(A) Mammary adenocarcinoma incidence in PyMT/RAG1+/− and PyMT/RAG1−/− mice (n = 15 and 18 mice/group, respectively) is depicted as percentage of tumor-free mice. Mice were considered to be tumor free until a palpable mass (>4.0 mm) persisted for longer then 4 days. No statistical differences between cohorts were observed as evaluated by Wilcoxon test. (B–D) Total tumor burden of PyMT/RAG1 (B), PyMT/JH (C), and PyMT/CD4/CD8 (D) cohorts evaluated at both 95 and 110 days of age, shown as mm3 (n = > 20 mice per cohort). Tumor size was determined by caliper measurement and multiple tumors in one animal were added together. No statistical differences between groups were found as evaluated by Mann-Whitney test.
(E) Representative lung tissue sections depicting metastatic tumor burden from 110-day-old PyMT/RAG1−/−, PyMT/JH−/−, PyMT/CD4−/−/CD8−/− mice following hematoxylin and eosin (H&E) staining (5× magnification).
(F) Quantification of metastatic foci/lung section/mouse from 110-day-old PyMT/RAG1, PyMT/JH, PyMT/CD4/CD8, PyMT/CD4, PyMT/CD8 cohorts. Each lung was serially sectioned and six sections 100 mm apart were H&E stained and total number of metastatic foci (greater then 5 cells) quantified. Each of the six sections was averaged per mouse (n = > 20 mice per cohort).
(G) Circulating carcinoma cells were analyzed by flow cytometry and counted as the number of cytokeratin+/CD45− cells in blood from 110-day-old PyMT/RAG1+/− (n = 10), PyMT/RAG1−/− (n = 10), PyMT/CD4+/− (n = 20), PyMT/CD4−/− (n = 15), or 110-day old PyMT mice treated with anti-CD4 depleting IgG (n = 8) or IgG control (n = 6) for 18 days. Data are depicted as the mean number of carcinoma cells per milliliter of blood.
(H) PyMT mRNA expression in circulating blood cells. RNA from whole blood cells of 110-day-old PyMT/RAG1+/− and /RAG1−/− mice was evaluated for PyMT mRNA gene expression by RT-PCR (25 cycles) (n = 8 mice/group). Results from ethidium bromide stained gels are depicted following quantification of pixel density using GelDoc software.
(I) Average number of metastatic foci/lung section/mouse from 110-day-old PyMT/CD4+/− mice treated with anti-CD4 depletion antibody (GK1.5) versus IgG control (CD4+/− IgG) or PyMT/CD4−/−CD8−/− mice following adoptive transfer of naive CD4+ T cells (CD4+ rescue). Each lung was serially sectioned and assessed as described above. Twenty mice were used for PyMT/CD4−/−CD8−/−, six mice for CD4+ rescue, and eight mice for CD4+/− IgG or GK1.5 groups. (B–I) SEM is shown and asterisk denotes p < 0.05 by Mann-Whitney.