Establishment and characterization of novel autologous pair cell lines from two Indian non‑habitual tongue carcinoma patients

Abstract

Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India due to very high percentage of patients with habits of smoking and chewing tobacco and associated products. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To understand disease progression in an improved way, knowledge of cross-talk between tumor stroma and the tumor cells becomes indispensable. Patient-derived in vitro cell line models are helpful to understand the complexity of diseases. However, they have very low efficiency of establishment from the tumor samples, particularly the cancer-associated fibroblasts (CAFs). In the present study, two novel autologous pairs were immortalized spontaneously from non-habitual, HPV-positive patients, who presented with OTSCC. The epithelial and fibroblast cell lines had typical polygonal and spindle-shaped morphology, respectively. Positive staining with epithelial specific Pan-cytokeratin (PanCK) and fibroblast specific protein (FSP-1) further confirmed their epithelial and fibroblast origin. Unique Short Tandem Repeat (STR) profile of the cultures confirmed their novelty, while the similarity of the STR profiles between the epithelial and fibroblast cells from the same patient, confirmed their autologous nature. DNA analysis revealed aneuploidy of the established cultures. An increase in the tumorigenic potential of the established epithelial cultures upon treatment with CAF-conditioned medium proved the 'CAF-ness' of the established fibroblast cells. The established cultures are the first of their kind which would serve as a useful platform in understanding the tumor-stroma cross-talk in tongue cancer progression.

Establishment and Characterization of Three Novel CAF Cell Lines from HNSCC Patients

“…As shown in the Fig. 1B, the cells exhibited a typical elongated, spindle shaped morphology (23,24), consistent with the fibroblast morphology as reported earlier (20).…”

“…In this study along with the previous one (20), we report a total of 5 CAFs from 4 sites of lesions of 5 HNSCC patients.…”

“…MhCT12-E and MhCT08-E epithelial cells which were previously established and characterized by Dwivedi et. al (20) were used as a neg tive control for FSP-1 staining and positive control for EpCAM staining. Overall, the results conclude and confirms the purity and origin of established CAF cell lines.…”

“…According to a recent study, different media can be used to promote the populations of fibroblasts and epithelial cells to develop at different rates (42). To avoid any phenotypic or genotypic changes brought on by patient-derived xenograft generations, the cultures described in the present investigation were grown in RPMI-1640 complete media as also in the previous report (20). These cultures did not require any feeder cells or viral vectors to be stabilized spontaneously.…”

“…Hayes et al previously produced 16 cell lines from patients with HNSCC to better understand the effects of various mutations on tumor behavior (19). However, establishment of CAF cell lines, so far, have only been reported from this group in June 2022 (20).…”