Our previous study showed that radiation-induced senescent-like fibroblasts promote invasive growth of cocultured MDA-MB-231 cells (8
). We asked in the present study whether such an effect of senescent-like fibroblasts could affect the radio- and chemosensitivity of breast cancer cells. The factors we previously identified as mediating the effects of radiation-induced senescent-like fibroblasts are secretory matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) and thereby indirectly influence surrounding epithelial cells. To determine whether factors other than MMPs could mediate the effects of fibroblasts on epithelial cells, we first cocultured fibroblasts and breast carcinoma cells in the absence of ECM. MDA-MB-231 cells were labeled with GFP and fibroblasts were immunostained with an anti-fibroblast antibody so that the two cell types could be distinguished visually under a fluorescence microscope. With available evidence indicating a secretary phenotype of senescent fibroblast that could affect nearby epithelial cells, we maintained confluent cultures of human mammary normally growing fibroblasts or senescent-like fibroblasts in low-serum (1% FBS) medium (LSM) to facilitate detecting the activity of secreted factors. Under these conditions, normally growing fibroblasts were gradually arrested after 3 days (not shown). MDA-MB-231 cells were also adapted in LSM for 3 days and then plated on top of the fibroblast lawn. The cocultures were maintained for an additional 7 days. Visual inspection under a phase-contrast microscope revealed a significant increase in cell numbers in the coculture with senescent-like fibroblasts compared to that with normally growing fibroblasts (, left panels). This increase in cell numbers appeared to result from an increase in MDA-MB-231 cells as shown by marked increase of green (GFP-labeled) cells (, right panels). To determine the carcinoma cell numbers, MDA-MB-231 cells were isolated from cocultures by magnetic cell sorting (MACS) and cells were counted using a Coulter counter. The results indicated a significant increase in the MDA-MB-231 cell population in a time-dependent manner, with an increase in carcinoma cell numbers of approximately twofold at day 3 and fourfold at day 7 in the cocultures with senescent-like fibroblasts relative to those with normally growing fibroblasts (). The increased cell numbers appeared to result from an elevated cell proliferation, because MDA-MD-231 cells exhibited a greater proliferation rate when cocultured with senescent-like fibroblasts than with normally growing fibroblasts (). The number of either senescent-like fibroblasts or normally growing fibroblasts in the cocultures was not detectably different (not shown).
FIG. 1 Senescent-like fibroblasts promote proliferation of cocultured MDA-MB-231 cells. Panel A: Representative images of cocultures of MDA231 cells with either normally growing fibroblasts or senescent-like fibroblasts as visualized under a phase-contrast microscope (more ...)
To probe this MMP-independent growth-promoting activity of radiation-induced senescent fibroblasts, we profiled the gene expression pattern of MDA-MB-231 cells. When we compared the gene expression profiles of MDA-MB-231 cells that were cocultivated with either normally growing fibroblasts or senescent-like fibroblasts, we identified 71 genes that were differentially (more than twofold) expressed. This set of genes was grouped into six clusters according to their distinct temporal expression patterns (not shown). Interestingly, seven out of the 14 genes that showed an early induction (subcluster 1) of genes encoding proteins important for the execution of mitosis, including HEC, a gene involved in spindle checkpoint signaling (15
), MPHOSPH1, a gene encoding a kinesin-related protein required for cytokinesis (16
), ECT2, a gene encoding a transforming protein that regulates cytokinesis (17
), TOP2A, a topoisomerase (18
), SMARCE1, a gene involved in chromatin remodeling (19
), and CENNFs, a gene responsible for chromosome segregation during mitosis (20
). We focused on this group of genes for further validation. Using quantitative RT-PCR to determine the mRNA levels, we verified the increased expression of these genes (). The increased expression of these genes would predict an accelerated cell cycle progression and cell division in MDA-MB-231 cells. To test this, we identified mitotic populations of MDA-MB-231 cells by immunostaining with the mitotic marker phospho-histone H3. The results revealed a marked increase in the percentage of mitotic MDA-MB-231 cells in cultures that were cocultured with senescent-like fibroblasts relative to those with normally growing fibroblasts (), confirming the data obtained from gene profiling. The increased number of mitoses is consistent with the elevated proliferation rate of MDA-MB-231 cells grown in the presence of senescent-like fibroblasts.
FIG. 2 Senescent-like fibroblasts stimulate mitosis of MDA-MB-231 cells in 2D cocultures. Panel A: The relative increase represents the mRNA level of MDA-MB-231 cultured with senescent-like fibroblasts normalized to that with normally growing fibroblasts. Data (more ...)
We next asked whether the growth promoting activity of senescent-like fibroblasts could modulate the sensitivity of cocultured breast cancer cells to either radio- or chemotherapy. MDA-MB-231 cells were cultured alone (homotypic culture) or with either normally growing fibroblasts or senescent-like fibroblasts in LSGM for 3 days and then treated with varying doses of radiation. The cocultures were allowed to recover for 24 h and then the breast cancer cells were isolated from the cocultures for the standard colony formation assay. The results indicated that senescent-like fibroblasts exerted a radioprotective effect on the cocultured MD-MB-231 cells, as shown by an apparent difference in the slopes of the radiation survival curves (). Notably, low doses of radiation (<20 cGy) appeared to stimulate the survival of carcinoma cells in the presence of senescent-like fibroblasts (). A similar result was seen in the cocultures of cells of another breast cancer cell line, MCF-7 (), indicating that the protective effect of senescent fibroblasts is not limited to one cell type. To examine whether this protective effect extended to chemotherapeutic agents, we cocultured MDA-MB-231 cells with either normally growing or senescent-like fibroblasts for 3 days and then treated them with adriamycin for 72 h. The cultures were fixed and stained with the Annexin V-FITC Apoptosis Detection Kit (Sigma, St. Louis, MO). MDA-MD-231 cells were differentiated from fibroblasts by staining with an epithelial cell marker (cytokeratin). Quantitative analysis of apoptosis indicated that, in the presence of senescent-like fibroblasts, MDA-MB-231 cells were less sensitive to adriamycin than MB-23 cells cocultured with normally growing fibroblasts (). The protective effect was also evident, albeit to a lesser extent, when conditioned medium was used (), supporting a critical role of secreted factors from senescent fibroblasts. The clonogenic cell survival assay showed that senescent-like fibroblasts rendered cocultured MDA-MB-231 cells more resistant to adriamycin (). Taken together, these results indicate that senescent fibroblasts render nearby malignant MECs resistant to cancer therapeutic agents.
FIG. 3 Senescent-like fibroblasts render MDA-MB-231 cells more radioresistant in 2D cocultures. MDA-MB-231 (panel A) or MCF-7 (panel B) cells were cultured alone (homotypic culture) or with either normally growing or senescent-like fibroblasts in LSGM for 24 (more ...)
To further substantiate the findings described above, we employed a three-dimensional coculture system that allows recapitulating cell-cell and cell-extracellular matrix (ECM) interactions to assess the influence of senescent stromal fibroblasts on the radiosensitivity of cocultured breast carcinoma cells in a tissue-like setting. MDA-MB-231 cells were cocultivated with normally growing or senescent-like fibroblasts in a 3D matrix for 4 days so that the interaction between epithelial cells and stromal fibroblasts became fully developed. The cocultures were then treated with single doses of radiation and maintained for an additional 10 days. Consistent with our previous results (8
), senescent-like fibroblasts induced invasive growth of cocultured MDA-MB-231 cells in 3D ECM. Radiation did not significantly inhibit such invasive growth unless the dose was greater than 0.5 Gy (). To analyze the effects quantitatively, single cells were recovered from the ECM gels after digestion with dispase and trypsin. MDA-MB-231 cells were isolated from HMFs using MACS and the cell numbers were quantified. The results indicate that the MDA-MB-231 cells cocultured with senescent-like fibroblasts displayed reduced sensitivity to radiation compared with the cells cocultured with normally growing fibroblasts. Of interest is that low-dose radiation (<20 cGy) appeared to stimulate the survival of cocultured MDA-MB-231 cells with senescent-like fibroblasts (). Again, a similar protective effect from senescent fibroblasts was also observed in MCF-7 cell cocultures ().
FIG. 4 Senescent-like fibroblasts render MDA-MB-231 cells more radioresistant in 3D cocultures. Panel A: MDA-MB-231 cells were cocultured with normally growing or senescent-like fibroblasts in LSGM for 4 days, after which the coculture was irradiated. Shown (more ...)
Our previous study showed that senescent-like fibroblasts induced invasive growth of MDA-MB-231 cells through the activation of the PI3K/Akt pathway (8
). Given the available evidence indicating that PI3K/Akt pathway enhances the viability of irradiated cells (21
), we asked whether the PI3K/Akt-dependent survival could contribute to the senescent-like fibroblast-induced radioresistance of MDA-MB-231 cells. For this, we tested cells of a MDA-MB-231 cell line overexpressing a dominant-negative Akt (K179M), which blocked the phosphorylation of Akt () and also partially attenuated the radioprotective effects of senescent-like fibroblasts (). We did not observe any detectable effect of dominant negative Akt in cocultures of normally growing fibroblasts with MDA-MB-231 (not shown). Together, our results indicate that the radio-responsiveness of cancer cells can be significantly modulated by alterations in the stromal microenvironment and that senescent stromal fibroblasts can markedly decrease the radiosensitivity of associated breast carcinoma cells.