# Barcoded Orthotopic Patient‐Derived Head & Neck Squamous Cell Carcinoma Model Demonstrating Clonal Stability and Maintenance of Cancer Driver Mutational Landscape

## TL;DR

A new barcoded mouse model for head and neck cancer maintains genetic stability and allows studying gene effects in a cost-effective way.

## Contribution

A novel barcoded orthotopic PDX model for HNSCC with high engraftment rates and preserved genetic landscapes.

## Key findings

- An 88.9% engraftment rate was achieved using purified tumor cells.
- Lingual injection of 100,000 cells achieved 100% orthotopic engraftment.
- Genetic and clonal stability was maintained through the model.

## Abstract

To illustrate a new barcoded orthotopic patient‐derived xenograft (PDX) mouse model where one can investigate phenotypic effects of single‐cell level gene manipulation in a pooled format. To address some concerns of current PDX mouse models of head and neck squamous cell carcinoma (HNSCC): (1) genomic evolution with passage by generating high‐purity cancer cells, which can also be utilized for other downstream applications, including cell culture‐based studies, and (2) cost‐effectiveness of current PDX models.

Two‐millimeter tumor cubes from nine patients were implanted into immunodeficient mouse flanks subcutaneously. Purified tumor cells were obtained from subcutaneous xenografts. Various numbers of purified tumor cells were then injected into the lingual tissue of immunodeficient mice, and the lowest amount of cells needed to achieve a 100% orthotopic engraftment rate were identified. Clonal stability was tested using a lentiviral barcoding system. The orthotopic PDXs' genetic landscapes were characterized using whole exome sequencing.

This approach yielded an overall engraftment rate of 88.9%. The purification process increased cancer cell purity from 34% to 92%. Lingual injection of 100,000 purified tumor cells achieved a 100% orthotopic engraftment rate from purified subcutaneous PDX tumor cells while maintaining clonal and genetic stability.

Our study presents a barcoded orthotopic patient‐derived xenograft model for head and neck squamous cell carcinoma with clonal stability. This model provides a way to study phenotypic effects of single cell level gene manipulation in a pooled format. The method can be adapted for in vitro work as well.

Barcoded orthotopic HNSCC PDX with high engraftment rate, clonal stability, and its genetic landscapes as an alternative method to obtain high quantities of purified tumor cells for downstream work.