# Competitive cell transplantation in rat mammary tissue and its use for radiation-induced cell competition and tumor clonality assays

## TL;DR

This study establishes a competitive cell transplantation method in rat mammary tissue to assess radiation effects and tumor clonality using fluorescent markers.

## Contribution

The study introduces a rat-based competitive transplantation protocol for radiation biology and tumor clonality analysis.

## Key findings

- Radiation exposure (4 Gy) did not affect the repopulating ability of transplanted cells.
- Tumors in recipients were polyclonal, indicating multiple cell origins.
- Successful repopulation occurred only in double transgenic recipients, not in wild-type ones.

## Abstract

Competitive transplantation of cells expressing distinct fluorescent proteins is an attractive method for assessing cell competition and tumor clonality, albeit its use has been limited to mouse models. Rat mammary cancer is an alternative model to mice due to its similarity to human breast cancer. This study aims to implement competitive transplantation in the context of radiation biology of the rat mammary gland and to provide a detailed protocol on key techniques. Dissociated mammary cells were obtained from wild-type and transgenic rats expressing either green (EGFP) or red (Discosoma sp. red fluorescent protein [DsRed]) fluorescent protein. The cells were exposed to γ rays or left untreated, mixed at a specific ratio and then transplanted into the cleared fat pads of wild-type or double transgenic rats. We evaluated the expression of fluorescent proteins in epithelial outgrowths using confocal imaging of cleared tissues or on histological sections. Transplanting transgenic cells into wild-type recipients induced inflammation and rejection of the donor cells. Conversely, transplanting into double transgenics resulted in successful repopulation and observation of the fluorescent markers. A competitive assay between irradiated EGFP-expressing cells and unirradiated DsRed-expressing cells revealed that radiation exposure (4 Gy) did not affect repopulating ability, despite the theoretical sensitivity of the assay, suggesting the absence of strong competition between these cells. Analysis of tumors revealed the polyclonal nature of adenomas that developed in recipients exposed to radiation or 1-methyl-1-nitrosourea. Thus, this study developed a method to track cells in the rat mammary gland that is useful in radiation biology.