Mesenchymal Stem/Stromal Cell Engulfment by Breast Cancer Cells Generates a Hybrid Cancer Cell Population with Dormancy and Chemoresistance

Abstract

IntroductionDrug‐resistant dormant tumor cells are a major causes of breast cancer relapse and metastasis. It has been reported that in the tumor microenvironment breast cancer cells (BCC) can engulf mesenchymal stem/stromal cells (MSCs) and acquire dormancy. We previously documented the presence of BCC that engulf MSCs in clinical samples of breast cancer metastasis and showed that MSC engulfment leads to a more aggressive breast cancer phenotype, with an upregulated gene signature consisting of MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1. However, the mechanisms of MSC engulfment, and the phenotypic features of hybrid breast cancer cells are still unclear. Here, we tested the hypothesis that MSC engulfment generates hybrid cancer cells exhibit a multinucleated phenotype and increased survival advantage, dormancy, and chemoresistance, and that the genes we identified may mediate MSC engulfment.MethodMSC labeled with DS‐RED and MDA‐MB‐231 labeled with GFP (GFP‐231) were co‐cultured in microfluidic cell pairing devices, and treated with antibodies against MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1 or IgG control to quantify the percentage of MSC engulfing on‐chip. The conditioned media was subjected to a cytokine array. Live Imaging Microscopy was used to characterize the hybrid cancer cells in co‐cultures compared the GFP‐231. In Co‐cultures of DS‐RED‐MSC with GFP‐231 stably transduced with shWNT5A, shMSR1 or lentivirus control, and treated with Doxorubicin or untreated, we analyzed the percentage of DS‐RED+/GFP+ hybrid population, Ki‐67 low cells in G1, multinucleated cells (N4+), and the percentage of apoptotic cells using annexin V/PI.ResultsNeutralizing antibodies or shRNAs against WNT5A and MSR1 significantly reduced the percentage of MSC engulfment by MDA‐MB‐231 cells, and changed the cytokine profile in the conditioned medium of co‐cultures. In co‐cultures with DS‐RED‐MSCs, DS‐RED+/GFP+ hybrid cells had a higher percentage of Ki‐67 low in G1 and higher percentage of aneuploidy compared to GFP‐231+ cells, which were reversed by WNT5A and MSR1 shRNA downregulation. Doxorubicin treatment increased the percentage of DS‐RED+/GFP+ hybrids cells and decreased the percentage of apoptosis in the co‐cultures when compared to untreated controls.ConclusionsMSC engulfment by BCCs results in a hybrid multinucleated cell population with increased percentage of aneuploidy than BCC controls and a dormant phenotype characterized by higher of Ki‐67 low in G1 compared to controls. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of pro‐metastatic proteins including thrombosponding‐1, UPAR, and VEGF. Hybrid cancer cells show increased resistance to Doxorubicin compared to control BCCs, which can be rescued by downregulation of WNT5A and MRS1.Support or Funding InformationRole of EZH2 Breast Cancer Progression – R01 to C. Kleer. Role of CCN6 (WISP3) in the progression and metastasis – R01 to C. Kleer‐ AIRC Investigator Grant (IG#18602) to S. Andò‐PRIN2015#2015B7M39T to S. Andò