Advances in Immunocompetent Mouse and Rat Models

Abstract

Rodent models of breast cancer have played critical roles in our understanding of breast cancer development and progression as well as preclinical testing of cancer prevention and therapeutics. In this article, we first review the values and challenges of conventional genetically engineered mouse (GEM) models and newer iterations of these models, especially those with inducible or conditional regulation of oncogenes and tumor suppressors. Then, we discuss nongermline (somatic) GEM models of breast cancer with temporospatial control, made possible by intraductal injection of viral vectors to deliver oncogenes or to manipulate the genome of mammary epithelial cells. Next, we introduce the latest development in precision editing of endogenous genes using in vivo CRISPR-Cas9 technology. We conclude with the recent development in generating somatic rat models for modeling estrogen receptor-positive breast cancer, something that has been difficult to accomplish in mice.

Fig 1:

Intraductal injection of lentivirus to engineer genetic alterations in mammary cells in mice. (A) Photos showing a successful intraductal injection. Bromophenol blue spreads throughout the ductal tree. (B) Photos showing whole-mount GFP imaging of a control gland un-injected with virus and a gland injected by Lenti-GFP (3.6×10⁶ IUs) three days earlier. (C) Photos showing whole-mount GFP imaging of a control gland un-injected with virus and a gland injected by Lenti-HrasQ61L-GFP (2.4×10⁶ IUs) eight days earlier. Several GFP+ precancerous early lesions can be seen, and the largest one is noted by an arrow.

Fig 2.

RCAS-TVA technology for delivering genetic alterations into selected mammary cells in mice. In a Tva-transgenic mouse line, intraductal injection delivers RCAS to mammary epithelial cells expressing TVA. The number of infected cells varies from a few (Bu et al. 2013; Bu et al. 2019) to tens of thousands (Du et al. 2006) depending upon both the TVA+ cells available for infection and the amount of virus injected. Stable expression of an oncogene from LTR following RCAS integration causes infected cells to expand to form atypical lesions, which evolve into cancer.

Fig 3:

The CRISPR method for introducing indels and point mutations into endogenous genes in their native loci. (A) In a Cas9 transgenic mouse line, intraductal injection of AAV carrying gRNA causes DSBs at the targeted loci, which are repaired by non-homologous end joining (NHEJ), leading to small insertions and deletions (indels). This method is most commonly used for loss of function studies, such as tumor suppressor genes. (B) We have found that a modified AAV vector carrying both gRNA and HDR can lead to precise point mutations. This approach can be used to test gain of function mutations of proto-oncogenes. Of note, indels still occur in this approach, but cells that suffered gain of function changes have competitive advantages and are thus selected for in tumor evolution. (C) Example of efficient infection of mammary epithelial cells by serotype 9 of AAV.

Fig 4.

HRASQ61L causes ovarian hormone-dependent ER+ adenocarcinoma in rats but metaplastic carcinoma with heavy squamous differentiation in mice. Lenti-HrasG61L was intraductally injected into adult FVB/n mice and Sprague Dawley rats. Mammary tumors appear within a few weeks in both groups of animals. Tumors in mice are metaplastic carcinoma with heavy squamous differentiation (also ER-negative). However, tumors in rats are adenocarcinoma with no evidence of metaplasia, but with strong positivity for both ERα and PR (not shown)(Bu and Li 2020). These ER+/PR+ tumors are also ovarian hormone dependent (Bu and Li 2020).

Fig 5.

The Nf1 CRISPR KO rat model of ER+/PR+ breast cancer. AAV9 carrying gRNA targeting Nf1 was intraductally injected into Cas9 rats. Tumors were palpated within two weeks to three months. These tumors are adenocarcinomas positive for both ERα and PR.

Fig 6:

ER+/PR+ DCIS, invasive adenocarcinoma in a rat model of breast cancer. Lenti-PIK3CAH1047R was intraductally injected into adult Sprague Dawley rats. These rats develop ER+/PR+ DCIS, followed by invasive ER+/PR+ adenocarcinoma with a median latency of approximately 3.5 months (Bu and Li 2020).