Probiotics and postbiotics in colorectal cancer: Prevention and complementary therapy

Monika Kvakova, Anna Kamlarova, Jana Stofilova, Veronika Benetinova, Izabela Bertkova

Abstract Colorectal cancer (CRC) is a leading cause of human mortality worldwide. As conventional anticancer therapy not always being effective, there is growing interest in innovative “drug-free” cancer treatments or interventions that improve the efficacy of established therapy. CRC is associated with microbiome alterations,a process known as dysbiosis that involves depletion and/or enrichment of particular gut bacterial species and their metabolic functions. Supplementing patient treatment with traditional probiotics (with or without prebiotics), nextgeneration probiotics (NGP), or postbiotics represents a potentially effective and accessible complementary anticancer strategy by restoring gut microbiota composition and/or by signaling to the host. In this capacity, restoration of the gut microbiota in cancer patients can stabilize and enhance intestinal barrier function, as well as promote anticarcinogenic, anti-inflammatory, antimutagenic or other biologically important biochemical pathways that show high specificity towards tumor cells. Potential benefits of traditional probiotics, NGP, and postbiotics include modulating gut microbiota composition and function, as well as the host inflammatory response. Their application in CRC prevention is highlighted in this review, where we consider supportive in vitro, animal, and clinical studies. Based on emerging research, NGP and postbiotics hold promise in establishing innovative treatments for CRC by conferring physiological functions via the production of dominant natural products and metabolites that provide new host-microbiota signals to combat CRC. Although favorable results have been reported, further investigations focusing on strain and dose specificity are required to ensure the efficacy and safety of traditional probiotics, NGP, and postbiotics in CRC prevention and treatment.

Key Words: Colorectal cancer; Traditional probiotics; Next-generation probiotics;Postbiotics; Gut microbiota

INTRODUCTION

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females worldwide; thus a significant change in lifestyle is required to facilitate effective CRC prevention[1]. CRC is a heterogeneous disease of the intestinal epithelium, comprising the colon, rectum, and anus. It is characterized by a dysregulated immune response, accumulation of stem cell mutations,intestinal barrier disruption, and dysbiosis, which is often regarded as an unfavorable alteration in gut microbiota composition and function. Up to 90% of CRC risk is thought to be lifestyle-dependent,primarily due to dietary or environmental factors including feeding patterns that modulate consumption of fiber, red and processed meat or alcohol consumption, and low omega-3 fatty acids and vitamin D intake. Obesity, lack of physical activity, and smoking are also significant risk factors that promote CRC-associated microbiota changes[2]. CRC is linked with microbiome alterations, which include depletion and/or enrichment in particular bacterial species that are present in CRC patients(extensively reviewed by Torres-Maravillaet al[3], Terneset al[4], Janneyet al[2], Fonget al[5], and Wirbelet al[6]).

The human microbiota is a complex ecosystem of bacteria, viruses, eukaryotes, and archea, which can regulate a variety of host physiological functions including digestion, immune response, metabolism,disease pathogenesis, elimination of toxins, and biosynthesis of key compounds such as essential vitamins and cofactors. Microbiota can even modulate gut-brain axis function to alter, for example,anxiety and mood. Symbiotic bacteria that colonize the human gut can be classified into several phyla comprising Bacteroidetes and Firmicutes, followed by Proteobacteria, Fusobacteria, Actinobacteria,Verrucomicrobia, and Spirochaetes. Microbiome composition varies between healthy individuals, as well as in CRC patients[7]. CRC-associated bacteria that have been identified to date include enrichment ofFusobacterium nucleatum,Enterococcus faecalis,Streptococcus gallolyticus, entero-toxigenicBacteroides fragilis,Escherichia coli,Peptostreptococcusspp., andRuminococcusspp. By contrast,Lactobacillusspp.,Bifidobacteriumspp.,Faecalibacteriumspp.,Roseburiaspp.,Clostridiumspp.,Granulicatellaspp.,Streptococcus thermophilus, and other species ofLachnospiraceaefamily are depleted in CRC (Table 1)[2-5]. These altered microbiota signatures can potentially be used to provide future diagnostics, and their presence/absence may contribute to the pathogenesis or prevention/treatment of CRC. However, the pathophysiological role of dysbiosis in CRC still remains unclear, since microbiota changes may reflect changes in host health status and some bacteria may even confer protection as a compensatory response to disease progression. This complexity is clearly evident by the report of specific bacteria associated with tumor initiation phase (driver bacteria) whereas other bacteria are associated with tumor development during progressive stages of CRC (passenger bacteria). Driver bacteria reportedly contribute to the formation of a tumor microenvironment that is comprised of normal epithelial cells and cancer cells. In this milieu, secreted microbial metabolites trigger damage to normal host cells, thus reprograming their metabolism to change the intestinal microenvironment and microbiome profile towards a more “CRC supportive” composition[8-10]. Potential driver bacteria includeBacteroides fragilis,Escherichia coli,Enterococcus Faecalis,Bacillus,Bradyrhizobium,Methylobacterium,Streptomyces,Shigella,Citrobacter,Salmonella,Intrasporangiaceae, andSinobacteraceae. On the other hand, passenger bacteria occupy an existing tumor microenvironment where they are thought to either promote or inhibit CRC progression. Reported passenger bacteria include speciesFusobacterium,Parvimonas,Peptostreptococcus,Campylobacter,Streptococcus,Schwartzia,Burkholderiales,Caulobacteraceae,Delftia,Oxalobacteraceae,Faecalibacterium, andSutterella[8-11]. The host gut microbiota and immune system play important roles in CRC prevention and development. Therefore, probiotics, next-generation probiotics(NGP), or postbiotics could be used as weapons to prevent CRC, to support the treatment and to improve the clinical outcomes in CRC patients.

Table 1 Overview of the most relevant bacteria related to colorectal cancer

This minireview summarizes recent CRC findings from clinical, animal andin vitrostudies, and discusses the efficiency of probiotics, NGP, and postbiotics in CRC prevention and therapy.

TRADITIONAL PROBIOTICS

Probiotics are defined as “l(fā)ive, non-pathogenic microorganisms that, when administered in adequate amounts, may confer a health benefit on the host”[12]. Probiotics have a centuries-long history of safe use as prevention and adjuvant therapy in combating human diseases. They are also promising candidates in modulating human gut microbiota composition and function in CRC patients. Traditional widely used probiotics mainly belong toBifidobacteriumspp.,Lactobacillusspp. and other lactic-acidproducing bacteria, including species belonging toStreptococcus, Enterococcus, andLactococcus, complemented by yeasts of the genusSaccharomyces. The beneficial effects of probiotics, functioning in a species and/or strain-specific manner, include sustaining a healthy microbiome, reversing dysbiosis,preventing pathogenic infections and mucosal adhesion of pathogens, stabilizing and enhancing intestinal barrier function. Probiotic bacteria may achieve these beneficial functions in part by producing anti-carcinogenic, anti-inflammatory, anti-mutagenic and other biologically important compounds such as short-chain fatty acids (SCFAs), vitamin K, or B-group vitamins[5,7,13,14].

Current research builds on a foundation of work demonstrating that gut microbiota modulation through administration of probiotics and/or prebiotics plays an important role in CRC prevention and therapy. In a randomized, double-blinded, placebo-controlled trial, 60 patients underwent surgical CRC resection, of whom 29 received the probiotic powder (Bifidobacterium animalissubsp.lactisHY8002 [1 ×108CFU],Lactobacillus caseiHY2782 [5 × 107CFU], andLactobacillus plantarumHY7712 [5 × 107CFU]) and 31 placebo, for 4 wk, starting at 1 wk preoperatively. The treatment group receiving probiotic powder showed an increase in abundance ofBifidobacterium,Akkermansia,Parabacteroides,Veillonella,Lactobacillus,Erysipelatoclostridiumand a reduction in bacteria associated with CRC, such asPrevotella,Alloprevotella,Fusobacterium, andPorphyromonas. Lower serum zonulin, improved postoperative bowel function, and postoperative recovery were evident in the probiotic group compared with placebo[15]. In another randomized clinical trial, a group of 31 CRC patients received probiotic supplementBifidobacterium longumBB536 (5 × 1010CFU/2 g/daily) preoperatively for 7–14 d and postoperatively for 2 wk.Attenuated postoperative inflammatory responses (high-sensitivity C-Reactive protein), reduced risk of postoperative infectious complications, and accelerated health recovery after colorectal resection were evident in the treatment group. Hospital stay was significantly shortened and correlated significantly with increased Actinobacteria and decreased Firmicutes after probiotic intervention[16]. Aisuet al[17]administered BIO THREE?2 mgEnterococcus faecalisT110, 10 mgClostridium butyricumTO-A, and 10 mgBacillus mesentericusTO-A to 75 CRC patients 15 d prior to the surgery. Incidence of postoperative complications and superficial incisional infections were lower, and these health effects were as shown to associate with an increased mean proportion of beneficialBifidobacterium, postoperatively, even though this organism was not administered as part of the probiotic regime. The change in microbial diversity and improved integrity of the mucosal barrier were also observed by Liuet al[18] afterLactobacillus plantarumCGMCC 1258,Lactobacillus acidophilusLA-11,Bifidobacterium longumBL-88 (2.6 × 1014CFU/2 g/daily) administration 6 d preoperatively and 10 d postoperatively to CRC patients. The numbers of beneficial bacteria, includingBifidobacteriaandLactobacilli, increased in the probiotic group after surgery,whereas they decreased in the placebo group. By contrast, Enterobacteriales andPseudomonas, were decreased in the probiotic group whereas they increased in the placebo group. Based on a number of clinical trials, the preoperative oral intake of probiotics combined with the postoperative treatment in patients who need gastrointestinal surgery is potentially recommended. Larger rigorously controlled clinical trials are required to endorse these preliminary positive outcome studies since avoidance of probiotic use has also been recommended in patients with immunodeficiency and dysbiosis. More studies and the key outcomes are listed in Table 2.

Table 2 Efficiency of probiotics in colorectal cancer prevention and therapy-clinical trials

NGP

One potential approach to achieve CRC prevention and treatment is through NGP administration. As described above, the most frequently used probiotics belong toBifidobacteriumspp. andLactobacillusspp.However, recent studies using metagenomic approaches have revealed the importance of further identification and characterization of commensal species, mainly anaerobic ones, residing in the gastrointestinal tract that play an important role in regulating the immune system and maintaining overall gut health. Growing evidence suggests that dysbiosis may contribute to CRC progression as well as several other diseases[19-22]. Although there is no official definition of NGP, it is generally defined as live microorganisms identified on the basis of comparative microbiota analyses between healthy and sick individuals/animals that, when administered with strain-specificity and in dose dependent manner, confer health benefits on the host[23,24]. Compared with healthy individuals, patients with CRC possess a different compositional structure and physiological activity of the gut microbiota with SCFAs-producing bacteria being depleted. This suggests that SCFAs-producing bacteria might potentially exhibit anti-inflammatory and anticarcinogenic properties, as well as being NGP candidates in CRC prevention and therapy. SCFAs, primarily acetate, propionate, and butyrate, are key physiological metabolites of the microbial fermentation of dietary fiber in the colon. Butyrate is the major energy source for colonocyte homeostasis, promoting growth stimulation and production of protective cytokines that maintain gut barrier integrity and function[14,25-27]. Furthermore, increasing levels of SCFAs in the gut helps to create a favorable microenvironment for beneficial bacteria by inhibiting the growth and adhesion of pathogens, and by enhancing vitamin bioavailability, mineral absorption and promoting mucosal integrity. Most butyrate-producing bacteria in the human colon belong to the Firmicutes phylum, clostridial clusters IV and XIVa, the most dominant species beingFaecalibacterium prausnitziiandEubacterium rectale,followed byEubacteriumspp. as well asAnaerostipesspp. andRoseburiaspp. In addition to butyrate-producing bacteria, other NGP candidates with important regulatory effects on gut homeostasis includeAkkermansia muciniphila, non-toxigenicBacteroides fragilis,Propionibacterium freudenreichii,and some strains ofBacillusspp. andClostridiumspp.,which belong to Generally Recognized As Safe microorganisms[7,28,29].

Chronic oral administration ofButyricicoccus pullicaecorumBCRC 81109 (butyrate producing bacteria)to BALB/cByJNarl male mice decreased colon tumor progression over 9 wk. This protection against CRC clinical outcomes was linked to activation of the SCFAs transporter solute carrier family 5 member 8 and/or G-protein-coupled receptor (GPR) 43[30]. Chenet al[25] also observed in anin vivoanimal study that application of butyrate producing bacteriaClostridium butyricumATCC 19398 (2 × 109CFU/0.2 mL 3 times a week for 12 wk) inhibited intestinal tumor development by an increasing apoptosis of CRC cells, by modulating the Wnt/β-catenin signaling pathway. There was also a reduction in pathogenic bacteria and bile acid-biotransforming bacteria, whereas an increase in beneficialLactobacillusspp. and SCFAs-producingRumincoccaceaeandEubacteriumspp. was evident.Thus, reduction in colonic secondary bile acids increased cecal SCFAs levels and activated G-protein coupled receptors, GPR43 and GPR109A, which were mechanistically implicated. Growth of CRC cell lines (HCT-116 and SW1116) was significantly inhibited by strainsBacillus subtilisATCC 23857 andClostridium butyricumATCC 19398, and by their main metabolites bacitracin and butyrate. mRNA levels of important receptors and transcriptional factors related to inflammation for example, TLR4, MYD88,nuclear factor-kappa B (NF-κB), interleukin 22 (IL-22), and survivin were decreased and expression of p21WAF1was increased after treatment of SW1116 cells withBacillus subtilisandClostridium butyricumNGP[31]. Purified components produced by NGP cells were also studied and inhibition of human cancer cell proliferation by controlling the cell cycle was detected. Polysaccharide A purified fromBacteroides fragilisNCTC9343 (non-toxigenic) induced the production of the pro-inflammatory cytokine IL-8[32] and aspartic protease Amuc_1434 (recombinant enzyme) fromAkkermansia muciniphilaupregulated the expression of tumor protein 53, increased mitochondrial reactive oxygen species (ROS)levels and promoted apoptosis of LS174T cells[33]. Pahleet al[34] employedClostridium perfringensenterotoxin (CPE) in CPE gene therapy to selectively target claudin-3 and claudin-4 expressing colon carcinomasin vitroandin vivoby using a translation optimized CPE expressing vector. Elevated toxicity of the optimized CPE expressing vector was evident in claudin-positive cells 48 h after the transfection,with toxicity rates of 76%–92% and rapid cytotoxic effects such as membrane disruption and necrosis.Furtherin vivostudies focused on the efficiency of NGP application in CRC are listed in Table 3 and postbiotics derived from NGP are considered below.

POSTBIOTICS

Postbiotics is an extensively researched subject that remains a largely understudied topic in CRC. Due to the phenomenal number and variety of metabolites produced by bacteria, it has been an enormous challenge to isolate and characterize the specific compound/s responsible for the therapeutic efficacy.Moreover, defining safety profiles and appropriate application doses of particular postbiotics in the preclinical and clinical settings may require regulatory guidelines and approvals[5]. The InternationalScientific Association for Probiotics and Prebiotics (ISAPP) offers expertise in microbiology, microbial physiology, gastroenterology, nutrition, food science and pediatrics. ISAPP recently provided the clear definition and scope of postbiotics to include “preparation of inanimate microorganisms and/or their components that confer a health benefit on the host”[35]. Postbiotics, which exert desired physiological effects to the host, include inactivated microbial cells or cell components (cell surface proteins, endo- or exo-polysaccharides, peptidoglycan-derived muropeptides and teichoic acids) or important metabolites secreted by gut microbiota through a fermentation process or released under certain conditions such as a change in intestinal environment or after lysis (SCFAs including acetate, propionate and butyrate;enzymes; bacteriocins; reuterin; acetoin; organic acids,etc)[5,35,36]. Therefore, the isolation and characterization of new postbiotics is a growing field and requires careful biochemical characterization of beneficial mechanisms. Supplementation with postbiotics, can in some cases be an effective and safer strategy to prevent and/or treat diseases, compared with ingestion of viable probiotic bacteria[5].

Table 3 Efficiency of next-generation probiotics in colorectal cancer in vivo

Microbial metabolites undoubtedly play an important role in CRC pathogenesis. Certain postbiotics exert antitumor activity, including selective cytotoxicity against tumor cells suggesting their therapeutic potential (Figure 1)[5]. For example, SCFAs are well-known inhibitors of epigenetic enzymes histone deacetylases, which play a central role in gene regulation; thus, SCFAs have the ability to induce cell cycle arrest, and/or apoptosis in many cancer cell lines[37]. Cell-free supernatants (CFS) of differentLactobacillusandBifidobacteriumstrains have been shown to induce apoptosis or inhibit proliferation of CRC cell lines[38-40]. Chenet al[41] demonstrated that supernatants ofLactobacillus johnsoniiBCRC17010 andLactobacillus reuteriBCRC14625 strains in high concentrations were able to damage HT-29 cell membranes causing elevated lactate dehydrogenase release. A recent study has reported a potent selective cytotoxicity effect of postbiotic metabolites fromLactobacillus plantarumstrainsviaanti-proliferative effects and induction of apoptosis in HT-29 cells whilst sparing the normal cells[42]. Cousinet al[43] showed that metabolites fromPropionibacterium freudenreichiiITG-P9, namely propionate and acetate, had induced intrinsic apoptosis of CRC cells,viathe production and release of SCFAs acting on mitochondria. Moreover, CFS or SCFAs in combination with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), increased the pro-apoptotic gene expression (TRAIL-R2/DR5), decreased the anti-apoptotic gene expression of FLIP and XIAP in HT-29 cancer cells and enhanced the cytotoxicity in CRC cells compared to human healthy intestinal epithelial cells. Further control studies are required to delineate specific molecular targets in these models since enhanced toxicity to fermentation induced acidic pH shifts remains a potential protective mechanism.

Figure 1 Examples of postbiotics and their proposed activity in patients with colorectal cancer. SCFAs: Short-chain fatty acids.

As inflammation is undeniably linked to carcinogenesis, any postbiotic that inhibits inflammation is also an important candidate acting as anti-tumor agent. It was shown thatLactobacillus rhamnosusGGderived protein p40 can play a role in the prevention of CRC by suppressing intestinal epithelial inflammation, inhibiting epithelial cells apoptosis and by promoting IgA production[44-46]. CFS derived from several other probiotic strains, such as,Lactobacillus acidophilus,Lactobacillus casei,Lactobacillus rhamnosusGG andBifidobacterium breve, were able to downregulate inflammation, exhibit antioxidant activity or maintained intestinal barrier integrity[47-49].

To date, only a few animal studies have been performed to evaluate the effectiveness of postbiotics in CRC prevention and therapyin vivo. The stage is now set to expand this work with the use of translationalin vivomodels and clinical trials, which are essential to demonstrate efficacy. Sharma and Shukla[50] observed that CFS fromLactobacillus rhamnosusMD 14 MH656799 containing acetamide, acetate,propionate, butyrate, thiocyanic acid and oxalic acid attenuated early colon carcinogenesis in Sprague–Dawley rats (n= 36). The protective mechanism was linked to reduced fecal procarcinogenic enzymes, oxidants, aberrant crypt foci, vis-à-vis downregulating oncogenes (β-catenin, K-ras, Cox-2, NFκB) and upregulating tumor suppressor p53 gene leading to an almost healthy colon histology. De Moreno de LeBlancet al[51] evaluated the effect of the enzyme catalase as a postbiotic from catalaseproducingLactococcus lactishtrA-NZ9000 on the prevention/regression of 1,2-dimethylhydrazine(DMH) induced CRC in BALB/c mice (n= 180-210). Catalase-producingLactococcus lactisincreased catalase activity in DMH-treated mice and reduced H2O2levels compared with the control group. Using the histopathological grading scale of chemically induced CRC, mice that received catalase-producingLactococcus lactishad significantly less colonic damage and inflammation (2.0 ± 0.4) compared to control animals that received non-catalase-producingLactococcus lactis(4.0 ± 0.3) or placebo-treated animals (4.7± 0.5). Increased antioxidant activity reduced levels of H2O2and ROS involved in CRC onset and progression.

There are also promising results from studies of postbiotics derived from NGP. Recently, numerousin vitrostudies showed that supernatant from SCFAs-producing bacteria, such asButyricicoccus pullicaecorumBCRC 81109[30],Clostridium butyricumATCC 19398[25],Propionibacterium freudenreichiiTL142[52],Propionibacterium acidipropioniciCNRZ80,Propionibacterium freudenreichiisubsp.freudenreichiiITG18,Propionibacterium freudenreichiisubsp.shermaniiSI41[53] suppressed CRC cells proliferation and induced apoptosis. The same results were documented by Zhaoet al[54], where single strain CFS from humanBacillusstrains BY38, BY40, BY43, BY45 exhibited inhibitory effects on the proliferation of CRC cells in a dose-dependent manner through the induction of cell apoptosis. These results suggest that NGP could represent novel and promising anti-tumor agents against CRC. Furtherin vitrostudies focused on the activity of postbiotics derived from different probiotic strains in CRC cell lines are listed in Table 4.

CONCLUSION

Traditional probiotics have utility in the management of CRC as adjuvant treatment, mainly to reduce postoperative complications and to alleviate the side effects of chemotherapy. Antitumorigenic mechanisms of probiotics include the modification of intestinal microbiome, improvement of intestinal barrier integrity, immune potentiation and maintaining gut homeostasis. However, it is well known that the efficiency of probiotics is strain specific. The available clinical data indicate that CRC patients mostoften benefit from combined administration of strainsLactobacillus acidophilus,Lactobacillus casei,Bifidobacterium lactis, andBifidobacterium longum. Use of their combination or in combination with other species is more effective than individual supplementation. Nevertheless, consideration of each CRC patient’s health status is still strictly recommended before administering viable probiotics. The gut microbiota is emerging as a contributing factor in the etiopathology of CRC. It is necessary to consider gut microbiota-drug interactions, including composition and metabolic activity of gut microbiota, which can both positively and negatively affect the outcome of CRC therapy. And even though research in this area is still in its infancy, it can be assumed that future clinical treatment and prevention of CRC will focus on supplementing the microbiome with commensal species (NGP candidates) that are predominantly anaerobic. Recent studies indicate that SCFAs-producing bacteria, especially butyrate producers,such asAkkermansia muciniphila,Propionibacterium freudenreichi, andButyricicoccus pullicaecorumbelong to beneficial NGP that may have applicability in CRC therapy. Furthermore, it was discovered that strains previously defined as potential pathogens appear to possess probiotic properties when these lack key virulence factors, for example non-toxigenicBacteroides fragilisNCTC9343 has positive effects on patient's health. A significant disadvantage of NGP is, above all, their safety as this has not yet been sufficiently confirmed in animal and clinical studies. Safety validation is of particular importance before administering NGP to oncology patients. Although NGP research is experimentally demanding,emerging data shows great potential. Therefore, it is necessary to continue and explore new possibilities of NGP use in the therapy or prevention of diseases, including CRC, especially through clinical trials.Supplementation with postbiotics should be favorable in CRC therapy, because postbiotics have the ability to stimulate immune responses, inhibit cancer cell proliferation, induce apoptosis and necrosis,and they can shape microbiome composition in CRC patients. The advantage of postbiotics is that they do not pose a risk of unwanted infection to the patient, although screening for product contamination will be important. Moreover, it is possible to accurately determine and verify administered doses of a particular postbiotic. However, this emerging research area currently lackin vivoor clinical data to assess feasibility. In conclusion, the administration of traditional probiotics, NGP or postbiotics,supported by various experimental studies, is an efficient complementary therapeutic approach to combat CRC. A protective effect of probiotics and postbiotics against CRC onset is also indicated,however, lifestyle changes are recommended as a first line of defense in CRC prevention.

ACKNOWLEDGEMENTS

We are thankful to Tor C Savidge, PhD for the editorial assistance.

FOOTNOTES

Author contributions:All authors contributed equally to the conceptualization and design of the manuscript;Kvakova M prepared and finalized the manuscript; Kamlarova A, Stofilova J, and Benetinova V reviewed the literature, and prepared the tables and figure; Bertkova I as senior author revised the manuscript; all authors approved the final manuscript.

Supported byScientific Grant Agency of the Ministry of Education of Slovak Republic and Academy of Sciences VEGA, No. 1/0393/20; and the Operational Program Integrated Infrastructure Within the Project: Demand-Driven Research for the Sustainable and Innovative Food, Co-financed by the European Regional Development Fund, No.Drive4SIFood 313011V336.

Conflict-of-interest statement:All the authors report no relevant conflicts of interest for this article.

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Country/Territory of origin:Slovakia

ORCID number:Monika Kvakova 0000-0002-9659-5584; Anna Kamlarova 0000-0001-7871-5338; Jana Stofilova 0000-0002-9409-4153; Veronika Benetinova 0000-0001-9523-8203; Izabela Bertkova 0000-0003-3076-2711.

S-Editor:Fan JR

L-Editor:Filipodia

P-Editor:Fan JR