Axitinib increases the infiltration of immune cells and reduces the suppressive capacity of monocytic MDSCs in an intracranial mouse melanoma model

Abstract

Melanoma patients are at a high risk of developing brain metastases, which are strongly vascularized and therefore have a significant risk of spontaneous bleeding. VEGF not only plays a role in neo-angiogenesis but also in the antitumor immune response. VEGFR-targeted therapy might not only have an impact on the tumor vascularization but also on tumor-infiltrating immune cells. In this study, we investigated the effect of axitinib, a small molecule TKI of VEGFR-1, -2, and -3, on tumor growth and on the composition of tumor-infiltrating immune cells in subcutaneous and intracranial mouse melanoma models. In vivo treatment with axitinib induced a strong inhibition of tumor growth and significantly improved survival in both tumor models. Characterization of the immune cells within the spleen and tumor of tumor-bearing mice respectively showed a significant increase in the number of CD3+CD8+ T cells and CD11b+ cells of axitinib-treated mice. More specifically, we observed a significant increase of intratumoral monocytic myeloid-derived suppressor cells (moMDSCs; CD11b+Ly6ChighLy6G-). Interestingly, in vitro proliferation assays showed that moMDSCs isolated from spleen or tumor of axitinib-treated mice had a reduced suppressive capacity on a per cell basis as compared to those isolated from vehicle-treated mice. Moreover, MDSCs from axitinib-treated animals displayed the capacity to stimulate allogeneic T cells. Thus, treatment with axitinib induces differentiation of moMDSC toward an antigen-presenting phenotype. Based on these observations, we conclude that the impact of axitinib on tumor growth and survival is most likely not restricted to direct anti-angiogenic effects but also involves important effects on tumor immunity.

Keywords: BLI, bioluminescent imaging; DCs, Dendritic Cells; FDA, US Food and Drug Administration; IL-2, interleukin-2; MDSC; MDSC, myeloid-derived suppressor cells; OT-1, CD8+ T-cells with transgenic receptor specific for the H-2Kb-restricted ovalbumin (OVA) peptide SIINFEKL; PD-1, programmed death 1; PD-L1, programmed death 1 ligand; PFS, progression-free survival; TKI, Tyrosine Kinase Inhibitor; TNFα, Tumor Necrosis Factor alfa; Treg, regulatory T cells; VEGF, Vascular Endothelial Growth Factor; angiogenesis; axitinib; brain metastasis; grMDSC, granulocytic MDSC, IFNγ: interferon gamma; immune cells; melanoma; moMDSC, monocytic MDSC.

Figures

Figure 1.

In vivo treatment with axitinib reduces tumor growth and increases survival in a subcutaneous and intracranial mouse model. Tumor growth and survival were monitored in subcutaneous and intracranial MO4-bearing mice that were treated with axitinib at 25 mg/kg twice daily (bid) or vehicle by oral gavage for 7 d. One representative of two independent experiments is shown. (A). Mean tumor volume of subcutaneous tumors of mice treated with vehicle or with axitinib. (5 mice per group, N = 2 ) (B). Survival curve of subcutaneous MO4-bearing mice treated with vehicle or axitinib. (5 mice per group, N = 2 ) (C). Bioluminescence imaging to monitor intracranial tumor volume of MO4-FLuc tumor-bearing mice treated with vehicle or axitinib is shown in the left panel. In the right panel an example comparison of difference in tumor growth between vehicle- and axitinib-treated mice 7 days after tumor inoculation. (6 mice per group, N = 2 ). (D). Mean tumor volume represented as bioluminescence signal (radiance) of intracranial MO4-FLuc-bearing mice treated with vehicle or axitinib. (6 mice per group, N = 2 ). (E). Survival curve of intracranial MO4-FLuc-bearing mice treated with vehicle or axitinib. (6 mice per group, N = 2 ).

Figure 2

(see previous page). In vivo treatment with axitinib increases the number of CD8+ T cells in the spleen. Different immune cell populations were analyzed in the spleen of subcutaneous MO4-bearing mice after treatment with axitinib at 25 mg/kg bid or vehicle by oral gavage. On the 7th day of treatment mice were sacrificed and single cell suspensions of the spleen were made and subsequently analyzed through flow cytometry. The total number of CD45+ cells was significantly increased in axitinib-treated mice as compared to vehicle-treated mice (B.). Within this CD45+ cell population there was a significant increase of CD3+CD8+ T cells (C.). (A). Gating strategy for different immune cell populations. (B). Percentage of CD45+ cells within the spleen of vehicle- and axitinib-treated mice. (C). Percentage of CD45+CD3+ T cells within the CD45+ cell population. Within the CD3+ cell population the percentage CD3+CD4+ and CD3+CD8+ T cells was determined. (D). Percentage of CD25+CD4+ (Treg) and CD25-CD4+ T cells as shown within the total population of CD4+ T cells. (E). Percentage of CD11c+ cells (DCs) within the CD45+ cell population. The percentage of CD80 and CD86 expression within the CD45+CD11c+ population was also determined. (F). Percentage of CD11b+ cells (myeloid cells) within the CD45+ cell population. Further differentiation in different subsets of MDSCs within the CD45+CD11b+ population was determined through expression of Ly6C and Ly6G: Ly6ChighLy6G- (moMDSC) and Ly6CintLy6G+ (grMDSC). Three independent experiments were performed (3 mice per group) and results are presented as mean ± SEM.

Figure 3.

Histological screening of tumors and increased infiltration of OT-1 T cells after axitinib treatment. (A–B). Histochemistry. (A). Subcutaneous tumor. Upper panel. Vehicle-treated mice. Amorf necrotic material with apoptic cells, nuclei and pigment (I.); viable tumor cells surrounding necrotic region (II.) and normal subcutaneous tissue (III.) (H&E, x40). Lower panel. Axitinib-treated mice. Amorf necrotic material with apoptic cells, nuclei and pigment (I.); transition between necrotic tissue and viable tumor: presence of cell and nuclear debris, and lymphocytic infiltrate (II); viable tumor cells surrounding necrotic region (III.) (H&E, x20). (B). Intracranial tumor. Upper panel. Vehicle-treated mice. Viable pleomorphic tumor cells. Solid growth pattern (I.). Lymphocytic infiltration less pronounced than in subcutaneous tumor (arrow)(H&E, x20). Lower panel. Axitinib-treated mice. Reduced tumoral cellularity. Subnecrotic tissue in between tumor cells. Lymphocytes surrounding cell and nuclear debris (arrow)(H&E, 20x) C-D. OT-1 infiltration. MO4 cells were subcutaneously (C.) or intracranially (D.) inoculated in CD45.1 transgenic mice and treated with vehicle or axitinib at 25mg/kg bid by oral gavage for 7 d. On the first day of axitinib-treatment, a lymphodepleting dose of cyclophosphamide was given (2mg diluted in 100μL of PBS per mouse, intraperitoneally). On the third day of axitinib-treatment, CD45.2 OT-1 T cells were infused intravenously. On the last day of treatment (5 d after the adoptive transfer), mice were sacrificed and single cell suspensions of the subcutaneous tumors were made. The percentage of CD45.2 OT-1 T cells within the tumor was subsequently analyzed with flow cytometry. In axitinib-treated mice there was a significant increased tumor infiltration (subcutaneous of CD45.2 OT-1 T cells (C.). In the intracranial tumors the tumor infiltration of CD45.2 OT-1 T cells was increased (not significant, D.) and two independent experiments were performed (3 mice per group) and results are presented as mean ± SEM.

Figure 4

(see previous page). In vivo treatment with axitinib increases the number of tumor-infiltrating immune cells, especially CD11b+ cells, in subcutaneous and intracranial tumor models. Determination of tumor-infiltrating immune cells in subcutaneous and intracranial MO4-bearing mice on the 7th day of treatment with axitinib at 25 mg/kg bid or vehicle by oral gavage. After sacrifice, single cell suspensions of the subcutaneous or intracranial tumors were made and subsequently analyzed by flow cytometry. A-E. Subcutaneous tumor. The total number of CD45+ cells was increased in axitinib-treated mice as compared to vehicle-treated mice (A.). This increase was due to an increase of CD11b+Ly6ChighLy6G- cells (E.). A. Percentage of CD45+ cells within the tumor. (B.) Percentage of CD45+CD3+ T cells within the CD45+ cell population. Within this population, the percentage of CD4+ and CD8+ T cells was determined. (C). Regulatory T cell population (Treg) within the CD4+ T cell population determined as CD4+CD25+ cells. (D). Percentage of CD11c+ cells (DCs) within the CD45+ cells and expression of CD80 and CD86 maturation markers on their surface. (E). Percentage of CD11b+ cells within the CD45+ cells. Within this population further characterization of the MDSCs: Ly6ChighLy6G- (moMDSC) and Ly6CintLy6G+ (grMDSC). Three independent experiments were performed (3 mice per group) and results are presented as mean ± SEM. F-J. Intracranial tumor. Similar to the subcutaneous tumor, the total number of CD45+ cells was increased (F.) in axitinib-treated mice as compared to vehicle-treated mice. That was also due to an increase of CD11b+ Ly6ChighLy6G- (J.) F. Percentage of CD45+ cells within the intracranial tumor. (G.) Percentage of CD3+CD45+ T cells within the CD45+ cell population. CD3+CD4+ and CD3+CD8+ T cells were determined within the CD3+ T cell population. (H.) Percentage of CD4+CD25+ cells (Treg) within the CD45+CD4+ T cell population. (I.) Percentage of CD11c+ population (DCs) within the CD45+ cell population and expression of the maturation markers CD80 and CD80 within this CD11c+ population. (J.) Percentage of CD11b+ cells (myeloid cells) within the CD45+ population. Further determination of the different MDSC subpopualtions through expression of Ly6C+ and Ly6G+: Ly6ChighLy6G- (moMDSC) and Ly6CintLy6G+ (grMDSC). Three independent experiments were performed (3 mice per group). Intracranial tumors were pooled from each group and results are presented as mean ± SEM.

Figure 5.

Axitinib reduces the suppressive capacity of splenic CD11b+Ly6ChighLy6G- cells. CD11b+Ly6ChighLy6G- (moMDSCs) and CD11b+Ly6CintLy6G+ (grMDSC) cells were sorted from the spleens of subcutaneous MO4-bearing mice on the 7th day of treatment with axitinib at 25 mg/kg bid or vehicle by oral gavage and were used in a suppression assay. Controls included T cells cultured in the absence of MDSCs with and without T-cell stimulation. CD11b+Ly6ChighLy6G- cells sorted out of axitinib-treated mice had a reduced suppressive capacity. This effect was observed in a 1:1 (CD11b+Ly6ChighLy6G- cell: T cell) ratio (A.) and was more distinct on CD8+ T cells (A. right panel) (A.) Overview of the percentage suppression of CD4+ T-cell proliferation (left panel) and CD8+ T-cell proliferation (right panel) cultured in the presence of different ratios of Ly6ChighLy6G- MDSC (moMDSC) and (B.) in the presence of different ratios of Ly6CintLy6G+ MDSC (grMDSC). (C.) IFNγ, TNFα, and IL-2 production by splenocytes was determined after 3 d of co-culture with different ratios of Ly6ChighLy6G– MDSC (moMDSC) and (D.) in the presence of different ratios of Ly6CintLy6G+ MDSC (grMDSC). Three independent experiments were performed (4 mice per group, spleens were pooled per group before cell sorting) and results are presented as mean ± SEM.

Figure 6.

Axitinib reduces the suppressive capacity of tumor-infiltrating CD11b+Ly6ChighLy6G- cells. CD11b+Ly6ChighLy6G– cells were sorted from the subcutaneous and intracranial tumors of MO4-bearing mice on the 7th day of treatment with axitinib at 25 mg/kg bid or vehicle by oral gavage and were used in a suppression assay. Controls included T cells cultured in the absence of MDSCs with and without T-cell stimulation.(A–B.) Subcutaneous tumor. The suppressive capacity of CD11b+Ly6ChighLy6G- cells sorted from axitinib-treated mice (A.) was strongly reduced as compared to those sorted from vehicle-treated mice, especially on CD8+ T-cell proliferation (A. right panel) (A.) Overview of the percentage of suppression of CD4+ T-cell proliferation (left panel) and of CD8+ T-cell proliferation (right panel) cultured in the presence of different ratios of Ly6ChighLy6G– MDSC (moMDSC) (B.) IFNγ, TNFα, and IL-2production by splenocytes was determined after 3 d of co-culture with different ratios of Ly6ChighLy6G- MDSC (moMDSC). Three independent experiments were performed (4 mice per group, tumors were pooled per group before cell sorting) and results are presented as mean ± SEM. (C–D.) Intracranial tumor. The suppressive capacity of CD11b+Ly6ChighLy6G- cells sorted from axitinib-treated mice on CD8+ T cell proliferation is significantly reduced as compared to those sorted form vehicle-treated mice (C.) Overview of the percentage suppression of CD4+ T-cell proliferation (left panel) and of CD8+ T-cell proliferation (right panel) cultured in the presence of Ly6ChighLy6G- MDSC (moMDSC) at a 1:10 ratio. D. IFNγ-, TNFα, and IL-2 production by splenocytes was determined after 3 d of co-culture with Ly6ChighLy6G- MDSC (moMDSC) at a 1:10 ratio. Two independent experiments were performed (4 mice per group, tumors were pooled per group before cell sorting) and results are presented as mean ± SEM.

Figure 7.

Axitinib induces differentiation of CD11b+Ly6ChighLy6G– cells to an antigen-presenting phenotype in a subcutaneous, but not in an intracranial tumor model. CD11b+Ly6ChighLy6G– cells were sorted from the subcutaneous and intracranial tumors of MO4-bearing mice on the 7th day of treatment with axitinib at 25 mg/kg bid or vehicle by oral gavage and used in an allogeneic mixed lymphocyte reaction. Controls included T cells cultured in the absence of MDSCs with and without T-cell stimulation. CD11b+Ly6ChighLy6G- cells sorted from subcutaneous tumors of axitinib-treated induced significant more proliferation (B.), and a significantly increased IFNγ, TNFα, and IL2-secretion was found in the supernatant of those proliferating C3H CD8+ T cells as compared to those sorted out of vehicle-treated mice (C.). (A). Representative FACS profile of the proliferation of CD8+ T cells (C3H mouse) in the presence of different ratios of Ly6ChighLy6G- MDSC (moMDSC). (B–C.) Subcutaneous tumor model. (B.) Overview of the percentage of proliferation of the CD8+ T cells (represented as mean fold increase) as induced through co-culture with Ly6ChighLy6G- (moMDSC) in different ratios. (C.) IFNγ, TNFα, and IL-2 production (represented as mean fold increase) by CD8+ T cells was determined after 3 d of co-culture with different ratios of Ly6ChighLy6G- MDSC (moMDSC). Three independent experiments were performed (4 mice per group, tumors were pooled per group before cell sorting) and results are presented as mean ± SEM. (D.) Intracranial tumor model. (D.) Overview of the proliferation of the CD8+ T cells (mean fold increase) as induced through co-cultures with Ly6ChighLy6G- (moMDSC) in different ratios. Two independent experiments were performed (4 mice per group, tumors were pooled per group before cell sorting) and results are presented as mean ± SEM.