Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: A subset of oropharyngeal squamous cell carcinoma is driven by human papilloma virus (HPV-OPSCC). Compared to their HPV-negative counterparts, HPV-OPSCC generally harbor good prognoses. Despite this, many patients require multiple modalities of therapy leading to significant morbidity. A significant proportion of patients die from locoregional and distant disease. Surgery, chemotherapy, and radiation are all treatment options, but no accepted treatments that leverage anti-HPV immunity exist. Establishing a robust model to investigate anti-viral immunity among these tumors is critical to identifying novel therapeutic approaches that leverage viral drivers of disease. Primary HPV-OPSCC cultures are uncommon in the literature and few published protocols exist. In this study, primary tumor and tumor-infiltrating lymphocyte (TIL) cultures were established from resected tumor specimens with simple and cost-effective methods.
Methods: Fresh HPV-OPSCC primary tumor specimens were obtained as a part of a universal biorepository protocol. All patients had previous biopsies consistent with P16+ HPV-OPSCC. Tumors were placed directly into ice-cold MACS buffer within 90 minutes of resection. Bulk tumor was dissociated with commercially available enzymes using gentleMACS tissue dissociator. Bulk tumor digest was placed into either 1. Pneumacult stem cell culture media (for primary tumor culture) or 2. T cell expansion media containing RPMI, 10% fetal bovine serum, IL-2, and anti-CD3/CD28 stimulating antibodies (for TIL culture). Tumor cultures were treated with interferon-gamma treatments using 100ng/mL for 24 hours. HLA-I, HLA-II, and beta-2-microglobulin (B2M) were detected using flow cytometry with FITC, APC, and PE fluorochromes, respectively. Cell types in TIL cultures were confirmed using flow cytometry.
Results: 15 total cases were collected representing 12 tonsil and 3 base of tongue tumors. Primary tumor culture failed in two cases. Primary tumor and TIL cultures were successfully derived from the remaining 13 cases with several cultures ongoing.
For primary tumor cultures, DNA from HPV-related proteins E2 and E6 was able to be detected via RT-PCR. 7 of the cell lines were then tested for inducible HLA expression in response to interferon-gamma. After treatment with interferon-gamma, all cell lines showed increased expression of HLA-I, HLA-II, and B2M compared to control conditions.
For 4 TIL cultures, samples were processed using flow cytometry cell makeups were averaged yielding 22% CD4 T cells, 45% CD8 T cells, and 7% NK cells.
Conclusion: In this study, we demonstrate that primary tumor and TIL cultures can be established from HPV-associated oropharyngeal tumor specimens with a high degree of success. HPV DNA can be detected in resulting tumor cell cultures, suggesting HPV infection continues in culture. Tumor cell lines have HLA and beta-2-microglobulin expression that is inducible. This is critical when planning immune-based assays that act through T-cell receptors. TIL cultures were established that contained CD4 T cells, CD8 T cells, and NK cells. Using this model, further investigation into anti-HPV immunity in HPV-OPSCC can be performed.