RNAscope is a novel quantitative in situ gene expression assay that allows single molecule visualization and preservation of spatiotemporal and cell specific information across intact tissue samples. This method has utility to measure gene expression using intact tissue; thus, allowing quantification in a spatial and cell type specific manner, which can be valuable to complement single cell sequencing approaches that use disaggregated tissue. The accurate quantitative capacity of RNAscope depends on retention of RNA integrity which can be lost with extensive or poor tissue handling procedures. This protocol outlines a method of processing and carmine staining mammary gland whole mounts, which is commonly used to identify developmental and reproductive morphologies or disease lesions, followed by excision of tissue areas of interest and RNAscope on tissue sections. This method preserves both RNA integrity and accurate quantification of the RNAscope technology, while bypassing a need for labor-intensive screening of serial sections to identify unpalpable disease lesions induced by environmental exposures at various stages of reproductive development.

Rattus norvegicus (a.k.a. laboratory rat or Brown Rat) Mammary carcinoma susceptibility 1b (Mcs1b) is a concordant ortholog of a female breast cancer risk allele at human 5q11.2. Previously, Mcs1b was delimited to a 1.8 Mb interval of RNO2 and Map3k1 along with Mier3 were determined to be Mcs1b-nonminated genes. This conclusion was based on shared synteny with human 5q11.2 and differential gene expression between cancer susceptible and Mcs1b resistant mammary glands. In this study, targeted genome sequencing of cancer susceptible and Mcs1b resistance associated alleles was used to identify three Mcs1b-nominated quantitative trait nucleotides (QTNs) in noncoding DNA. In vitro approaches, luciferase activity and electromobility shift assays, were used to suggest these variants reside in potential gene regulatory elements. One of these variants, UL-A74-SNV-17, resulted in luciferase activities that were 2.6× higher for the susceptibility associated variant compared to the resistance associated variant. These results recapitulated Mcs1b nominated gene transcript level differences between Mcs1b genotypes in mammary epithelial cells (MECs), where Map3k1 and Mier3 were 1.5- to 2.0-fold higher for the susceptible genotype compared to the Mcs1b resistance-associated genotype. Evidence of a chromatin loop in Mcs1b that may position Mcs1b QTNs near distal genes was uncovered using chromosome confirmation capture (3C). Rat Mcs1b was also functionally characterized by determining that Mcs1b genotype had effects on the amount of luminal MECs in adult mammary glands. In conclusion, UL-A74-SNV-17 is a priority candidate Mcs1b QTN with a hypothesized mechanistic role in the differential regulation of Mcs1b nominated genes, Mier3 and Map3k1.

PDF file - 69K, Primers to amplify and sequence Mcs1b region ORFs

<p>PDF file - 43K, C5ORF35 RT-PCR and lack of conservation</p>

PDF file - 53K, Mcs1b potential candidate gene QPCR probes and primers

<p>PDF file - 74K, RN02 genetic marker information</p>

PDF file - 53K, Mcs1b potential candidate gene QPCR probes and primers

<p>PDF file - 190K, Ectopic expression of eGFP-MIER3 in breast cancer cell lines</p>

<p>PDF file - 74K, RN02 genetic marker information</p>

<p>PDF file - 35K, Mier3 QPCR in non-mammary gland tissues</p>

PDF file - 74K, Uninformative genetic markers at the distal end of Mcs1b

PDF file - 43K, C5ORF35 RT-PCR and lack of conservation

<p>PDF file - 35K, Mier3 QPCR in non-mammary gland tissues</p>

Genome-wide association studies of breast cancer susceptibility have revealed risk-associated genetic variants and nominated candidate genes; however, the identification of causal variants and genes is often undetermined by genome-wide association studies. Comparative genomics, utilizing Rattus norvegicus strains differing in susceptibility to mammary tumor development, is a complimentary approach to identify breast cancer susceptibility genes. Mammary carcinoma susceptibility 3 (Mcs3) is a Copenhagen (COP/NHsd) allele that confers resistance to mammary carcinomas when introgressed into a mammary carcinoma susceptible Wistar Furth (WF/NHsd) genome. Here, Mcs3 was positionally mapped to a 7.2-Mb region of RNO1 spanning rs8149408 to rs107402736 (chr1:143700228-150929594, build 6.0/rn6) using WF.COP congenic strains and 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis. Male and female WF.COP-Mcs3 rats had significantly lower body mass compared to the Wistar Furth strain. The effect on female body mass was observed only when females were raised in the absence of males indicating a socioenvironmental interaction. Furthermore, female WF.COP-Mcs3 rats, raised in the absence of males, did not develop enhanced lobuloalveolar morphologies compared to those observed in the Wistar Furth strain. Human 15q25.1-25.2 was determined to be orthologous to rat Mcs3 (chr15:80005820-82285404 and chr15:83134545-84130720, build GRCh38/hg38). A public database search of 15q25.1-25.2 revealed genome-wide significant and nominally significant associations for body mass traits and breast cancer risk. These results support the existence of a breast cancer risk-associated allele at human 15q25.1-25.2 and warrant ultrafine mapping of rat Mcs3 and human 15q25.1-25.2 to discover novel causal genes and variants.

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

The p21-activated kinase 1 ( PAK1) gene encodes a serine/threonine kinase that is overexpressed in a subset of human breast carcinomas with poor prognosis. The laboratory rat ( Rattus norvegicus) orthologous gene is located at Mammary carcinoma susceptibility 3 ( Mcs3) QTL on rat chromosome 1. We used quantitative PCR to determine effects of Mcs3 genotype and 7,12-dimethylbenz(a)anthracene (DMBA) exposure on Pak1 expression. There was no effect of Mcs3 genotype; however, there was a 3.5-fold higher Pak1 level in DMBA-exposed mammary glands (MGs) than in unexposed glands ( P < 0.05). Sequence variants in Pak1 exons did not alter amino acid sequence between Mcs3-susceptible and -resistant strains. Protein expression of PAK1/Pak1 in human breast carcinomas and DMBA-exposed rat mammary glands was detected using immunohistochemistry (IHC). Rat mammary glands from 12-wk-old females unexposed to DMBA were negative for Pak1, whereas 24% of carcinogen-exposed mammary glands from age-matched females stained positive for Pak1. The positive mammary glands exposed to carcinogen had no pathological signs of disease. Human breast carcinomas, used as comparative controls, had a 22% positivity rats. This was consistent with other human breast cancer studies of PAK1 expression. Similar frequencies of human/rat PAK1/Pak1 expression in female breast carcinomas and carcinogen-induced rat mammary glands, showing no visible pathogenesis of disease, suggests aberrant PAK1 expression is an early event in development of some breast cancers. Laboratory rats will be a useful experimental organism for comparative studies of Pak1-mediated mechanisms of breast carcinogenesis. Future studies of PAK1 as a diagnostic marker of early breast disease are warranted.

Background: RNA in situ hybridization is an extremely useful gene expression analysis technique that preserves the spatiotemporal nature of tissue and allows for evaluation of specific cell populations and morphologies. This technique is especially useful in evaluating expression changes in disease progression models. Tissue processing procedures used to identify pathological disease morphologies in situ could compromise RNA integrity and the reproducibility and quantitative accuracy of RNA in situ hybridization assays. Methods: A combinatorial approach to pre-screen rat mammary gland tissue whole mounts for hyperplastic and malignant lesions that were not visually discernible without staining was used. This pre-screening process was followed by an RNA in situ hybridization analysis method known as RNAscope. Results: We show that there are no differences in the quantitative nature of RNAscope assays between tissue that was immediately formalin-fixed and paraffin embedded (FFPE), which is recommended by the manufacturer, and tissue that was whole mounted and pre-screened for lesions of interest prior to RNAscope. Conclusions: Preserving the integrity of RNA and quantitative nature of the RNAscope assay is important, as it allows unpalpable lesions to be directly identified, bypassing a need for labor-intensive serial sectioning of FFPE tissues to find lesions. This method is applicable to any epithelial-based disease progression model using whole-tissue mounts.

Background: Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344. WKY-Nat2(rapid/slow) rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility.Methods: Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats.Results: Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01).Conclusions: A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

Human breast and rat mammary cancer susceptibility are complex phenotypes where complete sets of risk associated loci remain to be identified for both species. We tested multiple congenic rat strains to physically confirm and positionally map rat Mammary carcinoma susceptibility 3 (Mcs3)-a mammary cancer resistance allele previously predicted at Rattus norvegicus chromosome 1 (RNO1). The mammary cancer susceptible Wistar Furth (WF) strain was the recipient, and the mammary cancer resistant Copenhagen (Cop) strain was the RNO1-segment donor for congenics. Inbred WF females averaged 6.3 carcinogen-induced mammary carcinomas per rat. Two WF. Cop congenic strains averaged 2.8 and 3.4 mammary carcinomas per rat, which confirmed Mcs3 as an independently acting allele. Two other WF. Cop congenic strains averaged 6.6 and 8.1 mammary carcinomas per rat, and, thus, did not contain Mcs3. Rat Mcs3 was delimited to 27.8 Mb of RNO1 from rs8149408 to rs105131702 (RNO1: 143700228-171517317 of RGSC 6.0/rn6). Human genetic variants with p values for association to breast cancer risk below 10(-7) had not been reported for Mcs3 orthologous loci; however, human variants located in Mcs3-orthologous regions with potential association to risk (10(-7), p, 10(-3)) were listed in some population-based studies. Further, rat Mcs3 contains sequence orthologous to human 11q13/14-a region frequently amplified in female breast cancer. We conclude that Mcs3 is an independently acting mammary carcinoma resistance allele. Human population-based, genome-targeted association studies interrogating Mcs3 orthologous loci may yield novel breast cancer risk associated variants and genes.