(A) D2A1 or D2.1 cells were implanted into the mammary fat pad of BALB/c mice (1 × 10⁶/mouse). Small, volume-matched D2A1 tumors (“Early D2A1”; n = 6), large, duration-matched D2A1 (“D2A1”; n = 4) tumors, or small D2.1 (n = 4) tumors were collected as indicated. (B) Final volume of tumors in A. (C) Flow cytometric analysis of total T cells (CD45⁺CD11b^–CD4⁺ or CD45⁺CD11b^–CD8β⁺) among total immune cells (CD45⁺) in tumors at time of euthanasia. (D) Percentage of CD8⁺ cells among total CD45⁺ cells. (E) Percentage of CD4⁺ cells among total CD45⁺ cells. (F) Left: Flow plots of FoxP3⁺CD4⁺ cells in D2A1 and D2.1 tumors obtained on day 35 (shown gated on CD45⁺ cells). Right: Quantification of Tregs in tumors from A. (G) The CD8⁺ cell to Treg (FoxP3⁺CD4⁺) ratio in total T cells by flow cytometry. One-way ANOVA with Tukey’s post hoc analysis was used to compare 3 groups for B–G. (H) Representative IHC image of tumor border from endpoint D2A1 tumors. Dashed lines indicate stromal/tumor interface and arrows indicate positively stained CD8⁺ (top) or CD4⁺ (bottom) cells. (I) Representative images of sized-matched D2A1 or D2.1 tumor interiors stained for CD8 (top) or CD4 (bottom). (J and K) Quantification of CD8⁺ (J) or CD4⁺ (K) cells/mm² in D2A1 and D2.1 tumor interiors (n = 5 each). (L) Representative images (left) and quantification (right) of FoxP3⁺ cells in tumors from H–K. Scale bars: 50 μm (H, I, and L). Density analysis was performed on 5 random fields/tumor using ImageJ software and comparisons were performed via 2-tailed t test (J–L). (M and N) Correlation of endpoint volume and CD8⁺ density (M) or FoxP3⁺ density (N) in D2.1 tumors of different sizes (n = 17). Pearson’s correlation is displayed in each plot. Data are presented as mean ± SEM.