CD276 Mouse

What is CD276 (B7-H3) and where is it expressed in mouse tissues?

CD276, also known as B7-H3, is a type I transmembrane protein and a member of the B7 family of co-stimulatory proteins. In mice, CD276 is expressed weakly on activated lymphocytes, macrophages, dendritic cells, nasal and airway epithelial cells, osteoblasts, and some tumor cell lines . A soluble form of CD276 is also secreted by monocytes, dendritic cells, and activated T cells . Single-cell RNA sequencing has demonstrated CD276 expression across multiple cell types in mouse models, including epithelial cells, fibroblasts, endothelial cells, T cells, and myeloid cells . Importantly, CD276 is prominently expressed in various tumor tissues but rarely expressed in normal tissues, making it a promising target for cancer research .

What are the known biological functions of CD276 in mouse immune regulation?

CD276 primarily functions as a negative regulator of immune responses, though its complete biological role remains under investigation . Multiple studies have demonstrated that CD276 mediates tumor immune evasion through several mechanisms, including:

• Reduction of CD8+ T cells and natural killer (NK) cells in the tumor microenvironment

• Suppression of T helper type 1-mediated immune responses

• Promotion of efferocytosis (clearance of apoptotic cells) by tumor-associated macrophages

• Suppression of MHC class II expression, resulting in decreased CD4+ and CD8+ T cell infiltration

These findings have been validated through multiple experimental approaches, including CD276 knockout mouse models, which show enhanced anti-tumor immunity with increased infiltration of cytotoxic immune cells . The MJ18 antibody has been developed to block CD276 in mice, further supporting research into its immunoregulatory functions .

How is CD276 expression regulated during cancer progression in mouse models?

CD276 expression shows significant upregulation during cancer progression in mouse models. In esophageal squamous cell carcinoma (ESCC) models, CD276 expression gradually increases from grade I and II to grade III tumors . Similarly, the expression of CD276 is significantly elevated in late-stage compared to early-stage disease . Immunohistochemistry (IHC) studies have demonstrated that CD276 protein is stepwise upregulated during malignant transformation .

In bladder cancer models induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), CD276 expression is detected in tumor tissue but virtually absent in normal bladder epithelium . Temporal analysis shows that CD276 expression appears early in tumor development and increases as tumors progress to more advanced stages .

How are CD276 knockout mouse models generated and what phenotypes do they display?

Researchers have developed several types of CD276 knockout mouse models using genetic engineering approaches:

• Whole-body knockout (CD276-wKO): These mice have CD276 deleted in all tissues through CRISPR/Cas9 technology or traditional gene targeting approaches . CD276-wKO mice are born at expected Mendelian ratios and develop normally with no obvious physical abnormalities . They are fertile and viable with a normal life span .

• Conditional knockout models:

  • Epithelial-specific knockout: Generated by crossing CD276 floxed mice with K14CreER mice (CD276-cKO)

  • Myeloid-specific knockout: Created by crossing CD276fl/fl mice with LysM-Cre; Rosa-tdTomato mice

Validation of knockout efficiency is typically performed through immunofluorescence staining, immunohistochemistry, or Western blotting to confirm the absence of CD276 protein expression in target tissues .

When challenged with carcinogens or tumor cell lines, CD276-wKO mice show remarkable resistance to tumor development. They exhibit smaller and fewer lesions in 4-nitroquinoline 1-oxide (4NQO)-induced esophageal cancer models and significantly improved survival in BBN-induced bladder cancer models . Tumors in CD276-wKO mice typically show decreased cell proliferation (measured by Ki67) and increased apoptosis (measured by active-Caspase3) .

What experimental approaches are used to study CD276 in mouse tumor models?

Researchers employ diverse experimental approaches to study CD276 in mouse tumor models:

• Carcinogen-induced tumor models:

  • 4NQO-induced esophageal squamous cell carcinoma: Administration in drinking water induces ESCC that recapitulates human disease

  • BBN-induced bladder cancer: Creates models resembling human muscle-invasive bladder cancer

• Transplantable tumor models:

  • Subcutaneous injection of tumor cell lines (e.g., MB49 bladder carcinoma cells) into syngeneic mice

  • Comparison of wild-type vs. CD276-knockout tumor cells

  • Comparison of tumor growth in wild-type vs. CD276-knockout host mice

• Analysis techniques:

  • Histopathological assessment of tumor grade, stage, and invasiveness

  • Immunohistochemistry for CD276 expression, proliferation markers, apoptosis markers, and immune cell infiltration

  • Flow cytometry for quantitative analysis of immune cell populations

  • Single-cell RNA sequencing to analyze cellular heterogeneity and gene expression changes

  • In vivo blocking experiments using antibodies against CD276 or other targets

These complementary approaches allow researchers to investigate CD276's functions from molecular mechanisms to potential therapeutic applications.

How can CD276 function be effectively blocked or inhibited in mouse models?

Several strategies have been developed to block or inhibit CD276 function in mouse models:

• Monoclonal antibodies:

  • The MJ18 monoclonal antibody specifically targets mouse CD276 and blocks its function when administered in vivo

  • Anti-CD276 antibodies can be used alone or in combination with other immunotherapies

• Genetic knockout approaches:

  • Whole-body knockout completely eliminates CD276 expression in all tissues

  • Conditional knockout using Cre-loxP system allows tissue-specific deletion of CD276

  • CRISPR/Cas9-mediated knockout in cell lines creates CD276-deficient cells for transplantation studies

• Combination approaches:

  • CD276 blockade combined with PD-1 inhibition has shown superior efficacy compared to either agent alone

  • Other effective combinations include:

    • Anti-CXCL1 antibodies

    • Anti-Ly6G antibodies (targeting neutrophils)

    • Anti-NK1.1 antibodies (targeting NK cells)

    • GSK484 inhibitors (targeting NETosis)

Each approach has specific advantages depending on the research question and model system. The effectiveness of these strategies in preclinical models suggests promising translational potential for human applications.

How does CD276 affect tumor growth and progression in mouse cancer models?

CD276 significantly promotes tumor growth across multiple mouse cancer models, as demonstrated through both genetic knockout studies and antibody blockade experiments:

• In esophageal squamous cell carcinoma (ESCC) models:

  • 4NQO-induced ESCC in CD276 knockout mice showed dramatically reduced tumor development

  • CD276-wKO mice exhibited smaller size and lower number of esophageal lesions compared to control mice

  • The formation of high-grade ESCC was greatly reduced in CD276-wKO mice

  • Epithelial-specific CD276 knockout (CD276-cKO) similarly led to decreased tumor size and number

• In bladder cancer (BLCA) models:

  • BBN-induced bladder cancer in CD276-wKO mice showed significantly improved survival and reduced tumor burden

  • Bladder indices (ratios of bladder weight to body weight) and tumor volume were dramatically reduced after global knockout of CD276

  • CD276-wKO mice showed delayed cancer development, with carcinoma in situ detected only after 24 weeks of carcinogen treatment compared to 16 weeks in wild-type mice

  • Myeloid-specific CD276 knockout also reduced bladder cancer development

These findings consistently demonstrate that CD276 promotes tumor growth and progression across different cancer types and experimental models, highlighting its potential as a therapeutic target.

What are the mechanisms by which CD276 promotes tumor development in mice?

CD276 promotes tumor development through several complementary mechanisms:

• Immune evasion:

  • CD276 facilitates tumor immune evasion by suppressing anti-tumor immune responses

  • It reduces CD8+ T cell and NK cell infiltration and activity in tumors

  • In CD276 knockout mice, increased intratumoral infiltration of CD8+ T cells (showing a 3-4 fold increase) and NK cells (5.5%±1.4 in CD276-wKO mice vs 1.9%±0.5 in WT mice) has been observed

• Modulation of tumor-associated macrophages (TAMs):

  • CD276 expression in TAMs promotes efferocytosis and suppresses MHC class II expression

  • This creates an immunosuppressive microenvironment that inhibits T cell function

  • Myeloid-specific CD276 deletion leads to more infiltrated CD8+ T cells and less infiltrated TAMs in tumors

• Chemokine regulation:

  • CD276 affects the expression of chemokines that recruit immune cells

  • Single-cell RNA sequencing revealed that CD276 knockdown in epithelial cells significantly downregulates CXCL1 chemokine

  • This alteration may change immune cell recruitment patterns in the tumor microenvironment

• Cell proliferation and apoptosis:

  • Studies have shown decreased cell proliferation (measured by Ki67) and increased apoptosis (measured by active-Caspase3) in tumor tissues of CD276 knockout mice

  • This suggests that CD276 may directly promote cancer cell proliferation and survival

These multifaceted mechanisms collectively contribute to CD276's tumor-promoting effects and highlight its potential as a therapeutic target for cancer immunotherapy.

How does CD276 expression correlate with tumor grade and patient outcomes in translational models?

Studies in both mouse models and human cancer samples have shown a strong correlation between CD276 expression, tumor grade, and patient outcomes:

These correlations between CD276 expression and cancer progression provide valuable insights for understanding CD276's role in human cancers and support its potential as both a biomarker for disease progression and a target for therapeutic intervention.

How does CD276 influence T cell and NK cell activity in the tumor microenvironment?

CD276 exerts significant effects on T cell and NK cell activities in the tumor microenvironment, primarily acting as a negative regulator of these immune cell populations:

These findings highlight the potential of targeting CD276 to enhance anti-tumor immunity by releasing the suppression on T cells and NK cells, potentially improving responses to other immunotherapeutic approaches.

What is the role of CD276 in tumor-associated macrophages (TAMs)?

CD276 plays several important roles in tumor-associated macrophages (TAMs), contributing to their immunosuppressive functions in the tumor microenvironment:

• Promotion of efferocytosis:

  • CD276 expressed by TAMs enhances efferocytosis, the process by which macrophages engulf and clear apoptotic cells

  • This function can contribute to an immunosuppressive environment by removing dying tumor cells before they can release danger signals that might activate anti-tumor immunity

• Suppression of MHC class II expression:

  • CD276 in TAMs has been shown to suppress MHC class II expression, which is crucial for antigen presentation to CD4+ T cells

  • This suppression results in decreased CD4+ T cell activation and consequently reduced CD8+ T cell activation and infiltration

• Impact on tumor growth through myeloid-specific CD276 deletion:

  • Studies using myeloid-specific CD276 conditional knockout mice (LysM-Cre; CD276fl/fl) have demonstrated that deletion of CD276 specifically in myeloid cells leads to :

    • Lower bladder indices in BBN-induced bladder cancer

    • Lower incidence of invasive tumor formation

    • Increased apoptosis in tumor cells

    • More infiltrated CD8+ T cells and less infiltrated TAMs in tumors

How does CD276 affect the cellular composition of the tumor microenvironment?

CD276 profoundly influences the cellular composition of the tumor microenvironment, as revealed by single-cell RNA sequencing and flow cytometry analyses:

• Alteration of immune cell populations:

  • Single-cell RNA sequencing of tumors from CD276 knockout and wild-type mice has revealed significant changes in immune cell composition :

    • Decreased proportion of neutrophils in CD276 knockout tumors

    • Increased proportion of T cells and NK cells in CD276 knockout tumors

    • Reduced infiltration of TAMs in CD276 knockout tumors

• Changes in chemokine expression:

  • CD276 affects the expression of chemokines that orchestrate immune cell recruitment

  • CXCL1 chemokine is significantly downregulated in epithelial cells of CD276 knockout mice, as shown by single-cell RNA sequencing and confirmed by immunohistochemistry

  • This differential expression was one of the most significant changes observed in the transcriptome following CD276 depletion

• Cell type-specific effects:

  • Epithelial CD276 deletion leads to increased activated CD8+ T cells and reduced neutrophils in the tumor microenvironment

  • Myeloid-specific CD276 deletion similarly increases CD8+ T cell infiltration while reducing TAM presence

  • The number of CD4+ regulatory T cells and macrophages remains comparable between some knockout models and wild-type mice

• Translational relevance:

  • Comparison of mouse and human single-cell RNA sequencing data has shown high similarity in cell type-specific transcriptional profiles

  • This suggests that findings from mouse models may have translational relevance for human cancers

These comprehensive effects on the tumor microenvironment underscore the multifaceted role of CD276 in cancer development and highlight its potential as a therapeutic target for remodeling the immune landscape in tumors.

How can CD276 be targeted for cancer immunotherapy in mouse models?

CD276 represents a promising target for cancer immunotherapy in mouse models, with several strategies showing efficacy:

• Monoclonal antibody blockade:

  • Anti-CD276 antibodies such as MJ18 have been used to block CD276 function in vivo

  • These antibodies can inhibit tumor growth by enhancing anti-tumor immune responses

  • The MJ18 antibody has been validated for in vivo CD276 blockade in multiple experimental settings

• Genetic approaches:

  • While not directly translatable to therapy, genetic studies using knockout models have provided proof-of-concept for CD276 targeting

  • Both global and tissue-specific deletion of CD276 have shown significant anti-tumor effects

  • These findings support the therapeutic potential of CD276 blockade

• Cell-specific targeting strategies:

  • Targeting CD276 in tumor-associated macrophages: Myeloid-specific CD276 deletion reduces tumor growth and enhances anti-tumor immunity

  • Targeting epithelial CD276: Epithelial-specific CD276 deletion also shows anti-tumor effects

  • These findings suggest that targeting CD276 in specific cell populations may provide therapeutic benefits

• Targeting downstream pathways:

  • Inhibiting CXCL1: CD276 regulates CXCL1 expression, and anti-CXCL1 antibodies have shown efficacy in mouse models

  • Targeting neutrophil recruitment: Anti-Ly6G antibodies (targeting neutrophils) can enhance anti-tumor effects

  • Inhibiting NETosis: GSK484 inhibitors have shown efficacy in combination with CD276 blockade

These approaches demonstrate the potential of CD276 as a target for cancer immunotherapy in mouse models and provide a foundation for translating these findings to human clinical applications.

What combination therapies involving CD276 blockade show promise in preclinical models?

Several combination therapies involving CD276 blockade have demonstrated promising results in preclinical mouse models:

• CD276 blockade + PD-1 inhibition:

  • The combination of anti-CD276 and anti-PD-1 antibodies has shown superior efficacy compared to either agent alone

  • This combination effectively restrains tumor growth by targeting complementary immune evasion mechanisms

  • The dual blockade may help overcome resistance to single-agent PD-1 inhibition, which is a common clinical challenge

• CD276 blockade + anti-CXCL1 therapy:

  • Studies have used the 4NQO-induced mouse model to evaluate the effects of anti-CXCL1 antibodies in combination with CD276 blockade

  • Since CD276 regulates CXCL1 expression, this combination targets both the immune checkpoint molecule and its downstream effector

• CD276 blockade + neutrophil-targeting strategies:

  • Anti-Ly6G antibodies: These antibodies deplete neutrophils, which may be pro-tumorigenic in some cancer contexts

  • The combination of CD276 blockade with neutrophil depletion has shown efficacy in mouse models

• CD276 blockade + NETosis inhibition:

  • GSK484 inhibitors: These target processes related to neutrophil extracellular traps (NETs)

  • The combination of CD276 blockade with GSK484 inhibitors has shown efficacy in mouse models

• CD276 blockade + NK cell modulation:

  • Anti-NK1.1 antibodies: These have been used to assess the role of NK cells in responses to CD276 blockade

  • Understanding this interaction could inform combination strategies that enhance NK cell-mediated tumor killing

These combination approaches highlight the potential for CD276-targeted therapies to enhance the efficacy of existing immunotherapies and overcome resistance mechanisms. The diversity of effective combinations also underscores the multifaceted role of CD276 in tumor immune evasion.

What are the latest research directions and unanswered questions in CD276 mouse studies?

Recent research on CD276 in mouse models has expanded in several innovative directions, with several important questions remaining to be addressed:

• Mechanistic understanding of CD276 function:

  • What is the precise molecular mechanism by which CD276 regulates immune cell function?

  • Which receptor(s) does CD276 interact with on different immune cell types?

  • How does CD276 regulate CXCL1 and other downstream effectors?

• Single-cell technologies to dissect CD276's effects:

  • Recent studies have employed single-cell RNA sequencing to comprehensively analyze changes in the tumor microenvironment following CD276 depletion

  • These approaches have revealed cell type-specific effects and identified new downstream targets

  • Further application of these technologies will provide deeper insights into CD276's functions

• Cell type-specific roles of CD276:

  • Research using conditional knockout models has begun to dissect the relative contributions of CD276 in different cell types

  • Comparing the effects of epithelial vs. myeloid CD276 deletion provides insights into cell type-specific functions

  • Future studies may explore CD276's role in other stromal cell types, such as fibroblasts and endothelial cells

• Translational research questions:

  • How do findings from mouse models translate to human cancers?

  • What biomarkers can predict response to CD276-targeted therapies?

  • How can CD276-targeted approaches be optimized for clinical application?

• Beyond cancer applications:

  • What role does CD276 play in non-cancer contexts, such as inflammatory or autoimmune conditions?

  • How does CD276 function in normal tissue homeostasis and response to infection?

These research directions reflect the growing recognition of CD276 as an important regulator of immune responses and a promising therapeutic target. Future studies will likely focus on translating these findings to human applications and developing optimal strategies for targeting CD276 in the clinic.

What are the key findings supporting CD276 as a therapeutic target in cancer?

CD276 (B7-H3) has emerged as a compelling therapeutic target for cancer immunotherapy based on several key findings from mouse models:

• Consistent anti-tumor effects of CD276 blockade: Global knockout, conditional knockout, and antibody blockade of CD276 all result in significant reduction of tumor growth across multiple cancer models .

• Immune regulatory functions: CD276 negatively regulates anti-tumor immunity by suppressing CD8+ T cells and NK cells while promoting immunosuppressive functions of tumor-associated macrophages .

• Synergy with established immunotherapies: Combination of CD276 blockade with PD-1 inhibition shows superior efficacy compared to either agent alone, suggesting potential to overcome resistance to existing immunotherapies .

• Correlation with poor outcomes: High CD276 expression correlates with worse survival and treatment resistance in both mouse models and human cancer samples, indicating its clinical relevance .

• Specificity for tumor tissues: CD276 is prominently expressed in tumor tissues but rarely in normal tissues, potentially allowing for tumor-specific targeting with minimal off-target effects .

These findings collectively support the continued development of CD276-targeted therapies for cancer treatment, with promising translational potential from mouse models to human applications.

What methodological approaches are most critical for researchers studying CD276 in mice?

For researchers studying CD276 in mice, several methodological approaches have proven particularly valuable:

• Genetic mouse models: Both global knockout and conditional knockout (tissue-specific) models provide powerful tools for mechanistic studies of CD276 function . These include:

  • CD276 whole-body knockout (CD276-wKO)

  • Epithelial-specific knockout (K14CreER; CD276fl/fl)

  • Myeloid-specific knockout (LysM-Cre; CD276fl/fl)

• Carcinogen-induced cancer models: These recapitulate human disease development and progression:

  • 4NQO-induced esophageal squamous cell carcinoma

  • BBN-induced bladder cancer

• Single-cell RNA sequencing: This technique has proven invaluable for comprehensively analyzing changes in the tumor microenvironment following CD276 depletion, revealing cell type-specific effects and identifying new downstream targets .

• Antibody-based approaches: The MJ18 monoclonal antibody enables in vivo blockade of CD276 function, allowing for therapeutic studies without genetic manipulation .

• Multiparameter flow cytometry: This approach enables detailed characterization of immune cell populations in tumors and lymphoid organs, critical for understanding CD276's effects on the immune landscape .

• Combinatorial treatment approaches: Testing CD276 blockade in combination with other immunotherapies or targeted agents provides insights into potential clinical applications and synergistic mechanisms .

Researchers should consider employing multiple complementary approaches to gain comprehensive insights into CD276's functions and therapeutic potential.

How might findings from CD276 mouse studies translate to human clinical applications?

Findings from CD276 mouse studies show strong translational potential for human clinical applications:

• Similar expression patterns: CD276 exhibits similar expression patterns in mouse and human cancers, with high expression in tumor tissues compared to normal tissues . The upregulation of CD276 during malignant transformation is observed in both species .

• Conserved functional mechanisms: The mechanisms by which CD276 promotes tumor development, including immune suppression and alterations of the tumor microenvironment, appear conserved between mice and humans .

• Comparable transcriptional profiles: Single-cell RNA sequencing analyses have shown high similarity between mouse and human tumor cell transcriptional profiles, supporting the relevance of mouse findings to human disease .

• Correlation with clinical outcomes: The association between high CD276 expression and poor clinical outcomes is observed in both mouse models and human patients .

• Therapeutic implications:

  • Antibody-based therapies targeting CD276 are being developed for human use, based on promising results from mouse studies

  • Combination approaches validated in mice, particularly CD276 blockade with PD-1 inhibition, provide rational strategies for human clinical trials

  • Biomarkers identified in mouse studies may help stratify patients for CD276-targeted therapies