Research ArticleOriginal Article
Open Access

Apatinib enhances targeted immunotherapy via the IL-6-gp130-PI3K pathway in HER2-positive gastric cancer

Ruoxi Tian, Ziyue Sha, Shasha Zhang, Miao Gong, Jianhua Wu, Juntao Lu, Wei Guo, Zhaoxu Zheng and Zhanjun Guo
Cancer Biology & Medicine July 2026, 20250687; DOI: https://doi.org/10.20892/j.issn.2095-3941.2025.0687

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  • Part 1: We demonstrated that apatinib enhanced the trastuzumab-induced inhibition of HER2-positive GC in in vitro and in vivo. Part 2: Bioinformatics analysis implicated effects of apatinib action on cytokine signaling pathways. Subsequent cytokine profiling identified IL-6 as the predominant differentially regulated cytokine, a finding further validated by qRT-PCR. Mechanistic studies revealed that apatinib suppressed HER2-positive GC via the IL-6-gp130-PI3K pathway. Part 3: To evaluate translational relevance, we established an hHSC-CDX model of HER2-positive GC. Using this model, we confirmed the synergistic effect of apatinib with trastuzumab targeted immunotherapy. Given that apatinib suppressed HER2-positive GC via IL-6, we also confirmed that tocilizumab (a monoclonal antibody targeting IL-6R) significantly potentiated apatinib’s efficacy with targeted immunotherapy in hHSC-CDXs. Potential mechanisms included decreased angiogenesis, M2-TAMs, and Tregs, and increased cytotoxic CD8+ T cell infiltration in the TME. Part 4: Clinical validation in 3 patients with HER2-positive GC corroborated the preclinical synergy, by demonstrating the synergistic effect of apatinib with the targeted immunotherapy combination. CCK-8, Cell Counting Kit 8; co-IP, co-immunoprecipitation; EdU, 5-ethynyl-2′-deoxyuridine; GC, gastric cancer; gp130, glycoprotein 130; HER2, human epidermal growth factor receptor 2; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; ICIs, immune checkpoint inhibitors; IF, immunofluorescence; IHC, immunohistochemical; IL-6, interleukin-6; M2-TAMs, M2-like tumor-associated macrophages; PI3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TME, tumor microenvironment; Tregs, regulatory T cells; VEGFR, vascular endothelial growth factor receptor. Figure created with Xunjie Sketching (www.liuchengtu.com).
    StudyFlow

    Part 1: We demonstrated that apatinib enhanced the trastuzumab-induced inhibition of HER2-positive GC in in vitro and in vivo. Part 2: Bioinformatics analysis implicated effects of apatinib action on cytokine signaling pathways. Subsequent cytokine profiling identified IL-6 as the predominant differentially regulated cytokine, a finding further validated by qRT-PCR. Mechanistic studies revealed that apatinib suppressed HER2-positive GC via the IL-6-gp130-PI3K pathway. Part 3: To evaluate translational relevance, we established an hHSC-CDX model of HER2-positive GC. Using this model, we confirmed the synergistic effect of apatinib with trastuzumab targeted immunotherapy. Given that apatinib suppressed HER2-positive GC via IL-6, we also confirmed that tocilizumab (a monoclonal antibody targeting IL-6R) significantly potentiated apatinib’s efficacy with targeted immunotherapy in hHSC-CDXs. Potential mechanisms included decreased angiogenesis, M2-TAMs, and Tregs, and increased cytotoxic CD8+ T cell infiltration in the TME. Part 4: Clinical validation in 3 patients with HER2-positive GC corroborated the preclinical synergy, by demonstrating the synergistic effect of apatinib with the targeted immunotherapy combination. CCK-8, Cell Counting Kit 8; co-IP, co-immunoprecipitation; EdU, 5-ethynyl-2′-deoxyuridine; GC, gastric cancer; gp130, glycoprotein 130; HER2, human epidermal growth factor receptor 2; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; ICIs, immune checkpoint inhibitors; IF, immunofluorescence; IHC, immunohistochemical; IL-6, interleukin-6; M2-TAMs, M2-like tumor-associated macrophages; PI3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TME, tumor microenvironment; Tregs, regulatory T cells; VEGFR, vascular endothelial growth factor receptor. Figure created with Xunjie Sketching (www.liuchengtu.com).

  • Apatinib enhances trastuzumab-related inhibition of HER2-positive GC in vitro. (A) CCK-8 assay showing the effects of HER2-siRNA, apatinib, and/or trastuzumab on proliferation in NCI-N87 and SNU-216 cells. (B) Colony formation assay analyzing the clonogenicity of NCI-N87 and SNU-216 cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. (C) Transwell assay measuring the migratory ability of NCI-N87 and SNU-216 cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. Scale bar: 200 μm. (D) Western blot analysis of key proteins in the MAPK, PI3K/AKT, and JAK2/STAT3 pathways in GC cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular regulated protein kinases; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.
    Apatinib enhances trastuzumab-related inhibition of HER2-positive GC in vitro. (A) CCK-8 assay showing the effects of HER2-siRNA, apatinib, and/or trastuzumab on proliferation in NCI-N87 and SNU-216 cells. (B) Colony formation assay analyzing the clonogenicity of NCI-N87 and SNU-216 cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. (C) Transwell assay measuring the migratory ability of NCI-N87 and SNU-216 cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. Scale bar: 200 μm. (D) Western blot analysis of key proteins in the MAPK, PI3K/AKT, and JAK2/STAT3 pathways in GC cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular regulated protein kinases; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.
    Figure 1

    Apatinib enhances trastuzumab-related inhibition of HER2-positive GC in vitro. (A) CCK-8 assay showing the effects of HER2-siRNA, apatinib, and/or trastuzumab on proliferation in NCI-N87 and SNU-216 cells. (B) Colony formation assay analyzing the clonogenicity of NCI-N87 and SNU-216 cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. (C) Transwell assay measuring the migratory ability of NCI-N87 and SNU-216 cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. Scale bar: 200 μm. (D) Western blot analysis of key proteins in the MAPK, PI3K/AKT, and JAK2/STAT3 pathways in GC cells after HER2 knockdown, and apatinib and/or trastuzumab treatment. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular regulated protein kinases; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.

  • Apatinib enhances trastuzumab-associated inhibition of HER2-positive GC in BALB/c-nu xenografts. (A) Images of NCI-N87 derived tumor tissues and tumor growth curves for BALB/c nu mice treated with vehicle, apatinib, trastuzumab, or both. (B) MVD quantified by CD31 immunohistochemistry. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Western blot analysis of key proteins in the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3; MVD, micro-vessel density.
    Figure 2

    Apatinib enhances trastuzumab-associated inhibition of HER2-positive GC in BALB/c-nu xenografts. (A) Images of NCI-N87 derived tumor tissues and tumor growth curves for BALB/c nu mice treated with vehicle, apatinib, trastuzumab, or both. (B) MVD quantified by CD31 immunohistochemistry. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Western blot analysis of key proteins in the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3; MVD, micro-vessel density.

  • Functional enrichment and cytokine profiling reveal that apatinib is associated with IL-6. (A) KEGG and GO pathway enrichment analysis in lung adenocarcinoma cells (GSE129221: post- vs. pre-apatinib treatment); (B) KEGG and GO pathway enrichment analysis in liposarcoma cells (GSE185783: post- vs. pre-apatinib treatment); (C) KEGG and GO pathway enrichment analysis in patients with HER2-positive GC (GSE220917: long-term responders vs. fast progressors during trastuzumab maintenance). (D). Flow fluorescence immune-microbead assay quantifying cytokine levels in NCI-N87 and SNU-216 cells after treatment with HER2-siRNA, apatinib, and/or trastuzumab. (E) qRT-PCR analysis of IL-6 mRNA expression in NCI-N87 and SNU-216 cells treated with HER2-siRNA, apatinib, and/or trastuzumab. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; IL-2, interleukin-2; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
    Functional enrichment and cytokine profiling reveal that apatinib is associated with IL-6. (A) KEGG and GO pathway enrichment analysis in lung adenocarcinoma cells (GSE129221: post- vs. pre-apatinib treatment); (B) KEGG and GO pathway enrichment analysis in liposarcoma cells (GSE185783: post- vs. pre-apatinib treatment); (C) KEGG and GO pathway enrichment analysis in patients with HER2-positive GC (GSE220917: long-term responders vs. fast progressors during trastuzumab maintenance). (D). Flow fluorescence immune-microbead assay quantifying cytokine levels in NCI-N87 and SNU-216 cells after treatment with HER2-siRNA, apatinib, and/or trastuzumab. (E) qRT-PCR analysis of IL-6 mRNA expression in NCI-N87 and SNU-216 cells treated with HER2-siRNA, apatinib, and/or trastuzumab. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; IL-2, interleukin-2; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; qRT-PCR, quantitative reverse transcription polymerase chain reaction.
    Figure 3

    Functional enrichment and cytokine profiling reveal that apatinib is associated with IL-6. (A) KEGG and GO pathway enrichment analysis in lung adenocarcinoma cells (GSE129221: post- vs. pre-apatinib treatment); (B) KEGG and GO pathway enrichment analysis in liposarcoma cells (GSE185783: post- vs. pre-apatinib treatment); (C) KEGG and GO pathway enrichment analysis in patients with HER2-positive GC (GSE220917: long-term responders vs. fast progressors during trastuzumab maintenance). (D). Flow fluorescence immune-microbead assay quantifying cytokine levels in NCI-N87 and SNU-216 cells after treatment with HER2-siRNA, apatinib, and/or trastuzumab. (E) qRT-PCR analysis of IL-6 mRNA expression in NCI-N87 and SNU-216 cells treated with HER2-siRNA, apatinib, and/or trastuzumab. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; IL-2, interleukin-2; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

  • Apatinib suppresses HER2-positive GC cells via IL-6. (A) CCK-8 assay assessing the proliferation of NCI-N87 and SNU-216 cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (B) Colony formation assay analyzing the clonogenicity of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (C) EdU assay quantifying DNA synthesis of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. (D) Transwell migration assay on GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, control group; TCZ, tocilizumab; IL-6, interleukin-6; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; EdU, 5-ethynyl-2′-deoxyuridine.
    Apatinib suppresses HER2-positive GC cells via IL-6. (A) CCK-8 assay assessing the proliferation of NCI-N87 and SNU-216 cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (B) Colony formation assay analyzing the clonogenicity of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (C) EdU assay quantifying DNA synthesis of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. (D) Transwell migration assay on GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, control group; TCZ, tocilizumab; IL-6, interleukin-6; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; EdU, 5-ethynyl-2′-deoxyuridine.
    Apatinib suppresses HER2-positive GC cells via IL-6. (A) CCK-8 assay assessing the proliferation of NCI-N87 and SNU-216 cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (B) Colony formation assay analyzing the clonogenicity of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (C) EdU assay quantifying DNA synthesis of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. (D) Transwell migration assay on GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, control group; TCZ, tocilizumab; IL-6, interleukin-6; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; EdU, 5-ethynyl-2′-deoxyuridine.
    Figure 4

    Apatinib suppresses HER2-positive GC cells via IL-6. (A) CCK-8 assay assessing the proliferation of NCI-N87 and SNU-216 cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (B) Colony formation assay analyzing the clonogenicity of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. (C) EdU assay quantifying DNA synthesis of GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. (D) Transwell migration assay on GC cells after treatment with apatinib, trastuzumab, IL-6, or TCZ. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, control group; TCZ, tocilizumab; IL-6, interleukin-6; HER2, human epidermal growth factor 2; GC, gastric cancer; CCK-8, Cell Counting Kit 8; EdU, 5-ethynyl-2′-deoxyuridine.

  • Apatinib suppresses HER2-positive GC cells via the IL-6-gp130-PI3K pathway. (A) Western blot analysis of key proteins in the PI3K/AKT/STAT3 pathway in GC cells treated with apatinib, trastuzumab, IL-6, and TCZ. (B) Co-immunoprecipitation assay assessing the interaction between gp130 and HER2 or PI3K in GC cells across treatment groups. (C) Western blot analysis of phosphorylated-HER2 (Tyr1248) levels in GC cells. (D) Immunofluorescence staining showing co-expression of gp130, phosphorylated-HER2 (Tyr1248), and phosphorylated-PI3K p85α (Tyr607) in GC cells. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; TCZ, tocilizumab; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Apatinib suppresses HER2-positive GC cells via the IL-6-gp130-PI3K pathway. (A) Western blot analysis of key proteins in the PI3K/AKT/STAT3 pathway in GC cells treated with apatinib, trastuzumab, IL-6, and TCZ. (B) Co-immunoprecipitation assay assessing the interaction between gp130 and HER2 or PI3K in GC cells across treatment groups. (C) Western blot analysis of phosphorylated-HER2 (Tyr1248) levels in GC cells. (D) Immunofluorescence staining showing co-expression of gp130, phosphorylated-HER2 (Tyr1248), and phosphorylated-PI3K p85α (Tyr607) in GC cells. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; TCZ, tocilizumab; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Apatinib suppresses HER2-positive GC cells via the IL-6-gp130-PI3K pathway. (A) Western blot analysis of key proteins in the PI3K/AKT/STAT3 pathway in GC cells treated with apatinib, trastuzumab, IL-6, and TCZ. (B) Co-immunoprecipitation assay assessing the interaction between gp130 and HER2 or PI3K in GC cells across treatment groups. (C) Western blot analysis of phosphorylated-HER2 (Tyr1248) levels in GC cells. (D) Immunofluorescence staining showing co-expression of gp130, phosphorylated-HER2 (Tyr1248), and phosphorylated-PI3K p85α (Tyr607) in GC cells. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; TCZ, tocilizumab; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Figure 5

    Apatinib suppresses HER2-positive GC cells via the IL-6-gp130-PI3K pathway. (A) Western blot analysis of key proteins in the PI3K/AKT/STAT3 pathway in GC cells treated with apatinib, trastuzumab, IL-6, and TCZ. (B) Co-immunoprecipitation assay assessing the interaction between gp130 and HER2 or PI3K in GC cells across treatment groups. (C) Western blot analysis of phosphorylated-HER2 (Tyr1248) levels in GC cells. (D) Immunofluorescence staining showing co-expression of gp130, phosphorylated-HER2 (Tyr1248), and phosphorylated-PI3K p85α (Tyr607) in GC cells. Scale bar: 200 μm. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. HER2, human epidermal growth factor 2; GC, gastric cancer; NC, negative control; TCZ, tocilizumab; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.

  • Apatinib enhances targeted immunotherapy against HER2-positive GC in hHSC-CDXs. (A) Images of NCI-N87-derived tumor tissues and tumor growth curves for hHSC-CDXs treated with tislelizumab, apatinib, and trastuzumab. (B) CD31 immunohistochemistry for MVD quantification. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Flow cytometry analysis of the human CD45+ cell proportion in tumor tissues. (D) M2-TAM (CD68+CD163+) proportion in CD45+ pre-gated cells. (E) CD8+ T cell (CD3+CD8+) proportion in pre-gated CD45+ cells. (F) CD8+ T cell subset (GZMB+TIM-3− cytotoxic; GZMB−TIM-3+ exhausted) proportion in pre-gated CD3+CD8+ T cells. (G) Treg (CD4+Foxp3+) proportion in the pre-gated CD3+CD8− T population. (H) Flow fluorescence immunomicrobead assay for IL-6 in tumor tissues. (I) Western blot analysis of the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; MVD, microvessel density; TAM, tumor-associated macrophage; Treg, regulatory T cell; GZMB, granzyme B; TIM-3, T-cell immunoglobulin and mucin-domain containing molecule 3; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Apatinib enhances targeted immunotherapy against HER2-positive GC in hHSC-CDXs. (A) Images of NCI-N87-derived tumor tissues and tumor growth curves for hHSC-CDXs treated with tislelizumab, apatinib, and trastuzumab. (B) CD31 immunohistochemistry for MVD quantification. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Flow cytometry analysis of the human CD45+ cell proportion in tumor tissues. (D) M2-TAM (CD68+CD163+) proportion in CD45+ pre-gated cells. (E) CD8+ T cell (CD3+CD8+) proportion in pre-gated CD45+ cells. (F) CD8+ T cell subset (GZMB+TIM-3− cytotoxic; GZMB−TIM-3+ exhausted) proportion in pre-gated CD3+CD8+ T cells. (G) Treg (CD4+Foxp3+) proportion in the pre-gated CD3+CD8− T population. (H) Flow fluorescence immunomicrobead assay for IL-6 in tumor tissues. (I) Western blot analysis of the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; MVD, microvessel density; TAM, tumor-associated macrophage; Treg, regulatory T cell; GZMB, granzyme B; TIM-3, T-cell immunoglobulin and mucin-domain containing molecule 3; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Figure 6

    Apatinib enhances targeted immunotherapy against HER2-positive GC in hHSC-CDXs. (A) Images of NCI-N87-derived tumor tissues and tumor growth curves for hHSC-CDXs treated with tislelizumab, apatinib, and trastuzumab. (B) CD31 immunohistochemistry for MVD quantification. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Flow cytometry analysis of the human CD45+ cell proportion in tumor tissues. (D) M2-TAM (CD68+CD163+) proportion in CD45+ pre-gated cells. (E) CD8+ T cell (CD3+CD8+) proportion in pre-gated CD45+ cells. (F) CD8+ T cell subset (GZMB+TIM-3− cytotoxic; GZMB−TIM-3+ exhausted) proportion in pre-gated CD3+CD8+ T cells. (G) Treg (CD4+Foxp3+) proportion in the pre-gated CD3+CD8− T population. (H) Flow fluorescence immunomicrobead assay for IL-6 in tumor tissues. (I) Western blot analysis of the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; MVD, microvessel density; TAM, tumor-associated macrophage; Treg, regulatory T cell; GZMB, granzyme B; TIM-3, T-cell immunoglobulin and mucin-domain containing molecule 3; IL-6, interleukin-6; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.

  • Tocilizumab enhances targeted immunotherapy in HER2-positive GC hHSC-CDXs. (A) Images of NCI-N87-derived tumor tissues and tumor growth curves for hHSC-CDXs treated with tislelizumab, apatinib, trastuzumab, and TCZ. (B) CD31 immunohistochemistry for MVD quantification. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Flow cytometry analysis of the human CD45+ cell proportion in tumor tissues. (D) M2-TAM (CD68+CD163+) proportion in CD45+ pre-gated cells. (E) CD8+ T cell (CD3+CD8+) proportion in pre-gated CD45+ cells. (F) CD8+ T cell subset (GZMB+TIM-3− cytotoxic; GZMB−TIM-3+ exhausted) proportion in pre-gated CD3+CD8+ T cells. (G) Treg (CD4+Foxp3+) proportion in the pre-gated CD3+CD8− T population. (H) Flow fluorescence immunomicrobead assay for IL-6 in tumor tissues. (I) Western blot analysis of the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; MVD, microvessel density; TAM, tumor-associated macrophage; Treg, regulatory T cell; GZMB, granzyme B; TIM-3, T cell immunoglobulin and mucin-domain containing molecule 3; PI3K, phosphatidylinositol 3-kinase; TCZ, tocilizumab; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Tocilizumab enhances targeted immunotherapy in HER2-positive GC hHSC-CDXs. (A) Images of NCI-N87-derived tumor tissues and tumor growth curves for hHSC-CDXs treated with tislelizumab, apatinib, trastuzumab, and TCZ. (B) CD31 immunohistochemistry for MVD quantification. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Flow cytometry analysis of the human CD45+ cell proportion in tumor tissues. (D) M2-TAM (CD68+CD163+) proportion in CD45+ pre-gated cells. (E) CD8+ T cell (CD3+CD8+) proportion in pre-gated CD45+ cells. (F) CD8+ T cell subset (GZMB+TIM-3− cytotoxic; GZMB−TIM-3+ exhausted) proportion in pre-gated CD3+CD8+ T cells. (G) Treg (CD4+Foxp3+) proportion in the pre-gated CD3+CD8− T population. (H) Flow fluorescence immunomicrobead assay for IL-6 in tumor tissues. (I) Western blot analysis of the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; MVD, microvessel density; TAM, tumor-associated macrophage; Treg, regulatory T cell; GZMB, granzyme B; TIM-3, T cell immunoglobulin and mucin-domain containing molecule 3; PI3K, phosphatidylinositol 3-kinase; TCZ, tocilizumab; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.
    Figure 7

    Tocilizumab enhances targeted immunotherapy in HER2-positive GC hHSC-CDXs. (A) Images of NCI-N87-derived tumor tissues and tumor growth curves for hHSC-CDXs treated with tislelizumab, apatinib, trastuzumab, and TCZ. (B) CD31 immunohistochemistry for MVD quantification. Black scale bar: 100 μm; red scale bar: 30 μm. (C) Flow cytometry analysis of the human CD45+ cell proportion in tumor tissues. (D) M2-TAM (CD68+CD163+) proportion in CD45+ pre-gated cells. (E) CD8+ T cell (CD3+CD8+) proportion in pre-gated CD45+ cells. (F) CD8+ T cell subset (GZMB+TIM-3− cytotoxic; GZMB−TIM-3+ exhausted) proportion in pre-gated CD3+CD8+ T cells. (G) Treg (CD4+Foxp3+) proportion in the pre-gated CD3+CD8− T population. (H) Flow fluorescence immunomicrobead assay for IL-6 in tumor tissues. (I) Western blot analysis of the PI3K/AKT/STAT3 pathway in tumor tissues. Data are presented as mean ± S.D. *P ≤ 0.05, **P ≤ 0.01. NC, negative control; HER2, human epidermal growth factor 2; GC, gastric cancer; hHSC-CDXs, humanized hematopoietic stem cell and tumor cell line-derived xenografts; MVD, microvessel density; TAM, tumor-associated macrophage; Treg, regulatory T cell; GZMB, granzyme B; TIM-3, T cell immunoglobulin and mucin-domain containing molecule 3; PI3K, phosphatidylinositol 3-kinase; TCZ, tocilizumab; AKT, protein kinase B; STAT3, signal transducer and activator of transcription 3.

  • Apatinib enhances the efficacy of targeted immunotherapy by inhibiting the IL-6/gp130/PI3K pathway in HER2-positive GC cells. Left: HER2 induces upregulation of IL-6 mRNA, potentially through NF-κB signaling (1), thereby leading to excessive secretion of IL-6 into the extracellular space. The binding of extracellular IL-6 to IL-6R activates gp130 by promoting its dimerization (2). Activated gp130 induces PI3K/AKT/STAT3 signal transduction, thus resulting in an immunosuppressive TME characterized by increased angiogenesis, M2-TAM polarization, and Treg infiltration, and decreased CD8+ T cell infiltration (3–4). Right: Apatinib enhances trastuzumab’s inhibition of IL-6 and further inhibits the gp130/PI3K/AKT/STAT3 signaling pathway (5–7), thus reversing the immunosuppressive TME and facilitating immunotherapy in HER2-positive GC (8). Apatinib also directly inhibits angiogenesis (9). AKT, protein kinase B; GC, gastric cancer; HER2, human epidermal growth factor 2; ICIs, immune checkpoint inhibitors; IL-6, interleukin-6; NF-κB, nuclear factor kappa-B; PI3K, phosphatidylinositol 3-kinase; STAT3, signal transducer and activator of transcription 3; TAM, tumor-associated macrophage; TME, tumor microenvironment; Treg, regulatory T cell; VEGFR2, vascular endothelial growth factor receptor 2. Figure created with BioRender (www.biorender.com).
    Figure 8

    Apatinib enhances the efficacy of targeted immunotherapy by inhibiting the IL-6/gp130/PI3K pathway in HER2-positive GC cells. Left: HER2 induces upregulation of IL-6 mRNA, potentially through NF-κB signaling (1), thereby leading to excessive secretion of IL-6 into the extracellular space. The binding of extracellular IL-6 to IL-6R activates gp130 by promoting its dimerization (2). Activated gp130 induces PI3K/AKT/STAT3 signal transduction, thus resulting in an immunosuppressive TME characterized by increased angiogenesis, M2-TAM polarization, and Treg infiltration, and decreased CD8+ T cell infiltration (3–4). Right: Apatinib enhances trastuzumab’s inhibition of IL-6 and further inhibits the gp130/PI3K/AKT/STAT3 signaling pathway (5–7), thus reversing the immunosuppressive TME and facilitating immunotherapy in HER2-positive GC (8). Apatinib also directly inhibits angiogenesis (9). AKT, protein kinase B; GC, gastric cancer; HER2, human epidermal growth factor 2; ICIs, immune checkpoint inhibitors; IL-6, interleukin-6; NF-κB, nuclear factor kappa-B; PI3K, phosphatidylinositol 3-kinase; STAT3, signal transducer and activator of transcription 3; TAM, tumor-associated macrophage; TME, tumor microenvironment; Treg, regulatory T cell; VEGFR2, vascular endothelial growth factor receptor 2. Figure created with BioRender (www.biorender.com).

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