Obesity causes systemic low-grade inflammation, which in turn increases cancer incidence and mortality. In breast tissue, obesity also results in increased infiltration of macrophages that contribute to inflammation. Many studies have shown that cancer cells can cause delipidation and cancer-association of adipocytes in the breast cancer microenvironment7,23. The ability of established tumors to stimulate M2 conversion in macrophages is also well documented24. However, the effects of nascent pro-inflammatory M1 macrophages on breast cancer-adipocyte crosstalk in early tumorigenesis is largely unknown.
Macrophage conditioned media has varying effectiveness upon different types of breast cancer cells.
To more accurately model the human breast cancer microenvironment, we developed a novel co-culture system that utilized only human derived cells and could model the breast cancer-adipocyte crosstalk in an obese and lean patient. Our model could also examine the contribution of infiltrating macrophages. The luminal A cell line, MCF-7, was principally used since it is hormone receptor positive and not metastatic. This non-aggressive cell line is a better representation of early tumorigenesis, which is usually less aggressive than an established tumor. MCF-7 cells grown in co-culture with macrophage CM showed higher proliferation and migration compared to breast cancer cells in co-culture without macrophage CM (Fig. 3). MCF-7 cells were also cultured alone in macrophage CM, which did not cause increased cell proliferation (Fig. 3A). Therefore, this effect is not due solely to the pro-inflammatory cytokines secreted by the macrophages, such as IL-1β and TNF-α (Fig. 2C), which can benefit tumor cell proliferation. Instead, the macrophage secreted factors affect the adipocytes, which in turn affect the breast cancer cells. MCF-7 cells grown in co-culture without macrophage CM also showed higher proliferation than the cancer cells alone, which agrees with previous studies that show increased tumor growth as a result of breast cancer-adipocyte crosstalk7,9. However, our study shows this effect is even stronger when macrophage CM media is added. This indicates macrophages that infiltrate into breast adipose tissue could aid in early tumorigenesis, before the tumor cells stimulate an M1 to M2 conversion.
This study’s main focus is on the effect of M1 macrophage infiltration in priming an inflammatory microenvironment in early tumorigenesis. However, to see if this would also affect a more aggressive, established tumor, the breast cancer proliferation and migration studies were repeated using the MDA-MB-231 cell line (Fig. S3). MDA-MB-231 is a triple-negative, highly aggressive and metastatic breast cancer cell line. In co-culture, adipocytes again promoted the growth of the cancer cells. However, in contrast to MCF-7 cells, macrophage CM did not appreciably increase MDA-MB-231 cell growth or migration. One possible explanation for the observed difference is the estrogen receptor status of these cell lines. MCF-7 cells are estrogen receptor positive, while MDA-MB-231 cells are estrogen receptor negative. Adipocytes synthesize estrogen through aromatase mediated metabolism of androgen precursors in post-menopausal women30. Macrophage derived proinflammatory mediators can also induce aromatase and estrogen dependent gene expression in adipocytes31. Therefore, estrogen overexpression by adipocytes could be one mechanism by which cancer-associated adipocytes contribute to breast cancer progression.
Macrophage conditioned media increases the pro-inflammatory character of adipocytes.
The expression levels of several cytokines associated with inflammation and breast cancer were highly up-regulated by astrocytes, both in co-culture and alone, in response to macrophage CM. For instance, there was marked increases in expression of the pro-inflammatory cytokines, IL-1β, TNF-α, and IL-6. This is consistent with another study by Permana et al., which also showed that murine RAW 264.7 macrophage conditioned media could induce 3T3-L1 adipocyte inflammation29. The co-culture system used in our study consisted of only human cell lines, establishing that these effects are present in human cell interactions, and can have important implications in human breast cancer progression and prognosis.
Curiously however, increased expression of adiponectin, an anti-inflammatory cytokine that is secreted solely by mature adipocytes and inversely correlated to fat mass, seemed to be more dependent upon co-culture with breast cancer cells than addition of macrophage CM. Increased adiponectin is an indicator of delipidation, which is one of the hallmarks of adipocyte cancer-association7,20. TNF-α has also been reported to cause delipidation of adipocytes and may promote the expression of adiponectin23,24. Yet when adipocytes were cultured alone in macrophage CM, there was no significant increase in adiponectin, unlike in co-culture, even though TNF-α was increased. Consequently, it would seem additional breast cancer cell related factors play a substantial role in adiponectin expression, and all three cell types work in concert to cause adipocyte cancer-association and promote tumor aggressiveness.
Macrophage conditioned media increases matrix metalloproteinase expression in adipocytes.
Macrophage CM also increased MMP-3 and MMP-11 expression by adipocytes in adipocyte-breast cancer co-culture (Fig. 4B). MMP-11 is a negative prognosis marker in several human cancers28 and can also cause delipidation of adipocytes20. Andarawewa et al. found that MMP-11 was expressed in human adipocytes proximally located to invasive cancer cells, but not distally located adipocytes20. In addition, this same study found these adipocytes at the tumor invasive front that expressed MMP-11 were reduced in size. In studies examining the role of MMPs in normal adipogenesis, MMP-11 and MMP-3 deficient mice developed more adipose tissue, indicating a negative regulatory role21,22. The increased expression on MMP-11 and MMP-3 further indicate that macrophage CM can increase adipocyte delipidation and cancer-association. Finally, while MMP-11 expression by adipocytes, both in co-culture and alone, was similarly affected by macrophage CM, there appeared to be an additive affect on MMP-3 expression. Although MMP-3 and MMP-11 are structurally and functionally similar, both being stromelysins and negative regulators of adipogenesis, their expression may require different signaling mechanisms.