Tracking Biodistribution of Myeloid-Derived Cells in Murine Models of Breast Cancer

Abstract

A growing tumor is constantly secreting inflammatory chemokines and cytokines that induce release of immature myeloid cells, including myeloid-derived suppressor cells (MDSCs) and macrophages, from the bone marrow. These cells not only promote tumor growth, but also prepare distant organs for tumor metastasis. On the other hand, the myeloid-derived cells also have phagocytic potential, and can serve as vehicles for drug delivery. We have previously identified thioaptamers that bind a subset of MDSCs with high affinity and specificity. In the current study, we applied one of the thioaptamers as a probe to track myeloid cell distribution in the bone, liver, spleen and tumor in multiple murine models of breast cancer including the 4T1 syngeneic model and MDA-MB-231 and SUM159 xenograft models. Information generated from this study will facilitate further understanding of tumor growth and metastasis, and predict biodistribution patterns of cell-mediated drug delivery.

Keywords: biodistribution; breast cancer; myeloid-derived suppressor cell; thioaptamer.

Figure 1

Schematic view on study design and myeloid cell separation. (a) Schematic view of research procedure. (b) Gating strategy for detection of aptamer-positive CD45⁺CD11b⁺Ly6G⁺Ly6C^low granulocytes, CD45⁺CD11b⁺Ly6G⁻Ly6C^high M-MDSCs, and CD45⁺CD11b⁺F4/80⁺ macrophages.

Figure 2

Analysis of aptamer-binding myeloid cells in murine model of primary 4T1 mammary gland tumor. Mice bearing primary 4T1 tumors (n = 3 mice/group) were treated with Cy5-labeled T1 or scramble aptamer. They were euthanized 4 h later, and single cells were prepared from bone marrow, spleen and tumor. Flow cytometry was performed to detect aptamer-positive cells in the granulocytes, M-MDSC and macrophage populations. Pink: CD11b⁺Ly6C^lowLy6G⁺ granulocytes; Blue: CD11b⁺Ly6C^highLy6G⁻ M-MDSCs; Green: CD11b⁺F4/80⁺ macrophages. Data is presented as mean ± s.d. P values are calculated with a two-tailed student t-test. *, p < 0.05; **, p < 0.01; ns, not significant.

Figure 3

Analysis of aptamer-binding myeloid cells in a murine model of MDA-MB-231 and SUM159 xenograft tumors. Mice bearing primary (a) MDA-MB-231 or (b) SUM159 primary tumors (n = 4 mice/group) were treated with Cy5-labeled T1 or scramble aptamer. They were euthanized 4 h later, and single cells were prepared from bone marrow, liver, spleen and tumor tissues. Flow cytometry was performed to detect aptamer-positive cells. Pink: CD11b⁺Ly6C^lowLy6G⁺ granulocytes; Blue: CD11b⁺Ly6C^highLy6G⁻ M-MDSCs; Green: CD11b⁺F4/80⁺ macrophages. Data is presented as mean ± s.d. p values are calculated with a two-tailed student t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; *****, p < 0.00001; ns, not significant.

Figure 4

T1 aptamer preferentially binds human bone marrow cells. (a) Human bone marrow cells were incubated with increasing concentrations of Cy5-labeled T1 or SCR aptamers, and flow cytometry was performed to detect aptamer-binding cells. (a) Separation of scramble- and T1 aptamer-binding human bone marrow hematopoietic cells. Aptamer concentration: 1.56 nM. (b) T1 aptamer concentration-dependent binding of human bone marrow hematopoietic cells.