B7 immune-checkpoints as targets for the treatment of neuroendocrine tumors

in Endocrine-Related Cancer
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  • 1 Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA
  • 2 Department of Surgery, Rutgers Cancer Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA
  • 3 Department of Genetics, Yeshiva University Albert Einstein College of Medicine, Bronx, New York, USA
  • 4 Microbiology and Immunology, Yeshiva University Albert Einstein College of Medicine, Bronx, New York, USA
  • 5 Medicine, Albert Einstein College of Bronx, New York
  • 6 Department of Surgery, oy J and Lucille A. Carver University of Iowa College of Medicine, Iowa City, Iowa, USA

Correspondence should be addressed to S K Libutti: steven.libutti@cinj.rutgers.edu
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The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1α overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor β-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia-inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet β-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.

Supplementary Materials

    • Supplemental Figure 1. Expression levels of immune check-point proteins HHLA2 and B7x are correlated with clinical and pathological parameters. The expressed positive cell rates of HHLA2 and B7x are statistically higher in nonfunctional (42.6&#xF0B1;12.8% and 45.2&#xF0B1; 7.9%) and functional neuroendocrine tumors (NETs) (15.9&#xF0B1;5.7% and 22.8&#xF0B1;4.8%) compared to nonfunctional case matched adjacent nonnoplastic tissues, 3.5&#xF0B1;1.9% and 4.9&#xF0B1;2.3%, respectively (P<0.001), and functional case matched adjacent nonnoplastic tissues, 1.8&#xF0B1;0.3% and 4.2&#xF0B1;1.1%, respectively (P<0.001) by immunohistochemistry (IHC) staining (A1 and A2); the expressed positive cell rates of HHLA2 and B7x are significantly higher in primary tumor tissues with metastasis (80.5&#xF0B1;5.4% and 90.3&#xF0B1;8.2%) compared to tumor tissues without metastasis (A1 and A2); similar results are observed in mRNA expression by real-time RT-PCR analysis (A3 and A4). (N: Normal controls; T: Tumors). The average across all samples &#x00B1; SD was used for statistics in the histograms. Statistically significant P values are indicated in the figures with asterisks: ***, P <0.001. There is a correlation between the expressed positive cell rate of HHLA2 and B7x with tumor size in nonfunctional neuroendocrine tumors (NETs) (B1 and B2) and functional NETs (B5 and B6). There is also a correlation between the expressed positive cell rate of HHLA2 and B7x with tumor cell proliferation as measured by IHC with the Ki67 marker in nonfunctional NETS (B3 and B4) and functional NETs (B7 and B8).
    • Supplemental Figure 2. Control normal tissues from Men1 wild type (WT) and knockout (KO) mice were analyzed for B7x expression by immunohistochemistry (IHC). H&E staining of the control normal tissues (brain, heart, liver, spleen, kidney, and muscle) from Men1 WT (A1-A6) and KO mice (B1-B6). Negative IHC staining of B7x in the control normal tissues (brain, heart, lung, liver, spleen, kidney, and muscle) from Men1 WT (C1-C6) and KO mice (D1-D6).
    • Supplemental Figure 3. B7x expression in the pancreatic neuroendocrine tumors (PNETs) with wild type (WT) menin from rat insulin promoter-T antigen (RIP-Tag) mice. H&E staining of the normal pancreatic tissue from WT control mice (A1) and PNET from RIP-Tag mice (A2). Negative immunohistochemistry (IHC) staining of B7x is shown in the normal pancreatic tissue from WT control mice (B1) and positive staining of B7x is shown in the PNET tissue from RIP-Tag mice (B2).
    • Supplemental Figure 4. Anti-B7x mAb increased expression of cytokines in the tumor microenvironment. The levels of interferon-&#xF067; (IFN-&#xF067;) (B) and tumor necrosis factor-&#xF061; (TNF-&#xF061;) (D) are significantly increased in the tumor microenvironment in the B7x treatment group compared with the IgG control (A and C) as shown by immunofluorescence (IF) staining, immunostaining of insulin (pink), IFN-&#xF067;/TNF-&#xF061; (green), and nuclei counterstained with DAPI (blue). The images were taken with the 10X objective.
    • Supplemental Figure 5. The expression levels of HIF-1&#xF061; and B7x in N134 cells. The expression levels of HIF-1&#xF061; and B7x were not induced in the islet tumor &#x03B2; cells following HIF-1&#xF061; siRNA treatment after 48 hours by western blot. We provided the entire films for the three antibodies in the Supplemental Figure 5.
    • Supplemental Figure 6. HIF-1&#xF061; binds to the PFKFB4 promoter. HIF-1&#xF061; can bind to the PFKFB4 promoter in N134 islet tumor &#x03B2; cells using a chromatin immunoprecipitation (ChIP)-PCR analysis. The HIF-1&#xF061; immunoprecipitation (IP) products were amplified using quantitative real-time RT-PCR with primers corresponding to the promoter regions of the PFKFB4 gene and the relative amount of DNA was calculated using fold enrichment compared to the IgG Ab control. Statistically significant P values are indicated in the figures with asterisks: ***, P <0.001.
    • Supplemental Figure 7
    • Supplemental Table 1: Clinical characteristics and the expression of B7x and HHLA2 in the patients with neuroendocrine tumors. The expression of B7x and HHLA2 were analyzed in the patients with neuroendocrine neoplasms by immunohistochemistry (IHC) staining. The expression of B7x and HHLA2 were significantly increased in the later stage cancer to compared to early stage cancer (P=0.0014), but there is a significantly correlation between the expression of B7x and HHLA2 with age (P=0.5389) or gender (P=0.8523).
    • Supplemental Table 2: The expression of B7x and HHLA2 in the NET patients with and without metastases. The expressed levels of B7x and HHLA2 were analyzed in the human NET tissues with and without metastasis (Lymph node and distant Metastasis) by IHC staining. The expression of B7x and HHLA2 were significantly increased in the NET tissues with metastasis compared to the NET tissues without metastasis (P<0.01).
    • Supplemental Table 3: Primer sequences for quantitative real-time RT-PCR.
    • Supplemental Table 4: Primer sequences for chromatin immunoprecipitation (ChIP)-PCR.
    • Supplemental Table 5: Clinical characteristics and expression of B7x and HHLA2 in the individual patients with neuroendocrine tumors.

 

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