Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

Abstract

Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts - the ability to alter epithelial differentiation - that might also explain the loss of tissue function and organization that is a hallmark of aging.

Fig. 1

Epithelial tumors stimulated by senescent human fibroblasts progress to full malignancy. (A) (i and ii) Morphology (40× magnification), and (iii and iv) cytokeratin (red) and vimentin (green) immunostaining (400× magnification), of SCp2-P (i and iii) and SCp2-T (ii and iv) cells in 3D Matrigel culture. (B) Invasion and migration of SCp2-P, SCp2-T and MDA-MB-231 (positive control) cells through basement membrane components (Matrigel) in Boyden chamber assays. Error bars indicate the s.e.m. from three fields. (C) Tumorigenicity of SCp2-T cells injected alone (solid black lines) into immunocompromised mice. Tumor cells were injected and tumor size was measured at the indicated intervals after injection. Tumors formed by SCp2-P cells co-injected with senescent fibroblasts (gray lines) is shown for comparison (Krtolica et al., 2001).

Fig. 2

Senescent human breast fibroblasts attenuate mammary epithelial alveolar differentiation. (A) MCF-10A cells co-cultured with presenescent (Presen, left) or senescent (Sen, right) hBFs in 3D on Matrigel. Low (40×, upper panels) and high (100×, lower panels) magnifications of the cultures are shown. (B) α6 integrin immunostaining (green) of MCF-10A cells co-cultured on Matrigel with presenescent (Presen, left) or senescent (Sen, right) hBFs. Nuclei were counterstained with DAPI (blue); images shown at 200× magnification. (C) E-cadherin immunostaining (green) of MCF-10A cells co-cultured on Matrigel with presenescent (Presen, left) or senescent (Sen, right) hBFs. Nuclei were counterstained with DAPI (blue); images shown at 1000× magnification. (D) GM-130 immunostaining (green) of a representative MCF-10A alveolus co-cultured on Matrigel with presenescent (Presen, left) or senescent (Sen, right) hBFs. Nuclei were counterstained with DAPI (blue). Merged images show the apical polarity of GM130; images shown at 400× magnification. (E) Tubulin (green) and actin (red) immunostaining of representative MCF-10A alveoli co-cultured on Matrigel with (Presen, left) or senescent (Sen, right) hBFs. Nuclei were counterstained with DAPI (blue), and images shown at 400× magnification.

Fig. 3

Senescent human breast fibroblasts attenuate mammary epithelial functional differentiation. (A) Ki67 immunostaining (green) of MCF-10A cells co-cultured on Matrigel with presenescent (Presen, left) or senescent (Sen, right) hBFs. Nuclei were counterstained with DAPI (blue); images shown at 200× magnification. (B) MCF-10A alveoli formed in the presence of presenescent (Presen) or senescent (Sen) hBFs were scored for having <5 (gray bars) or >5 (black bars) Ki67-positive nuclei per alveolus. (C) Sizes of DAPI-stained MCF-10A alveoli formed on Matrigel in the presence of presenescent or senescent hBFs were determined by digital quantification of images. A minimum of 50 alveoli per condition was analyzed. Shown is the average area occupied by alveoli in presenescent (Presen) or senescent (Sen) co-cultures. Error bars indicate s.e.m. (D) Western analysis of β-casein and cytokeratin 18 expression by Eph4 cells cultured on Matrigel plus lactogenic hormones in the presence of presenescent (Presen) or senescent (Sen) hBFs. Signals were quantified by densitometry and values from the presenescent culture were set arbitrarily at 1. Shown is the mean and standard deviation from four experiments. (E) β-casein immunostaining (red) of EpH4 alveoli cultured in the presence of presenescent (left panels) or senescent (right panels) hBFs. Nuclei were counterstained with DAPI (blue); images shown at 400× magnification.

Fig. 4

Senescent mouse fibroblasts stimulate mammary epithelial branching morphogenesis. Primary organoids were embedded in collagen and plated in the upper chambers of transwells. Presenescent (Presen) or senescent (Sen) mBF were plated in the transwell lower chambers. Error bars show s.e.m. of triplicate wells. Shown is one of four experiments, all of which gave similar results. (A) Morphology and color coded explanation of branching classification of organoids cultured with presenescent (i and iii) or senescent (ii and iv) mBF. Shown are the core areas (white), and the primary (1ary, blue), secondary (2ary, yellow) and tertiary (3ary, red) branches. (B) Average organoid size (Total), length of branches (Branches) and core areas (Core) of organoids co-cultured with presenescent or senescent mBF. (C) Average number of primary (1ary), secondary (2ary) and tertiary (3ary) branches of organoids co-cultured with presenescent or senescent mBF. (D) Average length of primary (1ary), secondary (2ary) and tertiary (3ary) branches of organoids co-cultured with presenescent or senescent mBF. (E) Average number of epithelial nuclei, quantified by DAPI fluorescence, in organoids co-cultured with presenescent or senescent mBF.

Fig. 5

Senescent fibroblast-produced factors stimulate epithelial branching. Collagen-embedded organoids were cultured in the presence of conditioned medium from presenescent (Presen) or senescent (Sen) mBF. The conditioned medium was either unsupplemented (−) or preincubated with HGF blocking antibody (anti-HGF), an MMP-3 blocking peptide (MMP-3i) or an MMP-2 inhibitor (MMP-2i). Error bars show s.e.m. of triplicate wells. (A) Average size (Total), branch length (Branches) and core area (Core) of organoids cultured with conditioned media lacking or containing an HGF blocking antibody or MMP-3i. (B) Average number of primary (1ary), secondary (2ary) and tertiary (3ary) branches in the organoids analyzed in A. (C) Organoids in A were analyzed for average primary and secondary branch lengths in the presence of HGF blocking antibody or MMP-3i, and expressed as change relative to presenescent or senescent-derived conditioned medium lacking antibody or inhibitor. The asterisks indicate a statistically significant reduction in branch length by HGF neutralization, compared to medium lacking HGF antibody, as determined by a Student’s t-test. Branching length in presenescent and senescent conditioned media was affected similarly by HGF neutralization. (D) Average number of epithelial cells per organoid, quantified by DAPI fluorescence of nuclei. Asterisks indicate a statistically significant change in proliferation, as determined by a Student’s t-test. (E) Percentage of organoids having primary (1’ Only) or higher level (secondary and tertiary; Side Branching) branches in the absence or presence of MMP-2i. (F) Casein zymography of conditioned medium from presenescent and senescent mBF. A 50 kDa marker and MMP-3 are indicated. Densitometry was performed on the reverse image to determine the relative expression; the signal from presenescent conditioned medium was set arbitrarily at 1.

Fig. 6

Role of MMP-3 in branching stimulated by senescent fibroblasts. (A) Morphology of representative collagen-embedded organoids after culture in the presence of conditioned medium from presenescent (Presen) mBF containing the indicated amounts of recombinant MMP-3. An organoid cultured with conditioned medium from senescent (Sen) (X-irradiated) mBF is shown for comparison. (B) Percentage of organoids that undergo primary (1’ only) or higher level (secondary and tertiary; Side branching) branching in the presence of presenescent mBF-conditioned medium containing the indicated concentrations of recombinant MMP-3. The effect of conditioned medium from senescent (Sen) mBF is shown for comparison. (C) Proliferation of organoids, determined by DAPI fluorescence, in the presence of presenescent mBF-conditioned medium containing the indicated concentrations of recombinant MMP-3. The effect of conditioned medium from senescent (Sen) mBF is shown for comparison.