Fig 1.
Anal tumor development in KC mice.
At age 9 months, anorectal tissue was excised for histological analysis. In male (A) and female (B) C57BL/6J wild type mice, normal anorectal histology was present. Additionally, 9-month-old KC male mice (C) demonstrated normal anorectal histology. In contrast, large perianal tumors were grossly evident in 9-month-old female KC mice with invasive anal SCC present on histologic examination. This is indicated by the bracket (D). Scale bars equal 200 μm.
Fig 2.
Anal tumor progression over time.
In female KC mice, anal tumors are visible as an area of congestion at 4 months of age, with mild erythema around the anal region. By age 5 months, an anal tumor is generally evident. By 9 months of age, the anal tumors are significant in size. Despite the large size, the mice are able to maintain weight, consume food, and defecate normally. No mice experienced obstructive symptoms.
Table 1.
Sex-dependent incidence of skin and anal lesions.
Fig 3.
Anal squamous cell carcinomas in KC mice were negative for MmuPV1 infection.
(A) No viral signatures were detected in representative anal tumors from KC mice stained via RNAScope ISH with a probe specific to the MmuPV1 E4 region of the genome. MmuPV1-infected and mock-infected Nod-scid IL2Rγnull (NSG) mouse anal tissues were included as positive and negative controls, respectively. Scale bars equal 100 μm. (B) DNA was recovered from FFPE slides of representative KC female anal squamous cell carcinomas and MmuPV1-infected and mock-infected anal tissues, and PCR was performed using primers specific to the MmuPV1 genome. KC lesions were negative for MmuPV1 DNA.
Fig 4.
Pdx1 expression and Cre-recombinase expression in mouse anus.
Anal tissue was excised from male and female wild type mice and male and female KC mice at 9 and 5 months of age. mRNA was isolated and RT-PCR performed to evaluate for Pdx1 and Cre-recombinase expression. Male and female C57BL/6J WT mice demonstrate Pdx1 expression in anal tissue at both 5 and 9 months, as did male and female KC mice (A, B, D, E). At 9 months age, KC females express significantly higher amounts of Cre mRNA (C) due to the presence of tumor tissue. At 5 months, male and female KC mice express similar amounts of Pdx1 and Cre mRNA (E, F).
Fig 5.
Tdtomato expression indicates Pdx1-Cre expression and mutant Kras expression in both male and female mice.
Anal tissue was excised from 3 month old Ai14; LSL-KrasG12D; Pdx1-Cre (AiKC) female (A, B, C) and male (D, E, F) mice, fixed and frozen embedded and sectioned for H&E analysis (A and D). Additional adjacent sections were prepared for immunohistochemistry (IHC) to identify tdTomato protein at 4x and 10x (B, C, E and F). Scale bars equal 100 μm. Positive IHC signal for tdTomato reveals the location of Cre expression (Pdx1-Cre), which serves as a marker for the location of mutant KrasG12D expression.
Fig 6.
Activated KrasG12D is present in the anal SCC anal tumor tissue.
The activated KrasG12D mutation is detected in the female anal tumor tissue, and the male anal tissues. Activated Kras refers to the successful removal of the Lox-stop-Lox codon preceding the KrasG12D. The activated mutation is present in the pancreas due to extensive Pdx1 expression, and absent in tail samples, which lack Pdx1 expression. Positive and negative controls are shown in S2 Fig.
Fig 7.
Castrated and ovariectomized KC mice and assessment for anal tumor.
Male KC mice were castrated and female KC mice were ovariectomized at 6–7 weeks of age, and the anal tissue evaluated at age 9 months. Macroscopic examination revealed no abnormalities in castrated KC males and most ovariectomized KC females (representative images, A and C). Concordantly, histopathologic examination revealed no microscopic tumor formation in male KC mice or macroscopically normal ovariectomized KC female mice (representative images B and D). Scale bars equal 100 μm.
Table 2.
Sex-dependent incidence of skin and anal lesions after castration or ovariectomy.
Fig 8.
Estrogen dosed castrated KC male develop anal SCC.
Castrated KC male mice and ovariectomized KC female mice were treated with 17-beta estradiol (E2) or sham (sesame oil). Sham dosed KC males did not develop anal SCC (A), while E2 dosing induced anal SCC in KC males (B, shown by bracket). Similarly, sham dosed KC females did not develop anal SCC (C) while E2 dosing rescued the anal SCC phenotype in KC females (D, shown by bracket). Scale bars equal 200 μm.
Table 3.
Incidence of anal SCC in beta-estradiol dosed KC mice.
Fig 9.
Estrogen Receptor alpha (ERα) is present in the KC anal tissue.
The presence of ERα was detected using immunofluorescence IHC. ERα was found to be present in the anal SCC of intact KC female mice (I), in the anal epithelium of ovariectomized KC female anus lacking tumor formation (II) and in the KC male anus (III). Panels A and B show the fluorescent staining of the receptor at 10x and 40x respectively. Panel C in each group shows the tissue with only the use of the secondary antibody confirming no off target staining. Group IV shows ERα staining of mouse arcuate nucleus as the positive control [15].