Anlotinib Induces a T Cell-Inflamed Tumor Microenvironment by Facilitating Vessel Normalization and Enhances the Efficacy of PD-1 Checkpoint Blockade in Neuroblastoma

Abstract

Purpose: Anlotinib has achieved good results in clinical trials of a variety of cancers. However, the effects of anlotinib on the tumor microenvironment (TME) and systemic immunity have not been reported. There is an urgent need to identify the underlying mechanism to reveal new opportunities for its application in neuroblastoma (NB) and other cancers. Understanding the mechanism will hopefully achieve the goal of using the same method to treat different cancers.

Experimental design: This study used bioinformatics, NB syngeneic mouse models, flow cytometry, RNA-seq, and immunofluorescence staining to explore the mechanisms of anlotinib on the TME, and further explored anlotinib-containing combination treatment strategies.

Results: We proved that anlotinib facilitates tumor vessel normalization at least partially through CD4+ T cells, reprograms the immunosuppressive TME into an immunostimulatory TME, significantly inhibits tumor growth, and effectively prevents systemic immunosuppression. Moreover, the combination of anlotinib with a PD-1 checkpoint inhibitor counteracts the immunosuppression caused by the upregulation of PD-L1 after monotherapy, extends the period of vascular normalization, and finally induces NB regression.

Conclusions: To our knowledge, this study is the first to dynamically evaluate the effect of a multitarget antiangiogenic tyrosine kinase inhibitor on the TME. These findings have very important clinical value in guiding the testing of related drugs in NB and other cancers. Based on these findings, we are conducting a phase II clinical study (NCT04842526) on the efficacy and safety of anlotinib, irinotecan, and temozolomide in the treatment of refractory or relapsed NB, and hopefully we will observe patient benefit.

Figure 1.

GPAGs are associated with vessel normalization and T-cell activation in patients with NB. A and B, The hierarchical clustering analysis of angiogenesis-related genes (GO: 0001525) associated with the prognosis showed two clusters of patients with differences in OS. The color scale represents row z-scores (A). C, GO terms that associated with GPAGs and PPAGs. The GPAGs were mostly related to positive regulation of vascular-associated smooth muscle cell proliferation, migration, contraction, and regulation of leukocyte adhesion to vascular EC and other pathways associated with vascular normalization. The PPAGs were mostly related to negative regulation of smooth muscle cell differentiation and positive regulation of the VEGFR signaling pathway and other pathways opposite to vascular normalization. The number of pathways is shown in parentheses. D, T-cell activation, activation of immune response, and adaptive immune response and other pathways analyzed by GSEA of GPAGs were significantly upregulated. Log-rank test (B).

Figure 2.

Anlotinib significantly inhibits tumor growth in 975A2 and 9464D syngeneic mouse models and reprograms the NB immunostimulatory microenvironment. A–D, Tumor growth curves and survival curves of 975A2 and 9464D syngeneic mouse models treated with different doses of anlotinib for 21 days as described in Supplementary Fig. S3A (n = 5 per group). Median (50%) survival (after treatment): vehicle, 27 days; 3 mg/kg, 51 days; 6 mg/kg, 69 days; and 12 mg/kg, 69 days (B). Median (50%) survival (after treatment): vehicle, 21 days; 3 mg/kg, 42 days; 6 mg/kg, 60 days; and 12 mg/kg, 60 days (D). E–L, RNA-seq expression analysis of tumor tissues from NB syngeneic mouse models treated with vehicle ± anlotinib (6 mg/kg) for 9 days as described in Supplementary Fig. S3A and genes differentially expressed were selected based on the following criteria: adjusted P < 0.05 and |log2 (fold change)| > 0.5. The top 10 GO terms of DEGs are shown with −log10 (P value) (n = 3 per group, E–H). Heatmaps of DEGs related to T-cell activation (GO: 0042110) and angiogenesis (GO: 0001525) and the color scale represents row z-scores (n = 3 per group, I–L). M–P, Tumor growth curves and survival curves of vehicle ± anlotinib (6 mg/kg) in nude mouse and C57BL/6 mouse tumor-bearing models (n = 5 per group). Median (50%) survival (after treatment): nude-vehicle, 27 days; C57BL/6-vehicle, 27 days; Nude-Anlo, 45 days; and C57BL/6-Anlo, 69 days (N). Median (50%) survival (after treatment): nude-vehicle, 21 days; C57BL/6-vehicle, 21 days; nude-Anlo, 30 days; and C57BL/6-Anlo, 60 days (P). Error bars represent the mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA (A, C, M, and O), log-rank test (B, D, N, and P).

Figure 3.

Anlotinib significantly impairs tumor angiogenesis and normalizes the remaining blood vessels, but a concomitant period of vascular normalization occurs. A–D, Representative immunofluorescence images of CD31 (red), α-SMA, lectin, dextran and CA-IX (green), and DAPI (blue) staining in tumor tissues from NB syngeneic mouse models treated with vehicle ± anlotinib (6 mg/kg) for 9 days and 21 days as described in Supplementary Fig. S3A. Scale bars, 100 μm. E–J, Quantitation results of immunofluorescent images (A–D). Relative proportions of α-SMA⁺ pericyte-covered blood vessels (E), lectin⁺ blood vessels (F), dextran⁺ blood vessels (G), and CA-IX⁺ cells (H) in NB syngeneic mouse models treated with vehicle ± anlotinib (6 mg/kg) for 9 and 21 days as described in Supplementary Fig. S3A. Relative tumor vessel length (I) and number of CD31⁺ cells (J) in NB syngeneic mouse models treated with vehicle ± anlotinib (6 mg/kg) for 9 days and 21 days as described in Supplementary Fig. S3A. Each dot indicates one tumor and represents the average of five images. n = 6 per group, including 975A2 (n = 3) and 9464D (n = 3). Error bars represent the mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA (E–J).

Figure 4.

Anlotinib significantly reprograms the NB immunostimulatory microenvironment, but it is affected by the concomitant period of vascular normalization. A–V, Flow cytometry analysis of tumor-infiltrating immune cells and tumor cells in the 975A2 syngeneic mouse model treated with vehicle ± anlotinib (6 mg/kg) for 9 and 21 days as described in Supplementary Fig. S3A, shown by the percentage of parent gates (A–B, D–M, and P–V). MFI indicates the mean fluorescence intensity of the indicated marker (N–O). Major immune cell panel, including leukocytes, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, Tregs, B cells, NK cells, TAMs, neutrophils, and DCs (A–B). Fold change relative to the vehicle mean (D9 or D21) in total cell abundance (cells/mg, C). T-cell function panel, including the expression of IFNγ, TNFα, and GzmB on T cells (D–H). TAM function panel, including the expression of CD206, MHC-II, iNOS, TNFα, costimulatory molecules (CD80 and CD86), and Arg1 on TAMs (I–N). DC function panel, including the expression of MHC-II and costimulatory molecules (CD80 and CD86) on DCs (O–Q). Tumor cell panel, including the expression of β₂M, H-2, and MHC-II on tumor cells (R–T). Antigen cross-presentation panel, including the expression of H-2K^b/SIINFEKL on TAMs and DCs (U–V). Each dot represents one mouse. n = 3 per group. Error bars, mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA (A–V).

Figure 5.

Efficacy of anlotinib combined with PD-1 checkpoint blockade in the treatment of NB models. A–H, Flow cytometry analysis of PD-1 and Ki67 expression on tumor-infiltrating T cells and PD-L1 expression on tumor cells, ECs, and immune cells in the 975A2 syngeneic mouse model treated with vehicle ± anlotinib (6 mg/kg) for 9 days and 21 days as described in Supplementary Fig. S3A, shown by the percentage of parent gates (A–F). MFI indicates the mean fluorescence intensity of the indicated marker (G–H). Each dot represents one tumor. n = 3 per group. I, Schematic of the experimental design in NB syngeneic mouse models, n = 6 per group. J and K, Mean tumor volume growth curves and survival curves. Median (50%) survival (after treatment): Isotype control, 31.5 days; α-PD1, 37.5 days; Anlo, 64.5 days; and Anlo + α-PD1, 86 days (K). L, Change in tumor volume compared with baseline. M–P, Individual tumor volume growth curves. Error bars represent the mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA (A–H and J), log-rank test (K).

Figure 6.

Anlotinib combined with PD-1 checkpoint blockade prolongs the period of vascular normalization. A–M, Flow cytometry analysis of tumor-infiltrating immune cells and tumor cells in 975A2 syngeneic mouse model treated as described in Fig. 5I, shown by the percentage of parent gates (A–B and D–M). Major immune cell panel, including leukocytes, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, Tregs, B cells, NK cells, TAMs, neutrophils, and DCs (A–B). Fold change relative to the vehicle mean (D9 or D21) in total cell abundance (cells/mg, C). T-cell function panel, including the expression of IFNγ, TNFα, and GzmB on T cells (D–H). TAM function panel, including the expression of CD206, MHC-II, and iNOS on TAMs (I–J). Tumor cell panel, including the expression of MHC-II, β₂M, and H-2 on tumor cells (K–M). Each dot represents one tumor. n = 3 per group. N–Q, Representative immunofluorescence images of CD31 (red), α-SMA, lectin, dextran, and CA-IX (green), and DAPI (blue) staining of tumor tissues in NB syngeneic mouse models treated with anlotinib combined with an anti–PD-1 antibody for 21 days as described in Fig. 5I. Scale bars, 100 μm. R–W, Quantitation results of immunofluorescent images (N–Q). Relative proportions of α-SMA⁺ pericyte-covered blood vessels (R), lectin⁺ blood vessels (S), dextran⁺ blood vessels (T), and CA-IX⁺ cells (U) in NB syngeneic mouse models treated as described in Fig. 5I. Relative tumor vessel length (V) and number of CD31⁺ cells (W) in NB syngeneic mouse models treated as described in Fig. 5I. Each dot indicates one tumor and represents the average of five images. n = 6 per group, including 975A2 (n = 3) and 9464D (n = 3). Error bars represent the mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA (A–M and R–W).

Figure 7.

Anlotinib induces tumor vessel normalization at least partially through CD4⁺ T cells. A–D, Tumor growth curves and survival curves of 975A2 and 9464D syngeneic mouse models treated as described in Supplementary Fig. S9Q (n = 5 per group). Median (50%) survival (after treatment): isotype control, 27 days; Anlo + α-CD8, 48 days; Anlo + α-CD4, 48 days; and Anlo, not reached (B). Median (50%) survival (after treatment): isotype control, 18 days; Anlo + α-CD8, 42 days; Anlo + α-CD4, 48 days; and Anlo, not reached (D). E–H, Representative immunofluorescence images of CD31 (red), α-SMA, lectin, dextran and CA-IX (green), and DAPI (blue) staining of tumor tissues in NB syngeneic mouse models treated as described in Supplementary Fig. S9Q. Scale bars, 100 μm. I–N, Quantitation results of immunofluorescent images (E–H). Relative proportions of α-SMA⁺ pericyte-covered blood vessels (I), lectin⁺ blood vessels (J), dextran⁺ blood vessels (K), and CA-IX⁺ cells (L) in NB syngeneic mouse models treated as described in Supplementary Fig. S9Q. Relative tumor vessel length (M) and number of CD31⁺ cells (N) in NB syngeneic mouse models treated as described in Supplementary Fig. S9Q. n = 6 per group, including 975A2 (n = 3) and 9464D (n = 3). Error bars represent the mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA (A, C and I–N), log-rank test (B and D).

Figure 8.

Schematic of the mechanism of interaction between anlotinib and anti–PD-1 therapy in NB syngeneic mouse models. Anlotinib facilitates tumor vessel normalization by inhibiting proangiogenic factor receptors (VEGFR, PDGFR, and FGFR) and reducing the expression of proangiogenic factors (Angpt2, Pdgfa, and Pdgfb). The pericyte coverage and perfusion of tumor vessels are increased, and blood vessel leakage and tissue hypoxia are reduced. The expression levels of immune cell adhesion molecules (Selp, Madcam1, and Icam1) and chemokines and their receptor (Cxcl9, Cxcl10, and Cxcr3) are increased. The infiltration of immune effector cells is increased, and their functions are enhanced. For example, the infiltration of CD4⁺ T cells is increased, and their abilities to secrete IFNγ and TNFα are enhanced; the infiltration of CD8⁺ T cells is also increased, and their abilities to secrete IFNγ, TNFα, and GzmB are enhanced; the proportion of M1 TAMs is increased, and the proportions of M2 TAMs and neutrophils are decreased. The densities of DCs and TAMs are increased, and their antigen presentation function is also enhanced. T cell–derived IFNγ promotes the expression of MHC-I and MHC-II in NB cells and improves antigen processing and presentation. Moreover, IFNγ enhances the expression of PD-L1 in the TME, and combined treatment with an anti–PD-1 antibody reverses the early exhaustion of T cells. Anti–PD-1 immunotherapy and antigen presentation enhance the activation of CD4⁺ T cells, forming a positive feedback loop of vascular normalization and immunostimulatory reprogramming, thereby prolonging the period of vascular normalization.