Hdc-expressing Myeloid-derived Suppressor Cells Promote Basal-like Transition And Metastasis Of Breast Cancer

Background and Objective:Breast cancer?BC?is the second leading cause of female cancer-associated mortality,and more than 90%of the cases are caused by metastasis.To form metastatic lesions,malignant cells should detach from the primary tumor,invade through the surrounding mesenchymal tissue,enter and survive in circulation system,and become circulating tumor cells?CTCs??also known as collective invasion?.These stages serve as an important window for clinical tailored strategies,because metastasis may occur before onset of clinical symptoms.BC tissues are histologically complex,containing a variety of cell types.Considerable advances have been made in elucidating the cellular diversity through transcriptome-based analysis and subdivide BC into three main types:luminal?hormone receptor-positive?,HER-2-overexp-ressing?hormone receptor-negative,HER2 positive?,and triple-negative?hormone receptor and HER2 negative?BC.Of note is the fact that the progression of BC is accompanied by an increase in cellular motility and matrix invasion.Cancer cells at the invasive front or in collective invasion exhibit different phenotypes compared to other tumor cells in animal models or clinical cases.Substantial evidence suggested that cytokeratin 14?CK14?,an essential protein for normal breast development,endows tumor cells with a conserved basal epithelial program.The migratory cells in the breast embryo placode display CK14⁺CK8⁺SMA^-.Normal breast stem cells and cancer cells with enhanced invasiveness,rather than relatively indolent malignant cells,express CK14.Although normal tubular cells show negativity,the reacquisition of CK14 expression in luminal tumor cells facilitates their metastasis.However,the underlying molecular mech-anisms are largely unknown.During the normal development,Wnt/?-catenin pathway contributes to the regulation of normal cellular behaviors,including stem cell pluripotency and differentiation.The aberrant activation of Wnt/?-catenin signaling leads to the breast dysplasia composed of CK14⁺CK5⁺CK8^-?SMA^-basal-like cells,suggesting the possibility that the expression of CK14 may be regulated by Wnt/?-catenin pathway.However,the exact cellular source of Wnts is unclear.Immune cells,widely appreciated as one of the most important regulators of microenvironment,play a central role during BC tumorigenesis and progression.At the early stages,BC tissues can release cytokines/chemokines such as TGF-?,IFN-?,and TNF-?,contributing to anticancer therapies.When chronic injury and pathogens persist,the delicate balance will be ruined by non-resolving inflammation and serves as the initiating event of the sequential neoplastic progression.BC cells can recruit myeloid-derived suppressor cells?MDSCs?into the tumor mass through mTOR signaling pathway or the secretion of granulocyte colony-stimulating factor?G-CSF?.MDSCs,deriving from the hematopoietic stem cells/progenitor cells?HSC/HSPCs?in the bone marrow or spleen,can be subdivided into two major types:the granulocytic MDSCs(PMN-MDSCs,CD45⁺CD11b⁺Ly6G^hi)and monocytic MDSCs(M-MDSCs,CD45⁺CD11b⁺Ly6C^hi).A recent study by our group suggested that Hdc can serve as a marker of the myeloid-biased HSC/HSPCs,which may differentiate into immature myeloid cells in the context of aging,injury,inflammation,and tumorigenesis.Hdc-expressing PMN-MDSCs promotes the progression of colonic caner in murine models via the recruitment of Foxp3⁺regulatory T cells.Considerable developments have been made to explore tailored strategies for inhibiting cancer-associated MDSCs over the last decade.However,several concerned issues are needed to be addressed to keep the promise of these strategies.Yet we have little definitive information about the exact entity of immune cells which promotes the evolution of BC mainly due to the heterogeneity of this population.In this study,we investigated the role and regulatory mechanisms of CK14 expression in the advanced stages of BC on the basis of transgenic murine models and clinical archives.The results show that Hdc⁺PMN-MDSCs specifically expanded in the setting of BC metastasis and expressed high levels of Wnts to abnormally activate Wnt/?-catenin signaling and enhance CK14 expression in adjacent tumor cells.Our data further unveil the interactions between Hdc⁺PMN-MDSCs and invasive tumor cells and propose that the inhibition or depletion of these cells with targeted strategies might facilitate anti-BC therapies.Methods:1.The archival blocks of patients with BC were collected and underwent tissue microarray?TMA?.Serial sections were evaluated by H&E and immunohistoch-emical stains for CK14.2.Metastatic BC murine models were established using Hdc-CreERT2;eGFP;MMTV-PyVT female transgenic mice.Immunohistochemical stains and FACS were employed to explore the number and percentage of MDSCs in metastatic tissues.3.The translocation of?-catenin in metastatic BC cells were observed by immunohistochemical stains to evaluated the situation of Wnt/?-catenin signaling.4.Hdc+PMN-MDSCs or Hdc+PMN-MDSCs-derived Wnts were deleted or inibhited by porcupineflox/flox or iDTR to explore the role of PMN-MDSCs-derived Wnts during the tumorigenesis and progression of BC,CK14 expression,and aberrant activation of Wnt/?-catenin signaling.Results:1.Of 112 patients with unmetastatic luminal BC,5 cases?4.5%?exhibited local positivity for CK14,and 33?28.2%?of 117 metastatic luminal BC cases showed strong and diffuse positivity for CK14.2.There was a significant tendency of CD15+PMN-MDSCs towards metastatic BC tissues in human cases.FACS analysis further confirmed an increased percentage of Hdc+PMN-MDSCs in CK14+metastatic BC tissues of transgenic animal models.3.RNA-Seq and RT-PCR results suggested that Hdc+PMN-MDSCs obtained from CK14+tumors expressed high levels of Wnts,including Wnt2,4,5a,and 7b.4.In vivo experiments via transgenic models ablated Hdc+meyloid cells or Hdc+meyloid cells-derived Wnts significantly inhibited the metastasis of BC and CK14 positive rate.Conclusion:Taken together,our study provided preliminary data to support the important role of MDSCs in BC progression.Hdc⁺PMN-MDSCs were recruited and expanded in CK14⁺metastatic tissues,followed by the secretion of high levels of Wnts,which aberrantely activated Wnt/?-catenin signaling with a paracrine pattern in CK14⁺BC cells.Our study keeps the perspective of tailed therepies on the basis of targeting Hdc⁺PMN-MDSCs.