The University Ethical Committee of Jamia Millia Islamia (JMI), New Delhi, and the Ethical Committee for Human Study of AIIMS (All India Institute of Medical Sciences), New Delhi, have officially approved the study. The experimental work had been undertaken with written consent obtained from each subject, and the study complies with the rules and standards set by the Ethics Code of the Medical Association of the world, which have been noted as per the Declaration of Helsinki as published in British Medical Journal (1964).

A total of 140 participants were included in the present case–control study. Cancer tissue from the breast and non-cancerous adjacent tissue were both obtained from the surgical oncology department of the collaborating institute (AIIMS). The samples were collected in three vials containing RNALater, phosphate-buffered saline (PBS), and formalin, respectively, for further processing.

The classification of breast cancer stages was done under the TNM staging system, and the histological grading of tumors was classified based on the Nottingham grade system. The exclusion criteria in the current study included familial cancer, any previous type of cancer, other metastasized cancer that has spread from different organs, and chemotherapy and radiotherapy exposure. In addition, included were various clinicopathological variables such as tumor distinctiveness [age, tumor size, metastasis at the lymph node level, TNM staging, grade of tumor, molecular subtype of tumor, hormonal receptor status (ER, PR, and Her2neu), and reproductive history (menopausal status parity)].

RNALater (Qiagen) was used to store excised tissues from normal and breast cancer patients, and then RNA was extracted by using the TRIzol method as per the manual. cDNA was synthesized using a Thermo Fisher verso kit from the extracted RNA. The qPCR is processed using Roche LightCycler® 96 machine with SYBR Green I Master mix reagent (Roche) with the help of FOXP3 primers (Fwd- 5′-TCC​CAG​AGT​TCC​TCC​ACA​AC-3′ and Rev-5′ATTGAGTGTCCGCTGCTTCT-3′) that give an amplified 122-bp product. The internal control used was GAPDH gene which was also amplified in the same PCR reactions. The program used for the amplification cycles was as follows: preheating at 95°C for 1 min, 30 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 15 s, extension at 72°C for 15 s, and further elongation at 72°C for 7 min. The experiments were repeated thrice.

The relative quantification of expression was calculated as the calibrator normalized ratio using LightCycler 96 (Roche) Software 1.5. The formula used, RQ = 2^–∆∆Ct, was according to MIQE.

The phenol-chloroform extraction method was used to isolate high-molecular-weight total gDNA from both tumor and normal tissues stored in PBS. The genomic DNA isolated was quantified on a Nanodrop spectrophotometer, and its quality was also assessed using an A260/280 ratio. It was further visualized on the 1% agarose gel stained with ethidium bromide under a UV transilluminator.

Isolated gDNA from the tissues were given bisulfite treatment using Zymo research EZ DNA Methylation-Gold™ Kit per the instructions. The treated gDNA was amplified using two sets of methylated and unmethylated primers for the FOXP3 promoter. MethPrimer tool was used to design the set of primers for methylation and unmethylation. (Li and Dahiya, 2002). The methylated primer pairs for the promoter region of the FOXP3 gene were: forward 5′- TGT​AGG​GGG​TGT​AGA​ATT​TTT​TTC-3′ and reverse 5′- AAA​CTA​AAT​TCC​CAA​AAA​CCT​CG-3′ and for the unmethylated were forward 5′- GTA​GGG​GGT​GTA​GAA​TTT​TTT​TTG​T-3 and reverse 5′- TAA​AAC​TAA​ATT​CCC​AAA​AAC​CTC​A-3′. The positive controls used in the experiment were commercially available Completely methylated and unmethylated human genomic DNA.

MS-PCR was performed under the following cycles: First denaturation at 95°C for 7 min, denaturation at 95°C for 30s 52.5°C annealing for both types of primers for 30 s, 72°C for 30 s and final elongation at 72°C for 7 min which was amplified for 35 cycles. Then, 2% agarose gel stained with EtBr was used to visualize and analyze the PCR product, which was finally photographed using the Bio-Rad Gel Documentation system. All experiments were conducted in triplicates.

The tissue biopsies of the breast carcinoma and adjoining non-cancerous tissue were conserved in formalin-fixed blocks. The blocks were then sectioned and engraved on slides coated with poly-L-lys and exposed to deparaffinization by dipping in different concentrations of xylene and further rehydrated with grades of ethanol. By using 0.3% H₂O₂ for 30 min, the endogenous peroxidase activity was blocked, and sodium citrate buffer (pH 9.0) was used for Ag retrieval.

The sections were treated for 30 min with TENG-T (10 mM Tris; 5 mM ethylenediaminetetraacetic acid, 0.15 mol/L NaCl, 0.25% gelatin, and 0.05% (v/v) Tween20 (pH 8.0) to block the samples. Bovine serum albumin was used to limit unspecific binding to the protein. The slides were treated with the primary antibody (mAbCam#ab22510 FOXP3 1:50) and incubated overnight at 4°C. The slides were then treated for 30 min with a biotinylated anti-mouse secondary antibody along with streptavidin–horseradish peroxidase conjugate. The DAB chromogen was finally used as a substrate to give a brown-colored precipitate. The slides were also treated with hematoxylin as a counterstain for better contrast.

Histopathologists interpreted the slides after immunohistochemistry; the slides were photographed under a light microscope at ×400 magnification. The pathologist further graded the expression on the number scale 0–4, with 0 as no expression and 4 as highest expression; the slides with >50% protein staining were considered in the highest scale.

SPSS version 22.0 for Windows was used for all the statistical correlations between the outcomes and the clinicopathological parameters. All data were expressed as mean ± standard error. Fisher’s exact test was used to obtain p-values between mRNA levels, methylation status, and protein expression with the clinicopathological parameters. Non-parametric Wilcoxon signed-rank test is used to estimate FOXP3/GAPDH mRNA expression levels significantly in both cancer and normal tissue samples. The p-values >0.05 were considered as significant.

FOXP3 mRNA expression revealed downregulation in 63.5% (89/140) of cases (Figure 1A), out of which nearly 86.5% (77/89) of the cases registered in the study were linked to histological grade type 1 and type 2. As per the fold change analysis, 89 cases out of 140 samples seemed to be downregulated (5.09-fold), while the expression pattern of FOXP3 at the level of mRNA, when normalized accordingly with the internal control GAPDH in tumor and non-tumor tissues, was 2.07 ± 0.7 (mean ± standard error) and 2.51 ± 0.4 (mean ± standard error) (Figure 1B), (p-value of <0.0001; Table 1) respectively. However, there was no significant association observed between FOXP3 mRNA level and various clinical variables.

The expression of FOXP3 at the protein level was found to be either low or absent in 95 cases of the total 140 samples involved in the study (67.85%) (Figure 2), while in the other 45 cases, the expression pattern was either in the high or moderate range as interpreted by a histopathologist on the basis expression scale (45/140, 32.14%). The protein expression pattern was in relation to the mRNA expression. The FOXP3 protein, as visualized by immunohistochemistry, was mainly located in the nuclear region.

As revealed by immunohistochemistry staining, the majority of FOXP3 proteins in the samples were found to be significantly downregulated. Moreover, when we tried to statistically associate the protein expression with the clinical parameters of the patients, we observed a significant correlation between TNM staging and FOXP3 protein expression (p < 0.035) (Table 2). However, with other parameters, no significant association was obtained statistically (Table 2), though most cases with tumor grades 1 and 2 seem to have protein loss (82/95, 86.3%).

Methylation at various CpG present in the upstream promoter area of FOXP3 gene was observed in 73 cases (73/140, 52.14%) (Figure 3) and, once linked with clinical parameters, revealed a significant association with the Nottingham histological grades 1 and 2 type tumors of breast cancer patients (0.031). Though no significant associations were seen with other parameters included in the study (Table 3), we did note a significantly higher number of methylated cases in metastatic lymph node (59/73, 80.8%), estrogen receptor-positive (52/73, 71.2%), and menopausal female patients (51/73, 69.8%) (Table 3).

The correlation study of methylated FOXP3 gene and its respective protein expression displayed the significant link, in which out of 95 cases with protein loss, 70 cases possessed methylation at the promoter region (70/95, 73.68%) (Table 4), whereas 25 cases were completely unmethylated (25/95, 26.3%) (Table 4). To add more, in 67 unmethylated samples (67/140, 47.8%), noticeably 62.6% (42/67) cases showed the presence of protein. Therefore, a potential statistical relation was observed between the FOXP3 protein expression and its promoter methylation (p = 0.0001) (Table A4).

The methylated promoter region of FOXP3 cases demonstrating either the absence or presence of protein exhibited a statistically significant relation with Her 2 neu receptor (p = 0.004) and metastatic lymph node tumors (p = 0.01) (Table 5). Moreover, 95.7% of methylated cases (67/70) with lymph node metastasis have protein loss. (Table 5). Additionally, the cases having protein loss exhibiting either a methylated or unmethylated FOXP3 promoter region shows a convincing association with positive Her-2 receptor (p = 0.03) and tumors of grades 1 and 2 (p = 0.01) (Table 5). Furthermore, it is seen that there is a strong statistical relation between FOXP3 protein loss and the promoter methylation with the various clinicopathological parameters (Table 6), where most of the features were associated in a highly significant manner (p-value < 0.05).