Abstract
Background/Aim: In the pursuit of translating stem cell therapy technology into clinical practice, ensuring the safety and efficacy of treatments is paramount. Despite advancements, the effectiveness of stem cell applications often falls short of clinical requirements. This study aimed to address the challenge of limited efficacy by investigating the safety and effectiveness of canine adipose tissue-derived mesenchymal stem cells (cATMSCs) preconditioned with deferoxamine (DFO). Materials and Methods: Different concentrations of DFO were used to evaluate its impact on cATMSC activity. The therapeutic potential of these preconditioned cells was validated using a mouse model of systemic inflammation. Comprehensive evaluations, including clinical hematological and radiological assessments before and after intravenous injection of preconditioned cells were conducted. Results: The study showed a notable reduction in inflammatory markers and an overall decrease in the inflammatory response in the mouse model. The data collected from the clinical hematological and radiological assessments provided essential insights. Conclusion: This study lays the groundwork for the future clinical deployment of DFO-preconditioned cATMSCs, demonstrating their potential to improve the efficacy and safety of stem cell therapies.
The rapid progress in stem cell therapy technology marks a pivotal breakthrough in modern medicine, providing innovative solutions for a broad spectrum of diseases. Its application in regenerative medicine and tissue engineering is particularly significant, with the potential to transform current methodologies for disease management and tissue restoration (1). However, translating these advancements into clinical settings faces numerous obstacles, particularly in achieving both efficacy and safety (2).
Mesenchymal stem cells (MSCs), as adult stem cells, are present in various tissues, including bone marrow, adipose tissue, placenta, umbilical cord blood, liver, and lungs (3, 4). Adipose tissue-derived MSCs (AT-MSCs) have emerged as a focal point of research in recent years, characterized by their ease of acquisition, diverse and abundant sources, extensive extraction capability, and maintenance of pluripotency (5). These features render AT-MSCs highly promising for widespread applications in regenerative medicine and tissue engineering. Recently, the treatment of small animal diseases has highlighted variations in the characteristics and therapeutic outcomes using stem cells derived from different sources (6). Bone marrow-derived MSCs have been widely used in the treatment of muscle injury, bone fracture and joint diseases. However, some studies have pointed out that compared with bone marrow MSCs, adipose tissue-derived MSCs have stronger angiogenic ability and immunomodulatory function. Therefore, different cell types should be selected for different therapeutic purposes (7, 8). In response, extensive research efforts have been dedicated to exploring various strategies to enhance the therapeutic potential of stem cells, ultimately aiming to improve the efficacy and efficiency of treatments for diseases of pets.
Within the field of regenerative medicine, the strategic preconditioning of canine AT-MSCs (cATMSCs) to enhance their paracrine functions has garnered significant interest (9, 10). This strategy aims to amplify their inherent therapeutic potential, offering promising avenues for advanced treatments. Simultaneously, several studies have identified deferoxamine (DFO) as a critical agent for enhancing cellular preconditioning (11-13). As an effective inhibitor of prolyl-hydroxylase, DFO stabilizes hypoxia-inducible factor 1-alpha (HIF-1α) in normoxic environments, promoting cell adaptability under various physiological conditions (14). This attribute is particularly advantageous for cATMSCs, as DFO not only bolsters their survival and proliferation rates but also significantly enhances their capacity for angiogenesis (15).
However, the safety profile of DFO-preconditioned cATMSCs remains largely unexplored. While the therapeutic advantages of DFO are clear, comprehensive studies assessing its safety implications in preconditioning of cATMSCs are essential. This gap in knowledge underscores the need for rigorous preclinical evaluations to establish the safety of DFO preconditioning in cATMSCs, ensuring their application in veterinary medicine is both effective and secure. This study focused on evaluating the effects of DFO preconditioning on cATMSCs. We aimed to systematically investigate the impact of different concentrations of DFO on the biological activities of these cells. Our approach included comprehensive in vivo experiments, along with detailed hematological and radiological assessments, to establish a foundational understanding of the role of DFO in modulating cATMSC properties.
Materials and Methods
Cell isolation and characterization. Adipose tissue was obtained from three healthy, 1-year-old dogs during ovariohysterectomy at Yanbian University Veterinary Medicine Teaching Hospital. The owners provided informed, written consent for research use. The procedure was approved by the Institutional Animal Care and Use Committee of Yanbian University and performed in accordance with approved guidelines (YD20240510013). cATMSCs were isolated and cultured as previously described (16). Before use in this study, cells were characterized by their expression of several stem cell markers using flow cytometry. Cells were suspended in 30 μl phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1 μl monoclonal antibodies against the following proteins: Fluorescein isothiocyanate (FITC)-conjugated cluster of differentiation 29 (CD29), CD31-FITC, phycoerythrin-conjugated CD34, phycoerythrin-conjugated CD73 (BD Biosciences, Franklin Lakes, NJ, USA), CD44-FITC, CD45-FITC, and CD90-allophycocyanin (eBiosciences, San Diego, CA, USA)-conjugated antibodies. Non-stained cells were used as controls for autofluorescence. Cells were analyzed using a BD FACSAria II system (BD Biosciences). Cellular differentiation was confirmed using PRIME-XV® Chondrogenic Differentiation Xeno-Free Serum-Free Medium, PRIME-XV® Osteogenic Differentiation Serum-Free Medium, and PRIME-XV® Adipogenic Differentiation Serum-Free Medium (all from Irvine Scientific, Santa Ana, CA, USA) according to the manufacturer’s instructions, followed by Alcian Blue staining, Alizarin Red staining, and Oil Red O staining, respectively (Sigma-Aldrich, St. Louis, MO, USA).
Cell proliferation assay. To evaluate the impact of DFO (Sigma-Aldrich) on the viability of cATMSCs, a range of DFO concentrations (25, 50, 100, 200, 400, and 800 μM) was applied to the cells, with PBS used as a control. After incubation for 24 h, cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay (Apexbio, Houston, TX, USA), and absorbance was measured at 450 nm using a spectrophotometer (Bio-Rad Microplate Reader Model 680; Bio-Rad Laboratories, Hercules, CA, USA). Following the determination of the optimal DFO concentration, its effect on cATMSC proliferation was further examined at 24, 48, and 72 h after treatment using CCK-8 assays.
Development of a mouse model of systemic inflammation. Twelve male C57BL/6 mice, each weighing approximately 20-25 g, were acquired from the Yanbian University animal center and were divided into four groups with three replicates in each group in the experiment: control (naïve), lipopolysaccharide (LPS)+PBS, LPS+cATMSC and LPS+DFO-treated cATMSC (DFO-cATMSC). All mice were maintained in a specific pathogen-free environment, with controlled temperature (20-22°C), humidity (50±5%), and a 12:12-h light-dark cycle.
Systemic inflammation was induced in the mice through an intraperitoneal injection of 10 mg/kg LPS sourced from Escherichia coli (O55; Sigma-Aldrich). Three hours post-LPS administration, mice in the LPS+PBS group received an injection of sterile PBS intraperitoneally. Additionally, the LPS+cATMSC and LPS+DFO-cATMSC groups were administered intraperitoneal injections of 5×106 native cATMSCs or cATMSCs preconditioned with DFO, respectively. The experiment concluded 24 h after the initial treatments, at which point all mice were euthanized for subsequent analyses.
Histopathological analyses. Following euthanasia, lung and liver tissues were collected for histopathological examination. These tissues were first fixed in 10% formaldehyde solution to preserve cellular details and structure, then embedded in paraffin to facilitate sectioning. For microscopic analysis, 5-μm sections were prepared from the embedded tissues. Each section was subsequently stained with hematoxylin and eosin to highlight cellular features and tissue architecture under microscopy.
RNA extraction and real-time quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from both in vivo mouse experimental tissues, namely the lung and liver, and from in vitro cell cultures of cATMSCs and DFO-preconditioned cATMSCs, using TRIzol™ Reagent (Invitrogen, Waltham, MA, USA). Subsequently, cDNA synthesis was performed employing a FastKing one-step kit (Tiangen Biotech, Beijing, PR China). The analysis of gene expression was carried out with SYBR premix SuperReal PreMix Plus Kit (Tiangen Biotech), applying 400 nM of both forward and reverse primers, as given in Table I. These procedures were executed in an Agilent Mx3000/5p thermocycler (Agilent Technologies, Santa Clara, CA, USA). The expression level of each gene was normalized to that of the reference gene glyceraldehyde 3-phosphate dehydrogenase according to the 2−CΔΔCt method. The relative gene expression was then calculated in comparison to the control group.
Primer sequences for quantitative real-time polymerase chain reaction amplification of canine and mouse target genes.
Enzyme-linked immunosorbent assay (ELISA). ELISA was performed to measure protein levels of tumor necrosis factor alpha-induced protein 6 (TSG6, TNFAIP6) in cell culture supernatant using Canine TSG-6 ELISA kit (MyBioSource, San Diego, CA, USA) according to the manufacturer’s instructions. Samples were added to antibody-coated wells, followed by incubation, addition of a detection antibody, and substrate for color development. The optical density was measured to determine protein concentrations relative to a standard curve.
Clinical trial procedures. To evaluate the safety profile of DFO-preconditioned cATMSCs, three healthy canines were selected from health screenings conducted at the Yanbian University Veterinary Medicine Teaching Hospital. The selected canines underwent intravenous administration of DFO-cATMSCs into the forelimb, with a cell dosage calibrated at 1×106 cells/kg. The cells, suspended in 15 ml of PBS, were administered over 15 min using an infusion pump to minimize the risk of thrombosis. Physical examinations, and hematological and serological assessments were conducted prior to and 1 h after the injection to identify any immediate adverse reactions or signs of systemic inflammation attributable to the cell therapy. Additionally, to assess long-term safety and investigate any potential oncogenicity of the injected cATMSCs, comprehensive follow-up ultrasound examinations were performed 3 months after administration.
Laboratory examinations. Blood samples were collected from the three canines as required for comprehensive hematological and biochemical analyses. The hematological parameters evaluated included white blood and red blood cell counts; hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell volume distribution width-coefficient of variation; differential count of eosinophils, basophils, lymphocytes and monocytes; and platelet count. Serum biochemistry assessments included measurements of C-reactive protein, albumin, total proteins, globulin, albumin/globulin ratio, total bilirubin, aspartate aminotransferase, alanine aminotransferase, amylase, creatine kinase, creatinine, blood urea nitrogen, glucose and triglycerides.
Statistical analysis. All experimental data were analyzed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). One-way analysis of variance or Student’s t-test was used for between-group differences. Statistical significance was set at p<0.05.
Results
Characterization of cATMSCs. To identify the expression of surface antigens on the isolated cells for cell characterization, this study utilized flow cytometry. cATMSCs were successfully cultured and expanded to passage 3, and the majority of the cells expressed the established stem cell markers CD29 (98.5%), CD73 (96%), and CD90 (75.2%), while very few cells expressed CD45 (2.1%) or CD34 (0.0%) (Figure 1A). The cATMSCs demonstrated multilineage plasticity, evident in their capacity for adipogenic, osteogenic, and chondrogenic differentiation (Figure 1B). After induction into the adipogenic lineage, the AT-MSCs stained with Oil Red O displayed abundant bright red circular lipid droplets within their cytoplasm. Following osteogenic differentiation induction, the cells stained with Alizarin Red revealed extensive red calcified nodules. Moreover, upon chondrogenic differentiation induction, the AT-MSCs stained with Alcian Blue exhibited a deep blue chondrogenic matrix, with cells presenting an irregular granular appearance.
Characterization of canine adipose tissue-derived mesenchymal stem cells (cATMSCs) based on surface marker expression and differentiation potential. (A) Flow cytometric histograms showing the expression of mesenchymal stem cell markers. (B) Potential of cATMSCs to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Magnification, 200×.
Effects of DFO on cATMSC proliferation. To determine the influence of DFO on cATMSCs, the proliferation rates of cells treated with different concentrations of DFO (0, 25, 50, 100, 200, 400, and 800 μM) were measured after a 24-h incubation period. Notably, cells exposed to 50 and 100 μM DFO showed the most pronounced increase in proliferation (Figure 2A). Figure 2B indicates that 50 μM might represent the optimal concentration for promoting cell growth. Moreover, Figure 2B revealed that the proliferation rate of DFO-treated cells was consistently and significantly elevated in comparison to naïve cells (p<0.01), underscoring the potential of 50 μM DFO as an effective preconditioning agent for enhancing cell proliferation. This sustained proliferative capacity indicates that DFO at this concentration may promote a favorable condition for cell growth without inducing cytotoxic effects over the tested duration.
Evaluation of deferoxamine (DFO) preconditioning on canine adipose tissue-derived mesenchymal stem cells (cATMSCs). (A) Cell viability assay results showing the effects of different concentrations of DFO on cATMSCs over a 24-h period compared to untreated cells. (B) Proliferation kinetics of cATMSCs treated with DFO at concentrations of 50 μM and 100 μM compared to untreated (naïve) cells. (C) Quantitative real-time polymerase chain reaction analysis of tumor necrosis factor alpha-induced protein 6 (Tsg6), interleukin-10 (Il10), and interferon-γ (Ifng) mRNA expression in cATMSCs following DFO treatment. (D) Enzyme-linked immunosorbent assay (ELISA) analysis of TSG6. Data are expressed as the mean±standard deviation obtained from three independent experiments. Values with different letters are significantly different at p<0.05 according to one-way analysis of variance. ns: Not statistically significant. Significantly different at *p<0.05, **p<0.01 versus the naive cells by Student’s t-test.
Effects of DFO on immunomodulation of cATMSCs. To elucidate the mechanisms behind the immunomodulatory effects of DFO preconditioning on cATMSCs, the gene expression of key immunoregulatory factors was assessed after 48 h of DFO treatment. The results indicated that DFO preconditioning significantly upregulated the gene expression of Tsg6 in AT-MSCs (Figure 2C). However, no significant enhancement was observed in the expression of the other immunoregulatory cytokines, Il10 and Infg. Following gene expression profiling, TSG6 protein concentration in the cell culture supernatant was quantified using an ELISA. Consistent with the upregulation of the mRNA level, a marked increase in TSG6 secretion was detected in the DFO-cATMSCs (Figure 2D).
Effects of DFO-cATMSCs on the mouse model of systemic inflammation. The lung and liver tissues from a mouse model of systemic inflammation were analyzed for histopathological alterations and gene expression changes to determine the modulatory effects of DFO-cATMSCs in vivo. Histopathological evaluation of the lung tissue sections (Figure 3A) indicated a disruption of alveolar architecture after LPS stimulation, characterized by extensive inflammatory cell infiltration, alveolar space occupation, and significant capillary dilation with hemorrhage. Treatment with cATMSCs markedly mitigated these pathological alterations, with DFO-preconditioned cATMSCs exhibiting the most profound effect in maintaining tissue integrity. Correspondingly, liver tissue specimens (Figure 3B) demonstrated similar patterns of histological improvement. The group treated with DFO-cATMSCs presented a notable reduction in inflammatory cell infiltration and tissue damage compared to that treated with LPS+PBS. Parallel to the histological assessments, gene expression analyses of pro-inflammatory cytokines showed a significant downregulation of Tnfa, Il1β, and Il6 in the cATMSC-treated groups in contrast to the LPS+PBS-treated group. The expression levels in the DFO-preconditioned group were lower than those in the group treated with cATMSCs without preconditioning, suggesting a potent anti-inflammatory modulatory capacity of DFO-preconditioned cATMSCs (Figure 3C and D).
Histopathological analysis and gene expression profiling in a mouse model of systemic inflammation. Histological examination of lung (A) and liver (B) tissues stained with hematoxylin and eosin at 200× and 400× magnification, showing structural changes across different treatment groups: Naïve, lipopolysaccharide (LPS+PBS), LPS+canine adipose tissue-derived mesenchymal stem cells (cATMSCs), and LPS+deferoxamine (DFO)-preconditioned cATMSCs. Quantitative real-time polymerase chain reaction analysis of gene expression of pro-inflammatory cytokines, tumor necrosis factor-α (Tnfa), interleukin-1β (Il1b), and interleukin-6 (Il6), in lung (C) and liver (D) tissues. Data are expressed as the mean±standard deviation. Values with different letters are significantly different at p<0.05 according to one-way analysis of variance.
Preclinical safety assessment of DFO-cATMSCs. To evaluate the preclinical safety profile of DFO-cATMSCs, the cells were injected into three healthy canines, followed by comprehensive physical, hematological and serological assessments. The results presented in Table II and Table III indicate that there were no notable changes in any of the parameters measured before and after the injections, suggesting no abnormalities attributable to the cell therapy. Further evaluation of vital parameters, including heart rate, respiratory rate, and body temperature, did not reveal any abnormal variations (Figure 4A). Additionally, C-reactive protein assays performed on blood samples showed no evidence of an inflammatory response (Figure 4B).
Complete blood count results before and after injection of deferoxamine-preconditioned canine adipose tissue-derived mesenchymal stem cells into three healthy canines. Data are the mean±standard deviation.
Serum chemistry results before and after injection of deferoxamine-conditioned canine adipose tissue-derived mesenchymal stem cells into three healthy canines. Data are the mean±standard deviation.
Preclinical safety evaluation of deferoxamine (DFO)-preconditioned canine adipose tissue-derived mesenchymal stem cells (cATMSCs). (A) Assessment of vital physiological indices namely heart rate, respiratory rate and temperature after injection of DFO-preconditioned cATMSCs. (B) Serum analysis of C-reactive protein (CRP) levels as an indicator of the inflammatory response following administration of DFO-preconditioned cATMSCs. (C) Ultrasonographic examination of the major abdominal organs, including the kidneys, urinary bladder, liver, and spleen. Data are expressed as the mean±standard deviation. ns: Not statistically significant by Student’s t-test.
Three months post-injection, ultrasonographic examination of the abdominal cavity provided comprehensive insights. The renal sonographic appearance for all three canines fell within normal criteria, characterized by kidneys with homogenous echotexture, smooth renal contours, and clearly delineated corticomedullary junctions. No ultrasonographic evidence of nephrolithiasis or neoplastic lesions was detected. The urinary bladder demonstrated normal wall thickness with a smooth, echogenic mucosal interface, devoid of intraluminal masses. The sonographic content of the bladder was unremarkable, with the trigone region free from mass effect. Hepatic sonograms revealed liver sizes within expected limits, showcasing a uniform, intermediate echogenic pattern with no vascular anomalies or focal lesions. Gallbladder sonography depicted well-distended lumens, walls without thickness variations, and an absence of cholecystic sediment or calculi. Visual inspection did not extend to the common bile duct. Spleen sonography affirmed normal organ size, with intact capsule integrity and typical anatomical positioning. The spleen exhibited homogeneous, finely granular parenchyma with echogenicity surpassing that of the renal cortex and liver, aligning with normal sonographic standards (Figure 4C).
Discussion
Current research on cATMSCs extends beyond merely understanding their biological attributes to focusing on significantly enhancing their therapeutic efficacy through various preconditioning strategies. Among these, cytokine and hypoxia preconditioning are prominent (17, 18). Of particular interest, DFO as a hypoxia mimetic offers distinct advantages due to its compatibility with the natural in vivo growth environment of stem cells and superior stability (19). Consequently, this study aimed to explore the effects of DFO preconditioning on cATMSCs and assess its potential to improve their therapeutic efficacy and preclinical safety.
Prior to evaluating the impact of DFO on cATMSCs, the isolated cells underwent a comprehensive characterization process. Flow cytometry revealed a significant expression of surface markers CD29, CD73, and CD90, coupled with minimal expression of CD45 and an absence of CD34 expression (Figure 1A). Moreover, the cells demonstrated multipotent differentiation capabilities (Figure 1B). Consequently, it was conclusively determined that the cells employed in this investigation were cATMSCs.
To identify the optimal concentration of DFO for preconditioning cATMSCs, cell proliferation ability was measured using the CCK-8 assay. As shown in Figure 2A and B, optimal cellular proliferation was achieved at a concentration of 50 μM. However, the efficacy of DFO did not exhibit a dose-dependent increase, and beyond a concentration of 100 μM, the proliferative response decreased. The optimal proliferation of cATMSCs at 50 μM DFO underscores the potential of mild stress induction, such as low-level hypoxia mimicry, in enhancing stem cell functions (20). These observations align with prior research demonstrating that moderate hypoxic conditions enhance MSC proliferation and stemness through the stabilization of hypoxia-inducible factor-1α and the activation of subsequent downstream signaling pathways (21). Moreover, high DFO concentrations might induce excessive iron chelation, disrupting essential cellular mechanisms, including electron transport and ATP synthesis, which might have a detrimental impact on cell viability and functionality (22). Additionally, an excess of DFO is known to trigger oxidative stress, disrupting the cellular equilibrium between pro-oxidants and antioxidants, thereby further diminishing proliferation rates (23).
To evaluate the promotive effect of DFO preconditioning on the immunomodulatory capabilities of cATMSCs, the expression profiles of key immunoregulatory genes were assessed via qRT-PCR. As depicted in Figure 2C, a marked elevation in Tsg6 gene expression was observed. TSG6 is widely recognized for its contribution to reducing inflammation and facilitating tissue repair (24, 25). This finding suggests that DFO preconditioning may prime cATMSCs towards a more potent immunosuppressive phenotype, which might be particularly advantageous for therapeutic applications in inflammatory and autoimmune conditions.
Previous studies have demonstrated that TSG6 is a crucial mediator in reducing inflammation, with its upregulation endowing DFO-cATMSCs with an enhanced immuno-suppressive capability (26, 27). Building on this foundation, we developed a mouse model of systemic inflammation, guided by earlier work, to assess the therapeutic potential of DFO-cATMSCs (28, 29). According to the results shown in Figure 3, cATMSCs preconditioned with DFO demonstrated enhanced therapeutic efficacy in the systemic inflammation model relative to cATMSCs without preconditioning. This was particularly evidenced by the pronounced reduction in the gene expression of pro-inflammatory cytokines, such as Tnfa, Il1b, and Il6 in the lung and liver tissues, which are organs notably susceptible to inflammatory insult (30, 31). These results suggest that successful management of systemic inflammation is discernible in these tissues, reflecting the systemic effect of the therapeutic intervention.
The in vivo study revealed that DFO-cATMSCs demonstrated enhanced efficacy in alleviating inflammatory responses. Therefore, it is imperative to extend our research to the clinical safety aspects of these cells to ensure the translational feasibility of this therapeutic approach. Our investigations into clinical safety encompass studies on immune responses and tumorigenic potential. Clinical outcome data reveal that a singular administration of DFO-cATMSCs elicited no adverse events in healthy subjects, and subsequent imaging evaluations 3 months post-injection failed to detect any aberrant alterations, including neoplastic formations, within the principal abdominal organs. These findings are pivotal, offering reassurance about the immediate and short-term safety of employing DFO-cATMSCs in therapeutic settings.
In the quest to refine the therapeutic potential of stem cell applications, this investigation into the preconditioning effects of DFO on cATMSCs represents a significant stride forward. This study not only elucidated that the efficacy of DFO-cATMSCs in mitigating inflammatory responses is enhanced but it also establishes a foundational understanding of their clinical safety profile, underscoring the translational feasibility of this innovative therapeutic approach.
Although the findings of this study are encouraging, it is important to acknowledge the limitations inherent to this research. The safety profile was primarily assessed in healthy animals with a single injection of DFO-cATMSCs, leaving unanswered questions regarding the potential side-effects of multiple administrations.
Conclusion
This study underscores the potential of DFO preconditioning as a viable strategy for enhancing the therapeutic efficacy of cATMSCs while maintaining a favorable safety profile. The journey towards clinical application is fraught with challenges, yet these promising outcomes observed provide a hopeful outlook for the future of regenerative medicine.
Acknowledgements
This research was supported by Chungbuk National University Korea National University Development Project (2022).
Footnotes
Authors’ Contributions
XPJ contributed to conceptualization, investigation, resources, and writing of the original draft; SY and HLT and JHA contributed to reviewing and editing; YZZ and XZL contributed to experimental advice and reviewing; SHC and QL contributed to supervising the procedures. All Authors have read and agreed to the published version of the article.
Funding
This work was supported by the Science and Technology Development Project in Jilin Province (YDZJ202201ZYTS436). This study was also supported by the Research Fund of Engineering Research Center of North-East Cold Region Beef Cattle Science & Technology Innovation, Ministry of Education and the “111” Project (D20034), P.R. China.
Conflicts of Interest
The Authors declare that they have no conflicts of interest
- Received June 7, 2024.
- Revision received July 27, 2024.
- Accepted August 16, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
