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Objective To investigate the expression of immune-related molecules and the infiltration of immune cells from mouse breast cancer stem cells (BCSCs). Methods Mouse BCSCs of 4T1 and 4T07 were enriched by spheroid culturing; the expression of immune checkpoint-and antigen presentation-associated genes in BCSCs was detected by RT-qPCR; the proportion of various types of immune cells in spleen or tumor tissue of orthotopic tumor model mouse was detected by flow cytometry. Results The mouse BCSCs 4T1 and 4T07 were successfully enriched by spheroid culturing; the expression of immune checkpoints-and antigen presentation-associated genes were significantly different as compared with the adherent cancer cells; the proportion of both CD4+ and CD8+ T cell increased. However, the proportion of macrophage (CD11b+/F480+) was decreased in tumors formed by spheroid cells as compared with that in the tumors formed by adherent cells. Conclusions The expression of immune checkpoint-and antigen presentation-associated genes in BCSCs were significantly different and the infiltration of T cells increased in tumors formed by BCSCs.