Recent breakthroughs in discovering novel immune signaling pathways have revolutionized different disease treatments. SERPINB9 (Sb9), also known as Proteinase Inhibitor 9 (PI-9), is a well-known endogenous inhibitor of Granzyme B (GzmB). GzmB is a potent cytotoxic molecule secreted by cytotoxic T lymphocytes and natural killer cells, which plays a crucial role in inducing apoptosis in target cells during immune responses. Sb9 acts as a protective mechanism against the potentially harmful effects of GzmB within the cells of the immune system itself. On the other hand, overexpression of Sb9 is an important mechanism of immune evasion in diseases like cancers and viral infections. The intricate functions of Sb9 in different cell types represent a fine-tuned regulatory mechanism for preventing immunopathology, protection against autoimmune diseases, and the regulation of cell death, all of which are essential for maintaining health and responding effectively to disease challenges. Dysregulation of the Sb9 will disrupt human normal physiological condition, potentially leading to a range of diseases, including cancers, inflammatory conditions, viral infections or other pathological disorders. Deepening our understanding of the role of Sb9 will aid in the discovery of innovative and effective treatments for various medical conditions. Therefore, the objective of this review is to consolidate current knowledge regarding the biological role of Sb9. It aims to offer insights into its discovery, structure, functions, distribution, its association with various diseases, and the potential of nanoparticle-based therapies targeting Sb9.

OBJECTIVE: In this research, we analyzed the expression of serpinB9 in hepatoblastoma and investigated the factors which enhance its expression.
METHODS: SerpinB9 expression in hepatoblastoma cell lines and macrophages co-cultured with each other or stimulated by anticancer agents was examined using RT-qPCR and western blotting. Immunohistochemistry for SerpinB9 in hepatoblastoma specimens was performed. Single-cell RNA-sequence data for hepatoblastoma from an online database were analyzed to investigate which types of cells express SerpinB9.
RESULTS: HepG2, a hepatoblastoma cell line, exhibited increased expression of SerpinB9 when indirectly co-cultured with macrophages. Immunohistochemistry for the specimens demonstrated that serpinB9 is positive not in hepatoblastoma cells but in macrophages. Single-cell RNA sequence analysis in tissues from hepatoblastoma patients showed that macrophages expressed SerpinB9 more than tumor cells did. Co-culture of macrophages with hepatoblastoma cell lines led to the enhanced expression of SerpinB9 in both macrophages and cell lines. Anticancer agents induced an elevation of SerpinB9 in hepatoblastomas cell lines.
CONCLUSIONS: In hepatoblastoma, SerpinB9 is thought to be more highly expressed in macrophages and enhanced by interaction with hepatoblastoma cell.

Serpinb9 is an inhibitor of granzyme B and is potentially involved in the immune escape of tumor cells. In the present study, bioinformatics analysis using open databases suggested that SerpinB9 is overexpressed in testicular embryonal carcinoma. Immunohistological analysis was performed on 28 cases of testicular germ cell tumors to investigate the relationship between SerpinB9 expression in testicular germ cell tumors and the tumor immune environment. SerpinB9 was significantly upregulated in the non-seminoma group and inversely correlated with the number of tumor-infiltrating CD8-positive cells. In addition, yolk sac tumors were characterized by the loss of human leukocyte antigen-class I expression. These findings suggest that SerpinB9 contributes to the immune escape of testicular germ cell tumors. Targeting therapy for SerpinB9 might therefore be useful in immunotherapy for testicular germ cell tumors resistant to immune checkpoint inhibitors.

Immunotherapies, in particular immune checkpoint blockade (ICB), have improved the clinical outcome of cancer patients, although many fail to mount a durable response. Several resistance mechanisms have been identified, but our understanding of the requirements for a robust ICB response is incomplete. We have engineered an MHC I/antigen: TCR-matched panel of human NSCLC cancer and T cells to identify tumor cell-intrinsic T cell resistance mechanisms. The top differentially expressed gene in resistant tumor cells was SERPINB9. This serine protease inhibitor of the effector T cell-derived molecule granzyme B prevents caspase-mediated tumor apoptosis. Concordantly, we show that genetic ablation of SERPINB9 reverts T cell resistance of NSCLC cell lines, whereas its overexpression reduces T cell sensitivity. SERPINB9 expression in NSCLC strongly correlates with a mesenchymal phenotype. We also find that SERPINB9 is commonly amplified in cancer, particularly melanoma in which it is indicative of poor prognosis. Single-cell RNA sequencing of ICB-treated melanomas revealed that SERPINB9 expression is elevated not only in cells from post- versus pre-treatment cancers, but also in ICB-refractory cancers. In NSCLC we commonly observed rare SERPINB9-positive cancer cells, possibly accounting for reservoirs of ICB-resistant cells. While underscoring SERPINB9 as a potential target to combat immunotherapy resistance, these results suggest its potential to serve as a prognostic and predictive biomarker.

Granzyme B (GrB) is a pivotal killer factor in immunotherapy whose application is limited by hyposensitivity and unsatisfactory cellular uptake by tumor cells. In this study, it was proved that SerpinB9 (Sb9) downregulation can enhance the GrB susceptibility of tumor cells. Moreover, a nanocarrier fused with M1 macrophage exosomes (M1 Exo) and photothermal sensitive liposomes was constructed to efficiently transport GrB and siRNA of Sb9 to the cells. The nanocarrier is characterized by cascade tumor targeting acquired by photothermal effect-triggered increased expression of vascular cell adhesion molecule-1 (VCAM-1) in tumor tissue. Furthermore, the innate cytokines in M1 Exo are capable of regulating the tumor microenvironment by repolarizing M2 macrophages to the M1 type. Collectively, the multifunctional nanoplatform (S+G@ELP) enhances the lethality of GrB to tumor cells, activates a widespread immune response uniting with photothermal therapy (PTT), restrains the tumor progression and metastasis effectively, which is expected to provide new insights into GrB-based combinational tumor therapy.

Protocatechuic acid (PCA) is a natural phenolic acid present in daily vegetables and fruits. Notably, PCA was demonstrated to inhibit the biological function of SerpinB9 (Sb9) and exhibit an excellent antitumor effect, showing great potential in cancer treatment. However, the short half-life time limits PCA\'s wide application against cancers. To overcome this shortage of PCA, we integrated PCA and another natural product with strong self-assembling properties, isoguanosine (isoG), to develop a novel multifunctional supramolecular hydrogel with good biocompatibility and injectability, which remarkably lengthens the releasing time of PCA and exerts considerable anticancer effects in vitro and in vivo. Besides, we surprisingly found that PCA could not only target Sb9 but also restrain cancer development through activating the JNK/P38 pathway, decreasing the ROS level, and repairing cancer stemness. In all, our results demonstrate that this PCA-based hydrogel could act as a multifunctional hydrogel system equipped with considerable anticancer effects, providing potential local administration integrating with targeted therapy and chemotherapy in one simple modality.

Multiple myeloma (MM) is a common hematological malignancy with poorly understood recurrence and relapse mechanisms. Notably, bortezomib resistance leading to relapse makes MM treatment significantly challenging. To clarify the drug resistance mechanism, we employed a quantitative proteomics approach to identify differentially expressed protein candidates implicated in bortezomib-resistant recurrent and relapsed MM (RRMM). Bone marrow aspirates from five patients newly diagnosed with MM (NDMM) were compared with those from five patients diagnosed with bortezomib-resistant RRMM using tandem mass tag-mass spectrometry (TMT-MS). Subcellular localization and functional classification of the differentially expressed proteins were determined by gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and hierarchical clustering analyses. The top candidates identified were validated with parallel reaction monitoring (PRM) analysis using tissue samples from 11 NDMM and 8 RRMM patients, followed by comparison with the NCBI Gene Expression Omnibus (GEO) dataset of 10 MM patients and 10 healthy controls (accession no.: GSE80608). Thirty-four differentially expressed proteins in RRMM, including proteinase inhibitor 9 (SERPINB9), were identified by TMT-MS. Subsequent functional enrichment analyses of the identified protein candidates indicated their involvement in regulating cellular metabolism, apoptosis, programmed cell death, lymphocyte-mediated immunity, and defense response pathways in RRMM. The top protein candidate SERPINB9 was confirmed by PRM analysis and western blotting as well as by comparison with an NCBI GEO dataset. We elucidated the proteome landscape of bortezomib-resistant RRMM and identified SERPINB9 as a promising novel therapeutic target. Our results provide a resource for future studies on the mechanism of RRMM.

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.

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (grB) that protects cytotoxic lymphocytes from grB-mediated death. In addition, Sb9 is also expressed in accessory immune cells, including dendritic cells (DCs), although its role is debated. Recently, we have demonstrated that Sb9 plays a grB-independent role in cross-presentation of antigens by CD8+ DCs. Here, using a mouse line expressing green fluorescent protein knocked in under the control of the Sb9 promoter, we demonstrate that Sb9 expression is highest in those tissue-resident and migratory DC subsets capable of cross-presentation. Further, we show that CD8+ DCs can be divided into two subsets based on Sb9 expression, and that only the subset expressing higher levels of Sb9 is capable of cross-presentation. These findings add support for role for Sb9 cross-presentation, and indicate that high Sb9 expression is a novel marker of cross-presentation capable DCs.

Patients who suffer from autoinflammatory disease (AID) exhibit seemingly uncontrolled release of interleukin (IL)-1β. The presence of this inflammatory cytokine triggers immune activation in absence of pathogens and foreign material. The mechanisms that contribute to \'sterile inflammation\' episodes in AID patients are not fully understood, although for some AIDs underlying genetic causes have been identified. We show that the serine protease inhibitor B9 (serpinB9) regulates IL-1β release in human monocytes. SerpinB9 function is more commonly known for its role in control of granzyme B. SerpinB9 however also serves to restrain IL-1β maturation through caspase-1 inhibition. We here describe an autoinflammatory disease-associated serpinB9 (c.985G>T, A329S) variant, which we discovered in a patient with unknown AID. Using patient cells and serpinB9 overexpressing monocytic cells, we show the A329S variant of serpinB9 exhibits unobstructed granzyme B inhibition, but compromised caspase-1 inhibition. SerpinB9 gene variants might contribute to AID development.