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Stress Keratin 17 mediates immune evasion in head and neck cancer [scRNA-seq]

ABSTRACT: We previously identified stress keratin 17 (K17) as a host factor that contributes to immune evasion in the context of mouse papillomavirus-induced disease. Analyses of head and neck squamous cell carcinoma (HNSCC) patient TCGA and tissue microarray data revealed that K17 can be overexpressed in both HPV+ and HPV- human cancer samples, with a more profound upregulation in HPV- HNSCC samples. In this report, we investigated the role of K17 in regulating immune response in HPV- head and neck cancer. To test whether K17 mediates immune evasion and resistance to immune checkpoint blockade (ICB) therapy in HPV- HNC, we used MOC2, a K17-expressing murine HNC cell line, and knocked out the K17 gene from it using CRISPR/Cas9 (K17KO MOC2 cells). Then we injected K17KO MOC2 cells or parental MOC2 cells into NSG and C57BL/6 mice and monitored tumor growth, immune cell infiltration, and response to ICB treatment in vivo. In NSG mice, the K17KO MOC2 cell lines grew as fast as parental MOC2 tumors. In C57BL/6 mice, K17KO MOC2 tumors grew significantly slower than parental MOC2 tumors, with 50% tumor rejection rate, in a T-cell dependent manner. Flow cytometry of tumor infiltrating T cells and bulk tumor RNA seq analyses show an active anti-tumor microenvironment in the K17KO MOC2 tumors, compared to parental MOC2 tumors. In addition, we observed that 5 out of 5 anti-PD1 + anti-CTLA4 treated K17KO MOC2 tumors completely regressed, while only 1 out of 5 parental MOC2 tumors similarly treated at the same size completely regressed (p<0.05). In exploring the role of chemokine CXCL9 in increased T cell infiltration, we blocked the receptor CXCR3, a receptor often upregulated on activated T cells, and saw a delay in the immune-mediated rejection K17KO MOC2 tumors. Single cell RNA seq analyses revealed a completely different immune landscape including both lymphoid and myeloid populations in K17KO MOC2 tumors. Overall, we demonstrated that K17 expression in HNSCC contributes to immune evasion and resistance to immune checkpoint blockade treatment through modulating the immune landscape in the tumor microenvironment.

ORGANISM(S): Mus musculus

PROVIDER: GSE182002 | GEO | 2022/07/06

REPOSITORIES: GEO

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Project description:We previously identified stress keratin 17 (K17) as a host factor that contributes to immune evasion in the context of mouse papillomavirus-induced disease. Analyses of head and neck squamous cell carcinoma (HNSCC) patient TCGA and tissue microarray data revealed that K17 can be overexpressed in both HPV+ and HPV- human cancer samples, with a more profound upregulation in HPV- HNSCC samples. In this report, we investigated the role of K17 in regulating immune response in HPV- head and neck cancer. To test whether K17 mediates immune evasion and resistance to immune checkpoint blockade (ICB) therapy in HPV- HNC, we used MOC2, a K17-expressing murine HNC cell line, and knocked out the K17 gene from it using CRISPR/Cas9 (K17KO MOC2 cells). Then we injected K17KO MOC2 cells or parental MOC2 cells into NSG and C57BL/6 mice and monitored tumor growth, immune cell infiltration, and response to ICB treatment in vivo. In NSG mice, the K17KO MOC2 cell lines grew as fast as parental MOC2 tumors. In C57BL/6 mice, K17KO MOC2 tumors grew significantly slower than parental MOC2 tumors, with 50% tumor rejection rate, in a T-cell dependent manner. Flow cytometry of tumor infiltrating T cells and bulk tumor RNA seq analyses show an active anti-tumor microenvironment in the K17KO MOC2 tumors, compared to parental MOC2 tumors. In addition, we observed that 5 out of 5 anti-PD1 + anti-CTLA4 treated K17KO MOC2 tumors completely regressed, while only 1 out of 5 parental MOC2 tumors similarly treated at the same size completely regressed (p<0.05). In exploring the role of chemokine CXCL9 in increased T cell infiltration, we blocked the receptor CXCR3, a receptor often upregulated on activated T cells, and saw a delay in the immune-mediated rejection K17KO MOC2 tumors. Single cell RNA seq analyses revealed a completely different immune landscape including both lymphoid and myeloid populations in K17KO MOC2 tumors. Overall, we demonstrated that K17 expression in HNSCC contributes to immune evasion and resistance to immune checkpoint blockade treatment through modulating the immune landscape in the tumor microenvironment.

Project description:Head and neck cancer (HNC) is the fifth most common malignancy worldwide with an annual mortality rate of 200,000. About 90% of HNC can be classified as head and neck squamous cell carcinomas (HNSCC), of which approximately 75% are attributed to alcohol and tobacco consumption and 25 are associated with human papillomavirus (HPV), predominantly HPV16. HPV-associated OPC have better prognosis and a more favorable response to therapy as compared to HPV-negative tumors. Viral oncoproteins are capable of transforming primary human keratinocytes from either genital or oral epithelia in vitro and most likely play the same role in vivo, by disrupting cell-cycle regulatory pathways leading to a genetic progression to ano-genital cancer and OPC. However, the precise mechanisms by which HPV mediates malignant transformation of keratinocytes in the upper digestive tract epithelia are not entirely clear. HPV E7-mediated inactivation of pRb results in overexpression of p16INK4A, which is commonly used as a clinical surrogate marker for HPV positivity/activity. However, high p16INK4A alone has insufficient sensitivity and specificity as a biomarker of HPV positivity in different mucosal sub-sites of HNC. Therefore, increasing emphasis is being placed on the assessment of viral load and E7 oncogene expression, resulting in further classification of HPV positive OPC as HPV-active and HPV-inactive. Differences in risk factors, age of presentation, clinical behavior and gene expression profiles indicate that HPV-positive and HPV-negative tumors develop via different molecular mechanisms and are biologically distinct. This study aimed to compare the gene expression profiles of HPV-active OPCs and normal benign uvula/tonsil tissues and determine what biological processes and pathways are affected in HPV-active tumors. ANALYSIS 5: Two-condition, one-color experiment: HPV-active oropharyngeal tumor samples and normal benign uvula/tonsil tissues. Biological replicates: 12 HPV active samples and 4 Normal samples.

Project description:Head and neck cancer (HNC) is the fifth most common malignancy worldwide with an annual mortality rate of 200,000. About 90% of HNC can be classified as head and neck squamous cell carcinomas (HNSCC), of which approximately 75% are attributed to alcohol and tobacco consumption and 25 are associated with human papillomavirus (HPV), predominantly HPV16. HPV-associated OPC have better prognosis and a more favorable response to therapy as compared to HPV-negative tumors. Viral oncoproteins are capable of transforming primary human keratinocytes from either genital or oral epithelia in vitro and most likely play the same role in vivo, by disrupting cell-cycle regulatory pathways leading to a genetic progression to ano-genital cancer and OPC. However, the precise mechanisms by which HPV mediates malignant transformation of keratinocytes in the upper digestive tract epithelia are not entirely clear. HPV E7-mediated inactivation of pRb results in overexpression of p16INK4A, which is commonly used as a clinical surrogate marker for HPV positivity/activity. However, high p16INK4A alone has insufficient sensitivity and specificity as a biomarker of HPV positivity in different mucosal sub-sites of HNC. Therefore, increasing emphasis is being placed on the assessment of viral load and E7 oncogene expression, resulting in further classification of HPV positive OPC as HPV-active and HPV-inactive. Differences in risk factors, age of presentation, clinical behavior and gene expression profiles indicate that HPV-positive and HPV-negative tumors develop via different molecular mechanisms and are biologically distinct. This study aimed to compare the gene expression profiles of HPV-negative and HPV-inactive OPCs from both African American and European American patients, and determine their biological and racial differences. ANALYSIS 4: Four-condition, one-color experiment: African American (AA) HPV-inactive, African American (AA) HPV-negative, European American (EA) HPV-inactive, European American (EA) HPV-negative oropharyngeal tumor samples. Biological replicates: 4 AA HPV Inactive samples, 8 AA HPV Negative samples, 4 EA HPV Inactive samples and 8 EA HPV Negative samples,

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Stress Keratin 17 mediates immune evasion in head and neck cancer [scRNA-seq]

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