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. 2021 Feb 26:12:609295.
doi: 10.3389/fimmu.2021.609295. eCollection 2021.

Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation

Huimin Tao  1   2 , Mingyu Liu  1   3 , Yuan Wang  1 , Shufeng Luo  1 , Yongquan Xu  1 , Bin Ye  4 , Limin Zheng  1 , Kun Meng  4 , Lian Li  1
Affiliations

Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation

Huimin Tao et al. Front Immunol. .

Abstract

Recent studies have demonstrated that splenic extramedullary hematopoiesis (EMH) is an important mechanism for the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissues, and thus contributes to disease progression. Icaritin, a prenylflavonoid derivative from plants of the Epimedium genus, has been implicated as a novel immune-modulator that could prolong the survival of hepatocellular carcinoma (HCC) patients. However, it is unclear whether icaritin achieves its anti-tumor effects via the regulation of MDSCs generated by EMH in HCC. Here, we investigated the anti-tumor potential of icaritin and its mechanism of action in murine HCC. Icaritin suppressed tumor progression and significantly prolonged the survival of mice-bearing orthotopic and subcutaneous HCC tumors. Rather than exerting direct cytotoxic activity against tumor cells, icaritin significantly reduced the accumulation and activation of tumoral and splenic MDSCs, and increased the number and activity of cytotoxic T cells. Mechanistically, icaritin downregulates the tumor-associated splenic EMH, thereby reducing the generation and activation of MDSCs. The inhibitory effects of icaritin on human MDSCs in vitro were verified in short-term culture with cord-blood derived hematopoietic precursors. Furthermore, icaritin synergistically enhanced the therapeutic efficacy of immune checkpoint blockade therapy in HCC mice. These findings revealed that icaritin dampens tumoral immunosuppression to elicit anti-tumor immune responses by preventing MDSC generation via the attenuation of EMH. Thus, icaritin may serve as a novel adjuvant or even a stand-alone therapeutic agent for the effective treatment of HCC.

Keywords: MDSC; extramedullary hematopoiesis; hepatocellular carcinoma; icaritin; immunotherapy.

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Conflict of interest statement

BY and KM are employees of Beijing Shenogen Biomedical Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Icaritin inhibits the growth of orthotopic and subcutaneous Hepa tumors. (A) Images of orthotopic tumors 26–28 days after inoculation (green dotted line indicates tumor margins). (B) Orthotopic tumor weights compared by Student's t-test. (C) Kaplan-Meier curves indicating the survival of mice with orthotopic tumors treated with and without icaritin. (D) Images of subcutaneous tumors 26–28 days after inoculation. (E) Mean subcutaneous tumor volume was recorded every other day and analyzed by two-way ANOVA corrected by Bonferroni's test. (F) Subcutaneous tumor weights compared by Student's t-test. (G) Kaplan-Meier curves indicating the survival of mice with subcutaneous tumors treated with and without icaritin. Data were pooled from two experiments and n = 8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle; HCC, hepatocellular carcinoma.
Figure 2
Figure 2
Icaritin increases both the number and activation status of tumor-infiltrating CTLs in Hepa mice. The frequencies of tumor-infiltrating CD3+ T cells (A) CD3+CD8+ T cells (B) IFN-γ+CD3+CD8+ cells in CD3+CD8+ T cells (C) and IFN-γ+CD3+CD8+ cells in CD45+ cells (D) from orthotopic Hepa tumors. The frequencies of tumor-infiltrating CD3+ T cells (E) CD3+CD8+ T cells (F) IFN-γ+CD3+CD8+ cells in CD3+CD8+ T cells (G) and IFN-γ+CD3+CD8+ cells in CD45+ cells (H) from subcutaneous Hepa tumors. Representative cytometric plots of tumor-infiltrating IFN-γ+CD3+CD8+ cells from orthotopic (I) and subcutaneous (J) Hepa tumors. Numbers in the flow cytometric plots indicate the proportions of the gated cell populations. Data were pooled from two experiments and n = 8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle.
Figure 3
Figure 3
Icaritin reduces both the number and activation status of tumor-infiltrating PMN-MDSCs. (A) Frequency and number of tumor-infiltrating myeloid cells (CD11b+Gr-1+), PMN-MDSCs (CD11b+Gr-1+Ly6G+Ly6Clow), and M-MDSCs (CD11b+Gr-1+Ly6G−Ly6Chigh) orthotopic Hepa mice. (B) Immunosuppressive activity of orthotopic tumor-infiltrating PMN-MDSCs on in vitro T cell proliferation (stimulated with anti-CD3- and anti-CD28) at the ratios of 1:2, 1:4, and 1:8. (C) Western blots indicating Arg-1 protein expression and STAT3 activation in PMN-MDSCs isolated from tumors of orthotopic Hepa mice. (D) Frequency and number of tumor-infiltrating myeloid cells (CD11b+Gr-1+), PMN-MDSCs (CD11b+Gr-1+Ly6G+Ly6Clow), and M-MDSCs (CD11b+Gr-1+Ly6G−Ly6Chigh) in subcutaneous Hepa mice. Numbers in the flow cytometric plots indicate the proportions of the gated cell populations. Differences between groups were analyzed using two-way ANOVA corrected by Bonferroni's test. Data were pooled from two experiments and n = 8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle; Arg-1, Arginase-1; M-MDSCs, mononuclear myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; p, phosphorylated; t, total.
Figure 4
Figure 4
Effects of icaritin on splenic PMN-MDSCs and CTLs. Images of subcutaneous tumors 26 days after inoculation (A). Mean tumor volume of subcutaneous Hepa tumor-bearing mice subjected to splenectomy with or without icaritin treatment (B). Differences between groups were examined for statistical significance by two-way ANOVA, and corrected by Bonferroni's test. *P < 0.05 and **P < 0.01 compared with “Veh” group. Frequencies and total numbers of splenic myeloid cells, PMN-MDSCs and M-MDSCs in orthotopic (C) and subcutaneous (D) Hepa mice. Frequency of splenic IFN-γ+CD3+CD8+ cells in CD45+ cells from orthotopic (E) and subcutaneous (F) Hepa mice. Numbers in the flow cytometric plots indicate the proportions of the gated cell populations. Differences between groups were analyzed using two-way ANOVA corrected by Bonferroni's test (C, D), or examined for statistical significance by Student's t-test (E, F). Data were pooled from two experiments and n = 8 mice per group. **P < 0.01, ***P < 0.001. SPx, splenectomy; Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle; M-MDSCs, mononuclear myeloid-derived suppressor cells; PMN-MDSCs, polymorphonuclear myeloid-derived suppressor cells; CTLs, cytotoxic T lymphocytes.
Figure 5
Figure 5
Icaritin reduces the accumulation of HSPCs in the spleen of Hepa mice. Spleen weight and total cell number from orthotopic (A) and subcutaneous (B) Hepa mice. Numbers of splenic LSK and LK cells from orthotopic (C) and subcutaneous (D) Hepa mice. Differences between groups were examined for statistical significance by Student's t-test. Data were pooled from two experiments and n = 8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. HSPCs, hematopoietic stem and progenitor cells; Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle; LSK, Linlo/−Sca-1+c-Kithi; LK, Linlo/−Sca-1−c-Kithi.
Figure 6
Figure 6
Icaritin inhibits the generation of human PMN-MDSCs in vitro. Freshly isolated human CD34+ cells from cord blood mononuclear cells were cultured in hematopoietic stem cell expansion media for 8–10 days. The expanded cells were cultured with combined IL-6 and G-CSF in complete medium with 0.1% DMSO (vehicle) or icaritin (2.5 μM) for 3 days. (A) Flow cytometric analysis of CD115 and HLA-DR expression on CD14+ or CD15+ cells. (B) Frequencies of PMN-MDSCs (CD15+CD115+) and M-MDSCs (CD14+HLA-DR−). Differences between groups were analyzed using one-way ANOVA, and corrected by Dunnett's test. Data are representative of three experiments. **P < 0.01. Veh, Vehicle; ICT, Icaritin.
Figure 7
Figure 7
Icaritin synergistically enhances anti-PD-1 efficacy in HCC mice. Mean tumor volume of subcutaneous Hepa (A) and subcutaneous H22 (B) tumor-bearing mice with and without anti-PD-1 and icaritin treatment. Differences between groups were examined for statistical significance by two-way ANOVA, and corrected by Bonferroni's test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with “Veh” group; #P < 0.05 compared with “αPD-1” group; &&&P < 0.001 compared with “ICT” group. Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle; αPD-1, anti-PD-1.
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