Mechanisms by which whole genome doubling drives breast tumor evolution [CUT&Run]

ABSTRACT: Triple-negative breast cancers (TNBC) account for 10-15% of all breast cancers. The combination of chemotherapy and Immunotherapy (ICI: immune checkpoint inhibitor) is now a standard in stages II-III TNBC while this regimen is used only in a subset of advanced TNBC tumors presenting a high expression of PD-L1. However, whole exome sequencing of tumors in clinical trials investigating ICI for treatment of various solid tumors other than breast cancer indicate that tumors which undergone a WGD (whole genome doubling) are more sensitive to immune checkpoints inhibitors. Almost all human cells are diploid. Nevertheless, a WGD happens when regulation system maintaining the diploid status fail. The WGD+ cells are tetraploid, oncogenic and facilitate tumorigenesis. The WGD occurs in 44% of breast cancers and has been correlated to advanced cases, lower survival and higher resistance to chemotherapies and targeted therapies. Therefore it is crucial todefine the role of WGD towards the immune escape/control of TNBC and response to ICI. The first step of this work was to generate different cells line in both WGD- and WGD+ status. Using a technique of membrane homofusion of parentals cells, we obtained a WGD in three different basal murine mammary tumor cell lines (67NR, 168fARN, TA3Hauschka) and one human TNBC cell (SUM 159). The acquisition of WGD was then confirmed by flow cytometry (FACS) (DNA quantity by Hoechst staining) and karyotyping. The TA3Hauschka cell line is of particular interest since it is the only murine cell line which is diploid in parental state. By injection of WGD+ or parental DP cells in mammary fat pads of syngeneic immunocompetent strain or immunocompromised (NSG) mice, we observed a plateau of tumoral growth only for immunocompetent mice injected with WGD- cells while the WGD+ tumors escaped to spontaneous immune control. No difference of growth was seen in NSG mice between WGD- and WGD+ tumors. On tumor sections, a much more pronounced necrosis was seen in WGD- tumors comparatively to WGD+ tumors in syngeneic mice while the necrosis was mild and similar for both WGD+ and WGD- tumors in NSG mice. Immunofluorescence staining demonstrated an important neutrophilic infiltration of WGD+ while the WGD- tumors had strong T-cell lymphoid infiltrates frankly superior to the lymphoid infiltration of WGD+ tumors. These differences of infiltrates have been confirmed on digested tumors by both FACS and single cells RNA expression (scRNA) analyses. The FACS analysis also showed that the WGD+ tumors had an immunosuppressive pattern with notably higher levels of Tregs cells. To validate the hypothesis that WGD- are controlled by the infiltration of cytotoxic CD8 cells (leading to higher necrosis) while WGD+ tumors induce an immunosuppressive field, we performed an in vivo experiment investigating depletion of CD8 and neutrophils (anti-CD8 and anti-Ly6G antibodies) and a treatment by anti-PD-L1 antibodies (ICI). The treatment by isotypes antibodies confirmed the immune control of only the WGD- tumors. Anti-CD8 treatment led to an important increase in WGD- tumors growth but hadn’t any impact on WGD+ tumors while anti-Ly6G antibodies had no significative impact on WGD- and WGD+ tumors. The treatment by anti-PD-L1 antibodies was significantly more efficient to reduce the growth of WGD+ tumors than WGD- tumors. ScRNA analysis of WGD- and WGD+ untreated tumors revealed that WGD+ tumors have a significative decrease of presentation of MHCI complex. Therefore, this study highlights the potential role of biomarker of response to immune checkpoint inhibitor of whole genome doubling in TNBC.

ORGANISM(S): Mus musculus

PROVIDER: GSE273085 | GEO | 2026/03/15

REPOSITORIES: GEO