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. 2017 Jan;66(1):75-85.
doi: 10.1016/j.jhep.2016.07.044. Epub 2016 Aug 9.

Successful chemoimmunotherapy against hepatocellular cancer in a novel murine model

Guangfu Li #  1   2 , Dai Liu #  1   2 , Timothy K Cooper  3 , Eric T Kimchi  1   2 , Xiaoqiang Qi  1   2 , Diego M Avella  4 , Ningfei Li  1 , Qing X Yang  4 , Mark Kester  5 , C Bart Rountree  6 , Jussuf T Kaifi  1   2 , David J Cole  1   2 , Don C Rockey  7 , Todd D Schell  8 , Kevin F Staveley-O'Carroll  1   2
Affiliations

Successful chemoimmunotherapy against hepatocellular cancer in a novel murine model

Guangfu Li et al. J Hepatol. 2017 Jan.

Abstract

Background & aims: We have established a clinically relevant animal model of hepatocellular cancer (HCC) in immune competent mice to elucidate the complex dialog between host immunity and tumors during HCC initiation and progression. Mechanistic findings have been leveraged to develop a clinically feasible anti-tumor chemoimmunotherapeutic strategy.

Methods: Intraperitoneal injection of carbon tetrachloride and intrasplenic inoculation of oncogenic hepatocytes were combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. Immunization and adoptive cell transfer (ACT) were used to dissect the tumor antigen-specific immune response. The ability of the tyrosine kinase inhibitor sunitinib to enhance immunotherapy in the setting of HCC was evaluated.

Results: This new mouse model mimics human HCC and reflects its typical features. Tumor-antigen-specific CD8+ T cells maintained a naïve phenotype and remained responsive during early-stage tumor progression. Late tumor progression produced circulating tumor cells, tumor migration into draining lymph nodes, and profound exhaustion of tumor-antigen-specific CD8+ T cells associated with accumulation of programmed cell death protein 1 (PD-1)hi CD8+ T cells and regulatory T cells (Tregs). Sunitinib-mediated tumoricidal effect and Treg suppression synergized with antibody-mediated blockade of PD-1 to powerfully suppress tumor growth and activate anti-tumor immunity.

Conclusion: Treg accumulation and upregulation of PD-1 provide two independent mechanisms to induce profound immune tolerance in HCC. Chemoimmunotherapy using Food and Drug Administration-approved sunitinib with anti-PD-1 antibodies achieved significant tumor control, supporting translation of this approach for the treatment of HCC patients.

Lay summary: In the current study, we have established a clinically relevant mouse model which mimics human liver cancer. Using this unique model, we studied the response of the immune system to this aggressive cancer. Findings from this trial have led to the development of an innovative and clinically feasible chemoimmunotherapeutic strategy.

Keywords: Cancer; Chemoimmunotherapy; Circulating tumor cell (CTC); Hepatocellular cancer (HCC); Immune checkpoint; Programmed cell death protein 1 (PD-1); Regulatory T cells (Tregs); Sunitinib.

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Figures

Figure 1
Figure 1. A novel murine model with typical features of human HCC
(a) The experimental design for establishing an innovative murine model of HCC. C57BL/6 mice were first treated with IP injection of CCl4 for three or four weeks to induce liver fibrosis. After 2 weeks, the CCl4-treated mice received normal-appearing hepatocytes isolated from young male MTD2 mice via ISPL inoculation. (b) Representative images of liver tumors in recipient mice with or without CCl4 treatment. (c) Representative images of liver sections with the indicated staining. Upper panel: hematoxylin and eosin (H&E) staining, asterisks depict forming nodules, arrowheads point to bile ductal proliferation. Second panel: picrosirius red staining; arrows point to abnormal collagen deposition. Third panel: Masson's trichrome staining. Fourth panel: α-SMA immunohistochemistry. Bottom panel: H&E staining, yellow arrows point to neutrophils, green arrows to macrophages, black arrows to lymphocytes, and the blue arrow to a plasma cell. (d) The number of tumorigenic foci in three cohorts is counted based on the MRI image seven weeks after treatment with CCl4. The cumulative results are presented; *p<0.05, **p<0.01; n=8; error bars represented mean ± SD. (e) A representative immunoblot demonstrates increased expression of AFP and GPC3 in tumor-bearing mice compared to normal mice.
Figure 2
Figure 2. Immune response of endogenous TAS CD8+ T cells in early-stage and late-stage tumor-bearing mice
Tumor-free mice and tumor-bearing mice with tumor volume <100 mm3 and > 400 mm3 received IP immunization with 3×107 B6/WT-19 cells. Splenocytes were harvested after seven days and stained directly with CD3, CD8, MHC Db/I tetramer and MHC Kb/IV tetramer. Parallel samples were stimulated with TAg epitope I or IV for 4 hours, then stained for intracellular IFN-γ and TNF-α in addition to CD3, CD8, MHC Db/I tetramer and MHC Kb/IV tetramer. Stained samples were evaluated by flow cytometry. (a) Representative images of livers in tumor-free and tumor-bearing mice with or without B6-WT-19 cell immunization. Yellow arrows and values indicate tumors and tumor sizes. Left panel: wild type C57BL/6 mice; middle panel: tumor-bearing mice with tumor size < 100 mm3 (early-stage tumor); right panel: tumor-bearing mice with tumor size > 400 mm3 (late-stage tumor). (b) The mean frequency of TAg epitope I-specific CD8+ T cells determined by Db/I tetramer staining. (c) The mean frequency of TAg epitope IV-specific CD8+ T cells determined by Kb/IV tetramer staining. (d) The mean frequency of TAg peptide I-specific CD8 T cells producing both IFN-γ and TNF-α. (e) The mean frequency of TAg peptide IV-specific CD8 T cell producing both IFN-γ and TNF-α. **p<0.01; n=5; error bars represent mean ± SD.
Figure 3
Figure 3. Immune response of exogenous TAS TCR-I T cells in early-stage and late-stage tumor mice
TAS TCR-I T cells were isolated from the spleen and LNs of 416 mice, and labeled with CFSE. 1×106 naïve CFSE-labeled TCR-I T cells were adoptively transferred into tumor-free and tumor-bearing mice at early and late stage, followed by immunization with 3×107 B6/WT-19 cells the next day. Seven days after immunization, lymphocytes were isolated from spleens and directly stained with CD8 and Db/I tetramer or stimulated in vitro with TAg epitope I peptide for 4 hours followed by staining for intracellular IFN-γ and TNF-α in addition to CD3, CD8, and with Db/I tetramer. Samples were evaluated by flow cytometry. (a) Representative plots showing the frequency of CSFEhi (top) and CSFE-diluted (bottom) CD8+ T cells; (b) Cumulative data showing the frequency of total CSFEhi and CSFE-diluted CD8+ T cells; (c) The mean frequency of IFN-γ-producing TCR-I T cells in tumor-free mice and early-stage tumor-bearing mice. (d) The mean frequency of IFN-γ-producing TCR-I T cells in tumor-free mice and late-stage tumor-bearing mice. Representative results for Fig 3c-3d are shown in Supplementary Fig 5. **p<0.01; n=5 mice per group; error bars represented mean ± SD.
Figure 4
Figure 4. Tumor cells home to local lymph nodes and circulate in the bloodstream of late-stage tumor-bearing mice
(a) The sensitivity and specificity of PCR for TAg detection. Lymphocytes were isolated from portal lymph nodes (LNs) of normal C57BL/6 and TAg-transgenic MTD2 mice. Varying numbers of lymphocytes from MTD2 mice were mixed with 106 lymphocytes from normal mice, and then genomic DNA was extracted from mixed lymphocytes and used as a template for PCR. (b) PCR detection of TAg in livers and LNs of the indicated mice. The draining portal LNs and liver were isolated from tumor-free, early-stage or late stage tumor-bearing mice. Genomic DNA was extracted from 106 lymphocytes or liver cells and used as templates for PCR. (c) Isolation and characterization of CTCs. Blood was collected from early-stage and late-stage tumor-bearing mice and CTCs enriched with a microfilter device. The enriched cells on the filters were stained by for immunofluorescence analysis with the indicated antibodies and representative images are shown.
Figure 5
Figure 5. The increase in the expression of PD-1 and frequency of Treg in late-stage tumor-bearing mice
Cell suspensions from spleen, liver and tumors of tumor-free mice, early-stage and late-stage tumor-bearing mice were stained for CD3, CD4, CD8, CD25, PD-1, CTLA-4, FoxP3, Gr-1 and CD11b and analyzed by flow cytometry. (a-d) Representative dot plots and cumulative results are presented for (a) the frequency of CTLA-4-expressing CD4+ T cells, (b) the frequency of PD-1-expressing CD4+ T cells, (c) the frequency of CTLA-4-expressing CD8+ T cells and (d) the frequency of PD-1-expressing CD8+ T cells. (e) Cumulative results showing the frequency of CD25+ and FoxP3+ CD4 T cells (Tregs; left) and CD11b+ and Gr-1+ cells (MDSC; right) among splenocytes. (f) Cumulative results showing the frequency of CD25+FoxP3+ CD4 T cells (Tregs) in liver-resident and tumor-resident lymphocytes (left) and Gr-1+CD11b+ MDSC in liver-resident and tumor-resident lymphocytes (right). Representative plots for Fig 5e-5f are shown in Supplementary Fig 6. n=3, * *p<0.01, error bars represent mean ±S.Ds.
Figure 6
Figure 6. Sunitinib synergizes with anti-PD-1 Ab to prevent tumor growth and activate anti-tumor immunity
(a) Experimental design of therapeutic approach in tumor-bearing mice. Size-matched tumor-bearing mice were randomly assigned to four groups and received the indicated monotherapy or combined therapy. (b) Mean tumor volume over the time course of the experiment as determined by MRI. (c) Representative images of MRI scans from the start and endpoints of the experiment. (d) Waterfall plots showing the change in tumor volume at the experimental endpoint relative to the starting tumor volume for each individual mouse. (e) Representative dot plots showing the frequency of CD25+FoxP3+ CD4+ T cells (Treg) among the indicated TILs. (f) Representative dot plots showing the frequency of PD-1-positive cells in gated CD8+ TILs. (g) Representative fold change in the frequency of CD8+ T cells producing IFN-γ. TILs were isolated from tumor-bearing mice given the indicated treatments and stimulated with epitope I or epitope IV peptide for 4 hours. After staining for CD3, CD8 and intracellular IFN-γ, the frequency of CD8+ T cells producing IFN-γ in gated CD3+ T cells was determined.
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