Potential activity of adiponectin-expressing regulatory T cells against triple-negative breast cancer cells through the cell-in-cell phenomenon

Abstract

Background: A population of regulatory T cells (Treg), which reside within thymic nurse cell complexes, express adiponectin and abrogate breast cancer development in transgenic mice. In this study, we examined whether adiponectin-expressing Treg could impair triple-negative breast cancer, which is defined by a lack of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor-2.

Methods: CD4- and CD25-positive cells were sorted from cultured T lymphocytes of a previously characterized experimental thymic tumor model composed of thymic nurse cells and abundant lymphoid stroma. These sorted cells were examined for FOXP3 and adiponectin immunoreactivity and subsequently exposed to triple-negative breast cancer MDA-MB-157 and -231 cells.

Results: Adiponectin-expressing Treg were obtained by CD4- and CD25-positive sorting and cell death was induced in triple-negative breast cancer cells through the cell-in-cell phenomenon.

Conclusions: Adiponectin-expressing Treg may be candidates for adoptive cell therapy against triple-negative breast cancer.

Keywords: Treg; adiponectin; adoptive cell therapy; cell-in-cell; triple-negative breast cancer.

FIGURE 1

Representative results of adiponectin‐expressing Treg harvesting. Lymphocytes were obtained from a culture of cells from a previously developed murine model of a human micronodular thymic tumor with lymphoid stroma., Control nonstained cells are shown in (a). Lymphoid cells were partially stained on the cell surface with anti‐CD4 and anti‐CD25 antibodies (b). CD4‐ and CD25‐positive lymphoid cells were sorted using an SH800S cell sorter (c). Subsequently, the cells were immunocytostained with antibodies. Nonimmunostained cells (stained with 4′,6‐diamidino‐2‐phenylindole [DAPI]) are shown in (d). Sorted cells exhibited FOXP3 and adiponectin immunoreactivity. In (e), the green signal indicates adiponectin immunoreactivity and the pink color indicates the merging of red FOXP3 immunoreactivity and blue DAPI staining. Scale bar, 20 μm. The immunoblotting results indicated that the sorted cells expressed high molecular weight (HMW) adiponectin, as indicated by the arrow (f; upper column). The glyceraldehyde‐3‐phosphate dehydrogenase (G3PDH) band is shown (f; lower column).

FIGURE 2

Limited effect of soluble adiponectin on apoptosis induction on triple negative MDA‐MB‐157 breast cancer cells. (a)–(d) MDA‐MB‐157 cells were incubated with eukaryotic recombinant adiponectin at various concentrations (a:0, b:0.05, c:0.5, d:5 μg/mL) for 36 h. Thereafter, the cells were examined using an annexin V‐PI assay. Even under 5 μg/mL adiponectin, less than 10% of cells were apoptotic. (e) *Indicates a 105 kDa T‐cadherin protein band, which is characterized as a mature receptor of high molecular weight (HMW) adiponectin. A weak 105 kDa T‐cadherin protein band was seen in lane 3 (MDA‐MB‐157), but not in lanes 1 (MDA‐MB‐330), 2 (MDA‐MB‐231), or 4 (MCF‐7). The glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) band is shown in the lower column.

FIGURE 3

Triple‐negative breast cancer cells exhibited cell death after engulfing adiponectin‐expressing Treg. MDA‐MB‐157 cells (a) were occulted due to adiponectin‐expressing Treg. After 16 h, numerous adiponectin‐expressing Treg attached to MDA‐MB‐157 cells (b). Note the integration of adiponectin‐expressing Treg into the cytoplasm (b and c, arrow) and destruction (c, arrowhead) of MDA‐MB‐157 cells. After 64 h of coculture, MDA‐MB‐157 cells exhibited cell death (d, arrow; insert indicates cell debris). Confocal laser imaging confirmed cytoplasmic integration, that is, the cell‐in‐cell phenomenon (e, f, and g) in MDA‐MB‐157 cells. MDA‐MB‐157 cells, the cytoplasm of which was stained red, engulfed adiponectin‐expressing Treg stained green. Note the attachment (e, arrowhead) and cytoplasmic integration (e, arrow) of adiponectin‐expressing Treg into MDA‐MB‐157 cells. Horizontal and vertical cell‐in‐cell images are shown in (f) and (g), respectively. MDA‐MB‐231 cells also demonstrated the cell‐in‐cell phenomenon by engulfing adiponectin‐expressing Treg as indicated by arrows in the horizontal (h) and vertical (i) images. The scale bar indicates 50 μm.