(A) The number of CD68⁺, CD20⁺, CD4⁺, and CD8⁺ cells was analyzed in patient samples of normal/hyperplastic breast tissue (n/h; n = 9), ductal carcinoma in situ (DCIS; n = 6), and invasive ductal carcinomas (IC; n = 150) using tissue microarrays. Representative 10× and 40× images are shown and the average number of positive cells as depicted reflects the mean number of cells in each disease stage, evaluated by counting all high power fields (20×) per tissue section (1.1 mm)/two sections/patient. *p < 0.05 by Mann-Whitney. (B, C) CD4⁺ and F4/80⁺ cell presence was evaluated during MMTV-PyMT mammary tumor development and is depicted by representative images in normal mammary tissue (-LM) and tumors from 76 and 110-day-old PyMT mice. CD4⁺ or F4/80⁺ cells were quantitatively assessed and data reflects the mean number of positive cell evaluated in 10 high-power fields (20×) per tumor, n = 4 mice per group.
Graphs are depicted as mean values and standard error of the mean (SEM) in all panels.

(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.

(A) Immunodetection of CD45⁺ cells in 95-day-old PyMT/RAG1^+/− and PyMT/RAG1^−/− mammary carcinomas. Representative 20× images are shown.
(B) Flow cytometric analysis of CD45⁺ cells in tumors from 95 and 110-day-old PyMT/RAG1 and PyMT/CD4 mice. Data are depicted as the mean percent of live cells ± SEM, n = 4 mice per cohort.
(C) Flow cytometric analysis of individual leukocyte populations as a percent of total CD45⁺ cells in mammary carcinomas of PyMT mice during progression and in day 95 tumors from RAG1-and CD4-deficient/PyMT mice. Data are depicted as the mean value from four mice/cohort ± SEM. No statistical differences were found between groups by Mann-Whitney test.
(D–L) Cytokine expression by TAM. Tumor-associated CD45⁺F4/80⁺Gr1⁻ macrophages were isolated by dual magnetic and flow sorting of mammary tumors from 95 day-old PyMT/CD4^+/− and PyMT/CD4^−/− mice (n = 3/cohort). Cytokine expression (TNF-α, IL-6, IL-12p40, IL-1β, IL-10) was assessed by ELISA of conditioned medium or by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR; Nos2, Arg1, Tgfβ and Vegf-a) from TAMs (50,000) following 18 hr of culture with or without exogenous recombinant IL-4 (10 ng/ml). Representative assays of mammary carcinomas from three or four mice evaluated independently in triplicate and depicted as mean ± SEM.
(M–Q) Analysis of tumor-derived IMC phenotype in PyMT/CD4^−/− mice. Tumor-associated CD45⁺ CD11b⁺Gr1^Hi IMCs were isolated by flow from mammary tumors of 95-day-old PyMT+/CD4^+/− and PyMT/CD4^−/− mice (n = 3/cohort). Isolated cells were lysed and RNAs assessed by qRT-PCR as described above. Representative assays from two independent cohorts each run at least in triplicate are depicted as mean values ± SEM. Asterisk denotes p < 0.05 by Mann-Whitney in all panels.

(A–H) Analysis of cytokine expression by tumor-associated CD4⁺ T cells. CD4⁺ T cells were isolated by flow sorting from LNs and tumors of 95-day-old PyMT mice and corresponding negative littermates (n = 4/cohort). Sorted cells were lysed and RNAs were assessed by qRT-PCR for GATA3, T-bet, FOXP3, IFNγ, IL-4, IL-13, IL-10, and IL-17a expression. Data are depicted as the mean fold change from the standardized sample (-LM LN).
(I) Cytokine analysis in CD4⁺ T cells ex vivo. Tumor-associated CD4⁺ lymphocytes were isolated by flow sorting from mammary tumors of 95-day-old PyMT/CD4^+/− mice (n = 3) and IL-4, IFNγ, and IL-17 expression assessed by ELISA after 18 hr of culture prior to (veh, white bars) or following TCR activation (black bars). Data are represented as the mean of three replicates.
(J, K) CD4⁺ T cells repress TAM M1 phenotype. Tumor-associated CD45⁺CD3⁺CD4⁺ T lymphocytes and CD45⁺F4/80⁺Gr1⁻ TAMs were isolated by flow sorting from mammary tumors of 95-day-old PyMT/CD4^+/− mice. TAMs were untreated (V, white bars), or cultured with IFNγ (5 ng/ml) and LPS (50 ng/ml) in the presence of control CD4⁺ T cells (treated with control IgGs, gray bars), or activated CD4⁺ T cells (black bar, Act-CD4). TNF-α and IL-12p40 expression in conditioned medium evaluated by ELISA after 18 hr of coculture. Representative data from two independent experiments are depicted.
(A–K) SEM is shown and asterisk denotes p < 0.05 by Mann-Whitney test.

(A) Quantitation of invasive organoids following coculture of TAMs. Stable wild-type MEC (nMEC) or PyMT-derived MEC (pMEC) organoids were allowed to form over 14–20 days and then cocultured with TAMs (48 hr). Representative immunofluorescent images of nMEC (a, c) and pMEC (b, d) organoids in the presence or absence of TAMs evaluated for cytokeratin 7 (green), F4/80 (red), and DAPI (blue) are shown. Representative bright field images of invasive pMEC organoids in coculture with TAMs are depicted at 20× magnification and 40× inset. TAMs are denoted by red arrows.
(B) Quantification of organoid disruption following coculture of TAM with pMEC spheroids in the presence of IL-4 (20 ng/ml), IL-13 (20 ng/ml), IL-10 (10 ng/ml), IFNγ (5 ng/ml), or LPS (50 ng/ml).
(C) Quantification of pMEC organoid disruption (formed over 14 days) following coculture (48 hr) with TAMs and/or tumor-associated CD3⁺CD4⁺ T cells. Cocultures were also exposed to exogenous recombinant mouse IL-4 (10 ng/ml) and/or an anti-mouse IL-4 neutralizing antibody (0.5 mg/ml; clone OP06) added 12 hr prior to leukocytes.

(A) Kaplan Meyer analysis of tumor incidence in PyMT/IL-4Rα^+/− and PyMT/IL-4Rα ^−/− mice (n = 15/group) depicted as percentage of tumor-free animals. No statistical differences between cohorts by generalized Wilcoxon test were found.
(B) Total mammary tumor burden of PyMT/IL-4Rα^+/− and ^−/− mice, and PyMT mice treated for 20 days with either IL-4 neutralizing Ig (11B11) or control IgG and evaluated at day 100, shown as mm³ (n = > 20 mice per cohort). Tumor size was determined by caliper measurement and multiple tumors in one animal were added together.
(C) Quantification of average number of metastatic foci/lung/mouse of 100-day-old PyMT/IL-4Rα and PyMT mice treated with either IL-4 neutralizing or control IgG. Each lung was serially sectioned, and six sections 100 μm apart were stained by H&E and total number of metastatic foci (greater then 5 cells) quantified. Each of the six sections was summed and each bar represents n ≥ 23 mice for all cohorts and ≥ 12 for Ig-treated groups.
(D and E) Tumor-associated CD45⁺F4/80⁺Gr1⁻ macrophages were isolated by flow sorting of mammary carcinomas from (D) PyMT/IL-4Rα or (E) PyMT mice treated with either IL-4 neutralizing IgG (11B11) or control IgG. ELISA was performed on conditioned medium from TAMs (50,000) after 18 hr of culture. Quantitative RT-PCR analysis was performed using the comparative threshold cycle method to calculate fold change in gene expression normalized to GAPDH as reference gene. Representative assays from three independent cohorts each run at least in triplicate are depicted as the mean fold change from the standardized sample. (B–E) Data are represented as mean ± SEM, and asterisk denotes p < 0.05 by Mann-Whitney test.

(A) EGF mRNA expression analysis from CSF-1- and IL-4-activated TAMs. TAMs were isolated from mammary tumors of 95-day-old PyMT/CD4^+/− or PyMT/CD4^−/− mice, and placed into culture with CSF-1 (10 ng/ml) and/or IL-4 (20 ng/ml) for 16 hr. Quantitative RT-PCR analysis of EGF mRNA expression is depicted as fold change from vehicle (CD4^−/− no treatment is set to 1.0) assessed by the comparative threshold cycle method normalized to reference gene expression.
(B) Quantification of pMEC organoid disruption following coculture of TAMs (48 hr) +/− IL-4 (10 ng/ml) or EFGR small molecule inhibitors PD153035 (0.1 μM) or BIBX1382 (10 nM). Invasive organoids were counted and data represented as a percentage of the total organoids (>100 replicate). Representative data from two independent experiments performed in quadruplicate are depicted.
(C) EGFR signaling regulates metastasis in PyMT mice. The 110 day-old PyMT mice were treated with the EGFR small molecule inhibitor PD153035 (25 mg/kg) versus vehicle (DMSO) by i.p. injection 5 hr prior to analysis (six mice/group). Presence of circulating carcinoma cells (cytokeratin⁺CD45⁻) was assessed by FACS evaluation of live cells in PB.
(A–C) Data are represented as mean ± SEM, and the asterisk denotes p < 0.05 by Mann-Whitney test.
(D) Schematic representation of T_H2 CD4⁺ T lymphocytes and their role in breast cancer metastasis. During early breast cancer development, increased presence of leukocytes in neoplastic stroma indicates establishment of a proinflammatory microenvironment. When immune cell infiltrates include high numbers of T_H2 CD4⁺ T lymphocytes that produce IL-4 and IL-13, M2-type TAMs and IMCs are activated and in turn produce EGF, thus resulting in activation of a paracrine-mediated enhancement of malignant cell invasion and dissemination into PB and pulmonary metastasis.