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. 2019 May;33(5):6596-6608.
doi: 10.1096/fj.201802067RR. Epub 2019 Feb 25.

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

Yang Zeng  1 , Binghao Li  1 , Yingying Liang  1 , Patrick M Reeves  1 , Xiying Qu  1 , Chongzhao Ran  2 , Qiuyan Liu  3 , Michael V Callahan  1 , Ann E Sluder  1 , Jeffrey A Gelfand  1 , Huabiao Chen  1 , Mark C Poznansky  1
Affiliations

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

Yang Zeng et al. FASEB J. 2019 May.

Abstract

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 (Treg)-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 Treg cells were decreased, and conversion of Treg 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.

Keywords: CXCR4 antagonist; combination immunotherapy; immune checkpoint inhibitor.

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Conflict of interest statement

The ID8 cell line was a kind gift from Dr. Kathy Roby (University of Kansas Medical Center, Kansas City, KS, USA). This work was supported by the Vaccine and Immunotherapy Center (VIC) Innovation Fund and the Massachusetts General Hospital (MGH) Research Scholar Award (to H.C., Y.Z., and M.C.P.). Cytometric findings reported here were performed in the MGH Department of Pathology Flow and Image Cytometry Research Core, which is supported by the U.S. National Institutes of Health (NIH) Shared Instrumentation Grants 1S10OD012027-01A1, 1S10OD016372-01, 1S10RR020936-01, and 1S10RR023440-01A1. M.C.P. is the Scientific Founder of AperiSys. The remaining authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
AMD3100 inhibits ID8-luc cell growth and migration in vitro. A) Representative data showing that CXCR4 and CXCL12 are highly expressed on ID8-luc cells (left). Comparative flow results showing that AMD3100 at 10 μg/ml down-regulated the expression of CXCR4 and CXCL12 (right). NC, negative controls (unstained cells). B) CyQuant proliferation assays. C) Representative images showing the inhibition of cancer cell invasion by AMD3100 proved by transwell assay. The blue color represents transmembrane cells. D) Results of transwell assay stimulated by different concentrations of AMD3100. E) Representative images showing the impact of AMD3100 on ID8-luc cell migration in the wound healing assay. F) Statistical results of wound healing assay. Error bars represent means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 2
Figure 2
Combination therapy augments tumor control and mouse survival. A) Representative IVIS images showing tumor growth at d 40 post–tumor inoculation among different treated groups. B) Tumor growth of ID8-luc cells in C57BL/6 mice treated with αPD-1, AMD3100, or in combination (n = 11–13). C) Survival curve showing most prolonged survival in AMD3100 in combination with αPD-1 therapy in comparison with mice receiving monotherapy or N-saline. D) Ascites volume in control and experimental groups at the time of euthanization 2 wk after last treatment (n = 6). E) ELISA results of CXCL12 in the ascites supernatant (n = 6). Error bars represent means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001.
Figure 3
Figure 3
Combination therapy enhanced intratumoral T-cell infiltration and effector T-cell function. A) Representative cytometric dot plots of CD3+CD8+ T cells in tumor (left) and the proportions of CD3+CD8+ T cells in tumor in different treatment groups (right). B) Representative cytometric dot plots of CD3+CD4+ T cells in tumor (left) and the proportions of CD3+CD4+ T cells in tumor in different treatment groups (right). C) Representative cytometric dot plots showing intratumoral CD8+ IFN-γ+ cells in saline and AMD3100 and αPD-1 combination groups (left), and percentage of intratumoral CD8+ IFN-γ+ cells showing that AMD3100 and αPD-1 combination could great enhance IFN-γ expressing on CD8+ cells (right). D) Representative dot plots showing intratumoral CD4+ IFN-γ+ cells (left), and percentage of intratumoral CD4+ IFN-γ+ cells (right). E, F) Percentage of ascites CD8+ IFN-γ+ (E) and CD4+ IFN-γ+ (F) cells showing that combination therapy could significantly enhance IFN-γ expressing on CD8+ and CD4+ T cells. G) Representative dot plots showing ascites CD8+ IL-2+ cells in saline and AMD3100 and αPD-1 combination groups (left), and percentage of ascites CD8+ IL-2+ cells (right). H) ELISA results showing IL-2 amount in the ascites (n = 6 for ascites). Error bars represent means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001.
Figure 4
Figure 4
Combination therapy enhanced intratumoral and ascites memory T cells. A) Representative cytometric dot plots of CD44+CD27+ memory T cells in intratumoral CD4 and CD8 T cells. B) Percentages of intratumoral CD4+CD44+CD27+ memory T cells in control and treatment groups. C) Percentages of intratumoral CD8+CD44+CD27+ memory T cells in different groups. D) Representative cytometric dot plots of CD44+CD27+ memory T cells in ascites CD4 and CD8 T cells. E) Percentages of ascites CD4+CD44+CD27+ memory T cells in different groups. F) Percentages of ascites CD8+CD44+CD27+ memory T cells in different groups. Error bars represent means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 5
Figure 5
AMD3100 inhibited Treg cells and enhanced CD4+FoxP3+CD25− T-helper–cell proportion and function. A) Representative cytometric dot plots of CD4+FoxP3+CD25+ Treg cells in tumor. B) Percentages of intratumoral Treg cells in treatment and control groups. B) Percentages of Treg cells in ascites cells of different groups. C) Percentages of intratumoral CD4+FoxP3+CD25− T-helper–like cells. D) Representative cytometric dot plots and percentages of CD40L+IL-2+ cells in intratumoral CD4+FoxP3+CD25− T-helper–like cells. E) Percentages of ascites CD4+FoxP3+CD25− T-helper–like cells. F) Percentages of CD40L+IL-2+ cells in ascites CD4+FoxP3+CD25− T-helper–like cells (n = 6 for ascites). G) Representative cytometric dot plots and percentages of intratumoral CD8+FoxP3+CD25− T cells in different groups. H) Representative cytometric dot plots and percentages of IFN-γ expression in CD8+FoxP3+CD25− T cells in different groups. Error bars represent means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 6
Figure 6
Combination immunotherapy resulted in a marked decrease in immunosuppressive intratumoral MDSCs. A) Representative cytometric dot plots of intratumoral CD11b+granulocyte antigen-1+ MDSCs in saline (top) and combination (bottom) treatment. B) Percentages of intratumoral MDSCs in total alive cells in tumor. C) Percentages of ascites MDSCs in whole alive cell population. D) ELISA results of IL-10, n = 6. E) ELISA results of IL-6, n = 6. Gr-1, granulocyte antigen 1. Error bars represent means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 7
Figure 7
Combination therapy showed beneficial antitumoral M1 macrophage polarization. A) Percentages of F4/80+CD11b+ macrophages in intratumoral CD45+ cell population. B) Representative dot plots of intratumoral M1 and M2 in saline (top) and combination (bottom) treatment. C) Percentages of M1 (CD163−MHC II+) in intratumoral F4/80+ CD11b+ cell population. D) Percentages of M2 (CD163+MHC II−) in intratumoral F4/80+CD11b+ cell population. E) Ratio of intratumoral M1:M2. F) Percentages of CD38+PD-L1+ cells in intramural CD45+ cell population. G) Percentages of CD38−PD-L1+ cells in intramural CD45+ cell population. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001.
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References

    1. Siegel R., DeSantis C., Virgo K., Stein K., Mariotto A., Smith T., Cooper D., Gansler T., Lerro C., Fedewa S., Lin C., Leach C., Cannady R. S., Cho H., Scoppa S., Hachey M., Kirch R., Jemal A., Ward E. (2012) Cancer treatment and survivorship statistics, 2012. CA Cancer J. Clin. 62, 220–241 - PubMed
    1. Bast R. C., Jr., Hennessy B., Mills G. B. (2009) The biology of ovarian cancer: new opportunities for translation. Nat. Rev. Cancer 9, 415–428 - PMC - PubMed
    1. Mantia-Smaldone G. M., Corr B., Chu C. S. (2012) Immunotherapy in ovarian cancer. Hum. Vaccin. Immunother. 8, 1179–1191 - PMC - PubMed
    1. Duraiswamy J., Freeman G. J., Coukos G. (2013) Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer. Cancer Res. 73, 6900–6912 - PMC - PubMed
    1. Conrad C., Gregorio J., Wang Y. H., Ito T., Meller S., Hanabuchi S., Anderson S., Atkinson N., Ramirez P. T., Liu Y. J., Freedman R., Gilliet M. (2012) Plasmacytoid dendritic cells promote immunosuppression in ovarian cancer via ICOS costimulation of Foxp3(+) T-regulatory cells. Cancer Res. 72, 5240–5249 - PMC - PubMed

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