Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis

Abstract

The mammary gland microenvironment during postlactational involution shares similarities with inflammation, including high matrix metalloproteinase activity, fibrillar collagen deposition, and release of bioactive fragments of fibronectin and laminin. Because inflammation can promote tumorigenesis, we evaluated whether the tissue microenvironment of the involuting gland is also promotional. Extracellular matrix was isolated from mammary glands of nulliparous rats or rats with mammary glands undergoing weaning-induced involution. Using these matrices as substratum, nulliparous matrix was found to promote ductal organization of normal mammary epithelial MCF-12A cells in three-dimensional culture and to suppress invasion of mammary tumor MDA-MB-231 cells in transwell filter assays. Conversely, involution matrix failed to support ductal development in normal cells and promoted invasiveness in tumor cells. To evaluate the effects of these matrices on metastasis in vivo, MDA-MB-231 cells, premixed with Matrigel, nulliparous matrix, or involution matrix, were injected into mammary fat pads of nude mice. Metastases to lung, liver, and kidney were increased in the involution matrix group, and correlated with a twofold increase in tumor vascular endothelial growth factor expression and increased angiogenesis. These data suggest that the mammary gland microenvironment becomes promotional for tumor cell dissemination during involution, thus providing a plausible mechanism to explain the high rate of metastases that occur with pregnancy-associated breast cancer.

Figure 1

Involution matrix shows attributes of provisional wound-healing stroma. a: Fibrillar collagen deposition is increased in the mammary glands of female rats undergoing weaning-induced involution (I) compared to nulliparous rats (N). Fibrillar collagen was detected histologically on 5-μm sections using Sirius Red F3Ba and Weigert’s iron hematoxylin counterstain. Arrows point to red-staining interlobular fibrillar collagen. b: Fibronectin (FN) and laminin (LN) levels are increased in involution matrix (I) compared to nulliparous matrix (N), as shown by Western blotting of mammary matrix preparations loaded by equal protein. A Coomassie Blue-stained gel is shown to demonstrate equal protein lane loading, and tissue actin is shown to demonstrate equal cellular content of the starting material from which the mammary matrix was extracted. c: MMP-2 and MMP-9 zymogen activities are enhanced in involution matrix (I) compared to nulliparous matrix (N). Matrix loaded by equal protein. Scale bar, 50 μm.

Figure 2

Quiescent and involution matrix differentially promote mammary epithelial cell organization and invasion. a: In three-dimensional culture assays, nontransformed human mammary epithelial MCF-12A cells form simple spheroid mammospheres on Matrigel (i), organize into duct-like structures with complex branching on nulliparous matrix (ii), and form simple mammospheres on involution matrix (iii). H&E-stained 5-μm section of MCF-12A organoid on nulliparous matrix shows highly organized duct-like structure with hollow central lumen (iv). MDA-MB-231 cells, derived from a highly aggressive human breast cancer, did not form mammospheres on any matrix and instead formed loosely organized clusters of cells (v, vi, vii) with invasive filopodia, as seen in the inset of vi. H&E-stained 5-μm section of a MDA-MB-231 organoid shows that these cells lack lateral adhesion junctions and are not hollow. b: In a transwell filter invasion assay, involution matrix (I) promoted MDA-MB-231 cell invasion more than either nulliparous matrix (N) or Matrigel (M). *Statistically significant difference between N and I; P = 0.010 (paired t-test). c: In an invasion assay modeling xenograft conditions (2 × 10⁶ cells in 20 μl of matrix), involution matrix preferentially promoted cell invasion. i: Matrigel/tumor cell bolus (M), and ii: nulliparous matrix/tumor cell bolus (N). The transwell filter pores appear as white circles, and the invasive cells stain purple (white arrow). In iii, more cells invaded in response to being precoated with involution matrix (I). iv: Involution matrix (I)-treated cells had lamellopodia (thick arrow) and filopodia (thin arrow). d: Physical interaction between the tumor cells and endogenous matrix in the xenograft model was confirmed by removal of ECM/tumor cell inoculates 72 hours after injection. The top panel shows representative image of cells adhered to matrix fibrils present in involution matrix (arrow) and nulliparous matrix (data not shown). Matrigel lacks a fibrillar component and thus did not show this type of cell-fibrillar matrix interaction. In the bottom panel, tumor cells are enveloped in nulliparous matrix (arrow), which is representative of all groups. Scale bars: 100 μm [a (i–iii, v–vii)]; 50 μm [a (iv, viii), c (i–iii), d]. Original magnifications: ×50 [a (i–iii, v–vii), c (i–iii)]; ×200 [a (iv, viii), c, iv]; ×400 [a, vii (inset), d].

Figure 3

Tumor growth and detection of metastasis. a: Tumor volumes of mammary fat pad tumors were calculated twice weekly. Nulliparous and involution matrix group tumor growth was identical. Matrigel group tumors were slightly smaller; however, this difference did not reach statistical significance. b: Tumor cell-specific vimentin was detected by immunohistochemistry. Left: Mouse mammary fat pad with human tumor cells positive for vimentin and normal mouse ductal epithelial cells (asterisk) negative for vimentin. Right: Vimentin-positive tumor cells, depicted by arrows, in the lung of an involution matrix group animal. c: Graph showing relationship between the size of primary tumor and likelihood of lung metastasis in individual animals, as determined by immunohistochemical detection of vimentin-positive cells and RT-PCR detection of human RNA using human-specific primers to β2M. There was a strong correlation between detection of metastases in lung and size of the primary tumor (data for Matrigel shown). For these analyses, RT-PCR and immunohistochemical signal intensities were divided into quartiles. Scale bar, 50 μm (b). Original magnifications, ×400 (b).

Figure 4

Involution matrix preferentially promotes metastasis to lung, liver, and kidney. a: In mice with mammary fat pad tumors less than 1 g, metastasis to lung was detected by RT-PCR using human-specific primers to β2M. Involution-group mice had more than twice the incidence of metastasis to lung. Each lane depicts signal from lung tissue of one animal. W, water control lane; −, negative control lane of mouse RNA only; and +, positive control lane of MDA-MB-231 cell RNA. M, Matrigel group mice; N, nulliparous group mice; and I, involution group mice. b: Real-time SYBR Green PCR on brain, kidney, liver, and lung RNA using human-specific primers to β2M with expression signal normalized to mouse β-actin. Involution group animals had more frequent metastasis to lung, liver, and kidney. Brain metastases were rare in all groups. c: Human Cot-1 DNA probe, labeled with Spectrum Red, was used to detect human tumor cells in mouse tissue. i: Dual image of red fluorescent human tumor cells and DAPI-stained nuclei at the tumor/mammary gland border from an involution group animal. Asterisk depicts tumor and arrow points to a tumor cell that has invaded the mammary fat pad. ii: Dual image of red fluorescent human tumor cells and DAPI-stained nuclei in the liver of the same animal. iii and iv: Only the signal from the human tumor cells are shown whereas v and vi show only the DAPI signal in mammary gland and liver, respectively. Scale bars: 60 μm (c, i, iii, and v); 20 μm (c, ii, iv, and vi).

Figure 5

Involution matrix promotes angiogenesis, fibroblast activation, and VEGF expression. To evaluate paths of metastasis, metastasis to mammary lymph nodes and vascularization at the tumor/mammary gland border were analyzed. a: i: Metastatic human MDA-MB-231 cells (arrows) in a mammary lymph node (asterisk depicts lymph cells within node). ii: Neovascularization at the tumor/mammary gland border in an involution group animal (I) at 72 hours after tumor cell injection. The border between the mammary gland and tumor bolus is depicted with green hatch marks. Numerous small vessels are evident at the border of the tumor bolus (arrow). At 6 weeks, focal angiogenic response was infrequent at the border of Matrigel group (iii) and nulliparous group tumors (iv) but was common in involution group tumors (v, arrows). Tumor/mammary gland borders shown are representative. b: Smooth muscle actin, a marker of reactive stroma, is present in fibroblasts in a representative involution group tumor (top) but is absent from the nulliparous group tumor stroma (bottom). The arrows show the tumor stroma border, asterisks depict tumor. c: Human tumor VEGF expression as a marker for angiogenesis was measured by quantitative real-time PCR. The average relative VEGF expression values, normalized to GAPDH expression levels, are reported for each group (open bars, left). The average tumor burden (g) per group is also shown (filled bars, left). VEGF expression in the involution group is statistically different from both nulliparous and Matrigel groups (Bonferroni P values: I versus M, P = 0.01; I versus N, P < 0.0001; M versus N, P = 0.15). In the right graph, VEGF expression levels normalized to tumor burden are shown (arbitrary units). Scale bars: 50 μm (a, i, and ii); 100 μm (a, iii, iv and v); 40 μm (b).