Blockade of immune‐checkpoint programmed cell death protein 1 (PD‐1) or programmed cell death ligand 1 can enhance effector T‐cell responses. However, the lack of response in many patients to checkpoint‐inhibitor therapies emphasizes the need for combination immunotherapies to pursue maximal antitumor efficacy. We have previously demonstrated that antagonism of C‐X‐C chemokine receptor type 4 (CXCR4) by plerixafor (AMD3100) can decrease regulatory T (T _reg )‐cell intratumoral infiltration. Therefore, a combination of these 2 therapies might increase antitumor effects. Here, we evaluated the antitumor efficacy of AMD3100 and anti‐PD‐1 (αPD‐1) antibody alone or in combination in an immunocompetent syngeneic mouse model of ovarian cancer. We found that AMD3100, a highly specific CXCR4 antagonist, directly down‐regulated the expression of both C‐X‐C motif chemokine 12 (CXCL12) and CXCR4 in vitro and in vivo in tumor cells. AMD3100 and αPD‐1 significantly inhibited tumor growth and prolonged the survival of tumor‐bearing mice when given as monotherapy. Combination of these 2 agents significantly enhanced antitumor effects compared with single‐agent administration. Benefits of tumor control and animal survival were associated with immunomodulation mediated by these 2 agents, which were characterized by increased effector T‐cell infiltration, increased effector T‐cell function, and increased memory T cells in tumor microenvironment. Intratumoral T _reg cells were decreased, and conversion of T _reg cells into T helper cells was increased by AMD3100 treatment. Intratumoral myeloid‐derived suppressor cells were decreased by the combined treatment, which was associated with decreased IL‐10 and IL‐6 in the ascites. Also, the combination therapy decreased suppressive leukocytes and facilitated M2‐to‐M1 macrophage polarization in the tumor. These results suggest that AMD3100 could be used to target the CXCR4‐CXCL12 axis to inhibit tumor growth and prevent multifaceted immunosuppression alone or in combination with αPD‐1 in ovarian cancer, which could be clinically relevant to patients with this disease.—Zeng, Y., Li, B., Liang, Y., Reeves, P. M., Qu, X., Ran, C., Liu, Q., Callahan, M. V., Sluder, A. E., Gelfand, J. A., Chen, H., Poznansky, M. C. Dual blockade of CXCL12‐CXCR4 and PD‐1‐PD‐L1 pathways prolongs survival of ovarian tumor‐bearing mice by prevention of immunosuppression in the tumor microenvironment. FASEB J. 33, 6596–6608 (2019). www.fasebj.org

Studies have demonstrated low expression of miR-145 associated with cell proliferation and migration in a wide variety of tumors. Here, we studied the expression of miR-145 in relation to the occurrence and development of breast cancer. Total RNA from breast cancer tissue and corresponding adjacent normal tissue was extracted and used to detect miR-145 expression by quantitative real-time polymerase chain reaction (qRT-PCR). We also transfected breast cancer cells with hsa-miR-145 mimics, hsa-miR-145 inhibitor, mimics negative control (mimics NC) or inhibitor negative control (inhibitor NC). Cell proliferation was analyzed by colony formation assays and methyl thiazolyl tetrazolium assays. Cell proliferation in breast cancer cells was decreased after overexpression of miR-145 and increased following miR-145 suppression. Cell migration and invasion were assessed using Transwell and wound healing assays, respectively, and were also decreased after overexpression of miR-145 and increased after miR-145 suppression in breast cancer cells. Finally, western blot assays showed that overexpression of miR-145 inhibited expression of transforming growth factor-β1 (TGF-β1). Collectively, these data suggest that miR-145 may inhibit TGF-β1 protein expression which may in turn contribute to tumor formation.

Mesenchymal stem cells (MSCs) have been demonstrated to be involved in tumor progression and the modulation of the tumor microenvironment, partly through their secretome. Extracellular vesicles (EVs) are membranous nanovesicles secreted by multiple types of cells and have been demonstrated to mediate intercellular communication in both physiological and pathological conditions. However, numerous questions still remain regarding the underlying mechanisms and functional consequences of these interactions. The purpose of this study was to investigate the effects of human umbilical cord mesenchymal stem cell-derived EVs (hUC-MSC-EVs) on the proliferation, migration and invasion of human breast cancer cells. We successfully generated and identified hUC-MSCs and hUC-MSC-EVs which were used in this study. The results revealed that treatment of the MDA-MB-231 and MCF-7 human breast cancer cells with medium containing hUC-MSC-EVs significantly enhanced the proliferation, migration and invasion of the cells in vitro. Treatment of the cells with medium containing hUC-MSC-EVs also reduced E-cadherin expression and increased N-cadherin expression, thus promoting the epithelial-mesenchymal transition (EMT) of the breast cancer cells. Treatment of the breast cancer cells with extracellular signal-regulated kinase (ERK) inhibitor prior to the interaction with hUC-MSC-EVs significantly reversed the enhanced proliferation, migration and invasion, as well as the EMT of the breast cancer cells induced by the hUC-MSC-EVs. On the whole, these data indicate that hUC-MSC-EVs promote the invasive and migratory potential of breast cancer cells through the induction of EMT via the ERK pathway, leading to malignant tumor progression and metastasis. Taken together, the findings of this study suggest that targeting pathways to reverse EMT may lead to the development of novel therapeutic approaches with which to combat breast cancer.

Mesenchymal stem cells (MSCs) are known to migrate to tumor tissues and to play an important role in cancer progression. However, the effects of MSCs on tumor progression remain controversial. The purpose of the present study was to detect the effects of human umbilical cord-derived MSCs (hUC‑MSCs) on the human breast cancer cell lines MDA-MB‑231 and MCF-7 in vitro and the underlying mechanisms. MSCs were isolated and identified from umbilical cord tissues. MDA-MB‑231 and MCF-7 cells were treated with conditioned medium (CM) from 10 and 20% umbilical cord MSCs (UC-MSCs), and the resulting changes in proliferation and migration were investigated. The 3-(4,5-dimethyl-2-thiazolyl)‑2,5-diphenyl‑2-H-tetrazolium bromide (MTT) and plate clone formation assays were used to assess the effect on proliferation, and the effects of CM on MDA-MB-231 and MCF-7 migration were assessed through scratch wound and Transwell migration assays. The expression of cell proliferation- and metastasis-related genes and proteins and activation of the ERK signaling pathway were analyzed by RT-PCR and western blot assays. UC-MSCs are characteristically similar to bone marrow MSCs (BM-MSCs) and exhibit multipotential differentiation capability (i.e., osteoblasts and adipocytes). The MTT, plate clone formation, scratch wound and Transwell migration assay results revealed that 10 and 20% CM promoted the proliferation and migration to higher levels than those observed in the control group. Our findings showed that UC-MSC-CM inhibited E-cadherin expression, increased the expression of N-cadherin and proliferating cell nuclear antigen (PCNA) and enhanced the expression of ZEB1, a transcription factor involved in epithelial‑to‑mesenchymal transition (EMT), through activation of the ERK pathway. U0126, an inhibitor of ERK, reversed the effects of UC-MSC-CM on breast cancer cell proliferation and migration. We conclude that UC-MSCs promote the proliferation and migration of breast cancer cell lines via activation of the ERK pathway.