Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy

•SerpinB9 protects cancer cells from their own granzyme B•SerpinB9 expression in CAFs, MDSCs, and TAMs can promote tumor growth•Deletion of serpinB9 in both the tumor and host suppresses tumor growth markedly•SerpinB9 inhibition can target cancer cells, CAFs, MDSCs, and TAMs simultaneously Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality. Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality. Serine proteases participate in a wide range of physiological processes, which are regulated by a large family of peptidase inhibitors referred to as serine protease inhibitors (serpins) (Silverman et al., 2001Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. et al.The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature.J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). Serpins act as a suicide substrate for a serine protease that results in a characteristic covalent inhibitory complex (Huntington et al., 2000Huntington J.A. Read R.J. Carrell R.W. Structure of a serpin-protease complex shows inhibition by deformation.Nature. 2000; 407: 923-926Crossref PubMed Scopus (910) Google Scholar; Mangan et al., 2008Mangan M.S. Kaiserman D. Bird P.I. The role of serpins in vertebrate immunity.Tissue Antigens. 2008; 72: 1-10Crossref PubMed Scopus (58) Google Scholar). In contrast to most serpins, which are extracellular, SerpinB9 (Sb9) (PI9 in human, Spi6 in mice) is a member of the ovalbumin family of serpins, which reside within the nuclei and cytoplasm of cells (Bird et al., 1998Bird C.H. Sutton V.R. Sun J. Hirst C.E. Novak A. Kumar S. Trapani J.A. Bird P.I. Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.Mol. Cell. Biol. 1998; 18: 6387-6398Crossref PubMed Google Scholar; Bots and Medema, 2008Bots M. Medema J.P. Serpins in T cell immunity.J. Leukoc. Biol. 2008; 84: 1238-1247Crossref PubMed Scopus (19) Google Scholar; Sun et al., 1996Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes.J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, Sun et al., 1997Sun J. Ooms L. Bird C.H. Sutton V.R. Trapani J.A. Bird P.I. A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).J. Biol. Chem. 1997; 272: 15434-15441Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Sb9 proteins are physiological inhibitors of granzyme B (GrB), which triggers apoptosis by activating caspases-3 and -8, following delivery into target cells by cytotoxic lymphocytes (CLs) (Pinkoski et al., 2001Pinkoski M.J. Waterhouse N.J. Heibein J.A. Wolf B.B. Kuwana T. Goldstein J.C. Newmeyer D.D. Bleackley R.C. Green D.R. Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway.J. Biol. Chem. 2001; 276: 12060-12067Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Sb9 has been shown to protect pro-inflammatory CLs from self-inflicted damage by their own GrB (Hirst et al., 2003Hirst C.E. Buzza M.S. Bird C.H. Warren H.S. Cameron P.U. Zhang M. Ashton-Rickardt P.G. Bird P.I. The intracellular granzyme B inhibitor, proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation, and its overexpression enhances CTL potency.J. Immunol. 2003; 170: 805-815Crossref PubMed Google Scholar; Sun et al., 1996Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes.J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Sb9 also protects other leukocytes, which are both pro-inflammatory (dendritic cells and neutrophils) (Medema et al., 2001bMedema J.P. Schuurhuis D.H. Rea D. van Tongeren J. de Jong J. Bres S.A. Laban S. Toes R.E. Toebes M. Schumacher T.N. et al.Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells.J. Exp. Med. 2001; 194: 657-667Crossref PubMed Scopus (173) Google Scholar; Rizzitelli et al., 2012Rizzitelli A. Meuter S. Vega Ramos J. Bird C.H. Mintern J.D. Mangan M.S. Villadangos J. Bird P.I. Serpinb9 (Spi6)-deficient mice are impaired in dendritic cell-mediated antigen cross-presentation.Immunol. Cell Biol. 2012; 90: 841-851Crossref PubMed Scopus (12) Google Scholar) or anti-inflammatory (regulatory T cells [Tregs] and myeloid-derived suppressor cells [MDSCs]) (Azzi et al., 2013Azzi J. Skartsis N. Mounayar M. Magee C.N. Batal I. Ting C. Moore R. Riella L.V. Ohori S. Abdoli R. et al.Serine protease inhibitor 6 plays a critical role in protecting murine granzyme B-producing regulatory T cells.J. Immunol. 2013; 191: 2319-2327Crossref PubMed Scopus (21) Google Scholar; Kumar et al., 2016Kumar V. Patel S. Tcyganov E. Gabrilovich D.I. The Nature of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.Trends Immunol. 2016; 37: 208-220Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar; Lindau et al., 2013Lindau D. Gielen P. Kroesen M. Wesseling P. Adema G.J. The immunosuppressive tumour network: myeloid-derived suppressor cells, regulatory T cells and natural killer T cells.Immunology. 2013; 138: 105-115Crossref PubMed Scopus (452) Google Scholar), from GrB that either originates from CL or is produced endogenously. Sb9 is also thought to protect tumor cells from GrB delivered by CLs, but this has not been tested directly in vivo (Bots and Medema, 2008Bots M. Medema J.P. Serpins in T cell immunity.J. Leukoc. Biol. 2008; 84: 1238-1247Crossref PubMed Scopus (19) Google Scholar; Mangan et al., 2008Mangan M.S. Kaiserman D. Bird P.I. 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Bacani J.T. Ingham R.J. Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs.Mol. Cancer. 2014; 13: 199Crossref PubMed Scopus (10) Google Scholar). Immunosuppressive tumor-associated macrophages (TAMs), MDSCs, and Tregs in the tumor microenvironment (TME) abet tumor progression and metastasis (Kumar et al., 2016Kumar V. Patel S. Tcyganov E. Gabrilovich D.I. The Nature of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.Trends Immunol. 2016; 37: 208-220Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar; Lindau et al., 2013Lindau D. Gielen P. Kroesen M. Wesseling P. Adema G.J. The immunosuppressive tumour network: myeloid-derived suppressor cells, regulatory T cells and natural killer T cells.Immunology. 2013; 138: 105-115Crossref PubMed Scopus (452) Google Scholar). The potential effect of GrB inhibition by Sb9 on both the anti-tumor cellular effectors (such as CL) and immunosuppressive components of the TME is not known (Quail and Joyce, 2013Quail D.F. Joyce J.A. Microenvironmental regulation of tumor progression and metastasis.Nat. Med. 2013; 19: 1423-1437Crossref PubMed Scopus (3401) Google Scholar). Aside from the potential for immunomodulation, whether inhibition of Sb9 results in beneficial elimination of tumors either by direct killing or by increased host immunity remains to be determined. In addition to its implications on immune cells and the intrinsic survival of tumor cells, the Sb9-GrB axis can also have a major impact on the tumor stroma as well. Stromal cells, including cancer-associated fibroblasts (CAFs), constitute a major cellular component of the diverse TME and play a critical role in tumor development (Kalluri, 2016Kalluri R. The biology and function of fibroblasts in cancer.Nat. Rev. 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Here, we show that genetic ablation of Sb9 sensitized tumors to killing by not only CL-derived GrB, but also from endogenously produced GrB, which together resulted in the control of cancer in mice. The role of Sb9 in the anti-tumor host response was examined in Sb9 KO mice, which exhibited increased resistance to tumors. This was a consequence of impaired survival of immunosuppressive TAMs, MDSCs, Tregs, and CAFs in the TME that resulted in increased activity of anti-tumor CL. We developed a specific small molecule inhibitor of Sb9 and showed that treatment of mice could control tumor growth through direct sensitization to GrB and the activation of protective immunity. We examined the expression of Sb9 and GrB in several tumors of human and mouse. Sb9 (Figure 1A) and GrB (Figure 1B) were expressed in primary human and mouse malignant melanoma, breast adenocarcinoma, and lung adenocarcinoma. We also analyzed single-cell RNA sequencing data from previous studies (Guo et al., 2018Guo X. Zhang Y. Zheng L. Zheng C. Song J. Zhang Q. Kang B. Liu Z. Jin L. Xing R. et al.Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing.Nat. Med. 2018; 24: 978-985Crossref PubMed Scopus (344) Google Scholar; Jerby-Arnon et al., 2018Jerby-Arnon L. Shah P. Cuoco M.S. Rodman C. Su M.J. Melms J.C. Leeson R. Kanodia A. Mei S. Lin J.R. et al.A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade.Cell. 2018; 175: 984-997Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar; Slyper et al., 2020Slyper M. Porter C.B.M. Ashenberg O. Waldman J. Drokhlyansky E. Wakiro I. Smillie C. Smith-Rosario G. Wu J. Dionne D. et al.A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.Nat. Med. 2020; 26: 792-802Crossref PubMed Scopus (64) Google Scholar) to map the detailed expression of Sb9 and GrB in melanoma, breast cancer, and lung cancer of human origin. Sb9 and GrB were detected in these tumor cells, and their expression levels were measured in comparison to tumor-infiltrating CD4+ and CD8+ T cells as a positive control (Figure S1A). Additionally, mouse cancer cell lines (B16, mouse melanoma; 4T1, mouse breast cancer; and LLC1, mouse lung cancer) and human cancer cell lines (A375, human melanoma; A549, human lung cancer; and SK-BR3, human breast cancer) also expressed both Sb9 (Figure S1C) and GrB (Figure S1D). Then, B16 cells and LLC1 cells were implanted into UBC-GFP transgenic mice (expressing GFP under the direction of the human ubiquitin C promoter), as previously described (Andersson et al., 2009Andersson A. Yang S.C. Huang M. Zhu L. Kar U.K. Batra R.K. Elashoff D. Strieter R.M. Dubinett S.M. Sharma S. IL-7 promotes CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.J. Immunol. 2009; 182: 6951-6958Crossref PubMed Scopus (71) Google Scholar; Barrett et al., 2012Barrett L.E. Granot Z. Coker C. Iavarone A. Hambardzumyan D. Holland E.C. Nam H.S. Benezra R. Self-renewal does not predict tumor growth potential in mouse models of high-grade glioma.Cancer Cell. 2012; 21: 11-24Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar; Kar et al., 2011Kar U.K. Srivastava M.K. Andersson A. Baratelli F. Huang M. Kickhoefer V.A. Dubinett S.M. Rome L.H. Sharma S. Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth.PLoS ONE. 2011; 6: e18758Crossref PubMed Scopus (75) Google Scholar), and the tumors were collected at day 17 post-implantation to compare GrB expression between the tumor and the host. Most GrB+ cells were GFP negative, indicating the presence of tumor-expressed GrB (Figure S1B). Inhibition of GrB by Sb9 via a classic serpin mechanism resulted in an SDS-resistant 62 kDa Sb9-GrB complex in B16 cells (Figures 1C and 1D; Mangan et al., 2016Mangan M.S. Bird C.H. Kaiserman D. Matthews A.Y. Hitchen C. Steer D.L. Thompson P.E. Bird P.I. A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP.J. Biol. Chem. 2016; 291: 3626-3638Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar; Zhang et al., 2006Zhang M. Park S.M. Wang Y. Shah R. Liu N. Murmann A.E. Wang C.R. Peter M.E. Ashton-Rickardt P.G. Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules.Immunity. 2006; 24: 451-461Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Western blot demonstrated that Sb9 was expressed mainly as a 47 kDa unbound monomer, while a smaller portion of Sb9 formed a 62 kDa Sb9-GrB complex in B16 cells (Figure 1C) (Mangan et al., 2016Mangan M.S. Bird C.H. Kaiserman D. Matthews A.Y. Hitchen C. Steer D.L. Thompson P.E. Bird P.I. A Novel Serpin Regulatory Mechanism: SerpinB9 IS REVERSIBLY INHIBITED BY VICINAL DISULFIDE BOND FORMATION IN THE REACTIVE CENTER LOOP.J. Biol. Chem. 2016; 291: 3626-3638Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). We observed that almost all of the GrB exists as a 62 kDa complex with Sb9, in contrast to the 26 kDa monomeric GrB found in B16 cells (Figure 1D). The GrB RNAscope staining demonstrates the gene expression of GrB (green) in both B16 and 4T1 cells (Figure 1E). Furthermore, a GranToxiLux assay, which measures GrB activity in live cells, demonstrated that the GrB in B16 is functionally active (Figure 1F).Figure S1Sb9 Is Required to Protect Melanoma Tumors from GrB-Induced Apoptosis, Related to Figure 1Show full captionA. Violin plots show the expression of Sb9 and GrB in cancer cells including human melanoma (n = 1751), human breast cancer (n = 1284), and human non-small cell cancer (n = 2150). Tumor-infiltrating CD4+ and CD8+ T cells were used as positive control.B. Fluorescence micrographs indicate the presence of GrB (red) in mouse melanoma and lung cancer cells (GFP-) implanted in UBC-GFP mice (GFP+). DAPI (blue) was the nuclei. Scale bar: 50 μm.C-D. Fluorescence micrographs demonstrate the presence of Sb9 (red) and GrB (green) in three mouse cancer cell lines–B16 (mouse melanoma), 4T1 (mouse breast cancer), and LLC1 (mouse lung cancer)–and three human cancer cell lines–A375 (human melanoma), SK-BR3 (human breast cancer), and A549 (human lung cancer). DAPI (blue) was the nuclei. Scale bar: 20 μm.E. CRIPSR/Cas9 gene editing system was used to disrupt the SERPINB9 gene in the B16 melanoma cells. We targeted the 20-nucleotide sequences upstream of the protospacer adjacent motif (PAM) sequences in exon 6 of the SERPINB9 gene.F. Western blotting confirms the knockout of Sb9 protein in the B16 melanoma cells. Sb9-GrB complex (com), unbound Sb9 (mono), and complex degradation products or cleaved Sb9 (deg) are indicated by arrows.G. Sb9 RNAscope staining demonstrates B16-Sb9 KO cells lack of Sb9 mRNA (red) compared to B16-WT. DAPI (blue) was the nuclei. Scale bar: 10 μm.H. Fluorescence micrographs show the staining of Sb9 antibody (red) in B16-WT and B16-Sb9 KO cells. DAPI (blue) was the nuclei. Scale bar: 20 μm.I. RT-qPCR analysis demonstrates that Ki67 mRNA expression in B16-Sb9 KO cells is similar to that in B16-WT cells. NS (no significant difference).J. Representative micrographs of Crystal Violet cell staining and quantitative analysis indicate that the cell proliferation rate of B16-Sb9 KO cells is similar to that of B16-WT cells. NS (no significant difference).K. GranToxiLux assay reveals significantly higher GrB activity in B16-Sb9 KO cells, as compared to B16-WT cells. ∗p < 0.05.L. RT-PCR results show that the gene expression of GrB by B16-WT cells is increased as IL-2 concentration increases (1, 5, and 20 ng/ml). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.M. Flow cytometric analysis reveals no significant difference in the percentages of apoptosis, as indicated by Annexin-V+ and viability- signature, among B16-Sb9&GrB KO cells treated with different concentrations of IL-2 (1, 5, and 20 ng/ml) for 48 h. NS (no significant difference).N. Flow cytometry quantitative analysis reveal that GrB inhibitor (368050) (10 μM) suppresses apoptosis (as indicated by Annexin-V+ and viability dye- population) in the treatment of B16-Sb9 KO cells with IL-2 (70 ng/ml) for 48 h. Cells treated with PBS alone were used as a negative control. ∗p < 0.05, ∗∗∗p < 0.001.O. Representative fluorescence micrographs demonstrate that treatment of B16-WT cells with IL-2 (70 ng/ml) versus PBS for 48 h results in higher GrB (red) expression in B16 mouse melanoma cells. DAPI (blue) was the nuclei. Scale bar: 20 μm.P. Representative fluorescence micrographs show that treatment of B16-Sb9 KO cells with IL-2 (70 ng/ml) for 48 h results in higher expression of cleaved caspase-3 (red) in comparison to B16-WT cells. Cells treated with PBS alone were used as a negative control. DAPI (blue) was the nuclei. Scale bar: 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A. Violin plots show the expression of Sb9 and GrB in cancer cells including human melanoma (n = 1751), human breast cancer (n = 1284), and human non-small cell cancer (n = 2150). Tumor-infiltrating CD4+ and CD8+ T cells were used as positive control. B. Fluorescence micrographs indicate the presence of GrB (red) in mouse melanoma and lung cancer cells (GFP-) implanted in UBC-GFP mice (GFP+). DAPI (blue) was the nuclei. Scale bar: 50 μm. C-D. Fluorescence micrographs demonstrate the presence of Sb9 (red) and GrB (green) in three mouse cancer cell lines–B16 (mouse melanoma), 4T1 (mouse breast cancer), and LLC1 (mouse lung cancer)–and three human cancer cell lines–A375 (human melanoma), SK-BR3 (human breast cancer), and A549 (human lung cancer). DAPI (blue) was the nuclei. Scale bar: 20 μm. E. CRIPSR/Cas9 gene editing system was used to disrupt the SERPINB9 gene in the B16 melanoma cells. We targeted the 20-nucleotide sequences upstream of the protospacer adjacent motif (PAM) sequences in exon 6 of the SERPINB9 gene. F. Western blotting confirms the knockout of Sb9 protein in the B16 melanoma cells. Sb9-GrB complex (com), unbound Sb9 (mono), and complex degradation products or cleaved Sb9 (deg) are indicated by arrows. G. Sb9 RNAscope staining demonstrates B16-Sb9 KO cells lack of Sb9 mRNA (red) compared to B16-WT. DAPI (blue) was the nuclei. Scale bar: 10 μm. H. Fluorescence micrographs show the staining of Sb9 antibody (red) in B16-WT and B16-Sb9 KO cells. DAPI (blue) was the nuclei. Scale bar: 20 μm. I. RT-qPCR analysis demonstrates that Ki67 mRNA expression in B16-Sb9 KO cells is similar to that in B16-WT cells. NS (no significant difference). J. Representative micrographs of Crystal Violet cell staining and quantitative analysis indicate that the cell proliferation rate of B16-Sb9 KO cells is similar to that of B16-WT cells. NS (no significant difference). K. GranToxiLux assay reveals significantly higher GrB activity in B16-Sb9 KO cells, as compared to B16-WT cells. ∗p < 0.05. L. RT-PCR results show that the gene expression of GrB by B16-WT cells is increased as IL-2 concentration increases (1, 5, and 20 ng/ml). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. M. Flow cytometric analysis reveals no significant difference in the percentages of apoptosis, as indicated by Annexin-V+ and viability- signature, among B16-Sb9&GrB KO cells treated with different concentrations of IL-2 (1, 5, and 20 ng/ml) for 48 h. NS (no significant difference). N. Flow cytometry quantitative analysis reveal that GrB inhibitor (368050) (10 μM) suppresses apoptosis (as indicated by Annexin-V+ and viability dye- population) in the treatment of B16-Sb9 KO cells with IL-2 (70 ng/ml) for 48 h. Cells treated with PBS alone were used as a negative control. ∗p < 0.05, ∗∗∗p < 0.001. O. Representative fluorescence micrographs demonstrate that treatment of B16-WT cells with IL-2 (70 ng/ml) versus PBS for 48 h results in higher GrB (red) expression in B16 mouse melanoma cells. DAPI (blue) was the nuclei. Scale bar: 20 μm. P. Representative fluorescence micrographs show that treatment of B16-Sb9 KO cells with IL-2 (70 ng/ml) for 48 h results in higher expression of cleaved caspase-3 (red) in comparison to B16-WT cells. Cells treated with PBS alone were used as a negative control. DAPI (blue) was the nuclei. Scale bar: 20 μm. To address the protection of Sb9 from GrB in melanoma cells, we disrupted the SERPINB9 gene using the CRISPR/Cas9 system. We targeted the 20-nucleotide sequence upstream of the protospacer-adjacent motif (PAM) sequence in exon 6 of the SERPINB9 gene (Figure S1E). Deletion of Sb9 in the B16 cells was confirmed by western blot (Figure S1F). The complex formation was hampered, suggesting that all GrB in the cell is no longer captured by Sb9. Residual Sb9 protein expression by B16-Sb9 knockout (KO) cells is not an infrequent observation (Figure S1F), due to common tetraploidy in these cells (Kendal et al., 1987Kendal W.S. Wang R.Y. Hsu T.C. Frost P. Rate of generation of major karyotypic abnormalities in relationship to the metastatic potential of B16 murine melanoma.Cancer Res. 1987; 47: 3835-3841PubMed Google Scholar; Smits et al., 2019Smits A.H. Ziebell F. Joberty G. Zinn N. Mueller W.F. Clauder-Münster S. Eberhard D. Fälth Savitski M. Grandi P. Jakob P. et al.Biological plasticity rescues target activity in CRISPR knock outs.Nat. Methods. 2019; 16: 1087-1093Crossref PubMed Scopus (69) Google Scholar). Sb9 RNAscope and immunofluorescence staining for Sb9 confirmed its lack of expression in B16-Sb9 KO cells (Figures S1G and S1H). However, Sb9 KO did not affect the proliferation of B16 cells, as evidenced by a colony formation assay and the expression of Ki67 gene (Figures S1I and S1J; Li et al., 2015Li L.T. Jiang G. Chen Q. Zheng J.N. Ki67 is a promising molecular target in the diagnosis of cancer (review).Mol. Med. Rep. 2015; 11: 1566-1572Crossref PubMed Scopus (268) Google Scholar). Sb9 KO resulted in 2-fold increase (p = 0.016) in GrB activity (Figure S1K) and a corresponding 2.3-fold (p = 0.017) increase in GrB-specific apoptosis in B16-Sb9 KO cells compared to B16-wild-type (WT) cells (Figure 1H). Previous studies have reported that interleukin (IL)-2 induces GrB expression in cytotoxic T cells (CTL) and natural killer (NK) CLs (Tamang et al., 2006Tamang D.L. Redelman D. Alves B.N. Vollger L. Bethley C. Hudig D. Induction of granzyme B and T cell cytotoxic capacity by IL-2 or IL-15 without antigens: multiclonal responses that are extremely lytic if triggered and short-lived after cytokine withdrawal.Cytokine. 2006; 36: 148-159Crossref PubMed Scopus (45) Google Scholar). We found that the mRNA level of GrB was significantly higher in IL-2-treated B16-WT cells and followed a dose-dependent response (Figure S1L). Additionally, the expression of GrB was also elevated significantly in IL-2-treated B16-WT and B16-Sb9 KO cells (Figures 1G and S1O). The cleaved caspase-3 (C-CAS3) expression was higher in B16-Sb9 KO cells compared to B16-WT and increased with IL-2 treatment (Figure S1P). The apoptosis rates (as indicated by the annexin-V+ and viability− population) of B16-WT and B16-Sb9 KO cells were significantly higher following treatment with multiple concentrations of IL-2 (Figure 1H), while B16-Sb9 and GrB KO cells showed no IL-2-induced apoptosis (Figure S1M). The GrB inhibitor 368050 also suppressed apoptosis following treatment with IL-2 (Figure S1N). Collectively, these results demonstrate that Sb9 is required to protect tumors from GrB-induced apoptosis. To investigate the role of Sb9 in tumor progression and metastasis in vivo, B16-WT or B16-Sb9 KO cells were injected subcutaneously into C57BL/6 mice, then tumor growth was monitored to the size end points. The Sb9-disrupted tumors were ∼4-fold smaller (p = 0.0001) than the B16-WT group at day 27 post-implantation (Figure 2A). The mice reached the size end points at 27 days and 39 days in the B16-WT and B16-Sb9 KO, respectively (Figure 2A). We also compared the sizes of B16-Sb9&GrB KO tumors with the B16-WT and B16-Sb9 KO tumors to assess the differential effects of tumor-derived and host-derived GrB in tumor growth. The B16-Sb9&GrB KO tumors grew faster than the B16-Sb9 KO tumors and slower than the B16-WT tumors (Figure S2A). The intermediate protection seen in B16-Sb9&GrB KO mice could be due to exposure to host-derived GrB. No difference was noted in the size of B16-Sb9 KO tumors derived from a single clone or those from a bulk cell population (Figure S2B). Staining of the melanoma sections with the melanoma marker MelanA at day 17 following implantation revealed a much smaller MelanA+ area in the B16-Sb9 KO group than the B16-WT group (Figures 2B and 2C). The median survival time (MST) of mice bearing B16-Sb9 KO tumors was significantly longer (p < 0.0001) than that of mice bearing B16-WT tumors (Figure S2C). B16-Sb9 KO tu