The mouse mammary carcinoma 4T1: characterization of the cellular landscape of primary tumours and metastatic tumour foci

Abstract

The murine mammary carcinoma 4T1 causes a leukemoid reaction with profound granulocytosis coincident with the production of tumour-derived growth factors. Here, we study the evolving cellular landscape of primary tumours and metastatic tumour foci and correlate haematopoietic cell infiltration with the production of tumour-derived chemokines. Flow cytometric analysis of enzyme digested primary tumours at different times after transplantation revealed a progressively increasing CD45(+) haematopoietic cell infiltrate consisting predominantly of CD11b(+) myeloid cells. Most of these cells had an F4/80(+)/CD11c(+) phenotype, many of which also stained Gr-1(+). Smaller numbers of Gr-1(+)CD11b(+) granulocytes and lymphoid cells were also identified. Progressive increases in Gr-1(+) granulocytes were observed in enzymatic digests of livers and lungs with metastatic tumour foci. Cultured 4T1 tumour cells expressed mRNA transcripts for the myeloid cell chemokines RANTES, MCP-1 and KC, and enzymatically digested cells from primary 4T1 tumours partially depleted of CD45(+) cells expressed transcripts for these chemokines and also MIP-1alpha and MIP-1beta. These data demonstrate that 4T1 tumour-bearing mice have mixed myeloid cell infiltrates of primary tumours and granulocytic infiltrates of metastatic organs. This pathologic presentation correlated with the expression of tumour-derived chemokines.

Figure 1

CD11b⁺ myeloid cells are the predominate infiltrating cells in primary 4T1 tumours. Primary tumours were digested with an enzyme cocktail, dissociated into single cell suspensions, stained with FITC-anti-CD45 and PE-anti-I-A^d, PE-Cy5-anti-CD3e, PE-Cy5-anti-Gr-1, PE-Cy7-anti-CD8a (a), FITC-anti-Gr-1, PE-anti-CD11b, PE-Cy5-anti-CD45 (b), and PE-Cy5-anti-CD45, PE-anti-CD11c, FITC-anti-Gr-1, PE-Cy7-streptavidin, and biotin-anti-F4/80 (c), and examined by flow cytometry. (a) Tumour cells were identified as CD45⁻/MHC II⁺ cells (92.8% of CD45⁻ population). (b) CD45⁺ infiltrating cells are predominantly CD11b⁺ myeloid cells. (c) CD45⁺ cells stained with both F4/80 and CD11c, and many stained with Gr-1. A population of Gr-1⁺ cells did not stain with either F4/80 or CD11c, together with a Gr-1 negative population that represented lymphoid cells.

Figure 2

The cellular landscape of the 4T1 tumour changes over time. (a) Flow cytometric dot plots of CD45⁺ tumour-infiltrating cells on days 15, 22, and 29 post-tumour transplant. (b) Single cell suspensions of primary tumour cells and infiltrates were labelled with FITC-anti-CD45, PE-anti-CD11b, PE-Cy5-anti-Gr-1, PE-Cy5-anti-CD3e and PE-Cy7-anti-CD8a. CD11b⁺ myeloid cells and lymphoid and other cells are shown as percentages of CD45⁺ tumour-infiltrating haematopoietic cells. Bars represent mean ± SD for groups of three mice in one of two similar experiments.

Figure 3

Chemokine mRNA expression in cultured 4T1 tumour cells and 4T1 tumour cells from primary tumours partially depleted of CD45⁺ cells. (a) Ethidium bromide-stained 1.5% agarose gels show expression of mRNA transcripts for the myeloid cell CC chemokines KC, RANTES and MCP-1 in 1 × 10⁶ cultured 4T1 cells and 1 × 10⁶ cells from enzymatically digested primary tumours partially depleted of CD45⁺ cells as described in the Materials and methods. (b) Expression of mRNA transcripts for the CC chemokines MIP-1α and MIP-1β in 4T1 tumour cells from enzymatically digested primary tumours partially depleted of CD45⁺ cells as described in the Materials and methods.

Figure 4

Granulocytic infiltrates in livers of 4T1 tumour-bearing mice. (a) H&E stained sections from livers of normal and 29-day tumour-bearing mice. (b) Bivariate flow cytometric dot plots showing the increase in infiltrating Gr-1⁺ granulocytes in livers of tumour-bearing mice. Livers were digested with an enzyme cocktail, made into a single cell suspension, stained with FITC-anti-CD45, PE-anti-CD4, PE-anti-CD19, PE-Cy5-anti-CD3e, PE-Cy5-anti-Gr-1 and PE-Cy7-anti-CD8a, and analysed by flow cytometry. Total liver cells were first gated on CD45, and then examined for Gr-1 staining. Numbers represent Gr-1⁺ cells as a percent of CD45⁺ cells in the liver. (c) Granulocytes (Gr-1⁺) and lymphocytes (CD3⁺ T lymphocytes and CD19⁺ B lymphocytes) as a percentage of CD45⁺ cells in livers of normal mice and mice at different times after tumour transplantation. Bars represent mean ± SD (four mice per group) from a representative experiment.

Figure 5

Granulocytic infiltrates in lungs of 4T1 tumour-bearing mice. (a) H&E stained sections from lungs of normal and 29-day tumour-bearing mice. (b) Bivariate flow cytometric dot plots showing the increase in infiltrating Gr-1⁺ granulocytes in lungs of tumour-bearing mice. Lungs were digested with an enzyme cocktail, made into a single cell suspension, stained with FITC-anti-CD45, PE-anti-CD4, PE-anti-CD19, PE-Cy5-anti-CD3e, PE-Cy5-anti-Gr-1 and PE-Cy7-anti-CD8a, and analysed by flow cytometry. Total lung cells were first gated on CD45, then Gr-1. Numbers represent Gr-1⁺ cells as a percent of CD45⁺ cells. (C) Granulocytes (Gr-1⁺) and lymphocytes (CD3⁺ T lymphocytes and CD19⁺ B lymphocytes) as a percentage of CD45⁺ cells in lungs of normal mice and mice at different times after tumour transplantation. Bars represent mean ± SD (four mice per group) from a representative experiment.