Abstract 2986: Conditional reprogramming of primary head and neck tumor cells to establish consistent and diverse cell line models
Authors: Li Daniel , Thomas Carlos , Shrivastava Nitisha , Gadsden Nicholas , Kawachi Nicole , Schiff Bradley A. , Smith Richard V. , Schlecht Nicolas F. , Prystowsky Michael B. , Rosenblatt Gregory , Augustine Stelby , Guha Chandan , Gavathiotis Evripidis , Burk Robert D. , Mehta Vikas , Ow Thomas J. 
Published: July 2021

Abstract
Introduction
Head and neck squamous cell carcinoma (HNSCC) most commonly arise in the oral cavity, pharynx, and larynx and exhibit diverse molecular characteristics and clinical behavior across sites. Current models of HNSCC largely rely on immortalized cell lines, which suffer from selection bias and clonality. Primary cell cultures, on the other hand, are difficult to maintain and are limited by their finite ability to proliferate. Here, we describe the establishment of a robust HNSCC cell line bank using a “conditional reprogramming” (CR) method which relies on treatment with a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival.
Methods
HNSCC tumors were acquired on an IRB-approved prospective tissue banking protocol. Tumor tissue and blood were collected from each patient, and tumor cells were digested and cultured using previously described CR methods. DNA was collected from tumor tissue and CR cultures and short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue. To verify that the cultures were of squamous origin, western blot analysis was used to detect cytokeratin 5, cytokeratin 6 and p63. Tumor cell phenotype and growth characteristics were examined with light microscopy. HPV testing was carried out on DNA from CR cultures and matched tumor samples using PCR and specific genotyping by oligonucleotide hybridization. HPV testing was correlated with clinical p16 testing results. Whole exome sequencing was carried out on a subset of blood/tumor/CR culture samples.
Results
Eighteen CR lines were successfully cultured and validated with STR genotyping from 25 sequentially procured tumors. 10/18 were derived from oral cavity squamous cell cancers (SCCs), 5/18 were derived from oropharyngeal SCCs, and 3/18 were derived from laryngeal SCCs. 18/18 lines were found to express p63 and either cytokeratin 5 or 6, verifying these cultures contained tumor cells of squamous origin. 4/5 tumors from oropharyngeal SCCs were p16-positive on clinical testing and considered HPV-mediated. These 4 tumors all tested positive for HPV DNA in both tumor samples and CR cultures. Preliminary comparison of exome sequencing results between CR cultures and primary tumors suggests that overall mutational profiles are preserved through the tumor “conditional programming” process. Tumor heterogeneity between original tumor and CR culture is being actively compared in ongoing analyses.
Conclusion
We have consistently generated primary tumor CR cultures from patients with HNSCC arising in three major anatomical subsites of HNSCC disease, including HPV-mediated tumors. CR methods can be readily applied to all HNSCC tumors regardless of disease site and molecular background, providing a translational research model that can capture the molecular and phenotypic breadth of HNSCC disease.
Citation Format: Daniel Li, Carlos Thomas, Nitisha Shrivastava, Nicholas Gadsden, Nicole Kawachi, Bradley A. Schiff, Richard V. Smith, Nicolas F. Schlecht, Michael B. Prystowsky, Gregory Rosenblatt, Stelby Augustine, Chandan Guha, Evripidis Gavathiotis, Robert D. Burk, Vikas Mehta, Thomas J. Ow. Conditional reprogramming of primary head and neck tumor cells to establish consistent and diverse cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2986.