Regulation of senescence escape by TSP1 and CD47 following chemotherapy treatment

Senescence is a tumor-suppressive mechanism induced by telomere shortening, oncogenes, or chemotherapy treatment. Although it is clear that this suppressive pathway leads to a permanent arrest in primary cells, this might not be the case in cancer cells that have inactivated their suppressive pathways. We have recently shown that subpopulations of cells can escape chemotherapy-mediated senescence and emerge as more transformed cells that induce tumor formation, resist anoikis, and are more invasive. In this study, we characterized this emergence and showed that senescent cells favor tumor growth and metastasis, in vitro and in vivo. Senescence escape was regulated by secreted proteins produced during emergence. Among these, we identified thrombospondin-1 (TSP1), a protein produced by senescent cells that prevented senescence escape. Using SWATH quantitative proteomic analysis, we found that TSP1 can be detected in the serum of patients suffering from triple-negative breast cancer and that its low expression was associated with treatment failure. The results also indicate that senescence escape is explained by the emergence of CD47low cells that express a reduced level of CD47, the TSP1 receptor. The results show that CD47 expression is regulated by p21waf1. The cell cycle inhibitor was sufficient to maintain senescence since its downregulation in senescent cells increased cell emergence. This leads to the upregulation of Myc, which then binds to the CD47 promoter to repress its expression, allowing the generation of CD47low cells that escape the suppressive arrest. Altogether, these results uncovered a new function for TSP1 and CD47 in the control of chemotherapy-mediated senescence.

Parental and emergent LS174T cells were injected subcutaneously in NOD/SCID mice. Tumors were recovered and histological specimen of LS174T or PLC tumors were stained (n=6). Area of necrosis are identified by hematoxylin and eosin staining, the purple dots correspond to tumor cells, pink regions correspond to necrosis, purple dots on pink regions correspond to inflammatory cells. In our experiments, the necrotic area is generally composed of dead cells and abnormal eosinophils as shown in one illustrative zoomed picture (B). The percentage of necrosis corresponds to the percentage of the necrotic region as compared to the total surface.

Supplementary Figure 3 Legend
A. Senescent cells were generated by treating LS174T cells with sn38 for 96h. Conditioned media (CM) were collected after 24h of serum starvation. Cell emergence was induced after treatment for 7 days using conditioned medium supplemented with 10% FBS. Clones were then counted using crystal violet staining (n=3). B. Migration assays were performed using Boyden inserts. Conditioned media obtained from LS174T parental or senescent cells stimulated with 10%FBS for 48h and supplemented with 3% FBS were placed at the well bottom. After 72h, migrating cells were stained with crystal violet (one image representative of three experiments). C. Migration assays were performed using Boyden inserts. Conditioned media obtained from MCF7 parental, senescent or emergent cells stimulated with 10% FBS for 48h and supplemented with 3% FBS were placed at the well bottom. After 48h, migrating cells were stained with crystal violet (one image representative of three experiments). D. Following emergence of LS174T cells, PLD and PLS cells were cell sorted and p21waf1 expression and β-galactosidase staining were evaluated (n=3, one representative image).

Supplementary Figure 4 Legend
Representative images of lung metastasis.

Supplementary Figure 5 Legend
Following senescence induction by sn38 or doxorubicin, emergent cells were generated from LS174T or MCF7 cells by adding 10% serum for 7 days. Cells were then serum-starved for 24h and supernatants were recovered. TSP1 concentration was evaluated by ELISA analysis (n=3+/-sd).

Supplementary Figure 6 Legend
A. Description of the PACS08 clinical trial, blood collection and mass spectrometry approach. See clinical trial ID: NCT00630032. Relapse was defined as: a local or regional relapse; a metastatic relapse, a contralateral breast cancer, or death from any cause. Note that blood samples were obtained before chemotherapy treatment. B. ELISA analysis of TSP1 seric levels in patients that suffer from triple negative breast cancers (n=22). C. Kaplan Meier analysis of the link between survival and TSP1 expression (n=225).

Supplementary Figure 7 Legend
A. Senescence was induced in LS174T or MCF7 cells and TSP1 expression was evaluated on emergent cells by immunofluorescence, either on the total population (left) or on senescent (middle) and dividing (right) subpopulations. Ki67 staining identifies the clones that have restarted proliferation in the middle of senescent cells (n=3, one representative image). B. Cells have been infected with a lentivirus expressing an shRNA directed against CD47 or a non targeting control. Clonogenic tests were then performed and cell growth was analyzed after 8 days (n=3 for LS174T, n=4 for MCF7 +/-sd. Kolmogorov-Smirnov test, *= p<0,05). C. LS174T cells have been infected with a lentivirus expressing an shRNA directed against CD47 or a non targeting control. Senescence was then induced for 4 days, 10% serum was added to start emergence and mRNAs were then extracted after two days to evaluate the expression of CD47 and TSP1 by RT-QPCR (n=3 +/-sd).