Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas

Abstract

A bigenic MUC1.Tg/MIN mouse model was developed by crossing Apc/(MIN/+) (MIN) mice with human MUC1 transgenic mice to evaluate MUC1 antigen-specific immunotherapy of intestinal adenomas. The MUC1.Tg/MIN mice developed adenomas at a rate comparable to that of MIN mice and had similar levels of serum MUC1 antigen. A MUC1-based vaccine consisting of MHC class I-restricted MUC1 peptides, a MHC class II-restricted pan-helper peptide, unmethylated CpG oligodeoxynucleotide and GM-CSF caused flattening of adenomas and significantly reduced the number of large adenomas. Immunization was successful in generating a MUC1-directed immune response evidenced by increased MUC1 peptide-specific anti-tumor cytotoxicity and IFN-gamma secretion by lymphocytes.

Figure 1. Incidence of adenomas in intestines of MUC1.Tg/MIN (M/M) and MIN mice

Mice were sacrificed on either day 40, day 75, or day 110 and intestines were dissected and adenomatous polyps were counted. M/M mice: Day 40, n=10; Day 75, n=11; Day 110, n=9. MIN mice: Day 40, n = 10; Day 75, n = 10; Day 110, n = 10.

Figure 2. Detection of human MUC1 protein in intestines

(A) Western blot analysis of human MUC1 protein in intestines. Lysates were prepared from colons (C) or small intestines (SI) of 110 day-old MUC1.Tg (MUC), MIN, and MUC1.Tg/MIN (Min/MUC) mice. Equal quantities of each sample (100 µg) were separated on a 5% SDS-PAGE gel, transferred to PVDF membrane and tested for reactivity with the anti-human MUC1 monoclonal antibody B27.29. MMT is a positive control for human MUC1 and is indicative of the highly glycosylated isoform of MUC1. (B) Immunohistochemical (IHC) detection of human MUC1 protein in intestines of MUC1.Tg, MIN, and MUC1.Tg/MIN mice. Specimens were obtained from intestines of 110 day-old animals and histological sections were prepared. Human MUC1 was detected by immune staining with the antihuman MUC1 monoclonal antibody B27.29. IHC demonstrates preferential MUC1 staining in adenomatous polyps, compared to non-adenomatous regions of the intestine (middle panels) or to similar regions of the MUC1.Tg mouse (right panels). MUC1 staining was not observed in polyps from MIN mice (left panels). IHC images were collected at 200X magnification. This figure is representative of at least 3 mice that were evaluated.

Figure 3. Comparison of adenoma morphology

(A) Section of adenoma from peptide-vaccinated (peptides + CpG-ODN + GM-CSF) MUC1.Tg/MIN mouse. Arrow indicates the flattened, indented morphology. (B). Section of adenoma from non-peptide-vaccinated (CpG-ODN + GM-CSF) MUC1.Tg/MIN mouse showing polypoid morphology and multiple cystic spaces. These samples were representative of the response observed in mice from each group. All sections were stained with routine hematoxylin and eosin. Images were collected at 200X magnification.

Figure 4. Peptide-based vaccine elicits a MUC1-specific immune response

(A) MUC1.Tg/MIN mice were vaccinated three times with either the peptide-based vaccine (peptides + CpG-ODN + GM-CSF) or the non-peptide vaccine (CpG-ODN + GM-CSF) at two week intervals. Splenocytes were then isolated and stimulated in vitro with the MUC1 TR peptides and the hepatitis B virus core antigen pan helper peptide for 48 h. Subsequently, supernatant was evaluated for IFN-γ production by ELISA. Splenocytes from individual mice from the peptide vaccine group (n=4) and the non-peptide vaccine group (n=3) were evaluated in triplicate. Data represent mean ± SD. (B) Cytolytic activity is MUC1-specific. Splenocytes from vaccinated MUC1.Tg/MIN mice were isolated and stimulated in vitro with the MUC1 TR peptides and the hepatitis B virus core antigen pan helper peptide for six days in the presence of 100 U/mL IL-2. Target cells were ⁵¹Cr-labeled MC38.MUC1 (murine colon carcinoma cell line expressing human MUC1) or MC38.neo (mock-transfected cell line not expressing human MUC1) plated at 5×10⁴ cells/well. Effector to target cell ratios (E:T) and p values of statistical evaluations at each E:T ratio are indicated on the x-axis.