anlotinib alters tumor immune microenvironment by downregulating PD-L1 expression on vascular endothelial cells

Abstract

Aberrant vascular network is a hallmark of cancer. However, the role of vascular endothelial cells (VECs)-expressing PD-L1 in tumor immune microenvironment and antiangiogenic therapy remains unclear. In this study, we used the specimens of cancer patients for immunohistochemical staining to observe the number of PD-L1+ CD34+ VECs and infiltrated immune cells inside tumor specimens. Immunofluorescence staining and flow cytometry were performed to observe the infiltration of CD8+ T cells and FoxP3+ T cells in tumor tissues. Here, we found that PD-L1 expression on VECs determined CD8+ T cells', FoxP3+ T cells' infiltration, and the prognosis of patients with lung adenocarcinoma. Anlotinib downregulated PD-L1 expression on VECs through the inactivation of AKT pathway, thereby improving the ratio of CD8/FoxP3 inside tumor and remolding the immune microenvironment. In conclusion, our results demonstrate that PD-L1 high expression on VECs inhibits the infiltration of CD8+ T cells, whereas promotes the aggregation of FoxP3+ T cells into tumor tissues, thus becoming an "immunosuppressive barrier". Anlotinib can ameliorate the immuno-microenvironment by downregulating PD-L1 expression on VECs to inhibit tumor growth.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. PD-L1 high expression on tumor VECs is closely related to the prognosis of patients.

a, b Typical images of CD34/PD-L1 (endothelial cells highly expressing PD-L1) immunostaining in lung adenocarcinoma (n = 35), kidney (n = 30), and colon cancer (n = 30), scale bars: 50 μm. Kaplan–Meier survival curves of OS c and PFS c of patients with lung adenocarcinoma exhibiting high (red) or low (green) VEC-PD-L1. d The correlation of PD-L1+ vessels and PD-L1+ tumor tissues (n = 106). Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Fig. 2. PD-L1 high expression on VECs is closely related to the infiltration of immune cells.

a, c Representative images of CD8+T cells and FoxP3+ T cells in sections taken from subjects with either PD-L1+ or PD-L1− vessels in lung, kidney, and colon cancer. Original magnification, ×200. The number of intratumoral CD8+ T cells and FoxP3+ T cells from patients’ sections (kidney or colon cancer) associated with the percentage of PD-L1+ vessels. The determination as high and low were grouped by the median values. Lung adenocarcinoma (n = 41) b, colon cancer (n = 50) e and kidney cancer (n = 32) d; scale bars: 100 µm. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Fig. 3. PD-L1 expression on endothelial cells affects the proliferation and activation of immune cells.

a PBMC was extracted from normal blood and treated with 2.5 µg/ml anti-CD3 antibody and IL-2 (100 U/ml) for 72 h, and co-cultured with tumor-derived endothelial cells (Td-EC) for 48 h. b HUVECs were treated with the supernatant of tumor cells (CM) for 48 h. c PBMC and Td-EC were treated with control, anlotinib (0.1 μm) or anti-PD-L1 antibody (4 μg/ml). d Detection of the CD8+ IFN-γ+, CD8+Ki67+, CD8+GranzymeB+, and FoxP3+ T cells by flow cytometry. e Quantification of the above positive cells in the immune cells. f Quantification of cytokine levels in co-culture supernatants (2 days) by liquid microarray. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Fig. 4. anlotinib can inhibit endothelial PD-L1 expression via the AKT pathway.

a Western blot analysis showed p-AKT and p-ERK levels in with- or without-anlotinib (0.1 µm) treated HUVECs at the indicated time points. b Western blot analysis showed PD-L1 expression levels in HUVECs treated by U0126 (ERK inhibitor,10 µm), Ly294002 (AKT inhibitor, 10 µm), or anlotinib (0.1 µm) at 24 h. c, e Western blot analysis showed p-AKT and PD-L1 expression in MRMEC and bEnd.3 treated as shown. d, f Western blot analysis showed p-AKT and PD-L1 expression in HUVECs treated as shown. (SC-79, one of activator of AKT). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Fig. 5. anlotinib inhibits endothelial PD-L1 expression and inhibits tumor growth.

a C57BL/6 mice were injected with 1 × 106 B16 cells and tumors grew. On day 11, mice were divided into four groups (n = 10 per group), and treated with bevacizumab, anlotinib, or both as shown. b Kaplan–Meier survival curves of B16 mice treated as indicated. c Representative images of B16 tumors in different groups. d Left: tumor growth curve of the various treatment groups; right: statistics of the weight of B16 tumors treated as indicated. e Representative flow images of CD31+ PD-L1+ cells taken from mice treated with anlotinib (n = 5), bevacizumab (n = 4), or both (n = 5). f Statistics of the percentage of CD31+ PD-L1+ cells in the B16 or MC38 tumors and the percentage of PD-L1+ non -VECs in B16 tumors. g Statistics of the percentage of CD45−CD31+ cells in the B16 or MC38 tumors. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Fig. 6. anlotinib improves the immune microenvironment via increasing the ratio of CD8/FoxP3.

a Representative flow images of CD4+ CD25+ FoxP3+ T cells taken from B16 tumors treated as above indicated. b, c Statistics of the percentage of CD4+T cells, CD8+ T cells, FoxP3+ T cells and ratio of CD8/FoxP3 in the B16 or MC38 tumors. d Representative images of CD31 immunostaining (red), 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (blue) and markers of immune cells (CD4, CD8, and FoxP3) (green) of B16 tumors (n = 5 per group, except for Bev group n = 4) treated as above indicated. Scale bars,100 mm. e Quantification of CD4+ T cells, CD8+ T cells, FoxP3+ T cells in B16 tumors. f Statistics of perivascular and without perivascular CD8+ T cells in the B16 tumors. g Statistics of ratio of CD8/FoxP3 in the B16 or MC38 tumors. All data are exhibited as mean ± SD. Statistical differences were assessed using the unpaired Student’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7. Endothelial PD-L1 expression affects the efficacy of anlotinib.

a C57BL/6 mice were injected with 1 × 106 B16 cells and tumors grew. On day 12, mice were divided into four groups (n = 8 per group), and treated with bEnd.3-vector, bEnd.3-CD274, PBS or anlotinib as shown. b Representative images of B16 tumor tissues treated as above indicated. c Left: tumor growth curve of the various treatment groups; right: Statistics of the weight of B16 tumors treated as indicated. d Representative flow images of CD4+ CD25+ FoxP3+ T cells taken from B16 tumors treated as indicated. e Statistics of the percentage of CD8+ T cells, CD8+ IFN-γ+ T cells, FoxP3+ T cells and ratio of CD8/FoxP3 in the tumors. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8. A schematic representation of effects of PD-L1 expression on VECs in tumor microenvironment.

a Pattern diagram of tumor immune microenvironment. b PD-L1 highly expressed on VECs in tumor tissues inhibits activation of CD8+ T cells and promotes immune escape of tumors. c anlotinib inhibits the expression of PD-L1 on VECs through the AKT pathway. d anlotinib improved the ratio of CD8/FoxP3 and broke the immune barrier by inhibiting the VEC-PD-L1.