# Establishment of a coculture system for Porphyromonas gingivalis and head and neck squamous cell carcinoma using spheroid culture and LATS inhibition

## TL;DR

Researchers created a new lab model to study how a mouth bacteria interacts with a type of throat cancer, using special cell growth and drug treatment techniques.

## Contribution

A novel coculture system was developed to maintain both Porphyromonas gingivalis and HNSCC cells under normoxic conditions using spheroid culture and LATS inhibition.

## Key findings

- Hippo pathway inhibition via LATS1/2 inhibitor enabled stable growth of HNSCC spheroids.
- Pg viability was maintained in HNSCC spheroids, especially with Hippo pathway inhibition.
- Coculture induced p38 activation and increased IL-1α and IL-8 expression in HNSCC cells.

## Abstract

Porphyromonas gingivalis (Pg) is a periodontal pathogen that has been implicated in the development and progression of head and neck squamous cell carcinoma (HNSCC). However, studying its interaction with HNSCC in vitro is challenging because of the obligatory anaerobic nature of Pg. To overcome this limitation, we developed a coculture system that enables the viability of both HNSCC cells and Pg using spheroid culture combined with Hippo pathway inhibition via treatment with a LATS1/2 inhibitor. In spheroid cultures, many HNSCC cell lines failed to grow in a normal medium. However, stable growth of these cells was achieved through Hippo pathway inhibition, which maintained the cells in an undifferentiated state. Furthermore, the addition of Pg to HNSCC spheroids maintained Pg viability in three out of four HNSCC cell lines, even after 3 days. Hippo pathway inhibition further enhanced Pg survival within the spheroids, likely by suppressing the differentiation‐induced expression of antimicrobial genes S100A8 and S100A9. Coculturing HNSCC cells with Pg did not promote spheroid growth but induced p38 activation, leading to increased expression of the proinflammatory cytokines IL‐1α and IL‐8. Database analysis using The Cancer Microbiome Atlas corroborated these findings, showing upregulation of p38 phosphorylation, IL‐1α, and IL‐8 in Porphyromonas‐positive HNSCC samples. These findings suggest that the established coculture system is a representative model of the clinical properties of Pg‐positive HNSCC and can serve as a valuable tool for investigating the long‐term interactions between HNSCC and viable Pg.

We established a spheroid coculture system enabling viable Porphyromonas gingivalis–HNSCC interactions under normoxic conditions. Inhibition of LATS1/2 maintains tumor cells in an undifferentiated state, which may promote spheroid growth and create a more permissive environment for bacterial persistence. The model recapitulates features of Porphyromonas‐positive HNSCC, including p38‐mediated IL‐1α and IL‐8 induction, facilitating studies of bacterium‐driven tumor modulation.