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. 2021 Nov 1;24(12):103392.
doi: 10.1016/j.isci.2021.103392. eCollection 2021 Dec 17.

Targeting myeloid-derived suppressor cells to attenuate vasculogenic mimicry and synergistically enhance the anti-tumor effect of PD-1 inhibitor

Yinan Li  1   2 , Kailiang Qiao  1   2 , Xiaoyun Zhang  1   2 , Haoyang Liu  1   2 , Heng Zhang  1   2 , Zhiyang Li  1   2 , Yanrong Liu  3 , Tao Sun  1   2
Affiliations

Targeting myeloid-derived suppressor cells to attenuate vasculogenic mimicry and synergistically enhance the anti-tumor effect of PD-1 inhibitor

Yinan Li et al. iScience. .

Erratum in

Abstract

Myeloid-derived suppressor cells (MDSCs) enhance the proliferation of endothelial cells to stimulate angiogenesis. However, many aggressive malignant tumors do not have endothelial cell-dependent blood vessels in the early stage and instead generate microcirculation by forming vasculogenic mimicry (VM). To date, the relationship between MDSCs and tumor cells remains the focus of ongoing studies. In this work, MDSCs were co-cultured with mouse melanoma cells and can enhance proliferation and VM formation of melanoma cells. For MDSCs targeting, doxycycline (DOX) was found to selectively suppress PMN-MDSCs but has no influence on T cells. In addition, DOX pretreatment substantially reduced the promoting ability of MDSCs for the VM formation of B16-F10 cells. DOX also inhibited tumor growth and enhanced the antitumor activity of PD-1 inhibitors in C57BL6 and BALB/c mice subcutaneously inoculated with B16-F10 and 4T1 cells, respectively. In conclusion, the combination of DOX and PD-1 inhibitor could be an anticancer strategy.

Keywords: Cancer; Genetics.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
MDSCs accumulate in B16-F10 tumor-bearing mice (A) Representative dot plots of CD11b+Gr-1+ MDSCs in the bone marrows (left two panels) and spleen (right two panels) of naive mice or B16-F10 tumor-bearing mice. (B) Unpaired Student’s t test was done for statistical analyses of MDSC ratio. (C) MDSCs of bone marrow from naive and B16-F10 tumor-bearing mice labeled by CD11b (red), Gr1 (green), and nucleus (blue) to detect the expression of MDSC makers. (D) Volcano plot of the differentially expressed proteins of MDSCs between native mice and B16-F10 tumor-bearing mice (n = 3). (E) Enrichment analysis of differentially expressed proteins by Metascape (https://metascape.org). (F–H) Immunosuppressive function analysis of MDSCs including ROS level, NO production, and Arg1 activity. All statistical data are represented as mean ± SEM and Unpaired Student’s t test was done for statistical analyses. ∗p < 0.05, ∗∗p < 0.01.
Figure 2
Figure 2
MDSCs promote proliferation and VM formation of B16-F10 cells in vitro (A) Representative CFSE histograms of co-cultured cells (B16-F10 and MDSCs) showing that the MDSCs from tumor-bearing mice dose dependently promoted the VM of B16-F10 cell line. (B) Statistical analysis of the ratio of proliferating cells. (C) Representative fields of tube formation of B16-F10 with or without MDSCs of different concentrations. (D) Statistical analysis of the relative tube number to evaluate the ability of VM formation for B16-F10 cells. (E) Living cell tracing for the co-culture of B16 cells labeled by GFP and MDSCs labeled by CM-DiI. (F) SEM image of VM showing that the co-culture of MDSCs and B16 promoted vascular mimicry and extracellular matrix secretion. (G) Immunofluorescence staining showing the expression of VM marker VE-cadherin. (H) Representative image of Transwell invasion assay. (I) Statistical analysis of the relative number of invaded cells. All statistical data are represented as mean ± SEM. Unpaired Student’s t test was done for statistical analyses. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 3
Figure 3
Doxycycline inhibits the promoting capability of MDSCs for VM formation in vitro (A) Flow cytometric analysis of the proportion of MDSCs in bone marrow from C57BL/6 tumor-bearing mice treated by different doses of doxycycline and 5-FU. (B) Statistical analysis of the ratio of MDSCs. (C) Flow cytometric analysis of the proportion of PMN-MDSCs (CD11b+Ly6G+Ly6Clow) and M-MDSCs (CD11b+Ly6G−Ly6Chigh) in bone marrow from C57BL/6 tumor-bearing mice treated by different doses of doxycycline and 5-FU. (D) Statistical analysis of the ratio of MDSCs subsets. (E) Representative CFSE histograms of PMN-MDSCs. (F) Statistical analysis of the ratio of proliferating cells. (G) Representative dot plots for apoptosis of T cells after DOX treatment. (H) Statistical analysis of the apoptosis ratio of T cells. (I) Western blot to detect the levels of Arg-1 and iNOS in MDSCs after different doses of DOX treatment. (J) Schematic showing the co-culture of B16 and MDSCs with or without DOX treatment. (K–L) Representative images and statistical analysis of tube formation to evaluate the ability of co-cultured cells. (M) Immunofluorescence image to detect the expression of VE-cadherin of B16 cells. All statistical data are represented as mean ± SEM. Unpaired Student’s t test was done for statistical analyses. ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 4
Figure 4
Doxycycline reduces the number of MDSCs and synergistically enhances the anti-tumor activity of PD1 in B16-F10 cell tumor-bearing model (A, C, and E) Dot plots of the proportion of MDSCs (CD11b+Gr-1+) in bone marrows, spleens, and tumor cells from control and DOX-treated B16-F10 tumor-bearing mice (n = 3). (B, D, and F) Unpaired Student’s t test was done for statistical analyses of ratio of MDSCs. All data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Tumor images of four groups to evaluate the anti-tumor effect of combination drug therapy of DOX and PD-1. (H) Tumor growth curves and statistical analysis of the four groups. (I) Survival curve of the four groups. Survival analysis was performed by using the Kaplan-Meier method with log rank test. ∗p < 0.05, ∗∗p < 0.01. (J) Volcano map of the up- and downregulated proteins of MDSCs. (K) Enrichment analysis of the differential proteins.
Figure 5
Figure 5
Doxycycline reduces the number of MDSCs and synergistically enhances the anti-tumor activity of PD1 in 4T1 tumor-bearing model (A, C, and E) Dot plots of the proportion of MDSCs (CD11b+Gr-1+) in bone marrows, spleens, and tumor cells from control and DOX-treated 4T1 tumor-bearing mice (n = 3). (B, D, and F) Unpaired Student’s t test was done for statistical analyses of ratio of MDSCs in 4T1 tumor-bearing model. All data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001. (G) Tumor images of four groups to evaluate the anti-tumor effect of combination drug therapy of DOX and PD-1 in 4T1 tumor-bearing model. (H) Tumor growth curves and statistical analysis of the four groups. The data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001(Student t-test). (I) Survival curve of the four groups. Survival analysis was performed by using the Kaplan-Meier method with log rank test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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