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Abstract

Objective

To investigate the influence of recombinant human CD40 ligand (rhCD40L) on the biological behaviour of breast cancer cells.

Methods

MDA-MB-23l and MDA-MB-435 treated with rhCD40L were observed for changes in the cell cycle, in membrane proteins, and in mRNA levels of B cell lymphoma-extra-large (Bcl-xl), Bcl-2 associated X protein (Bax) and regulated upon activation, normal T cell expressed and secreted (RANTES). Effects of rhCD40L on cell proliferation in the presence or absence of interferon (IFN)-γ (500 IU/ml) and/or doxorubicin (20 ng/ml) were also determined.

Results

rhCD40L dose-dependently inhibited cell proliferation. Combination of rhCD40L with IFN-γ or doxorubicin potentiated the inhibitory activity. After treatment, an increase in cells entering the G₁ phase of the cell cycle was observed, with a significant decrease in the number entering the S phase. Levels of several membrane proteins including CD95L and CD120a were also increased. Reverse transcription–polymerase chain reaction revealed an increase in the Bax/Bcl-xl mRNA ratio and an increase in RANTES.

Conclusion

rhCD40L treatment of breast cancer cells mediates a variety of anti-tumour effects, not only by direct cytotoxic activity but also by upregulation of adhesion molecules, co-stimulators and cytokines to rectify T cell immunity.

Keywords

rhCD40L breast cancer cell doxorubicin anti-tumour

Introduction

Data obtained from laboratory studies and clinical trials suggest that immune escape of transformed cells leads to the formation and development of tumours.^1,2 In many tumour models, CD40-ligand (CD40L)-mediated signals stimulate host anti-tumour immunity^3–4 and significantly increase tumour incidence in Cd40 gene knockout mice.⁵ Using CD40 as the target molecule, new biological therapies for cancer have been developed wherein CD40/CD40L signals are stimulated using CD40 antibody, CD40L recombinant products or tumour vaccines with cDNA in order to inhibit tumour growth in epithelial cells.^6–9 The mechanism of action and biological activity of these novel therapies does, however, require further study.

The present study aimed to stimulate the CD40 signal with recombinant human (rh)CD40L in the human breast cancer cell lines, MDA-MB-231 and MDA-MB-435; the study also investigated the effects of a combination of rhCD40L and the chemotherapy agents interferon (IFN)-γ and doxorubicin on the biological behaviours of these cells.

Materials and methods

Detection of cell proliferation

MDA-MB-231 and MDA-MB-435 breast cancer cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were inoculated at a density of 2 × 10⁵ cells/ml in 96-well plates containing RPMI 1640 medium (GIBCO® Cell Culture, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 IU/ml streptomycin. rhCD40L (PeproTech®, Rocky Hill, NJ, USA) at concentrations of 0.05–10 µg/ml was added and cells were cultured at 37℃ in an atmosphere of 5% CO₂ for 3 days. [³H]Thymidine (3.7 × 10⁴Bq/well) was added during the final 12 h of culture, after which cells were harvested and counts/min measured by β-liquid scintillation counting (Beckman Coulter, Brea, CA, USA). Experiments were performed in triplicate and untreated cells were used as a negative control.

Cell cycle analysis

MDA-MB-231 and MDA-MB-435 cells were permeabilized using 0.25% trypsin (Invitrogen, Carlsbad, CA, USA), washed three times using 10 mM cold sterile phosphate-buffered saline (PBS, pH 7.4), and adjusted to a concentration of 5 × 10⁵ cells/ml. Cells were then transferred into six-well plates, mixed with 2.5 µg/ml of rhCD40L and cultured at 37℃ in an atmosphere of 5% CO₂ before harvesting at 48 h and 72 h. Following harvesting, cells were washed twice with 0.01 mM cold PBS (pH 7.4) and centrifuged at 1006. g at 37℃ for 5 min. Centrifugation was repeated twice at 5 min intervals. The cell concentration was then adjusted to 1 × 10⁶ cells/ml and 0.5 ml propidium iodide solution (containing 100 mg/ml of RNAase and 0.1% of Triton X™-100) was added (Sigma-Aldrich, St Louis, MO, USA). Cells were kept in the dark at 37℃ for 30 min, then assayed by flow cytometer (Beckman Coulter Inc., Brea, CA, USA) after filtering through a 200-mesh sieve. The distribution ratio of each cell cycle phase was performed by normal fitting (G vs S vs G₂/M).

Immunofluorescence analysis of membrane molecules

MDA-MB-231 and MDA-MB-435 cells were treated with 2.5 µg/ml of rhCD40L for 72 h and changes in selected membrane molecules (see Results) were analysed using an indirect or direct immunofluorescence labelling method. For measurement of indirect immunofluorescence, cells were collected, centrifuged at 1006 g at 37℃ for 5 min and washed with precooled 10 mM PBS (pH 7.4). The cell concentration was adjusted to 5 × 10⁵ cells/ml and cells were fixed using 4% paraformaldehyde at 4℃ for 30 min. After washing twice with 10 mM PBS (pH 7.4), cells were blocked with PBS containing 1% bovine serum albumin (Sigma-Aldrich) at 37℃ for 30 min. After washing twice with PBS, the cells (100 µl) were incubated with l µg of mouse antihuman monoclonal antibodies (Jianmei Co., Shenzhen, China) at 4℃ for 30–45 min. Mouse immunoglobulin G1 (Immunotec, Westbrook, ME, USA) was used as an isotype control. Cells were then washed twice with 10 mM PBS (pH 7.4) containing 0.25% fetal calf serum and 0.01% sodium azide, and incubated with fluorescein isothiocyanate-labelled 0.01% goat antimouse secondary antibodies in secondary antibody dilution buffer (pH 7.4) (Beyotime Co., Haimen, China) at 4℃ for 30 min. After washing in 10 mM PBS (pH 7.4), changes in cell surface molecules were analysed by flow cytometry (Beckman Coulter). Methods were identical for measurement of direct immunofluorescence but cells were incubated with l µg of phycoerythrin-labelled mouse antihuman monoclonal antibodies (Jianmei Co.).

RT–PCR for analysis of Bcl-xl, Bax and RANTES mRNA

MDA-MB-231 and MDA-MB-435 cells (1 × 10⁵ cells/ml) were collected in the logarithmic phase and treated with 2.5 µg/ml of rhCD40L for 4 h and 24 h. Untreated cells served as a control. A single-step extraction method was used to obtain the total RNA with Trizol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was reverse transcribed to cDNA using 5 µl of cDNA as a template, diluted to 50 µl with reaction solution (QIAGEN® OneStep RT–PCR Kit, Hilden, Germany). Reverse transcription–polymerase chain reaction (RT–PCR) was performed using a S1000 thermal cycler (Bio-Rad, Hercules, CA, USA) by sequentially adding primers for B cell lymphoma-extra-large (Bcl-xl), Bcl-2 associated X protein (Bax), regulated upon activation, normal T cell expressed and secreted (RANTES, all 40 pmol) or ß-actin (10 pmol) and Taq-DNA polymerase (1.5 µl). Primer sequences are shown in Table 1. The cycling programme involved a total of 30 cycles of denaturation at 94℃ for 60 s, annealing for 90 s at 56℃ for Bcl-xl, RANTES and ß-actin or at 54℃ for Bax, and extension at 72℃ for 120 s, followed by a final extension step at 72℃ for 4 min. PCR products (10 µl) were loaded onto a 1.5% agarose gel and visualized using a Gel Doc™ 1000 ultraviolet imager (Bio-Rad). Semiquantitative analysis of Bcl-xl, Bax and RANTES expression was conducted using ß-actin as a reference.

Table 1.

Sequence-specific primers used for reverse transcription–polymerase chain reaction to determine changes in gene expression, following treatment of breast cancer cell lines with recombinant human CD40-ligand^a.

Gene	Primer pair
Bcl-xl	Forward: 5′-TAGATGGAGCCAACAG-3′
Bcl-xl	Reverse: 5′-TCAGGAAGCGGTTG AAGCC-3′
Bax	Forward: 5′-TGAATGGAGCCACTGC GCACAG-3′
Bax	Reverse: 5′-TCAGGAACCAGCGGT TGAAGCG-3′
ß-Actin	Forward: 5′-ACACTATACCCATCATA CAAAAAA-3′
ß-Actin	Reverse: 5′-ATGATGGAGTTGAAGG TAGTTTCATGGAT-3′
RANTES	Forward: 5′-CAGGTGCCATGAAG GTCTC-3′
RANTES	Reverse: 5′-CAGGTGCCATGAAG GTCTC-3′

Synthesized by Shanghai Shengong Biotechnology Company, Shanghai, China.

Bcl-xl, B cell lymphoma-extra-large; Bax, Bcl-2 associated X protein; RANTES, regulated upon activation, normal T cell expressed and secreted.

Cell proliferation determined by MTT assay

The effects of rhCD40L in combination with IFN- γ or doxorubicin (both from Sigma-Aldrich) on cell proliferation were determined by 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. MDA-MB-231 cells (1 × 10⁵/ml) were seeded in 96-well plates in 200 µl of RPMI 1640 medium. IFN-γ (500 IU/ml) and rhCD40L (0.1, 0.5, 1, 10 or 20 µg/ml) were added to each well and cells were cultured for 3 days at 37℃ in a humidified atmosphere containing 5% CO₂. Subsequently, 20 µl of MTT solution (50 mg/ml) was added to each well and incubated for 4 h. The supernatant fraction was then removed and 100 µl of acidified isopropanol was added to solubilize blue formazan particles. Optical density was measured at 570 nm using a microplate reader (PR3100 TSC, Bio-Rad). All experiments were performed in triplicate.

On determination of the optimal concentration of rhCD40L (see above), MDA-MB-231 cells were divided into six groups in order to assess the effects of rhCD40L in combination with doxorubicin, as follows: (i) control group (untreated cells); (ii) rhCD40L (2.5 µg/ml) alone; (iii) doxorubicin (20 ng/ml) alone; (iv) rhCD40L (2.5 µg/ml) plus doxorubicin (20 ng/ml); (v) rhCD40L (2.5 µg/ml) plus doxorubicin (20 ng/ml) and IFN-γ (500 IU/ml); (vi) blank control (medium only). Cell proliferation experiments were repeated as described above using the MTT assay.

Statistical analyses

All statistical analyses were performed using SPSS® statistical software, version 13.0 (SPSS, Inc., Chicago, IL, USA). Statistically significant differences were determined by Student’s t-test and a P-value of <0.05 was considered significant.

Results

Proliferation of MDA-MB-231 and MDA-MB-435 breast cancer cells expressing CD40 was significantly inhibited by rhCD40L in a dose-related manner compared with controls (P = 0.018, Figure 1).

Figure 1.

Effects of recombinant human CD40-ligand (rhCD40L) on proliferation of breast cancer cell lines after 3 days’ culture, determined by [³H]thymidine incorporation assay. Data presented as mean ± SD of three experiments. *P = 0.018 compared with untreated cells; Student’s t-test.

Following treatment with rhCD40L, MDA-MB-435 cells increased significantly at 48 h and 72 h during the G_l phase and decreased in the S (48 h only) and G₂ phases (48 and 72 h), compared with controls (P < 0.05; Table 2). There were no obvious cell cycle changes in MDA-MB-231 cells at 48 h (Table 3) but cells were significantly increased at 72 h in the G_l phase (P = 0.012) and significantly decreased in the S phase (P = 0.023).

Table 2.

Effects of recombinant human CD40-ligand (rhCD40L) on the percentage of MDA-MB-435 breast cancer cells in the G₁, S and G₂ phases of the cell cycle.

Time, h	Untreated cells, % cells			2.5 µg/ml rhCD40L, % cells
Time, h	G₁	S	G₂	G₁	S	G₂
0	41.4 ± 2.1	37.6 ± 6.7	21.0 ± 1.3	41.4 ± 2.1	37.6 ± 6.7	21.0 ± 1.3
48	44.7 ± 3.4	40.0 ± 2.3	15.3 ± 3.5	66.5 ± 5.6^a	23.3 ± 3.3^a	10.3 ± 2.1^a
72	39.1 ± 2.9	36.0 ± 3.4	24.9 ± 5.4	58.4 ± 4.8^a	30.0 ± 1.0	11.0 ± 6.1^a

Data presented as mean ± SD of three experiments.

P < 0.05 compared with untreated cells; Student’s t-test.

Table 3.

Effects of recombinant human CD40-ligand (rhCD40L) on the percentage of MDA-MB-231 breast cancer cells in the G₁, S and G₂ phases of the cell cycle.

Time, h	Untreated cells, % cells			2.5 µg/ml rhCD40L, % cells
Time, h	G₁	S	G₂	G₁	S	G₂
0	45.1 ± 1.5	43.4 ± 4.3	11.6 ± 3.1	45.1 ± 1.5	43.4 ± 4.3	11.6 ± 3.1
48	43.4 ± 4.9	48.2 ± 7.3	8.4 ± 4.7	46.0 ± 1.0	44.4 ± 2.4	9.7 ± 3.9
72	47.1 ± 4.7	40.0 ± 5.7	12.9 ± 4.2	54.2 ± 1.4^a	34.7 ± 4.7^b	11.2 ± 2.1

Data presented as mean ± SD of three experiments.

P = 0.012 compared with untreated cells; Student’s t-test.

P = 0.023 compared with untreated cells; Student’s t-test.

High levels of the following membrane molecules were determined in MDA-MB-231 cells: adhesion molecules, CD49e and CD44; the major histocompatibility complex (MHC) class 1 molecule, human leucocyte antigen (HLA)-ABC; the negative regulatory molecule, programmed cell death ligand-1 (PD-Ll). Low levels of CD19, CD62e, CD80, CD86, CD40L, tumour necrosis factor (TNF), HLA-DR and CD120a were determined. There were no significant changes in any of these molecules following treatment of MDA-MB-231 cells with rhCD40L (Table 4). A significant increase in CD54, CD95 (Fas) and CD120b was observed in the presence of rhCD40L (P < 0.05). CD95L levels were decreased (P = 0.013). In MDA-MB-435 cells, high levels of CD44, CD54, CD95 and HLA-ABC and low levels of CD19, CD62e, CD40L, CD80, CD86 and CD120b were observed in the presence and absence of rhCD40L. Significant increases in CD49e, CD95L, CD120a and HLA-DR were determined following treatment with rhCD40L (Table 4). There were no obvious changes in TNF after treatment with rhCD40L in either breast cancer cell line (Table 4).

Table 4.

Effects of treatment with recombinant human CD40-ligand (rhCD40L) for 72 h on the levels of various membrane proteins in MDA-MB-231 and MDA-MB-435 breast cancer cell lines.

Membrane protein	MDA-MB-231		MDA-MB-435
Membrane protein	Control, %	2.5 µg/ml rhCD40L, %	Control, %	2.5 µg/ml rhCD40L, %
CD19	5.2 ± 2.5	4.6 ± 3.5	1.2 ± 0.9	1.5 ± 0.2
CD49e	91.5 ± 3.1	90.5 ± 5.0	47.6 ± 4.7	63.9 ± 2.4^a
CD44	99.8 ± 0.1	99.7 ± 0.2	99.9 ± 0.1	100.0 ± 0
CD54	53.5 ± 5.6	73.9 ± 5.7^a	99.9 ± 0	99.9 ± 0
CD62e	4.2 ± 3.4	3.2 ± 2.1	2.9 ± 1.9	6.0 ± 3.0
CD80	3.6 ± 3.6	3.3 ± 2.3	14.0 ± 2.4	15.4 ± 2.5
CD86	2.6 ± 1.6	3.2 ± 1.3	1.4 ± 0.3	1.2 ± 0.8
CD95	42.4 ± 3.4	59.2 ± 2.8^a	98.7 ± 0.9	99.5 ± 0.1
CD95L	39.6 ± 5.7	21.6 ± 3.2^b	7.6 ± 2.7	17.0 ± 0.8^a
CD120a	6.0 ± 3.7	6.6 ± 4.2	6.6 ± 3.5	26.4 ± 0.6^a
CD120b	7.3 ± 5,3	28. 3 ± 3.8^a	20.7 ± 2.5	20.8 ± 3.4
TNF	9.3 ± 3.6	6.7 ± 3.0	3.0 ± 1.4	3.9 ± 2.3
CD40L	2.0 ± 1.5	6.6 ± 3.5	1.1 ± 0.8	2.5 ± 0.9
PD-LI	99.0 ± 0.9	98.4 ± 0.6	27.5 ± 4.5	29.0 ± 2.5
HLA-ABC	99.9 ± 0.1	99.8 ± 0.1	99.8 ± 0.1	100.0 ± 0
HLA-DR	4.5 ± 2.6	3.2 ± 1.9	64.7 ± 2.4	79.6 ± 5.4^a

Data are presented as mean ± SD.

P < 0.05 compared with controls; Student’s t-test.

TNF, tumour necrosis factor; PD-L1, programmed cell death ligand-1; HLA, human leukocyte antigen.

RT–PCR analysis revealed that the Bax/Bcl-xl mRNA ratio was increased in both MDA-MB-231 and MDA-MB-435 cells at 4 h and 24 h after treatment with 2.5 µg/ml rhCD40L compared with untreated cells (Figure 2).

Figure 2.

Representative reverse transcription–polymerase chain reaction analysis showing the effects of 2.5 µg/ml recombinant human CD40-ligand on Bcl-xl, Bax and RANTES mRNA in MDA-MB-231 and MDA-MB-435 breast cancer cells.

Results of the MTT cell proliferation assay demonstrated that rhCD40L dose-dependently inhibited the growth of MDA-MB-231 cells in culture. The degree of inhibition was potentiated by combining rhCD40L with IFN-γ (P < 0.05, Figure 3) or doxorubicin (P = 0.027, Figure 4). Addition of IFN-γ to the combination of rhCD40L and doxorubicin further increased the level of inhibition (P = 0.018, Figure 4).

Figure 3.

Effects of recombinant human CD40-ligand (rhCD40L) alone and in combination with 500 U/ml interferon-γ (IFN-γ) on proliferation of MDA-MB-231 cells in culture as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Data are shown as mean ± SD of three experiments. *P < 0.05 compared with rhCD40L alone as determined by Student’s t-test.

Figure 4.

Effects of 2.5 µg/ml recombinant human CD40-ligand (rhCD40L) alone, 20 ng/ml doxorubicin (ADM) alone, or a combination of rhCD40L, ADM and 500 U/ml interferon-γ (IFN-γ) on proliferation of MDA-MB-231 cells in culture as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Data are shown as mean ± SD of three experiments. *P < 0.05 compared with untreated MDA-MB-231 cells; **P < 0.05 compared with rhCD40L alone as determined by Student’s t-test.

Discussion

Published studies have confirmed that CD40 is expressed in a variety of malignant cells.¹⁰ Following the activation or ligation of CD40, surface co-stimulatory molecules or adhesion molecules are increased, leading to the cell becoming an effective antigen-presenting cell.¹¹ Some tumours, including Burkitt's lymphoma, are resistant to IFN-γ but stimulation of CD40 signals can increase MHC-1, transporter for antigen processing-1 (TAP1) and endogenous antigen processing functions, meaning that specific cytotoxic T lymphocytes can effectively identify lymphoma.^12,13 It has been reported that activation of CD40 by rhCD40L in two breast cancer cell lines (MDA-MB-231 and MDA-MB-435) is associated with an increase in HLA-DR, which leads to high expression of CD40.^14,15 CD44 and CD54 are highly expressed on tumour cells and can stabilize cell contact through combination with their respective ligands,^14,15 thereby enhancing immunogenicity and promoting T cell activation. A variety of factors can promote RANTES expression; it has been reported that the combination of CD40 and IFN-γ increase secretion of RANTES in peritoneal mesothelial cells by up to 20-fold, therefore promoting interleukin (IL)-15 secretion.^14,15 Furthermore, it has been reported that IL-8, RANTES, monocyte chemoattractant protein and intracellular adhesion molecule-1 are increased in airway epithelial cells of asthma patients following rhCD40L activation.¹⁶ Similarly, data from the present study suggest that activation of tumour epithelial cells by CD40 mediates an increase in the transcription of RANTES. Activation of CD40 in tumour cells may, therefore, stimulate T cell chemotaxis via the expression of RANTES. These observations reflect an indirect mechanism for the anti-tumour effect of CD40 in tumour cells.

It has previously been demonstrated that triggering of CD40 on B cell lymphoma and multiple myeloma cells can lead to growth inhibition and/or apoptosis.¹⁷ Results from the present study indicate that rhCD40L can affect growth and/or apoptosis of MDA-MB-231 and MDA-MB-435 cells. It was also demonstrated that rhCD40L improved the sensitivity of tumour cells to the actions of doxorubicin and IFN-γ. This finding indicates that the clinical dose of chemotherapy agents could be reduced if used in combination with CD40-targeted therapies, which would help to avoid the side effects caused by their extensive use. The synergistic action of rhCD40L, chemotherapy drugs and cytokines also theoretically suggests that the dose of rhCD40L could be reduced in order to avoid the systemic impact of this molecule. Cell cycle analysis indicated that MDA-MB-435 and MDA-MB-231 entered into cell arrest in the Gl phase at 48 h and 72 h, respectively, following treatment with rhCD40L. The mechanism of cell arrest varies depending on cell type and the protein kinases involved.¹⁸ In the present study, inhibition of MDA-MB-231 and MDA-MB-435 cell growth by rhCD40L was dose-dependent and most likely due to both cell cycle arrest and apoptosis; cell arrest in the G₁ phase may lead to apoptosis of tumour cells that are not able to enter the normal cell cycle. At the level of gene transcription, the present study demonstrated that activation of CD40 improved the ratio of Bax/Bcl-xl mRNA in MDA-MB-435 and MDA-MB-231 cells. This finding indicates that levels of the pro-apoptotic protein Bax may increase more than that of that of Bcl-xl in response to rhCD40L treatment, which could promote tumour cell apoptosis. The data also suggest that relative concentrations of anti-apoptotic and pro-apoptotic proteins could be considered as a rheostat in the cell suicide process.

It has been reported that in normal B cells and other solid tumour cells, one of the mechanisms associated with apoptosis could involve activation of the CD95 (Fas) signalling pathway and TNF receptor-1 (TNFR-1 or CD120a).¹⁹ In the present study, high levels of CD95 were detected in MDA-MD-435 cell membranes after activation of CD40 although no changes were detected in TNF levels. CD120a and CD95L were also increased, indicating that the apoptotic signal may be mediated via the CD95 pathway. This, in turn, may change the ratio of Bax/Bcl-xl, both of which proteins are involved in the regulation of tumour cell growth.^20,21 Taken together the findings from the present study suggest that activation of CD40 acts as a signal to inhibit cell growth or/and promote apoptosis through a variety of different mechanisms. Therefore, CD40 represents an important target molecule since it exhibits direct anti-tumour effects.

In conclusion, the activation of CD40 signals can mediate a variety of anti-tumour effects including changes in cell phenotype, increases in the production of immune-stimulating cytokines, increases in the expression of T cell co-stimulatory molecules, improvements in tumour antigen presentation, and indirect stimulation and expansion of anti-tumour immunity. Furthermore, by changing the expression of TNF/TNFR family members, activation of CD40 causes tumour cells to enter into G1 phase arrest. This increases the ratio of pro-apoptotic Bax/anti-apoptotic Bcl-xl, which may induce apoptosis and directly inhibit tumour cell growth. CD40 signal intervention may represent an important and novel target for the biological treatment of breast cancer.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflict of interests.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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Effects of CD40 ligation combined with chemotherapy drugs on human breast cancer cell lines

Abstract

Objective

Methods

Results

Conclusion

Keywords

Introduction

Materials and methods

Detection of cell proliferation

Cell cycle analysis

Immunofluorescence analysis of membrane molecules

RT–PCR for analysis of Bcl-xl, Bax and RANTES mRNA

Cell proliferation determined by MTT assay

Statistical analyses

Results

Discussion

Footnotes

Declaration of conflicting interest

Funding

References