Integration of genomics, metagenomics, and metabolomics to identify interplay between susceptibility alleles and microbiota in adenoma initiation.

BACKGROUND: Colorectal cancer (CRC) is a multifactorial disease resulting from both genetic predisposition and environmental factors including the gut microbiota (GM), but deciphering the influence of genetic variants, environmental variables, and interactions with the GM is exceedingly difficult. We previously observed significant differences in intestinal adenoma multiplicity between C57BL/6 J-ApcMin (B6-Min/J) from The Jackson Laboratory (JAX), and original founder strain C57BL/6JD-ApcMin (B6-Min/D) from the University of Wisconsin. METHODS: To resolve genetic and environmental interactions and determine their contributions we utilized two genetically inbred, independently isolated ApcMin mouse colonies that have been separated for over 20 generations. Whole genome sequencing was used to identify genetic variants unique to the two substrains. To determine the influence of genetic variants and the impact of differences in the GM on phenotypic variability, we used complex microbiota targeted rederivation to generate two Apc mutant mouse colonies harboring complex GMs from two different sources (GMJAX originally from JAX or GMHSD originally from Envigo), creating four ApcMin groups. Untargeted metabolomics were used to characterize shifts in the fecal metabolite profile based on genetic variation and differences in the GM. RESULTS: WGS revealed several thousand high quality variants unique to the two substrains. No homozygous variants were present in coding regions, with the vast majority of variants residing in noncoding regions. Host genetic divergence between Min/J and Min/D and the complex GM additively determined differential adenoma susceptibility. Untargeted metabolomics revealed that both genetic lineage and the GM collectively determined the fecal metabolite profile, and that each differentially regulates bile acid (BA) metabolism. Metabolomics pathway analysis facilitated identification of a functionally relevant private noncoding variant associated with the bile acid transporter Fatty acid binding protein 6 (Fabp6). Expression studies demonstrated differential expression of Fabp6 between Min/J and Min/D, and the variant correlates with adenoma multiplicity in backcrossed mice. CONCLUSIONS: We found that both genetic variation and differences in microbiota influences the quantitiative adenoma phenotype in ApcMin mice. These findings demonstrate how the use of metabolomics datasets can aid as a functional genomic tool, and furthermore illustrate the power of a multi-omics approach to dissect complex disease susceptibility of noncoding variants.

Evaluation of a Tumor-Targeting, Near-Infrared Fluorescent Peptide for Early Detection and Endoscopic Resection of Polyps in a Rat Model of Colorectal Cancer.

The goal of these studies was to use a tumor-targeting, near-infrared (NIR) fluorescent peptide to evaluate early detection and to guide surgical removal of polyps in a genetically engineered rat model of spontaneous colorectal cancer. This peptide, LS301, was conjugated to Cy7.5 and applied topically to the colon of adenoma-bearing Pirc rats. Ten minutes after administration, rats underwent targeted NIR laser colonoscopy. Rats were also evaluated by white light colonoscopy and narrow-band imaging, for comparison to the NIR technique. Unlike white light and narrow-band colonoscopy, NIR imaging detected unexpected flat lesions in young Pirc rats. NIR imaging was also used to assess resection margins after electrocauterization of polyps. Tumor margins remained negative at 5 weeks postsurgery, demonstrating successful polypectomy. The present studies show that NIR-targeted colonoscopy is an attractive strategy to improve screening for and resection of colorectal neoplasia.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

Monoallelic silencing and haploinsufficiency in early murine intestinal neoplasms.

Studies of tumors from human familial adenomatous polyposis, sporadic colon cancer, and mouse and rat models of intestinal cancer indicate that the majority of early adenomas develop through loss of normal function of the Adenomatous polyposis coli (APC) gene. In murine models of familial adenomatous polyposis, specifically the multiple intestinal neoplasia mouse (Min) and the polyposis in the rat colon (Pirc) rat, most adenomas have lost their WT copy of the Apc gene through loss of heterozygosity by homologous somatic recombination. We report that large colonic adenomas in the Pirc rat have no detectable copy number losses or gains in genomic material and that most tumors lose heterozygosity only on the short arm of chromosome 18. Examination of early mouse and rat tumors indicates that a substantial subset of tumors shows maintenance of heterozygosity of Apc in genomic DNA, apparently violating Knudson's two-hit hypothesis. Sequencing of the Apc gene in a sampling of rat tumors failed to find secondary mutations in the majority of tumors that maintained heterozygosity of Apc in genomic DNA. Using quantitative allele-specific assays of Apc cDNA, we discovered two neoplastic pathways. One class of tumors maintains heterozygosity of Apc(Min/+) or Apc(Pirc/+) RNA expression and may involve haploinsufficiency for Apc function. Another class of tumors exhibits highly biased monoallelic expression of the mutant Apc allele, providing evidence for a stochastic or random process of monoallelic epigenetic silencing of the tumor suppressor gene Apc.

Integrated multi-omic analyses provide insight into colon adenoma susceptibility modulation by the gut microbiota.

Colon cancer onset is strongly associated with the differences in microbial taxa in the gastrointestinal tract. Although recent studies highlight the role of individual taxa, the effect of a complex gut microbiome (GM) on the metabolome and host transcriptome is still unknown. We used a multi-omics approach to determine how differences in the GM affect the susceptibility to adenoma development in a rat model of human colon cancer. Ultra-high performance liquid chromatography mass spectrometry of feces collected prior to observable disease onset identified putative metabolite profiles that likely predict future disease severity. Transcriptome analyses performed after disease onset from normal colonic epithelium and tumor tissues show a correlation between GM and host gene expression. Integrated pathway analyses of the metabolome and transcriptome based on putatively identified metabolic features indicate that bile acid biosynthesis is enriched in rats with high tumors along with increased fatty acid metabolism and mucin biosynthesis. Targeted pyrosequencing of the Pirc allele indicates that the GM alters the mechanism of adenoma development and may drive an epigenetic pathway of tumor suppressor silencing. This study reveals how untargeted metabolomics identifies signatures of susceptibility and integrated analyses uncover pathways of differential mechanisms of loss of tumor suppressor gene function and for potential prevention and therapeutic intervention. IMPORTANCE The association between the gut microbiome and colon cancer is significant but difficult to test in model systems. This study highlights the association of differences in the pathogen-free gut microbiome to changes in the host transcriptome and metabolome that correlate with colon adenoma initiation and development in a rat genetic model of early colon cancer. The utilization of a multi-omics approach integrating metabolomics and transcriptomics reveals differences in pathways including bile acid biosynthesis and fatty acid metabolism. The study also shows that differences in gut microbiomes significantly alter the mechanism of adenoma formation, shifting from genetic changes to epigenetic changes that initiate the early loss of tumor suppressor function. These findings enhance our understanding of the gut microbiome's role in colon cancer susceptibility, offer insights into potential biomarkers and therapeutic targets, and may pave the way for future prevention and intervention strategies.

KSOM-R supports both mouse and rat preimplantation embryo development in vitro.

A modified KSOM for rat embryo culture (KSOM-R), which has enriched taurine, glycine, glutamic acid, and alanine, promoted rat embryo development in vitro. Since mice and rats share similar amino acid profiles in their female reproductive tracts, this study explored whether KSOM-R would also have a positive effect on mouse embryo development and if KSOM-R modifications could extend its shelf time at 2-8 °C for consistency. We first examined the effects of newly made (≤1 month at 2-8 °C) antibiotics-free KSOM-R (mKSOM-R), antibiotics-free KSOM (mKSOM) and KSOM on the development of in vivo or in vitro derived C57BL/6NJ zygotes. We then investigated the effect of extended shelf life (6 months at 2-8 °C) of mKSOM-R and mKOSM on the development of C57BL/6NJ mouse and Sprague Dawley (SD) rat embryos. The results showed that there were no significant differences in cleavage, blastocyst, and hatching rates of C57BL/6NJ embryos among the three freshly made media. After 6 months of storage at 2-8 °C, mKSOM-R and mKSOM were still able to support the development of in vivo C57BL/6NJ zygotes at comparable rates seen with newly made (≤1 month at 2-8 °C) KSOM (control) in terms of cleavage, blastocyst formation and hatching. There were also no significant differences in total cell numbers in day 4 blastocysts among the three groups. After surgical embryo transfers, C57BL/6NJ blastocysts cultured in mKSOM-R (6 months at 2-8 °C) and newly made (≤1 month at 2-8 °C) KSOM culture developed into live pups. These pups had no gross abnormalities in animal morphology and growth. SD zygotes cultured in mKSOM-R stored at 2-8 °C for 6 months developed at comparable rates in cleavage, blastocyst and hatching rates when compared to those cultured in newly made mKSOM-R (≤1 month at 2-8 °C). The data showed that, although no significant beneficial effects were observed on mouse embryo development, mKSOM-R was able to support both mouse and rat embryo development in vitro. Additionally, mKSOM-R and mKSOM can be stored at 2-8 °C for at least 6 months without significantly compromising quality. This study suggests that it is possible to reduce the media inventory by using only mKSOM-R to culture both mouse and rat embryos, and quality media with extended shelf life can be made through modifications.

Supplementation by vitamin D compounds does not affect colonic tumor development in vitamin D sufficient murine models.

Epidemiological studies indicate that sunlight exposure and vitamin D are each associated with a lower risk of colon cancer. The few controlled supplementation trials testing vitamin D in humans reported to date show conflicting results. We have used two genetic models of familial colon cancer, the Apc(Pirc/+) (Pirc) rat and the Apc(Min/+) (Min) mouse, to investigate the effect of 25-hydroxyvitamin D(3) [25(OH)D(3)] and two analogs of vitamin D hormone on colonic tumors. Longitudinal endoscopic monitoring allowed us to test the efficacy of these compounds in preventing newly arising colonic tumors and in affecting established colonic tumors. 25(OH)D(3) and two analogs of vitamin D hormone each failed to reduce tumor multiplicities or alter the growth patterns of colonic tumors in the Pirc rat or the Min mouse.

The pleiotropic phenotype of Apc mutations in the mouse: allele specificity and effects of the genetic background.

Familial adenomatous polyposis (FAP) is a human cancer syndrome characterized by the development of hundreds to thousands of colonic polyps and extracolonic lesions including desmoid fibromas, osteomas, epidermoid cysts, and congenital hypertrophy of the pigmented retinal epithelium. Afflicted individuals are heterozygous for mutations in the APC gene. Detailed investigations of mice heterozygous for mutations in the ortholog Apc have shown that other genetic factors strongly influence the phenotype. Here we report qualitative and quantitative modifications of the phenotype of Apc mutants as a function of three genetic variables: Apc allele, p53 allele, and genetic background. We have found major differences between the Apc alleles Min and 1638N in multiplicity and regionality of intestinal tumors, as well as in incidence of extracolonic lesions. By contrast, Min mice homozygous for either of two different knockout alleles of p53 show similar phenotypic effects. These studies illustrate the classic principle that functional genetics is enriched by assessing penetrance and expressivity with allelic series. The mouse permits study of an allelic gene series on multiple genetic backgrounds, thereby leading to a better understanding of gene action in a range of biological processes.

Characterization of the Rat Gut Microbiota via 16S rRNA Amplicon Library Sequencing.

It is becoming increasingly apparent that microbiota have measurable effects on numerous phenotypes in laboratory animals. This "second genome" has often been disregarded or ignored due to its commensal nonpathogenic nature, and the difficulty, expense, and analysis of sequence. Recent advances in sequencing methods and analyses of large datasets have made characterization of microbiota populations routine and have uncovered previously unknown relationships of microbial communities and host biological systems. The largest and most diverse microbial community in the laboratory rat is in the gut, and has been shown to affect the physiology of the whole animal, and genetic disease penetrance. We present here a cost-effective method for the characterization of the rat fecal microbiota through multiplexed 16S ribosomal sequencing and freely available software.

Colorectal tumor prevention by the progestin medroxyprogesterone acetate is critically dependent on postmenopausal status.

The large randomized placebo controlled trials of the Women's Health Initiative have shown that the combination of estrogen and progestin medroxyprogesterone acetate (MPA) protects from colorectal cancer in postmenopausal women. No effect was observed in women treated with estrogen alone. This suggests that progesterone, or more specifically the progestin MPA may have chemopreventive activity. The effect of MPA on colorectal carcinogenesis has been difficult to study in animal models. Most models are not affected by either depleting female hormones by ovariectomy or treatment with MPA. Importantly, an ovariectomy fails to reproduce one of the hall marks of the postmenopausal state in women with intact ovaries. That is, the continued production of androgens by the atrophic postmenopausal ovaries. Here we show that adenoma incidence is increased in the vinyl cylcohexene diepoxide (VCD) mouse model of the menopause compared to age matched fertile female mice. Treatment with MPA protected VCD treated mice from adenomagenesis, but had no effect on adenoma numbers in age-matched fertile female mice. Our data show that the protective effect of MPA depends on the postmenopausal state and suggest that MPA monotherapy may be studied as a chemopreventive agent in postmenopausal women.

Western diet enhances benzo(a)pyrene-induced colon tumorigenesis in a polyposis in rat coli (PIRC) rat model of colon cancer.

Consumption of Western diet (WD), contaminated with environmental toxicants, has been implicated as one of the risk factors for sporadic colon cancer. Our earlier studies using a mouse model revealed that compared to unsaturated dietary fat, the saturated dietary fat exacerbated the development of colon tumors caused by B(a)P. The objective of this study was to study how WD potentiates B(a)P-induced colon carcinogenesis in the adult male rats that carry a mutation in the Apc locus - the polyposis in the rat colon (PIRC) rats. Groups of PIRC rats were fed with AIN-76A standard diet (RD) or Western diet (WD) and received 25, 50, or 100 µg B(a)P/kg body weight (wt) via oral gavage for 60 days. Subsequent to exposure, rats were euthanized; colons were retrieved and preserved in 10% formalin for counting the polyp numbers, measuring the polyp size, and histological analyses. Blood samples were collected and concentrations of cholesterol, triglycerides, glucose, insulin and leptin were measured. Rats that received WD + B(a)P showed increased levels of cholesterol, triglycerides, and leptin in comparison to RD + B(a)P groups or controls. The colon tumor numbers showed a B(a)P dose-response relationship. Adenomas with high grade dysplasia were prominent in B(a)P + WD rats compared to B(a)P + RD rats and controls (p < 0.05). The larger rat model system used in this study allows for studying more advanced tumor phenotypes over a longer duration and delineating the role of diet - toxicant interactions in sporadic colon tumor development.

Fischer-344 Tp53-knockout rats exhibit a high rate of bone and brain neoplasia with frequent metastasis.

Somatic mutations in the Tp53 tumor suppressor gene are the most commonly seen genetic alterations in cancer, and germline mutations in Tp53 predispose individuals to a variety of early-onset cancers. Development of appropriate translational animal models that carry mutations in Tp53 and recapitulate human disease are important for drug discovery, biomarker development and disease modeling. Current Tp53 mouse and rat models have significant phenotypic and genetic limitations, and often do not recapitulate certain aspects of human disease. We used a marker-assisted speed congenic approach to transfer a well-characterized Tp53-mutant allele from an outbred rat to the genetically inbred Fischer-344 (F344) rat to create the F344-Tp53tm1(EGFP-Pac)Qly/Rrrc (F344-Tp53) strain. On the F344 genetic background, the tumor spectrum shifted, with the primary tumor types being osteosarcomas and meningeal sarcomas, compared to the hepatic hemangiosarcoma and lymphoma identified in the original outbred stock model. The Fischer model is more consistent with the early onset of bone and central nervous system sarcomas found in humans with germline Tp53 mutations. The frequency of osteosarcomas in F344-Tp53 homozygous and heterozygous animals was 57% and 36%, respectively. Tumors were highly representative of human disease radiographically and histologically, with tumors found primarily on long bones with frequent pulmonary metastases. Importantly, the rapid onset of osteosarcomas in this promising new model fills a current void in animal models that recapitulate human pediatric osteosarcomas and could facilitate studies to identify therapeutic targets.

Delayed and Aberrant Presentation of VX2 Carcinoma in a Rabbit Model of Hepatic Neoplasia.

A socially-housed New Zealand white rabbit presented with a large subcutaneous mass on the ventral thorax approximately 11 mo after the intrahepatic delivery of a suspension of VX2 carcinoma cells to induce hepatocellular carcinoma as part of a nanoparticle study. The mass and closely associated axillary lymph node were removed en bloc. Immunohistochemical staining identified the mass as an undifferentiated carcinoma. The rabbit demonstrated no appreciable pathology at the study end point at 16 mo after VX2 inoculation. An additional rabbit from the same VX2 injection cohort was found at necropsy to have an unanticipated intraabdominal mass, also identified as an undifferentiated carcinoma. This case report summarizes the molecular analysis of both tumors through a novel PCR assay, which identified the delayed and aberrant onset of VX2 carcinoma in an extended timeframe not previously reported.

Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota.

Recent studies investigating the human microbiome have identified particular bacterial species that correlate with the presence of colorectal cancer. To evaluate the role of qualitatively different but naturally occurring gut microbiota and the relationship with colorectal cancer development, genetically identical embryos from the Polyposis in Rat Colon (Pirc) rat model of colorectal cancer were transferred into recipients of three different genetic backgrounds (F344/NHsd, LEW/SsNHsd, and Crl:SD). Tumor development in the pups was tracked longitudinally via colonoscopy, and end-stage tumor burden was determined. To confirm vertical transmission and identify associations between the gut microbiota and disease phenotype, the fecal microbiota was characterized in recipient dams 24 hours pre-partum, and in Pirc rat offspring prior to and during disease progression. Our data show that the gut microbiota varies between rat strains, with LEW/SsNHsd having a greater relative abundance of the bacteria Prevotella copri. The mature gut microbiota of pups resembled the profile of their dams, indicating that the dam is the primary determinant of the developing microbiota. Both male and female F344-Pirc rats harboring the Lewis microbiota had decreased tumor burden relative to genetically identical rats harboring F344 or SD microbiota. Significant negative correlations were detected between tumor burden and the relative abundance of specific taxa from samples taken at weaning and shortly thereafter, prior to observable adenoma development. Notably, this naturally occurring variation in the gut microbiota is associated with a significant difference in severity of colorectal cancer, and the abundance of certain taxa is associated with decreased tumor burden.

The utility of Apc-mutant rats in modeling human colon cancer.

Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.

A simple, quantitative method using alginate gel to determine rat colonic tumor volume in vivo.

Many studies of the response of colonic tumors to therapeutics use tumor multiplicity as the endpoint to determine the effectiveness of the agent. These studies can be greatly enhanced by accurate measurements of tumor volume. Here we present a quantitative method to easily and accurately determine colonic tumor volume. This approach uses a biocompatible alginate to create a negative mold of a tumor-bearing colon; this mold is then used to make positive casts of dental stone that replicate the shape of each original tumor. The weight of the dental stone cast correlates highly with the weight of the dissected tumors. After refinement of the technique, overall error in tumor volume was 16.9% ± 7.9% and includes error from both the alginate and dental stone procedures. Because this technique is limited to molding of tumors in the colon, we utilized the Apc(Pirc/+) rat, which has a propensity for developing colonic tumors that reflect the location of the majority of human intestinal tumors. We have successfully used the described method to determine tumor volumes ranging from 4 to 196 mm³. Alginate molding combined with dental stone casting is a facile method for determining tumor volume in vivo without costly equipment or knowledge of analytic software. This broadly accessible method creates the opportunity to objectively study colonic tumors over time in living animals in conjunction with other experiments and without transferring animals from the facility where they are maintained.

A strategy to identify dominant point mutant modifiers of a quantitative trait.

A central goal in the analysis of complex traits is to identify genes that modify a phenotype. Modifiers of a cancer phenotype may act either intrinsically or extrinsically on the salient cell lineage. Germline point mutagenesis by ethylnitrosourea can provide alleles for a gene of interest that include loss-, gain-, or alteration-of-function. Unlike strain polymorphisms, point mutations with heterozygous quantitative phenotypes are detectable in both essential and nonessential genes and are unlinked from other variants that might confound their identification and analysis. This report analyzes strategies seeking quantitative mutational modifiers of Apc(Min) in the mouse. To identify a quantitative modifier of a phenotype of interest, a cluster of test progeny is needed. The cluster size can be increased as necessary for statistical significance if the founder is a male whose sperm is cryopreserved. A second critical element in this identification is a mapping panel free of polymorphic modifiers of the phenotype, to enable low-resolution mapping followed by targeted resequencing to identify the causative mutation. Here, we describe the development of a panel of six "isogenic mapping partner lines" for C57BL/6J, carrying single-nucleotide markers introduced by mutagenesis. One such derivative, B6.SNVg, shown to be phenotypically neutral in combination with Apc(Min), is an appropriate mapping partner to locate induced mutant modifiers of the Apc(Min) phenotype. The evolved strategy can complement four current major initiatives in the genetic analysis of complex systems: the Genome-wide Association Study; the Collaborative Cross; the Knockout Mouse Project; and The Cancer Genome Atlas.

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of Apc(Min) (/+) ROSA11 Gene Trap Mice.

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in Apc(Min) (/+) mice. The insertion site of the ß-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors.

A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer.

Progress toward the understanding and management of human colon cancer can be significantly advanced if appropriate experimental platforms become available. We have investigated whether a rat model carrying a knockout allele in the gatekeeper gene Adenomatous polyposis coli (Apc) recapitulates familial colon cancer of the human more closely than existing murine models. We have established a mutagen-induced nonsense allele of the rat Apc gene on an inbred F344/NTac (F344) genetic background. Carriers of this mutant allele develop multiple neoplasms with a distribution between the colon and small intestine that closely simulates that found in human familial adenomatous polyposis patients. To distinguish this phenotype from the predominantly small intestinal phenotype found in most Apc-mutant mouse strains, this strain has been designated the polyposis in the rat colon (Pirc) kindred. The Pirc rat kindred provides several unique and favorable features for the study of colon cancer. Tumor-bearing Pirc rats can live at least 17 months, carrying a significant colonic tumor burden. These tumors can be imaged both by micro computed tomography scanning and by classical endoscopy, enabling longitudinal studies of tumor genotype and phenotype as a function of response to chemopreventive and therapeutic regimes. The metacentric character of the rat karyotype, like that of the human and unlike the acrocentric mouse, has enabled us to demonstrate that the loss of the wild-type Apc allele in tumors does not involve chromosome loss. We believe that the Pirc rat kindred can address many of the current gaps in the modeling of human colon cancer.