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

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.


INTRODUCTION
Triple-negative breast cancer (TNBC) is a type of breast cancer characterized by the loss of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 (HER2). Accordingly, they are not sensitive to endocrine therapy or HER2 treatment. 1 Moreover, TNBC often demonstrates aggressive clinicopathological behavior; therefore, the standardization of treatment regimens for patients with TNBC is still in progress. 2 Obesity also increases the risk of postmenopausal breast cancer. 3 Recent advances have highlighted that adiponectin, a well-characterized insulin-sensitizing adipokine, and its receptors, especially T-cadherin (also known as H-cadherin or CDH13), may resist obesity-related carcinogenesis. 4 Paradoxically, the serum concentration of adiponectin is reduced in obesity. 5 The impaired expression of adiponectin or adiponectin receptors leads to mammary gland carcinogenesis. [6][7][8] Adiponectin induces autophagic cell death 9 and can induce apoptosis via fatty acid metabolic reprogramming in breast cancer. 10 However, T-cadherin, a receptor for high-molecular weights (HMW) adiponectin, 11 which is known as a highly active form of adiponectin, is often lost in breast cancer cells due to promoter hypermethylation, 6,12,13 worsening the prognosis of patients with TNBC. 14 Therefore, an approach for the efficient introduction of adiponectin into breast cancer cells should be developed.
A population of Treg cells residing within thymic nurse cell complexes expresses adiponectin. [15][16][17] Adiponectinexpressing Treg significantly reduced mammary carcinogenesis in transgenic mice model. 15 Recently, we succeeded in expanding the murine T cell fraction, which contains adiponectin-expressing Treg, using an experimental thymic model. 16,17 Notably, these adiponectin-expressing Treg exhibited cell-in-cell phenomenon to thymic stromal nursing cells, 16 similar to that of previously reported "HOZOT" cytotoxic Treg cells. [18][19][20] HOZOT cells have been used for tumor-specific intracellular delivery of "oncolytic adenoviruses" to gastric and colorectal cancer cells through cell-incell invasion. 21 In the present study, we examined whether adiponectinexpressing Treg function as effective adiponectin transporters in TNBC.

Cells and culture
Two TNBC cell lines, MDA-MB-157 and MDA-MB-231, and a lobular carcinoma cell line, MDA-MB-330, were obtained from the American Type Culture Collection (Manassas, VA, USA). Luminal A-type breast cancer cell line MCF-7 was purchased from the Japanese Collection of Research Biosources Cell Bank (Osaka, Japan). The breast cancer cells were passaged for no more than 6 months after resuscitation.
T cells consisting of adiponectin-expressing Treg were obtained from murine tumors, which mimic human micronodular thymic tumors with lymphoid stroma, and were maintained through coculture with thymic stromal cells, as previously described. 16,17 Cells were cultured in Dulbecco's modified Eagle's medium-high glucose (4500 mg/L) (Sigma-Aldrich) with 10% fetal bovine serum.
In several experiments, cells were labeled using Lumini-Cell Tracker-Cell labeling kit (Sigma-Aldrich).
Briefly, the cells were incubated with or without 1:100 diluted antibodies at 4 C for 1 h. The cells were then washed with phosphate-buffered saline (PBS), and also analyzed using a Guava EasyCyte cell analyzer (Hayward) and a confocal laser scanning microscope (Leica TCS SP8) as previously described. 23

Detection of apoptosis
Recombinant human eukaryotic adiponectin was purchased from R&D Systems Inc. The number of cells undergoing apoptosis was quantified using FITC-conjugated annexin V and propidium iodide (PI: PromoCell GmbH). Briefly, 1 Â 10 4 cells under different culture conditions were harvested, washed, resuspended in binding buffer, mixed with annexin V-FITC and PI, and analyzed as previously described. 24 Immunoblotting Immunoblotting was performed according to the method described by Towbin et al. with modifications as previously described. 24,25 Briefly, cell lysates were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Immobilon-P Transfer Membrane; Millipore). The membranes were blocked with Block Ace (blocking milk; Yukijirushi) and incubated with rabbit anti-adiponectin antibody (cat. no. GTX107737, GeneTex Inc.) and anti-T-cadherin antibody. 26 For immunodetection, horseradish peroxidaseconjugated anti-rabbit secondary antibody (1:2000; cat. no. 7074); Cell Signaling Technology Inc. and ultrasensitive HRP substrate (TaKaRa Bio) were used. Images were obtained using an Invitrogen iBright 1500 gel imaging system (Thermo Fisher Scientific). After stripping immunoreactivity, the membranes were incubated with an anti-GAPDH antibody (Sigma) to evaluate the input proteins.

Coculture of TNBC cells and adiponectinexpressing Treg
CD4-and CD25-positive T cells were harvested using an SH800S cell sorter (Sony Biotechnology). Subsequently, adiponectin-expressing Treg were cocultured with MDA-MB-157 or MDA-MB-231 cells. Time-lapse images of the cells were acquired using a CytoWatcher (ATTO) or confocal laser scanning microscope (Leica TCS SP8).

Harvest of adiponectin-expressing Treg
The detailed characterization of the murine tumor model from which we obtained adiponectin-expressing Treg cells has been previously documented. 16,17 Briefly, the murine model exhibited thymic tumors composed of thymic epithelioid tumor cells with abundant lymphoid stroma. As shown in Figure S1, the lymphoid stroma partially contained adiponectin-expressing and FOXP3-positive Treg cells. Using CD4-and CD25-positive sorting, we successfully harvested adiponectin-expressing Treg cells that exhibited FOXP3 nuclear staining. Immunoblotting also demonstrated that the sorted Treg cells expressed HMW adiponectin. The representative results are shown in Figure 1.

DISCUSSION
Here, we demonstrated that using CD4 and CD25 sorting, adiponectin-expressing Treg cells can be successfully harvested (Figure 1a-e) from cultured cells of an experimental murine thymic tumor model ( Figure S1). Notably, these Treg cells expressed HMW adiponectin (Figure 1f Previously, we noted that adiponectin-expressing Treg exhibit the cell-in-cell phenomenon to thymic stromal nursing cells. 16 In this study, we demonstrated that adiponectinexpressing Treg attached to and integrated into TNBC cells. As shown in Figure 3, most TNBC cells died after the engulfment of adiponectin-expressing Treg.  Previous studies have reported that a Treg cell line, designated HOZOT, exhibits the cell-in-cell phenomenon in several cancer cells. HOZOT cells were established from human umbilical cord blood through coculture with mouse stromal cells and were characterized as a cytotoxic Treg line with cell-in-cell activity. [18][19][20] HOZOT cells demonstrated tumor-specific intracellular delivery of "oncolytic adenoviruses" to gastric and colorectal cancer cells through cell-incell invasion. 21 Adiponectin is believed to play a protective role against carcinogenesis in various types of cancers. 4 Therefore, adiponectin-based therapy has been proposed as a potential pharmacological approach in both cancer prevention and management. Nevertheless, the intrinsic limitations of adiponectin, that is, the HMW of its active form and reduced stability, have hampered its therapeutic application in clinical settings, including its use for the treatment of cancer. Moreover, T-cadherin, a receptor of HMW adiponectin, is downregulated in many cancers, including breast cancer. 6,7 Notably, the Treg cells in the present study were shown to have a high level of HMW adiponectin in the cytoplasm ( Figure 1). Therefore, engulfment of adiponectin-expressing Treg cells may enable the enforced uptake of adiponectin to malignant tumors, whether or not adiponectin receptors are present.
Adiponectin-expressing Treg cells were obtained from T cells, which were maintained by coculture with murine thymic stroma cells. 16 Without direct cellular contact with thymic stromal cells, T cells undergo apoptosis. 17 Some Treg cells are also known to be engulfed by hepatocellular cell carcinoma cells. 30 We speculate that the adiponectinexpressing Treg in the present study may be a relative of HOZOT or other Treg subtypes that have cell-in-cell activity. However, further studies are necessary to verify this hypothesis and to unravel the molecular mechanisms that participate in the Treg-mediated cell-in-cell phenomenon.
In conclusion, in this study, we established that novel adiponectin-expressing Treg cells exhibit a cell-in-cell phenomenon, subsequently causing cell death in TNBC cells. We believe that they function as effective adiponectin transporters in TNBC cells.

AUTHOR CONTRIBUTIONS
CS and TT designed and conceived the study. YC, TH, HH, and YH performed the experiments. YK, MF, and NM interpreted the data. All authors participated in the data analysis. All authors read and approved the final manuscript.

ACKNOWLEDGMENT
This study was partially supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 20 K07406).

CONFLICT OF INTEREST STATEMENT
The authors declare no competing interests.