrix degradation (Fig. 5d). We also treated cultures with the cell permeable cathepsin B/L inhibitor, CA074Me, and found a 60 – 70 % reduction in matrix degradation (Fig. 5e). CA074Me has previously been shown to be a more efficacious inhibitor against cathepsins B and L than is E64d [35, 55]. We did not observe complete inhibition of proteolysis with the cysteine protease inhibitors, a finding in accord with previous findings that DCIS cells and CAFs secrete many non-cysteine proteases [31, 56, 57].

We have shown that CAFs promote the proliferation and migration of human breast DCIS cells. To determine whether CAF-stimulated migration and interaction with MCF10.DCIS cells could be inhibited by blocking IL-6, we co-cultured MCF10.DCIS cells with two human breast carcinoma-associated fibroblast lines WS-12Ti or CAF40TKi, in the presence of IL-6 nAb or an isotype control antibody. IL-6 nAb reduced the size of MCF10.DCIS:CAF multicellular structures when grown with either CAF cell line (Fig. 6a-e). Multicellular structure volume measurements confirmed a significant reduction when treated with IL-6 nAb (Fig. 6f-h).

To confirm the role of IL-6 as a key player in the formation of the multicellular structure interconnections, we utilized shRNA targeting IL-6 expression. In CAF40TKi and MCF10.DCIS cells, we achieved greater than 50 % reduction in secreted IL-6 (Additional file 14: Figure S8A). When we co-cultured CAF40TKi-shRNA control fibroblasts with MCF10.DCIS cells, we saw a phenotype similar to non-shRNA transduced cultures (Additional file 14: Figure S8B, cf. 3E). Knocking down CAF40TKi IL-6 expression in co-culture resulted in the formation of multicellular structures with uniform borders and few invasive processes (Additional file 14: Figure S8C). Co-culture with non-shRNA transduced CAF40TKi fibroblasts and shRNA-IL-6 transduced MCF10.DCIS cells showed greater MCF10.DCIS:CAF40TKi interaction and multicellular structure branching (Additional file 14: Figure S8D).

IL-6 signaling is propagated through either direct cell membrane receptor signaling or soluble receptor trans-signaling (TS). In the DCIS cell lines, we found that IL-6R expression was low and sometimes undetectable by qRT-PCR. Additionally, we detected very low levels of sIL-6R secreted from MCF10.DCIS cells and none from SUM102 cells. In contrast, CAFs had a higher level of IL-6R gene expression and high secretion of sIL-6R as verified by ELISA. These findings suggest that IL-6TS is a likely mechanism of IL-6 action in these DCIS cells [58] (Additional file 15: Figure S9).