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. 2024 Mar 21;76(3):301–311. doi: 10.1007/s10616-024-00618-1

Anti-cancer drug-mediated increase in mitochondrial mass limits the application of metabolic viability-based MTT assay in cytotoxicity screening

Abhishek Kumar 1, Yogesh Rai 1, Anant Narayan Bhatt 1,✉
PMCID: PMC11082113  PMID: 38736730

Abstract

The high-throughput metabolic viability-based colorimetric MTT test is commonly employed to screen the cytotoxicity of different chemotherapeutic drugs. The assay assumes a cell density-dependent linear correlation with the MTT spectral absorbance. Therefore, the present study aimed to compare the cytotoxicity assessment between the MTT assay and gold standard cell number enumeration. The cytotoxicity was induced by Cisplatin, Etoposide, and Doxorubicin in human lung epithelial adenocarcinoma cells (A549) and cervix carcinoma (HeLa) cell lines. The mitochondrial mass was estimated, and immunoblotting of succinate dehydrogenase (SDH-A) was performed following drug treatment in both cell lines. Student’s t-test paired analysis was employed to calculate the significance of the results, where the value p < 0.05 was considered statistically significant. The drug-induced cytotoxic response estimated by MTT absorbance did not show any significant difference with respect to control, and no correlation was observed with the enumerated cell number in both A549 and HeLa cells. Interestingly, per-cell metabolic viability was found to be increased by 1.18 to 3.26-fold (p < 0.05) following drug treatment. Further, mechanistic investigation revealed a drug concentration-dependent significant increase in mitochondrial mass (1.21 to 4.2-fold) and upregulation of SDH protein (50–70%) as well as enzymatic activity with respect to control in both A549 and Hela cells. The limitation of the MTT assay for drug-induced cytotoxicity assessment is due to increased mitochondrial mass and SDH upregulation in surviving cells, leading to enhanced formazan formation. This leads to a lack of correlation between cell number and MTT spectral absorbance, suggesting that the MTT assay may provide an erroneous conclusion for cytotoxicity assessment.

Keywords: Anti-cancer drugs, MTT, Metabolic viability, Mitochondrial biogenesis, Cell growth inhibition

Introduction

MTT (3-(4,5‐dimethyl-thiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide) is a high throughput assay designed to assess the cytotoxic profile of drugs directly in cell lines in a multi-well format (Mosmann 1983). MTT is one of the most commonly used assays for preliminary clinical screening of anti-cancer or anti-neoplastic drug development (Vistica et al. 1991; Scudiero et al. 1988). The tetrazolium salts used in these assays get reduced by NADPH‐dependent oxidoreductase or dehydrogenases of viable cells into purple‐colored formazan, which can then be solubilized for spectrophotometric analysis (Gerlier and Thomasset 1986). The total amount of formazan produced upon MTT reduction is directly proportional to the number of viable cells in the culture. The MTT assay is favored over other assays because of its increased sensitivity and its potential as a miniaturized high-throughput assay. This cell viability-based enumeration technology made a significant advancement by efficiently and cost-effectively replacing the radioactive isotope-based 3 H-thymidine incorporation assay (Tonder et al. 2015). With time MTT assay has undergone various modifications, such as the inclusion of DMF to dissolve the formazan in a water-based solution, or the removal of excess dye by PBS washing, followed by dissolving the formazan crystals in DMSO (Hansen et al. 1989; Rensburg et al. 1997). These changes have enhanced the simplicity and sensitivity of the assay. Ensuring precise and dependable outcomes from in-vitro cytotoxicity studies is crucial throughout the initial phases of preclinical investigation and the data can have a substantial influence on the likelihood of the drug candidate progressing to the development stage.

In our earlier study, we reported the limitation of the MTT assay in determining the radiation-induced cytotoxicity in different tissue-origin cell lines ascribed to mitochondrial biogenesis and metabolic hyperactivation (Rai et al. 2018). In the present study, we aimed to investigate the reliability of MTT assay in the context of chemotherapeutic drugs. We hypothesize that any exogenous stress to the cells, be it of a genotoxic or cytotoxic nature, should contribute towards the increased mitochondrial metabolic activity of the affected cell.

To gain the wide acceptance of our hypothesis we selected drugs based on their different class, structure, and extensive research in different malignancies. We screened the cytotoxic potential of the three most commonly used chemotherapeutic drugs (Fig. 1A), i.e., Cisplatin, Etoposide, and Doxorubicin in lung adenocarcinoma (A549) and cervix cancer cells (HeLa). Cisplatin is a chemotherapeutic drug classified as an alkylating agent (Dasari and Tchounwou 2014). It is utilized in the treatment of many forms of cancer including bladder, head and neck, lung, ovarian, and testicular cancers. The mechanism by which it operates is associated with its capacity to form crosslinks with the purine bases on the DNA, disrupting DNA repair systems, resulting in DNA damage, and ultimately triggering apoptosis in cancer cells. Etoposide, in contrast, inhibits DNA repair enzyme topoisomerase II, which impedes DNA synthesis and repair by inducing structural disturbances in double-stranded DNA (Baldwin and Osheroff 2005). Furthermore, Doxorubicin is also a critical chemotherapeutic in the treatment of breast cancer, childhood solid tumors, soft tissue sarcomas, and aggressive lymphomas. It belongs to the anthracycline class of chemotherapeutic drugs. Doxorubicin is a DNA intercalator that also hinders the activity of topoisomerase II, thereby impeding the proliferation of cancer cells (Rivankar 2014).

Fig. 1.

Fig. 1

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MTT assay tends to underestimate the drug-induced cell growth inhibition. A Diagram showing the chemical structures of Cisplatin, Etoposide, and Doxorubicin B The inhibitory effect of Cisplatin, Etoposide, and Doxorubicin was evaluated in A549 i–iii) and HeLa cells iv–vi using an MTT assay. C Cell number estimation was performed at the indicated time points for the respective drugs in A549 i–iii and HeLa cells iv–vi. D The Bar graph represents metabolic viability per cell count derived from MTT values (ΔOD) normalized with the respective cell number in both A549 i–iii and HeLa cells iv–vi. Data are expressed as mean ± SD (n = 4) and *p < 0.05

The aforementioned drugs are reported to induce mitochondrial biogenesis, and oxidative stress in cancer cells. The limitation of the MTT assay is linked to increased mitochondrial biogenesis, ROS production, and altered calcium homeostasis (Rai et al. 2018). Therefore, it is reasonable to speculate that these drugs may also influence the predominant subcellular site of MTT reduction i.e. mitochondria. To examine this hypothesis, we correlated the cell viability data obtained from the MTT assay with the actual cell count observed for the respective cytotoxic concentrations, in this study. The cell viability using the MTT does not correlate with the cell numbers obtained for the individual drug concentrations, which are ascribed to the increased mitochondrial biogenesis in the cells in response to cytotoxic stress induced by these drugs.

Methods

Cell line propagation

The human lung epithelial adenocarcinoma cells (A549; ATCC no: CCL-185) and cervix carcinoma (HeLa; ATCC no: CCL-2) cells were obtained from NCCS, Pune, India. The cells were cultured in High glucose Dulbecco’s modified essential medium (Sigma Aldrich, USA) supplemented with 10% fetal bovine serum (Gibco, South American origin) and antibiotics (Penicillin G & Streptomycin, Sigma, USA) in a 60 mm tissue culture petri dish (BD Falcon, USA). The culture was maintained in the exponential growth phase by sub-culturing them in a ratio of 1:3 every three days (Kumari et al. 2020).

MTT assay and Formazan Imaging

The cells of both the cell lines were plated in 24-well culture plates (Corning, USA) with a density of 0.05 × 106 cells/ml (500 µl per well) and incubated overnight in a CO2 Incubator (NuAire, USA) with 5% CO2 at 37 ℃. Drug treatments [Cisplatin-232,120, Etoposide-E1383, and Doxorubicin-D1515 (Sigma, USA)] were given the following day according to the experimental requirements. Further, 50 µl MTT (Sigma, USA) solution from the Stock (5 mg/ml) to each well was added at respective time points, followed by 2 h incubation in a CO2 incubator. After MTT incubation, the images of Formazan crystals were captured under a bright field (20× objective) with an inverted microscope (Olympus, Japan) (Rai et al. 2018). Further, the medium was aspirated, and Formazan crystals formed by the cells were dissolved using 500 µl of DMSO. The absorbance was read at 570 nm using 630 nm as a reference wavelength on a Multi-well spectrophotometer (Biotech Instruments, USA).

Cell counting

At each respective time point and for each drug concentration, cell number was counted with a Neubauer-improved counting chamber (Paul Marienfeld GmbH & Co. KG, Germany) under 10× objective and 10× eyepiece magnification with a compound light microscope (Olympus CH30, Japan) (Rai et al. 2021).

Measurement of mitochondrial content

Quantitative analysis of mitochondrial content was carried out using Mitotracker Green (Invitrogen, USA) at respective time points, post-drug treatment as described previously (Rai et al. 2018). Cells were incubated with 100 nM of Mitotracker Green (in PBS) for 15 min at 37 ℃, then washed twice with PBS before being re-suspended in PBS for Fluorescence-based quantitative analysis. The Fluorescence was estimated at 490/516 (ex/em) on Synergy H1 Multi-mode Microplate reader (BioTek Instruments, USA), followed by normalization of each group with its respective cell number.

Immuno-blotting

The protein levels of SDH-A, and the loading control β-Actin were determined in control and drug-treated cells (HeLa and A549) by immunoblot analysis as described earlier (Sah et al. 2021). Cells were seeded in a 60 mm Petri Dish with a density of 0.6 × 106/PD, incubated at 37 ℃ overnight in a CO2 incubator, and treated the following day. Post-treatment at 24 h, cells were harvested and lysed in ice-cold RIPA lysis buffer containing protease inhibitors. A BCA protein assay kit (Thermo Scientific, USA) determined the protein concentration in cell lysates. Protein (60 µg) was resolved on 10–15% SDS–PAGE (depending on the molecular weight) and electroblotted onto a PVDF membrane (MDI, India). The membrane was then incubated in 4% skim milk (according to the manufacturer’s protocol) for 2 h, followed by primary antibody incubation SDH-A (1:1000) from Cell Signaling Technology, USA, β-Actin (1:5000) from Santa Cruz Biotechnology. The membrane was washed and incubated with the appropriate HRP-conjugated secondary antibody for 2 h. After washing, the blots were developed using an ECL chemiluminescence detection reagent (Biological Industries, Israel). The signal was detected by ECL, and band intensities for each protein were quantified by densitometry, corrected for background staining, and normalized to the signal for β-Actin.

Statistical analysis

The arithmetic mean and the error bars are presented in every bar graphic. Unless otherwise specified, each experiment consisted of three independent runs (n = 3). To conduct the analysis, GraphPad Prism8 and a two-tailed Student t-test were utilized. Results were considered significant if they had p-values that were lower than 0.05.

Results

MTT-based cytotoxicity screening did not show a correlation with cell number

We investigated the cytotoxicity of three widely used anti-cancer drugs, Cisplatin, Etoposide, and Doxorubicin, using the MTT assay in A549 and HeLa cell lines. The dose-dependent effect of all three drugs on cellular cytotoxicity was examined with screening for the effective cytotoxic concentration, coupled with one lower and higher range. A comparative assessment was carried out using MTT and cell number enumeration. Our results did not show any correlation between MTT and cell number at all the tested concentrations of drugs in both cell lines. The colorimetric absorbance of MTT (ΔOD) showed minimal change at 24 and 48 h post-exposure to drugs (Cisplatin, Etoposide, and Doxorubicin) treatment. At the same time, a significant difference in cell number was quantified in concentration and time-dependent manner in both the cell lines (Fig. 1B–C).

Normalizing the MTT (ΔOD) ratio to cell number revealed that drug-treated A549 and HeLa cells had increased metabolic viability per cell count, most evident at 48 h post-drug exposure (Fig. 1D). In contrast to the cell number-dependent MTT reduction, a considerable correlation was observed between concentration-dependent change in metabolic viability per cell count and cell number at 48 h post-drug treatment in both A549 and HeLa cells. To better understand this interdependent cellular stress behavior, cell growth inhibition versus (vs.) change in metabolic viability per cell count was estimated in all three tested drugs for both A549 and HeLa cells at 48 h post-treatment.

A concentration-dependent change in cell growth inhibition vs. relative increase in metabolic viability per cell count in the A549 cell line was estimated for Cisplatin 5, 10, and 20 µM (23% vs. 1.34-fold; 44% vs. 1.81-fold; and 54% vs. 1.88-fold), Etoposide 3.5, 7 and 15 µM (10% vs. 1.43-fold; 35% vs. 1.98-fold; and 62% vs. 3.04-fold) and Doxorubicin 0.1, 0.2 and 0.5 µM (20% vs. 1.18-fold; 39% vs. 1.5-fold; and 69% vs. 2.69-fold) at 48 h post-treatment (Fig. 1B–D). Likewise, in HeLa cells these effects were quantified for Cisplatin 3.5, 7 and 15 µM (50% vs. 1.61-fold; 65% vs. 2.39-fold; and 68% vs. 2.9-fold), Etoposide 3.5, 7 and 15 µM (26% vs. 1.27-fold; 60% vs. 2.22-fold; and 73% vs. 3.22-fold), and Doxorubicin 0.1, 0.2 and 0.5 µM (58% vs. 2-fold; 70% vs. 2.34-fold; and 74% vs. 3.26-fold). Despite increased cell growth inhibition in the surviving cells, intracellular metabolic activities undergo profound alteration and are found significant at both the tested time points of 24 and 48 h in HeLa cells (Fig. 1D iv-vi). However, this effect was confined to mainly at 48 h in A549 cells which was most likely ascribed to the tissue origin and time-based inherent sensitivity to the tested drugs (Fig. 1D i-iii). Nevertheless, in response to exogenous cellular stress, the extent of change in the MTT index was minimal compared to the substantial time-dependent decrease in the enumerated cell number of both A549 and HeLa cells.

These results imply that exogenous stress, either radiation (earlier report) or drug (present study), modulates the metabolic activity of viable cells, eventually influencing MTT absorbance and hence notwithstanding the notion of its cell number-dependent reduction into formazan.

The limitation of the MTT assay is attributed to the anti-cancerous drug-induced mitochondrial mass and upregulation of SDH

In a prior study, we reported that in response to radiation exposure, differences in cell number and MTT absorbance are attributed to mitochondrial biogenesis and metabolic hyperactivation (Rai et al. 2018). Therefore, we aimed to determine if a similar intracellular mechanism is at play in A549 and HeLa cells treated with Cisplatin, Etoposide, or Doxorubicin. Interestingly, despite the consequences of the drug-induced changes, nearly all drug-treated cells showed an overall significant increase in the mitochondrial mass following drug treatment at both 24 and 48 h.

In A549 cells, Cisplatin showed comparable change with control at the lowest used concentration of 5 µM; however, at 10 and 20 µM a significant change was notified as 1.61 and 2.21-fold, respectively, with respect to control at 48 h post-treatment (Fig. 2A i). Whereas for Etoposide (3.5, 7, and 15 µM) and Doxorubicin (0.1, 0.2, and 0.5 µM), a concentration-dependent significant change was observed ranging from a minimum of 1.21 to a maximum of 2.78-fold and 1.26 to 3.12-fold increase respectively (Fig. 2A ii-iii). Likewise, HeLa cells for Cisplatin (3.5, 7, and 15 µM) showed a 1.88 to 2.92-fold increase, whereas Etoposide at these concentrations was found in the range of 1.36 to 3.83-fold, and Doxorubicin (0.1, 0.2 and 0.5 µM) estimated as an increase of 2.53 to 4.20-fold (Fig. 2B i-iii). Maximum gains in mitochondrial mass were seen at the higher concentrations of Cisplatin, Etoposide, or Doxorubicin in both cell lines, suggesting a clear association between the magnitude of the stress imposed on the cells and the mitochondrial response (Fig. 2A-B). Consistent with our previous work, we also observed an increase in the expression of mitochondrial protein SDH, a key MTT-reducing enzyme following drug treatment in A549 and HeLa cells (Fig. 2C).

Moreover, as we observed in previous results, there is an interdependent change in cell growth inhibition and metabolic viability per cell count, which is ascribed to a profound change in the intracellular metabolic activity of surviving cells following exposure to anti-cancer drugs. Similarly, it was intriguing to observe that in response to these drugs, there was also an increase in mitochondrial mass per cell count, which showed a similar apparent change in increased metabolic viability of these cells.

Considering the results of our previous and current findings, here it is reasonable to speculate that despite reduced cell number following drug treatment, an increase in metabolic viability per cell count is attributable to increased mitochondrial mass per cell count, i.e., mitochondrial biogenesis, which in turn leads to more intracellular MTT reduction.

Anti-cancer drug treatment augments MTT conversion to formazan in an SDH-dependent manner

It is well evident that intracellular MTT reduction is predominantly based on mitochondrial SDH activity (Garn et al. 1994; Peng et al. 2005). Therefore, next, we sought to examine whether the trend of increased SDH activity and formazan production observed in our previous work after radiation treatment remains true for anti-cancerous drugs. All the tested drug concentrations and time points studied showed a significant increase in drug-induced formazan production compared to the respective controls, where the maximum difference is attained at the highest used drug concentration observed at 48 h in both cell lines (Fig. 3A–B). Further microscopic examination of untreated and treated cells showed a substantial increase in the perinuclear accumulation of formazan in drug-treated cells (Fig. 3C–D), in line with our previous study reported with radiation treatment (Rai et al. 2018). Likewise, this increase in formazan accumulation showed a plausible outcome of drug-induced higher mitochondrial mass and SDH upregulation.

Fig. 3.

Fig. 3

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Anti-cancer drugs induced SDH activity leading to enhanced formazan formation A-B. The bar blot shows the change in SDH activity corresponding to formazan formation in A549 A i–iii and HeLa B i–iii cells. The formazan was quantified using MTT (ΔOD) and the formazan standard curve in the indicated treatment groups. C, D. The photomicrographs show the visual change in formazan formation at 24 h post-drug treatment in A549 C and HeLa cells D. The bar plot is the quantitative correlation of relative change in formazan formation in both cell lines. The bright-field images were captured using (20⨯ objective and 10⨯ eyepiece magnification). Data are expressed as mean ± SD (n = 4) and *p < 0.05

Moreover, according to these findings, it appeared like a significant increase in anti-cancer drug-induced metabolic viability per cell count might be the consequence of an increase in mitochondrial mass, which is accompanied by an upregulation of SDH together, eventually contributing to more MTT reduction to formazan in both A549 and HeLa cells. These findings corroborate a robust interdependent relationship between metabolic viability, mitochondrial mass, and formazan formation, all of which ultimately contribute to treatment-induced higher spectrophotometric absorbance irrespective of the reduced cell number.

Discussion

The MTT assay is often used as the principal assay for in-vitro cytotoxicity screening and IC-50 determination of various anti-cancer drugs; however, excessive reliance on the MTT test for screening such chemotherapeutics is a cause for concern since its limitations have been outlined by several studies (Ulukaya et al. 2008; Han et al. 2010). Considering the significance of pre-clinical investigations in drug development, the present study aimed to carry out an extrapolated investigation to our earlier reported study, which is a limitation of MTT assay in estimating radiation-induced cell growth inhibition. Previously, we reported that radiation-induced metabolic hyperactivation and mitochondrial biogenesis are accountable for limiting the cytotoxicity determination based on metabolic viability assay, i.e., MTT (Rai et al. 2018). In similar experimental settings, MTT assay was employed for the cytotoxicity examination of anti-cancer drugs like Cisplatin, Doxorubicin, and Etoposide in HeLa and A549 cell lines. We hypothesize that exogenous cellular stressors, either radiation or anti-cancer drugs, both have the potential to modulate mitochondrial function. Consequently, this causes a significant discrepancy between mitochondria-dependent metabolic viability tests and the gold standard cell growth assay. After treatment with Cisplatin, Etoposide, and Doxorubicin, both A549 and HeLa cells exhibited a slight concentration-dependent change in MTT absorbance. This indicates the minimal cytotoxicity at the tested drug concentrations. On the contrary, upon the cell number enumeration, a huge difference was observed in a drug concentration-dependent manner. Given the fact that a linear change in MTT absorbance is a cell density-dependent process; however, a significant shift in MTT absorbance (from 24 to 48 h) was observed particularly at higher drug concentrations, when the obtained cell number is either similar or even reduced from 24 h (Fig. 1B–C). This raises an inquiry for the assessment of the predominant subcellular site and factors associated with MTT to formazan conversion, which might be contributing to the obtained difference in cell number and the MTT absorbance. In cells with an active metabolism, mitochondrial dehydrogenases primarily succinate dehydrogenase (SDH), convert the tetrazolium salt MTT to a water-insoluble purple formazan crystal. Previous studies demonstrate that the predominant MTT reduction site, i.e., complex II (C-II) of mitochondria, commonly known as SDH, plays a crucial role in the reprogramming of metabolic and respiratory adaptability in response to a wide range of endogenous and exogenous stressors (Bezawork-Geleta et al. 2017). Overexpression of SDH-A protein in all the drug-treated cells and per-cell SDH activity confirmed the potential role of SDH in the MTT reduction to formazan. Nevertheless, the huge shift in the MTT absorbance from 24 to 48 h cannot be solely attributed to only upregulated SDH response in drug-treated cells. In this context increased mitochondrial mass per cell in both A549 and HeLa cells following drug treatment corroborated our earlier findings (Rai et al. 2018). These results suggest that irrespective of the reduced cell number in the surviving cells an increase in SDH activity is associated with a high number of mitochondria per cell contributing to the increased metabolic viability per cell and increased MTT to formazan conversion (Figs. 1D and 2A–B). The proteomic, metabolomic, genomic, and epigenomic control research revealed that C-II/SDH is a critical regulator of mitochondrial and cellular metabolism (Bezawork-Geleta et al. 2017). Since mitochondrial biogenesis and turnover are ubiquitous mitochondrial stress-adaptive mechanisms, they allow tumor cells to endure stress conditions like radiation and chemotherapy (Jin et al. 2022). Chemotherapeutic drugs used in the study are reported to induce mitochondrial biogenesis (Shen et al. 2018; Fu et al. 2008; Dornfeld et al. 2021), and we found that associated complex-II activity interferes with the treatment response contributing to change in the assessment of cell viability. Considering these facts and findings in the study it is pertinent to note that per cell metabolic activity may change to overcome the energy stress required to promote the cell survivability. Consistent with our previous study, the present study in tumor cells using chemotherapeutic drugs confirmed that both exogenous stressors (drug/radiation) induce mitochondrial biogenesis in these cells. We found that an increase in metabolic viability per cell is directly proportional to an increase in mitochondrial mass (Figs. 1 and 2), demonstrating that mitochondrial mass is predominant in reducing MTT to formazan. Therefore, on a similar experimental platform, the result variation between MTT and gold standard cell number enumeration has been observed consistently, notwithstanding the toxic response as reflected by the actual cell number.

Fig. 2.

Fig. 2

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Anti-cancer drugs induced mitochondrial biogenesis and upregulation of SDH A-B. The bar graphs represent the relative change in mitochondrial mass following Cisplatin, Etoposide, and Doxorubicin treatment at indicated time points in A549 A i–iii and HeLa B i–iii cells. C Immunoblots of SDH protein expression were presented at 24 h following Cisplatin, Etoposide, and Doxorubicin treatment in A549 i and HeLa ii cells. The bar plot shows the relative change in SDH expression with respect to control. Data are expressed as mean ± SD (n = 4) and *p < 0.05

Conclusion

The experimental observations in the study revealed that chemotherapeutic drugs, regardless of their different chemical and structural classifications, have a significant impact on mitochondria. This supports the notion that intracellular signals related to cell survival or death converge on mitochondria, ultimately determining the cell fate. The extent of MTT reduction to formazan predominantly depends on the number of mitochondria per cell and complex-II dehydrogenase evidenced by increased mitochondrial mass and SDH activity following drug treatment. Therefore, findings in the study also corroborate our earlier results and suggest that mitochondrial activity must be taken into consideration for metabolic viability-based cytotoxicity assessment following exposure to exogenous stressors either physical (radiation; previous study) or chemotherapeutic drug (present study). Our findings demonstrate that the MTT assay used for in-vitro anti-cancer drug cytotoxicity evaluation or IC-50 determination may provide an erroneous conclusion.

Acknowledgements

The work was funded by DRDO, Ministry of Defense, Gov. of India. Dr. Sudhir Chandna, director of the Institute of Nuclear Medicine and Allied Sciences, provided the research facilities and infrastructure. Abhishek Kumar received fellowship support from CSIR throughout the study.

Author contributions

Conceptualization: ANB; Investigation: AK; Validation: YR; Formal Analysis: AK, YR; Resources: ANB; Data curation: AK, YR; Writing—Original draft Preparation: AK, YR; Writing—Review & Editing: ANB; Visualization: AK, YR; Supervision and Project Administration: ANB.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

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