Adiponectin-expressing Treg-containing T cell fraction inhibits tumor growth in orthotopically implanted triple-negative breast cancer

Abstract

Background: In our previous study, we identified a population of adiponectin expressing regulatory T cells (Tregs) residing within thymic nurse cell complexes, which were capable of inhibiting the development of breast cancer in vitro. Triple-negative breast cancer (TNBC) with no proper treatment at present is characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2. In this study, we aimed to investigate the potential of a cultured T cell fraction comprising adiponectin-expressing Tregs, referred to as A-TregTF (adiponectin-expressing Treg-containing T cell fraction), in inhibiting the progression of TNBC in vivo.

Methods: The efficacy of a spontaneously expanding T cell fraction comprising adiponectin-expressing Treg in inhibiting tumor growth was analyzed in a murine orthotopic 4 T1-Luc TNBC model.

Results: The treatment with T cell fraction containing adiponectin-expressing Tregs significantly inhibited the growth and metastasis of orthotopically transplanted 4 T1-Luc tumor cells. Histopathological examination further revealed that the adiponectin-expressing Tregs infiltrated the tumor tissue via a cell-in-cell mechanism and were found to be specifically localized around the necrotic areas.

Conclusions: Based on our findings, the T cell fraction comprising adiponectin-expressing Tregs, represents a potential candidate for adoptive cell therapy against TNBC.

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

FIGURE 1

Characterization of cultured T cell fraction composed of adiponectin‐expressing Tregs, A‐TregTF. Upper panel: Control nonstained cells are indicated as mock cells. Representative cell surface staining of A‐TregTF with anti‐CD4 and anti‐CD25 antibodies (A‐TregTF). Lower panel: Cells were subjected to immunocytostaining with specific antibodies. A‐TregTF exhibits immunoreactivity to both FOXP3 and adiponectin. The green signal indicates adiponectin immunoreactivity and magenta represents the merging of red FOXP3 immunoreactivity and blue 4′, 6‐diamidino‐2‐phenylindole (DAPI) staining highlighting the cell nuclei. Scale bar: 20 μm.

FIGURE 2

A‐TregTF inhibits the growth of orthotopically transplanted 4 T1‐Luc tumor cells. Female BALB/c mice were implanted with 4 T1‐Luc cells via mammary fat pad inoculation on day 1 (indicated by black arrow). Thereafter, A‐TregTFs were injected at the 4 T1‐Luc transplantation site (day 4 and day 2 in the in the first second experiment, respectively, indicated by red arrow). In both the experiments, A‐TregTFs significantly suppressed the tumor growth of orthotopically transplanted 4 T1‐Luc cells (left; on day 14: right; on day 10, p < 0.05, student's t‐test).

FIGURE 3

A‐TregTF induces tumor necrosis in orthotopically transplanted 4 T1‐Luc cells. (a) Orthotopically transplanted control 4 T1‐Luc cells exhibit rapid growth in the absence of necrosis. (b) Orthotopically transplanted 4 T1‐Luc cells exhibit dispersed growth with significant necrosis. Note the cell‐in‐cell structure of 4 T1‐Luc cells as indicated by the arrow. Scale bar: 50 μm (a and b). (c) Representative immunofluorescence tissue staining. Merged signals of FOXP3 (red), adiponectin (green), and 4′, 6‐diamidino‐2‐phenylindole (DAPI) (blue) are visualized towards the left side of the necrotic area. Scale bar: 20 μm.