Reproductive tract dysbiosis, due to the action of pathogens and/or unhealthy lifestyle, has been related to many reproductive diseases and disorders in mammalian species. Classically, such a problem has been confronted by the administration of antibiotics. Despite their effectiveness for controlling disease, treatments with antibiotics may negatively affect the fertility of males and females and, mainly, may induce antibiotic resistance. Accordingly, safer alternatives for maintaining reproductive system eubiosis, such as probiotics, are required. The present review summarizes the current knowledge on the biodiversity of the microbiota at the reproductive tract, possible changes in the case of dysbiosis, and their relationships with adequate reproductive health and functioning in both females and males. Afterwards, mechanisms of action and benefits of different probiotics are weighed since the biological activities of probiotics may provide a promising alternative to antibiotics for maintaining and restoring reproductive eubiosis and function. However, at present, it is still necessary for further research to focus on: (a) identifying mechanisms by which probiotics can affect reproductive processes; (b) the safety of probiotics to the host, specifically when consumed during sensitive reproductive windows such as pregnancy; and (c) the hazards instructions and regulatory rules required for marketing these biological-based therapies with sufficient safety. Thus, in this review, to draw a comprehensive overview with a relatively low number of clinical studies in this field, we showed the findings of studies performed either on human or animal models. This review strategy may help provide concrete facts on the eligible probiotic strains, probiotics colonization and transfer route, and prophylactic and/or therapeutic effects of different probiotic strains. Plenty of unfriendly factors can adversely affect the community of the reproductive tract’s microbiome in both human beings and/or animals because of either modern lifestyle or industrialization and intensification of production, leading to reproductive tract dysbiosis and the need for restoring a eubiotic state.pro that means “for” or “in support”, and the second clause is originated from the Greek adjective biotic that means “life”, so together, they mean “in support of life”. Scientifically, probiotics refer to viable microorganisms that have beneficial effects on the consumers’ health. Reproductive dysbiosis has been related to several reproductive disorders and infertility. The use of antibiotics and the use of probiotics are basically two hypothesized approaches that could be used to control reproductive tract microbiota. In this context, several studies have confirmed the beneficial effects of antibiotics to tackle many reproductive microbial infectious diseases and related infertility ,2,3. On Many studies have shown the positive effects of probiotics-based therapies on reproductive health in human ,7,8,9 anThe present review sheds the light on: 1- the relationships between reproductive tract eubiosis/dysbiosis and reproductive health and functioning; 2- the use of probiotics for maintaining and restoring reproductive eubiosis and function with emphasis on their properties, mechanisms of action, and benefits for reproductive function; 3- the safety of probiotics to the host; and 4- the litigation and regulatory rules required for marketing these biological-based therapies.Lactobacillus species, as well as Acinetobacter, Pseudomonas, Comamonadaceae, Jonquetella, Fusobacterium, Bacteroidetes, and Preovotella [Escherichia coli, Trueperella pyogenes, Enterococci, Enterobacteriaceae, Streptococci, Ureaplasma, and Staphylococci are classified as pathogenic microbes and have been linked to several reproductive disorders [A direct relationship between the biodiversity of reproductive tract microbiota and reproductive health and the completion of different reproductive events has been confirmed. The microbial biodiversity of the reproductive tract is highly dynamic and is affected by several internal and/or external factors. However, for maintaining adequate reproductive functioning and health, the prevalence of beneficial microbes should be ensured. The prevalence of some microbial species has been related to sufficient reproductive health and functions. Some microbial species are classified as beneficial microbial strains, mainly ovotella . In contisorders ,16,17.Either in males or females, reproductive tract dysbiosis has been related to many reproductive dysfunction and infertility. Dysbiosis (the dominance of pathogen-driven microbes) of the reproductive tract can disrupt numerous vital reproductive functions such as gametogenesis, steroidogenesis, embryo development and implantation, and establishment of pregnancy . Atopobium vaginae, Acinetobacter, Cloacibacterium, Comamonadaceae, Escherichia, Porphyromonas, and Pseudomonas [Bacteroides, Mobiluncus, Trichomonas vaginalis, and Candida species was seen in vaginal smears [Propionibacterium, Streptococcus, Actinomyces, Bifidobacterium, and Propionibacterium in their follicular fluid had less embryo transfer and pregnancy outcomes following in vitro fertilization and embryo transfer [Lactobacillus [In women, many gynecological diseases are related to the dysbiosis of endometrial microbiome, i.e., chronic endometriosis , dysfuncudomonas ,24. In wl smears . The dystransfer . In womebacillus ,28.In women, reproductive tract microbial biodiversity is highly dynamic and affected by several factors, mainly hormonal changes related to the stage of the reproductive cycle. High estrogen levels, such as around ovulation time, have been found to contribute to the maintenance of reproductive tract eubiosis. Estrogens maintain the acidity of the vaginal fluids, which makes them suitable media for the growth of beneficial bacteria lactobacilli. On the other hand, low levels of estrogens and the increment in pH such as that induced around menstrual period can shift the reproductive tract from eubiosis to dysbiosis through lowering the number of lactobacilli and increasing the opportunity of undesirable microorganisms’ growth, increasing the susceptibility of infectious pathogens ,30. Mycoplasma hominis was seen in infertile men than in fertile men [Neisseria, Pseudomonas, and Klebsiella accompanied by a reduction of Lactobacillus has recently been linked to oligoasthenoteratozoospermia and seminal hyper viscosity [Proteobacteria and a lesser population of the friendly bacteria Lactobacilli compared to those of healthy men [In men, the disbiosis of the male reproductive tract’s microbiome leads to several inflammatory diseases , subfertility, and poor semen quality . For exatile men . An incriscosity . In anotlthy men .Bacteroides and Enterobacteriaceae have been shown in cows with reproductive disease compared to healthy cows [Bacteroidetes and decreased Parvimonas, Porphyromonas, unclassified Veillonellaceae, and Mycoplasma was associated with bovine necrotic vulvovaginitis [Bacteroidetes and Fusobacteria was associated with metritis [Campylobacter in vaginal microbiota was associated with increased rates of abortion [The relationship between the reproductive tract microbiota biodiversity and reproductive health has also been reported in animals. The dominance of certain genera in the vaginal microbiota in female bovine have been linked to reproductive disorders. For instance, increased relative abundance of thy cows . In boviaginitis . The premetritis . In sheeabortion .These findings highlight the role of reproductive tract eubiosis in achieving adequate reproductive health and fertility and the importance of fixing any dysbiosis that might be raised due to unpleasant factors.The manipulation of the microbiota existing in the reproductive tract of both males and females can be an effective tool to control many reproductive diseases and associated infertility. Reproductive outcomes in both humans ,40 and aThe negative effects of antibiotics on reproductive organs and functions can be related to the lack of the specificity of broad-spectrum antibiotics, which could not only impair the growth of dysbiotic bacteria but also symbiotic bacteria. Furthermore, the widespread use of antibiotics poses a serious health risk due to the development of multiple-antibiotic-resistant microbial species. Furthermore, antibiotics-based treatments are linked to high rates of recurrent infections after treatment and side effects derived from the clearance of naturally harbored flora in other body sites . These aLactobacillus species , Bifidobacterium species, and Bacillus species are the major representatives of probiotics for modulating fertility dysbiosis [The term ‘probiotics’ has come to be used to refer to the live microorganisms that, when administered in adequate amounts, confer a health benefit to the host . More reysbiosis . The proProbiotic microbial species should have certain criteria to be approved as being safe compounds for health and effective when they are used for prophylactic and/or therapeutic interventions. In this term, safety properties  and functional properties  should be tested before approving any microorganism as a potential probiotic ,50. ThesLactobacillus rhamnosus GR1 and Lactobacillus Reuteri RC14 may persist up to 19 days in the human vagina following intravaginal administration. Both strains survive at low pH, have high adherent ability to uroepithelial and vaginal cells, and are able to colonize the vagina when administered orally, whereas some strains such as Lactobacillus rhamnosus GG and Lactobacillus acidophilus are not well-suited to colonize the vagina or to produce H2O2 , explaining why both these strains fail to prevent the recurrence of urogenital infections [Functional properties of probiotics are also important to achieve effective probiotics-based therapy. The properties such as tolerance to acids, bile salts, and lysozyme and gastrointestinal survival are related to the ability of these microorganisms to survive through the gastrointestinal system and to transfer to other target sites in body such as reproductive organs. Thus, these properties may be very important for oral-based administration to ensure the transfer of the probiotics to other sites of action, conferring local and direct effects on target organs. On the other hand, adhesion capacity may be more important when probiotics are vaginally administrated to allow a direct and targeted colonization action of the probiotics for restoring unhealthy vaginal microbiota ,62. In fLactobacilli isolated from the vaginas of healthy women for their potential safety and functional properties. Of 119 assessed strains, 7 identified strains  have been found to typically fulfill most criteria of probiotics. In the same study, 6 out of 119 strains belonging to Lactobacillus crispatus and Lactobacillus gassseri species have been found to exhibit phenotypical resistance to vancomycin. According to the previous facts on the eligibility criteria of microorganisms to act as an effective probiotic strain for maintaining and restoring reproductive health and functioning, it is supposed that one important potential source of probiotic strains is the reproductive tract itself . InnovatLactobacillus plantarum, fermentum and rhamnosus) with potential probiotic properties. These bacterial species also exhibit phenotypical resistance to vancomycin-resistant strains and sensitivity to streptomycin, erythromycin, kanamycin, and chloramphenicol [Enterococci may preclude this fear, as the antimicrobial resistance of these strains is attributed to the synthesis of a modified cell-wall peptidoglycan precursor and is different from the transferable mechanism observed for other bacteria [El-Deeb and co-workers  isolatedphenicol . In factbacteria .Lactobacillus rhamnosus BPL005 (CECT 8800) against the bacterial colonization of primary endometrial epithelial cells infected with Atopobium vaginae, Gardnerella vaginalis, Propioni bacterium acnes, and Streptococcus agalactiae pathogens. In the same context, Lactobacillus crispatus is the most frequent colonizing species in healthy women with antimicrobial activity, and it is therefore nominated to be used as a probiotic therapeutic alternative to antibiotics [Weissella confusa, Enterococcus hirae, Leuconostoc lactis, and Ilyobacter polytropus. However, these strains showed different hydrophobicity, antibacterial activity, and adherence ability. Thus, the authors suggested that the use of a mixture of these strains can be a more efficient intervention for developing probiotic products that can prevent and treat reproductive tract infections in cows. Similarly, Ametaj and co-workers [Lactobacillus sakei FUA 3089, Pediococcus acidilactici FUA 3138, and FUA 3140, isolated from the vaginal tract of healthy cows to decrease the incidence of metritis, pyometra, and vaginal purulent discharges, as well as the number of cows with abnormal cervical size and uterine fluctuation. In addition, vaginal isolates of dromedary camels (Camelus dromedarius) have been found to contain Lactobacillus species belonging to Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus rhamnosus strains with potential probiotic properties. These bacterial species showed strong antimicrobial activity against Staphylococcus aureus, which is considered as one of the most common bacterial causes of endometritis in dromedary camels and other animal species [The probiotic properties of microbial species isolated from different sites of the reproductive tract has been confirmed in many studies. Chenoll and co-workers  confirmeibiotics . In anot-workers  proved t species . Overall, these studies performed either on human or animal models confirm that the reproductive tract microbial communities represent a suitable source of probiotic candidates that could be used in new functional formulates for maintaining and restoring reproductive tract eubiosis.Lactobacilli drop, whilst there is an overgrowth of other groups such as Gardnerella, Atopobium, Prevotella, and Streptococcus, among others [Lactobacilli, in the uterine lumen and vaginal microbiome help to maintain acidic milieu, which provides an efficient protective agent against pathogenic microorganisms [Probiotics can maintain and restore reproductive tract eubiosis through different mechanisms. Probiotics have been found to produce bioactive molecules that have direct effects on reproductive tract microbiota, as these bioactive molecules can act as antimicrobial agents. In a pathogenic process, the microbiome shifts to a dysbiotic state, in which pH is increased to values above 4.5 and g others . The prorganisms .Some probiotic species, in addition to the ability of many species to reduce the pH, are able to produce oxidative molecules such as hydrogen peroxide, as well as another group of molecules known as bacteriocins . 2+ or Cu2+; (2) the synthesis of their own antioxidant enzymes such as Fe- or Mn-superoxide dismutase and Catalase; (3) the production of metabolites with antioxidant activity, such as glutathione, butyrate, and folate; (4) controlling antioxidant signaling pathways ; and (5) the regulation of the enzymes producing reactive oxygen species such as NADPH oxidase complex [As shown in  complex .adrenal (HPA) axis. Some probiotic species such as Bifidobacterium infantis and Lactobacillus rhamnosus (JB-1) and farciminis have been found to exhibit anxiolytic effects and improve psychiatric-disorder-related behaviors, including anxiety, depression, acute stress disorders, and obsessive disorders [Other studies have highlighted another mechanism of action of probiotic that is mediated by the hypothalamic–pituitary–isorders ,72. Thesisorders .vaginal odor, burning with urination, and itching. Additionally, it is linked to the increased risk of early delivery (pre-term delivery) among pregnant women [Lactobacillus vaginal tablets revealed the disappearance of bacterial vaginosis symptoms and had a clinically normal vaginal microbiota after daily probiotic therapy application for just 7 days. Furthermore, long-term vaginal administration of Lactobacillus rhamnosus appears to be a useful, safe, and effective antibiotic complementary approach in the management of bacterial vaginosis [Lactobacillus rhamnosus GR1 and reuteri RC14 in pregnant women restores the normal vaginal flora and acidic pH and interrupts the infectious/inflammatory process due to bacterial vaginosis with recovery rates comparable to those of tocolytic therapies. Thus, probiotics are preferred over tocolytic therapy to reduce the adverse maternal and fetal outcomes [Bifidobacterium lactis V9 was an effective treatment for tackling polycystic ovary syndrome, a hormonal-disturbance-induced disease. As hypothesized by the authors of this study, the effect of Bifidobacterium lactis V9 is mediated by the gut–brain axis, as Bifidobacterium lactis V9 produces certain amounts of short-chain fatty acids, which consequently increase the release of ghrelin and peptide YY. These latter two molecules can restore the hormonal profile of sex hormones, mainly LH and prolactin, at the brain level by a manner similar to normal hormonal profiles.Among reproductive diseases in women, bacterial vaginosis represents one of the major pathogenic diseases in women of reproductive age. Bacterial vaginosis is caused by vaginal dysbiosis and predominance of pathogenic microorganisms . It is characterized by several clinical and uncomfortable symptoms such as the release of discharge with abnormal nt women . In manynt women ,13; in taginosis . In addioutcomes . Probiotoutcomes  found thLactobacillus rhamnosus HN001 (6 × 109 CFU/day) to women during early pregnancy and during the first 6 months of breastfeeding can reduce the rates of infant eczema and atopic sensitization at 1-year and maternal gestational diabetes mellitus, bacterial vaginosis, and depression and anxiety postpartum [Interestingly, probiotics can be safely used during pregnancy and breastfeeding to offer a unique opportunity to influence a range of important maternal and neonatal outcomes. The oral supplementation with the probiotic stpartum . The finThe positive effects of probiotics on female reproduction are not only observed in humans but also in animal models. Specifically, farm animals may be of particular importance, as maintaining their health, improving reproductive and productive performance, and eliminating drugs/antibiotics use represent crucial challenges. Considering these aspects, many probiotic microbial species have been tested either for treating reproductive diseases or for improving the productivity of healthy farm animals .Lactobacillus species  at the time of the insertion of fluorogestone acetate (FGA) sponges for estrus synchronization in ewes restored normal vaginal microflora and improved fertility . For example, the management of the estrous cycle in some farm animals  includes many protocols that depend on intravaginal progesterone devices and/or sponges. In many cases, the administration of these intravaginal means has been found to generate histological and cytological alterations  at the time of its withdrawal, which evoke inflammatory responses and provoke abnormal vaginal discharges, impacting animal welfare aspects and subsequent reproductive performance . In a reLactobacillus sakei FUA3089, Pediococcus acidilactici FUA3138, and Pediococcus acidilactici FUA3140 had smaller cross-sectional areas of gravid horn and uterine body on day 14 postpartum, earlier uterine involution, and resumed ovarian cyclicity earlier. Generally, the positive effects of probiotic treatments on reproductive performance and cows’ health might be ascribed to the ability of Lactobacillus species to decrease postpartum uterine infection and thus to decrease endotoxin translocation into the systemic circulation, decreasing the occurrence of inflammatory responses [In addition, probiotics-based treatments can be used to improve the postpartum health and reproductive performance of farm animals. Specifically, parturition and uterine involution during the postpartum period may expose females to many reproductive infectious diseases, mainly due to the associated disturbance in the metabolism and the increase in inflammatory responses ,77,78,81Clostridium butyricum  from day 90 of gestation to weaning at Day 21 postpartum could shorten the duration of farrowing and enhance the growth performance of suckling piglets. Moreover, 0.2% Clostridium butyricum administration to sows changed the composition of intestinal microbiota, specifically increasing the relative abundance of Prevotella [Bacillus species serve as safe and effective feed additives which can improve the reproductive performance of farm animals [Bacillus subtilis and/or Bacillus licheniformis improved the lipid and protein metabolism profiles of sows, the placental antioxidant capacity, and placental efficiency that resulted in greater piglet birth weight. In sheep, the administration of monensin or Saccharomyces cerevisiae improved the metabolic status and ovarian steroid production of dams, as well as the growth performance of lambs, highlighting the possibility of using probiotic Saccharomyces cerevisiae as an alternative to the antibiotic monensin [Focusing on improving the productivity of animals, in sows, the addition of dietary evotella . In addi animals ,83. Gu a animals  found thmonensin . The finA summary of some studies on the effects of probiotics on men’s reproductive health and fertility is shown in These positive effects have been seen in different infertility-related models. Infertility related to age , obesity9 CFU/mL of Lactobacillus acidophilus, Bifidobacterium spp., Bacillus coagulans, and Lactobacillus rhamnosus), for 63 consecutive days, reversed all negative effects of NAFLD on semen quality and redox homeostasis in rats. Other studies  [The candidate probiotic species, in addition to health safety as discussed above, should have functional and industrial properties, enabling them to be stable during pharmaceutical processing and storage and to survive afterwards in different biological environments, such as the gastrointestinal and reproductive tracts. In this context, the emergence of some recent industrial technologies such as micro/nanoencapsulation technology can help to improve the stability of probiotics . The emecterium)  and mushcterium)  in the fCurrently, the most recent research trends refer to the possibility of the use of postbiotics  as an alternative to probiotics itself. The use of postbiotics might have offered several advantages over probiotics, such as obtaining a specific mechanism of action and biological role, an easier production process for industrial-scale-up, and, lastly, a better availability and accessibility during marketing and application .Lactobacillus, Bifidobacterium, and Bacillus are the most adopted for modulating reproductive tract dysbiosis [The dysbiosis of the reproductive tract microbiota due to the action of pathogens and/or unhealthy lifestyle has been related to many reproductive diseases and disorders in both humans and animal models. Recent studies have focused on the possible roles of different probiotics in restoring the reproductive tract eubiosis. Among the different candidate species, ysbiosis . The bioysbiosis . A highlysbiosis ,52. Hence, future studies have to focus on identifying mechanisms by which probiotics can affect reproductive processes, the safety of probiotics to the host specifically when they are consumed during sensitive reproductive windows such as pregnancy, and finally, the litigation and regulatory rules required for marketing these biological-based therapies with sufficient safety and hazards instructions .


The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals. MSCs are present in numerous stem cell niches including bone marrow and adipose tissue and can be expanded in vitro by plating onto tissue culture polystyrene (TCPS), which causes them to adhere, adopt a spindle-like shape, and proliferate into fibroblastic colony-forming units2. Owing to their unique properties and ease of expansion, MSCs have been extensively studied and used in numerous clinical trials for the treatment of various medical disorders4.Mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of differentiating into cells that produce various mesodermal tissues, including osteoblasts, adipocytes, and chondrocytes5, which influence numerous functions including migration, differentiation, and paracrine signaling6. To regulate stem cell behavior outside of the body, biomaterials are used to recapitulate specific properties of tissue microenvironments. For example, ECM elasticity and tissue-level stiffness are strong drivers of cellular mechanosensing and phenotypic commitment in vitro7. Engler et al. showed that MSCs atop soft hydrogels that mimic the stiffness of brain tissue express neuronal biomarkers, whereas MSCs on rigid substrates produce osteocalcin, a bone tissue-specific protein secreted by osteoblasts7. On a molecular level, MSC mechanosensing is led by several mechano-transducer proteins that collectively induce changes in focal adhesion maturation9, cytoskeletal contractility/alignment11, and nuclear Yes-associated protein (YAP) localization12.In vivo, the multifunctional phenotype of MSCs is regulated by chemical and physical cues of the tissue microenvironment13, and donor variability between MSC lines derived from adult tissues is significant, resulting in inconsistent responses to engineered signals14. These challenges limit the possibility of manufacturing high quality, homogeneous MSC lines in large numbers needed for basic research and stem cell-based therapies15. When successfully reprogrammed, induced pluripotent stem cells (iPSCs) reset any possible mechanical memory that could bias their response to engineered materials by erasing historical epigenetic, transcriptional, and non-genetic information17. Indeed, iPSCs have recently been differentiated into functional MSCs (iMSCs), displaying phenotypic similarities with tissue derived MSCs19. However, there are only a limited number of studies that have explored the use of iMSCs20, and the effects of mechanical signals on iMSC mechanosensing is not well understood. In this study we sought to test two hypotheses: (1) iMSCs “feel” mechanical properties resulting in changes in morphology and intracellular mechanosensitive protein organization  comparable to MSCs, and (2) iMSC mechanosensitivity to substrate stiffness is more reproducible within iMSC populations and across iMSC lines as a consequence of the reprogramming process, which erases mechanical memories acquired during development and in response to engineered biophysical cues.Although engineered mechanical cues can regulate MSC mechanosensing in vitro, MSC populations are heterogeneous21. We report that iMSCs are more responsive to matrix stiffness than human MSCs derived from adult tissues, and that stiffness-mediated changes in cellular mechanosensing are more consistent across different iMSC lines.To test our hypotheses, we evaluated cell-material interactions of three human iMSC lines derived from iPSCs generated using a robotic, fully automated platform, which results in the production of highly reproducible iMSC lines21.Manual production of human iPSC lines is time consuming and can result in significant line-to-line variability. To enable consistent production of high-quality and highly reproducible iMSC lines, fibroblasts from human skin biopsies were reprogrammed into stem cells using the NYSCF Global Stem Cell Array®, a modular, robotic platform for high-throughput production, maintenance, and differentiation of iPSCs Fig. a. Three 23. Interestingly, in vitro expansion over ten passages also results in a reproducible and progressive increase in average cellular length, which is a phenomenon also observed in tissue derived MSCs  class II, which suggests iMSCs are more immunoprivileged than adult MSCs26. Notably, iMSCs express more integrin alpha 2 (CD49b), integrin alpha 3 (CD49c), and integrin alpha 4 (CD49d) than adult MSCs , intermediate (10 kPa), and stiff (20 kPa) HA hydrogels30. Thus, we sought to synthesize HA hydrogels with a stiffness range of 5–20 kPa to study the effects of mechanics on iMSC shape and mechanotransduction.While there are many macromers that can be used to synthesize hydrogels, hyaluronic acid (HA) hydrogels are highly biocompatible and amenable to extensive biochemical and biophysical modifications32. The amount of macromer (3 wt%) was kept constant and Low (5.19 ± 1.04 kPa), Med (9.58 ± 0.98 kPa), and High (19.27 ± 2.41 kPa) matrix stiffness hydrogels were formed by varying the amount of crosslinker added  macromers with thiols in DTT crosslinkers as previously reported2, respectively  is known to initialize at least two signaling pathways of MSC mechanosensing and plays an important role in controlling several cellular processes including cell spreading, migration, and focal adhesion maturation37. After three days in culture, all iMSC lines displayed an increase in focal adhesion maturation with increasing matrix stiffness. pFAK length for iMSCs on Low, Med, and High stiffness hydrogels was 3.94 ± 0.56, 5.98 ± 0.62, and 7.18 ± 0.56 µm, respectively  on Med stiffness hydrogels. Differences between iMSC and MSC morphology were not as pronounced on Low or High stiffness hydrogels  stiffness substrates, representative cell silhouettes of iMSCs Fig. a, top quHistograms of cell area show a homogeneous distribution for iMSCs Fig. b, top. INext, we examined scatter plots of nuclear YAP versus area of iMSC Fig. d, top anActin anisotropy values for iMSCs were also consistent across different lines while for MSCs actin anisotropy was significantly different across cell lines Fig. f. These 39. Due to their remarkable sensitivity and homogeneity, iMSCs could be a viable source for large scale manufacturing of human stem cells for both autogenic and allogeneic cell therapies. As we continue to increase our understanding of iMSC-material interactions, we also believe that iMSCs will emerge as a new class of cells for regenerative medicine and tissue engineering applications.In this study, we derive iMSCs from iPSCs and demonstrate that iMSCs are more homogeneous and mechanosensitive than MSCs isolated from adult tissues. This finding resulted from evaluating iMSC morphology and matrix mechanosensing on mechanically defined 2D hydrogels and motivates future studies that investigate iMSC-material interactions in more complex and physiologically relevant environments. To this end, the thiol-norbornene chemistry used here can be easily adapted to form hydrogels that support 3D cell culture and spatial patterning of biophysical and biochemical signals21. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by The New York Stem Cell Foundation Research Institute. Skin biopsies were shared as deidentified following written informed consent. Generated iPSC lines were characterized via confirmation of pluripotency markers OCT4 and TRA-1-60 using immunostaining and fluorescence imaging. Additional characterization was performed via global surface marker profiling using a BD Lyoplate Human Cell Surface Marker Screening Panel (BD Biosciences) per manufacturer’s instructions. Using chemically defined differentiation medium, iPSC lines were differentiated towards osteoblasts, chondrocytes, or adipocytes and evaluated using von Kossa , Alcian blue , or Oil red O (intracellular triglycerides), respectively.Fibroblasts from skin biopsies were programmed into iPSCs using the Global Stem Cell Array® as previously reported1H NMR spectra used to calculate percent of HA repeat units functionalized with Nor- and Me- is in Supplementary Fig. Sodium hyaluronate (NaHA) was first converted to its tetrabutylammonium salt (HA-TBA). To synthesize HANor, the carboxylic acid residues of HA-TBA were modified with 5-norbornene-2-methylamine . To synthesize HANorMe, the hydroxyl residues of HANor were modified with methacrylic anhydride . To biofunctionalize HANorMe with RGD, a Michael addition reaction between thiolated RGD (cRGD) peptide and methacrylates was performed . Representative 2). Individual hydrogels were removed from the molds and placed in 1 mL of PBS to swell overnight at 37 °C before mechanical testing. Compressive moduli were determined using a Shimadzu EZ-SX Mechanical Tester running at a constant strain rate of 10%/min. The modulus was calculated from the slope of the stress–strain curve between 10 and 20% strain (n ≥ 3).HANor was dissolved in phosphate buffer saline (PBS) at 3 wt% with varying amounts of DTT and 0.05 wt% I2959. The prepolymer solution (80 µL) was pipetted into a silicone mold  and irradiated with UV light .Stem cell lines from the New York Stem Cell Foundation Research Institute expanded to passage 4 in growth media  were used in all experiments. Human mesenchymal stem cell lines (Lonza) were expanded to passage 4 using the culture media described above. Cells were then cultured on top of Low, Medium, or High stiffness hydrogels , phalloidin (actin), and/or Hoescht (nuclei). Samples were then imaged using a Nikon A1 confocal microscope and ImageJ software was used to calculate morphology  and cellular mechanosensing  parameters.All data are from three independent biological experiments. At least 50 cells per treatment and biological experiment were quantified. For three group comparisons, one way ANOVA between groups (α = 0.05) was performed using GraphPad Prism. If the results of the ANOVA were found to be significant, post hoc analysis was performed using the Tukey multiple comparisons test to compare results among groups. Hierarchical clustering of surface marker data was generated using the analysis software Morpheus (Broad Institute) based on Euclidean distance.Supplementary Information.


Cip1/Waf1. A panel of eight neuroblastoma cell lines and a TH-MYCN mouse model were investigated for the expression of p21Cip1/Waf1 using RT-qPCR, Western blot, and immunofluorescence. This was followed by investigation of sensitivity towards cisplatin and the p21Cip1/Waf1 inhibitor UC2288. Whereas the cell lines and the mouse model showed low levels of un-phosphorylated p21Cip1/Waf1, the phosphorylated p21Cip1/Waf1 (Thr145) was highly expressed, which in the cell lines correlated to cisplatin resistance. Furthermore, the neuroblastoma cell lines showed high sensitivity to UC2288, and combination treatment with cisplatin resulted in considerably decreased cell viability and delay in regrowth in the two most resistant cell lines, SK-N-DZ and BE(2)-C. Thus, targeting p21Cip1/Waf1 can offer new treatment strategies and subsequently lead to the design of more efficient combination treatments for high-risk neuroblastoma.Platinum-based chemotherapies such as cisplatin are used as first-line treatment for the paediatric tumour neuroblastoma. Although the majority of neuroblastoma tumours respond to therapy, there is a high fraction of high-risk neuroblastoma patients that eventually relapse with increased resistance. Here, we show that one key determinant of cisplatin sensitivity is phosphorylation of the cyclin-dependent kinase inhibitor p21 Neuroblastoma (NB) is the most common extracranial solid tumour in early childhood. It is a heterogenous neoplasia with clinical presentation ranging from spontaneous regression of metastatic disease to a rapidly progressive course , 2. HighIn order to combat development of resistant cells, novel targeted strategies are being implemented for high-risk NB, including small-molecule inhibitors of anaplastic lymphoma kinase (ALK), radiolabelled somatostatin analogues, and monoclonal antibodies against the antigen GD2 , 6. EvenCip1/Waf1 (p21) has been linked to cisplatin resistance in testicular cancer  continues to be the cornerstone of systemic NB treatment. The platinum complex cisplatin is commonly included in first line therapies as adjuvant therapy, with the aim of inducing cytotoxicity and apoptosis , 8, 9. Ur cancer  and in or cancer .The p21 protein is transcriptionally regulated by p53-dependent and –independent pathways and is a key regulator of cell fate. Based on the intracellular localization, the p21 protein shows pleiotropic effects on cell growth and apoptosis in both malignant and non-malignant cells and tissue –16. In tFew small molecule inhibitors of p21, with the aim to inhibit p21 for potential cancer therapy, have been reported so far. These include butyrolactone I , LLW10 , sorafenThe possible role of p21 as an oncogene has previously not been studied in high-risk NB. Therefore, we have here mapped the endogenous expression of p21, both un-phosphorylated and phosphorylated, in a panel of eight high-risk NB cell lines and one NB mouse model. The effect of UC2288, either alone or in combination with cisplatin, was examined to investigate how functional modulation of p21 and p-p21 may alter NB cell viability. Here we report that increased cisplatin resistance was correlated to p-p21, and that combination treatment with UC2288 and cisplatin considerably decreased tumour cell viability. These results suggest an important role for p21 and p-p21 in preventing cisplatin-induced cell death in high-risk NB.TP53 wild type (wt) and five were TP53 mutated (mut) in order to compare between TP53 dependent and independent expression of p21. The non-tumourigenic fibroblast cell line MRC5 and the acute promyelocytic leukemia cell line HL-60 were included as controls (BE(2)-C and Kelly were obtained from ATCC . SK-N-SH, SH-SY5Y, IMR-32, SK-N-AS, SK-N-FI, SK-N-DZ, and MRC5 were a kind gift from professor Per Kogner at Karolinska Institute. HL-60 was a kind gift from Dr. Mohammad Hojjat Farsangi at Karolinska Institute. Eight high-risk NB cell lines were used, where three were TH-MYCN animals were obtained from the Mouse Model of Human Cancer Consortium Repository and kept on a 129X1/SvJ background . We here used tumours from hemizygous mice (MYCN+/−) for immunofluorescent staining for p21 and p-p21 according to the immunofluorescent protocol description.The transgenic ckground . TumoursTwenty-four hours after plating the protein from the NB cell lines, the MRC5, and the HL-60 cell lines were extracted for the baseline data. The protein extraction was done by using RIPA buffer  supplemented with a protease and phosphatase inhibitor  according to the manufacturer’s recommendations, separated electrophoretically and blotted on a nitrocellulose membrane  using standard procedures. All samples were loaded at 20 µg for equal loading. The p21 protein was detected using an anti-p21 antibody  or an anti-p-p21 (T145) antibody , and Cleaved-Caspase3 (C-Casp3) was detected using an anti-C-Casp3 antibody . GAPDH was stained with anti-GAPDH antibody . Protein bands were visualized with secondary antibodies diluted 1:15,000  using a ChemiDoc MP imaging system  or a LI-COR imaging system (LI-COR Biosciences UK Ltd). Analysis of band intensity was performed using ImageJ. All experiments were done in triplicate or quadruplicate, except for the HL-60 experiments which were done in duplicates.® Reverse Transcription manufacturer protocol . RT-qPCR was performed following QuantiText® SYBR® Green PCR manufacturer protocol . The primer sequences used can be found in Each cell line in logarithmic growth was plated onto petri dishes and incubated for 24 hours before samples for RT-qPCR were harvested. mRNA was harvested according to the RNeasy Mini Kit protocol . ß-mercaptoethanol was added at a volume of 10 µl/ml of Buffer RLT. mRNA concentration was measured using Nanodrop™ . Reverse transcription of mRNA into cDNA was performed according to the Quantitect3 in 1xPBS was added and the cells were incubated overnight at +4°C. The primary antibody was then removed, and cells were washed 3x5 min with 1xPBS before the secondary antibody, diluted in TNB buffer, was added. The cells were kept at room temperature for 2 hours before the secondary antibody was removed and the cover slips were washed 3x5 min with 1xPBS. The cells were mounted with Prolong Gold antifade with DAPI  to stain the cell nuclei. A Metafer® Slide Scanning Platform  was used to analyse the cells. The spatial localization of either the p21 or p-p21 expression was done by optical inspection by two independent observers. The mean intensity (mean fluorescence in arbitrary units per cell ± SD) was provided by the Metafersystem software and the low, intermediate, or high expression levels were based on equal range distributions of the values. Following the protocol for immunofluorescence, a triple staining was performed on all eight cell lines using antibodies for p21, the DNA damage marker phosphorylated ataxia-telangiectasia mutated (pATM), and the replication marker Ki67. All experiments were done in triplicate. Antibodies used can be found in Each cell line in logarithmic growth was plated onto round 13 mm #1 coverslips  in petri dishes and incubated for 24 hours before being fixated for 15 minutes using 4% paraformaldehyde . TNB buffer  block was added for 30 min at room temperature. Primary antibody diluted in 0.3% Triton X-100, 0.1% NaN50 , UC2288 at a dose of 10 µM or a combination of cisplatin and UC2288 followed by fixation for 15 min with 4% paraformaldehyde and treated according to the protocol for immunofluorescence or collected for protein according to the protocol for Western blot. All experiments were done in quadruplicates.Twenty-four hours after plating of Kelly and SH-SY5Y, UC2288 at a dose of 10 µM  diluted in dimethyl sulfoxide (DMSO) was added for 2 hours, 4 hours or 6 hours before the cells were fixated for 15 min with 4% paraformaldehyde and treated according to the protocol for immunofluorescence, or collected for protein or mRNA according to the protocol for either Western blot or RT-qPCR. UC2288 was added at a concentration of 10 µM based on the literature . All exp2. Seventy-two hours after treatment, 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed where 20 µl CellTiter 96® Aqueous One Solution Assay  was added to each well. Plates were incubated for 2.5 hours and metabolic activity was analysed in the microplate reader FLUOstar Omega  by measuring absorbance at 490 nm and 690 nm. All treatments were done in triplicate.Dose–response curves for cell viability were obtained after treatment for 72 hours with either UC2288 or cisplatin, in eight NB cell lines and one fibroblast cell line, MRC5, which was used as a non-tumourigenic control for drug toxicity. UC2288 was added as a single agent at concentrations of 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM, 0.05 µM or 0.01 µM and cisplatin at concentrations of 100 µM, 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM or 0.05 µM. For the combination treatments, cisplatin was added at concentrations of 100 µM, 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM or 0.05 µM either alone or in combination with UC2288 at a concentration of either 10 µM or 1 µM. The plates were incubated at 37°C, 5% CO3 cells), Kelly (10 × 103 cells), SK-N-DZ (10 × 103 cells) and BE(2)-C (5 × 103 cells) were seeded in 200 µl medium/well in a 96 well plate, and the edges were filled with medium to avoid edge effects. Twenty-four hours after seeding each cell line was treated with the corresponding IC50 value for cisplatin and 10 µM UC2288 for 48 hours. DMSO (mock) was used as negative controls. The plates were scanned by an IncuCyte S3 Live® Cell Analysis System  48 hours post treatment followed by medium change. The plates were thereafter scanned followed by medium change every 96 hours for a total of 30 days. The proliferation was determined by measuring the cell confluency. Once the cell lines reached 100% confluency, they were maintained for the duration of the experiment. Each treatment was done in 5-10 replicates and the data are presented as mean ± Standard Error of the Mean (SEM).SK-N-FI . In general, a CI of <1 is considered a positive and a CI of >1 a negative combined effect. More specifically, CI<0.70 was defined as synergy, CI>1.45 as antagonism, and values in between as additive effects, according to the recommendations of the ComboSyn software.To estimate the effects of the combination treatments the median-effect method of Chou   was appl50 value for each of the single treatments or for the combination treatment. The data was logarithmically transformed and normalized against 0% (no cell viability) and 100% (maximum cell viability) controls for the combination treatments. For determining IC50 values for the single treatments, the nonlinear regression analysis method log(inhibitor) vs normalized response was used. For determining IC50 values for combination treatment, the nonlinear regression analysis log(inhibitor) vs normalized response – variable slope was used. Linear regression analysis with Pearson’s correlation coefficient was used to investigate a possible relationship between the IC50 values of UC2288 and cisplatin, and between the fraction of p21 positive cells with either UC2288 or cisplatin. All analyses were performed in Graphpad Prism (version 9.3.1). Western blot data are presented as mean ± standard deviation (SD). Immunofluorescence data were analysed in RStudio and the percentage of positive cells followed by the mean ± standard deviation (SD) were calculated for each combination. Data was analysed using Student’s t-test or one-way ANOVA with Tukey post hoc test, as indicated in the text. Graphpad Prism (version 9.1.0) was used for generation of graphs.Following the MTS-assay, the data was analysed to obtain the ICTP53 wt cell lines or TP53 mut cell lines showed that among the TP53 wt cell lines SK-N-SH had the highest p21 mRNA expression followed by IMR-32 and among the TP53 mut cell lines SK-N-FI and Kelly showed the highest p21 mRNA expression  mRNA expression level of p21 (CDKN1A) within eight high-risk NB cell lines and one non-tumourigenic fibroblast cell line, MRC5. The MRC5 cell line had the highest p21 mRNA expression compared to the remaining cell lines (p<0.0001)  among all the tested cell lines  and IMR-32 , compared to SH-SY5Y (4.7% ± 0.9). In the TP53 mut cell lines, the fraction of p21 positive cells was generally lower with SK-N-FI  having a significantly higher fraction of positive cells compare to BE(2)-C (1.5% ± 0.3) and SK-N-DZ (0.1% ± 0.1). Cells positive for p21 were also detected in SK-N-AS (5.7% ± 0.6) and Kelly (4.9% ± 0.5)  and IMR-32 (2.1% ± 2.2). Among the TP53 mut cell lines, SK-N-FI (93.9% ± 9.1) and SK-N-DZ (97.6% ± 2.2) had significantly higher fractions of p-p21 positive cells compared to SK-N-AS, Kelly, and BE(2)-C cell lines which had moderate expression   compared to the three cell lines with the highest expression, SH-SY5Y (78.6% ± 17), SK-N-FI (93.9% ± 9.1), and SK-N-DZ (97.6% ± 2.2) (https://www.proteinatlas.org/ENSG00000124762-CDKN1A/cell+line). No p21 or p-p21 staining was detected in the HL-60 cell line when investigated with Western blot and immunofluorescence validating the specificity of the antibodies -C, which displayed high expression. Moderate cytoplasmic expression was observed in SK-N-SH, SK-N-FI, and SK-N-AS, followed by low or no detectable cytoplasmic staining in IMR-32, SH-SY5Y, Kelly, and SK-N-DZ , and the mice spontaneously develop NB at 5.6–19 weeks of age  and IMR-32  showed higher fraction of positive cells compared to SH-SY5Y (2.7% ± 0.4). Among the TP53 mut cell lines SK-N-FI (9.3% ± 0.8) had the highest fraction of cells expressing low levels of p21 followed by SK-N-AS (5.3% ± 0.5), Kelly (4.8% ± 0.5), BE(2)-C (1.2% ± 0.2), and SK-N-DZ (0.1% ± 0.1)  having a significantly higher fraction of positive cells compared to SH-SY5Y (0.2% ± 0.2). There was only one TP53 mut cell line with cells positive for high p21 expression, SK-N-FI (0.1% ± 0.1). The remaining TP53 mut cell lines SK-N-AS, Kelly, BE(2)-C, and SK-N-DZ showed no cells with high p21 expression , and the proliferation marker Ki67. First, each marker was analyzed on its own with the fraction of p21 positive cells being higher in Analysis of co-expression of the markers showed that within each cell line, a very small fraction of p21 expressing cells displayed co-expression of p21 and pATM (0-0.2%)  to 32.0 µM for BE(2)-C -C, displaying the highest IC50 values for cisplatin, were selected for further investigation of the cisplatin response. Each of the four cell lines were treated with the corresponding cisplatin IC50 concentration for 24 hours following analysis, using immunofluorescence, of DNA damage (pATM) and proliferation (Ki67) as a predictor of treatment outcome -C, had a decrease either in the fraction or the mean intensity of p21 positive cells. At the same time, three of the four tested cell lines, all but SK-N-FI, had an increase in either the fraction or the mean intensity of p-p21 positive cells, indicating that there is a preference of p-p21 activation, compared to p21, following cisplatin treatment.Taken together, the 50 values ranging between 4.3 µM to 53.9 µM -C. Regrowth (confluency) was mapped during a period of 30 days following a pulse treatment for 48 hours of mock or UC2288 at three different concentrations: 1 µM, 10 µM or the corresponding IC50 value for each cell line. SK-N-FI showed the highest sensitivity to the treatment, with a delay in growth at all three tested concentrations of UC2288 (50 dosing of UC2288 before an increase in confluency (regrowth) was observed  also had low IC50 values for cisplatin. On the other hand, BE(2)-C and SK-N-DZ had higher IC50 values for both UC2288 and cisplatin, implying that they are more resistant to single agent treatment with these drugs  for SK-N-FI. For BE(2)-C, the cisplatin IC50 value shifted from 29.1 µM to 40.4 nM and for SK-N-DZ from 30.2 µM to 31.2 nM  when combined with UC2288 at 10 µM. Moreover, a small shift in the cisplatin IC50 value was observed when combined with following UC2288 at 1 µM -C and SK-N-DZ. Due to the huge shift following combination treatment with UC2288 at 10 µM, the IC50, UC2288 10 µM, or combination of cisplatin IC50 and UC2288 10 µM for each corresponding cell line. Two cell lines, SK-N-DZ and BE(2)-C, showed delay in growth following combination treatment compared to a single cisplatin treatment  was mapped during a period of 30 days following a pulse treatment for 48 hours of mock, cisplatin ICTaken together, combination treatment with UC2288 and cisplatin reduced viability in both sensitive, Kelly and SK-N-FI, and resistant, BE(2)-C and SK-N-DZ, cell lines, even though the effect was more profound in the sensitive cell lines. Furthermore, combination treatment showed increase in cell death, indicated by C-Casp3, and a delay in regrowth compared to a single treatment with cisplatin in the two resistant cell lines, BE(2)-C and SK-N-DZ.TP53 wt and TP53 mut cell lines showed expression. However, there was discrepancies between the expression level of p21 mRNA and the p21 protein, both as un-phosphorylated and phosphorylated protein. This is in concordance with studies showing general poor correlation between expression levels of mRNA and protein -C, have MYCN amplification. The clinical prevalence of these genetic alterations is seen among 20-30% of NB tumours which have MYCN amplification, and TP53 mut are observed in a high proportion following relapse, indicating a mechanistic relevance in the development of therapy resistance -C cell line where a reduction of cytoplasmic p-p21 was observed when analyzed with Western blot but not when analyzed with immunofluorescence which detects both cytoplasmic and nuclear p-p21 expression, further validating the ability of UC2288 to inhibit mainly cytoplasmic p-p21. Further treatment of TP53 mut NB cell lines with UC2288 in combination with cisplatin showed a synergistic effect and a dramatically reduced the IC50 value, with an increase in cell death, indicated by C-Casp3 activation, suggesting UC2288´s potential as a drug against chemotherapy resistance .SSF contributed to conceptualization, data curation, methodology, project administration, resources, software, supervision, visualization, and funding acquisition. AS, VH and SSF performed the formal analysis, statistical analysis, investigation, and validation. AS, MW and SSF wrote the first draft of the manuscript. MW and SSF wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This research was funded by the Childhood Cancer Foundation grant numbers, TJ2019-0118, PR2019-0101, and PR2021-0037, the Swedish Cancer Society grant number 21-0313 SIA. Eva and Oscar Ahrén Foundation, Stockholm, and Magnus Bergvalls stiftelse grant number 2021-04262.The authors wish to thank Dr. Per Kogner for NB cell lines and Dr. Mohammad Hojjat Farsangi for the HL-60 cell line. We would also like to thank Dr. Ingrid Lilienthal and Hala Ibrahim Mohammad Habash for scientific discussions and technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-small cell lung cancer (NSCLC) patients acquire resistance to tyrosine kinase inhibitors (TKIs) via EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). In this study, we elucidated the mechanism of EGFR-TKI resistance mediated by VEGF/VEGFR in EGFR-mutated NSCLC cell lines and Erlotinib-resistant cell lines as compared to parental cell lines. Increased expression of VEGF, VEGFR-2, and NP1 was observed in Erlotinib-resistant cell lines. Furthermore, we observed an increased efficacy of Erlotinib in combination with a VEGFR-2 inhibitor in Erlotinib-resistant cell lines. Late-stage NSCLC patients with high expression of VEGFR-2 had shorter survival times compared to patients with low VEGFR-2 expression. These results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance that can be overcome with a combination treatment of Erlotinib and a VEGFR-2 inhibitor, which may serve as an effective treatment option for NSCLC patients with EGFR mutations.NSCLC treatment includes targeting of EGFR with tyrosine kinase inhibitors (TKIs) such as Erlotinib; however, resistance to TKIs is commonly acquired through T790M EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). We investigated the mechanisms of EGFR-TKI resistance in NSCLC cell lines with EGFR mutations or acquired resistance to Erlotinib. These studies showed upregulated gene and protein expression of VEGF, VEGFR-2, and a VEGF co-receptor neuropilin-1 (NP-1) in Erlotinib-resistant (1.4–5.3-fold) and EGFR double-mutant  NSCLC cells compared to parental and EGFR single-mutant (L858R) NSCLC cell lines, respectively. Immunofluorescence and FACS analysis revealed increased expression of VEGFR-2 and NP-1 in EGFR-TKI-resistant cell lines compared to TKI-sensitive cell lines. Cell proliferation assays showed that treatment with a VEGFR-2 inhibitor combined with Erlotinib lowered cell survival in EGFR double-mutant NSCLC cells to 9% compared to 72% after treatment with Erlotinib alone. Furthermore, Kaplan–Meier analysis revealed shorter median survival in late-stage NSCLC patients with high vs. low VEGFR-2 expression (14 mos vs. 21 mos). The results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance and that combined treatment of Erlotinib with a VEGFR-2 inhibitor may serve as an effective therapy in NSCLC patients with EGFR mutations. Lung cancer is the leading cause of cancer-related deaths. It was projected that, in 2021, 235,760 new lung cancer cases would be diagnosed and that 131,880 individuals would die from the disease in the United States alone . Among tEGFR is a receptor tyrosine kinase (RTK) overexpressed in 40–80% of NSCLC patients. When dysregulated, EGFR facilitates tumorigenic signaling ,6. ExpreNumerous tyrosine kinase inhibitors (TKIs) have been developed to act against activating mutations in EGFR. First-generation TKIs such as Erlotinib and second-generation TKIs such as Afatinib are used to overcome EGFR aberrant signaling by covalently binding to EGFR’s kinase domain . The devAs observed in lung cancer, dysregulation of the EGFR pathway results in upregulation of the human vascular endothelial growth factor (VEGF) pathway. Previous studies using cell culture models have shown that EGFR activation via epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) increases the production of VEGF ,16. VascNeuropilins (NPs)  are non-tyrosine kinase receptors that act as VEGF co-receptors. Prior studies have shown that co-expression of NPs is significantly correlated with poor prognosis and tumor progression in NSCLC . NeuropiTo better understand the mechanism of EGFR-TKI resistance in NSCLC and to develop more effective therapies against it, we studied three biomarkers linked to increased angiogenesis in NSCLC . We inveErlotinib hydrochloride (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine; C22H23N3O4•HCl) was obtained from LC laboratories , reconstituted in Dimethyl Sulfoxide (DMSO) at a concentration of 20 mM, and then stored in aliquots of 20 µL at −20 °C. ZM 323,881 hydrochloride (5-((7-Benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol hydrochloride), a selective inhibitor of VEGFR2, was obtained from Tocris Bioscience, a Bio-Techne brand  , reconstRabbit polyclonal antibody for VEGFR-2 (cat. no. SAB4501645-100UG) was obtained from Sigma-Aldrich. Rabbit polyclonal antibody for VEGFR-2 (cat. no. 07-716-I) was obtained from Millipore . Rabbit mouse monoclonal neuropilin-1 antibody (cat. no. Ab81321) was obtained from Abcam . Goat polyclonal antibody for neuropilin-1 (cat. no. sc-7239) was obtained from Santa Cruz . Mouse monoclonal VEGF (C-1) antibody (cat. no. sc-7269) was obtained from Santa Cruz . Bevacizumab, a humanized monoclonal VEGF-165 antibody (cat. no. NBP2-59637), was obtained from Novus Biologicals . Human antibodies CD309 (VEGFR-2/KDR)-APC (cat. no. 130-098-910) and CD304-PE (BDCA-4/Neuropilin-1) (cat. no. 130-098-876) were obtained from MACS Miltenyi Biotec . All primary antibodies were diluted according to the manufacturer’s instructions in TBST with 1% BSA. Secondary antibodies were prepared at a dilution of 1:1000 in TBST with 1% blocking grade milk (cat. no. 170-6404XTU) from Bio-Rad .2 and cultured according to ATCC’s instructions. H2170 and H358 cell lines have wild-type EGFR status, whereas H3255 (L858R) and H1975 (L858R and T790M) cell lines have mutated EGFR, as documented by ATCC. H2170 and H358 cell lines were cultured in increasing drug concentrations of Erlotinib to obtain EGFR-TKI-resistant cells. H2170-ER and H358-ER cells were obtained and cultured in the presence of erlotinib, as described previously [H2170, H358, H3255, and H1975 NSCLC cell lines  were purchased from American Type Culture Collection (ATCC) . Cell lines were grown in incubators at 37 °C with 6% CO(−Δ(ΔCt)) was calculated to evaluate the fold changes in gene expression. All primers  and H358-P/ER cells were plated in 35 mm petri dishes and allowed to adhere and grow for 24 h. Cells were then starved for 24 h and total RNA was collected using a PureLink RNA Mini Kit (cat. no. 12183018A) from Life Technologies, according to the manufacturer’s protocol. RNA was quantified using the Take3 NanoDrop method. Total RNA was used to perform qPCR with an Applied Biosystems 7300 Real Time-PCR system. For this procedure, a SuperScript™ III Platinum™ SYBR™ Green One-Step qRT-PCR Kit (cat. no. 11736059) from Life Technologies was employed and the manufacturer’s protocol was followed. The cycle threshold (Ct) values were recorded and normalized using GAPDH, and 2 primers  used forNSCLC cells were cultured as described previously . H2170-ETM 488 (1:250)  and Hoechst nuclear staining dye   diluted in 1X PBS with 1% BSA. Slides were then mounted and covered with a coverslip using fluorogel . Stained cells were observed using an Olympus Fv10i Fluoview Confocal Microscope and images were captured. Average fluorescence intensity of staining was quantitatively measured using Olympus Fluoview image analysis software.NSCLC cells were seeded on an eight-well chamber slide, fixed, permeabilized, blocked, and incubated at 4 °C overnight with primary antibodies for VEGFR-2 and NP-1. Primary antibodies were then removed and the cells were incubated in the dark at room temperature for an hour with anti-mouse/rabbit IgG secondary antibody conjugated with DyLightNSCLC cells were seeded in a 100 mm petri dish. After 48 h, the growth medium was removed. The cells were then washed with PBS and treated with Accutase Cell Detachment Solution  in an incubator for five minutes or until all of the cells were detached. Cells were collected in a 15 mL tube, pelleted using a centrifuge, and then re-suspended in flow cytometry buffer (PBS + 2 mM EDTA + 0.5% BSA). Cells were pelleted again and probed with fluorophore-labeled antibody for ten minutes on ice. The cells were washed with flow cytometry buffer to remove excess antibody, then suspended in FACS buffer for analysis with a BD FACS CALIBUR flow cytometer.NSCLC cells were seeded on a 96-well plate in RPMI medium with 10% FBS. After 24 h, cells were treated with TKIs, suspended in 100 µL starving media (RPMI + 0.5% BSA) per well, and incubated for an additional 72 h at 37 °C. Cells were then washed with RPMI media without phenol red and 100 µL of phenol red-free RPMI media was added to the cells. Subsequently, 10 µL of MTT reagent  was added to each well and plates were incubated for four hours at 37 °C. Finally, 100 µL of MTT solubilization reagent (acidic isopropanol) was added to each well and mixed to dissolve any and all formazan crystals formed. Absorbance was measured using a spectrophotometer at wavelengths of 570 nm and 630 nm, and percent cell viability was calculated with respect to controls. This experiment was performed in replicates of six for each treatment condition.2 vented flasks and allowed to adhere for 24 h in RPMI medium with 10% FBS. Medium was then removed, followed by two 1X PBS washes. Serum-free medium was added and incubated for 24 h. Then, the medium was pipetted into a micro-centrifuge tube and centrifuged for ten minutes at 4 °C with a LABNET Refrigerated Z326K centrifuge  at 1500 rpm. The supernatant was immediately aliquoted, avoiding the debris pelleted, and samples were stored at −70 °C. Collection of conditioned medium was repeated for 24 and 48 h time points. The commercial ELISA kit Human VEGF-A ELISA from RayBiotech  was used to detect and quantify the amount of VEGF in the NSCLC cell lines at two separate time points: 24 h and 48 h. The vendor’s protocol was followed to run the experiment, and each test was run in triplicate.NSCLC cells were plated in 25 cmWe analyzed 79 paraffin-embedded, formalin-fixed tissues from patients with NSCLC. All cases were retrieved from the pathology archives at Rockford Memorial Hospital and the Swedish American Hospital with institutional approval, in accordance with the Institutional Review Board protocol (351597-11 and 17 August 2018). Immunostaining procedures were conducted as described previously . Approprt-test was used to analyze the statistical significance of the data. A p-value of less than 0.05 was considered statistically significant throughout the study.All experiments were performed three to five times. The Student’s The gene expressions of potential EGFR-TKI-resistance modulators were analyzed in the wild type-EGFR NSCLC cell lines H358-P and H2170-P and compared to corresponding Erlotinib-resistant H358-ER and H2170-ER cell lines. H358 cell lines also possess a unique KRAS mutation. The results demonstrated an increase in gene expression of VEGF (2.5-fold), VEGFR-2 (3.5-fold), and NP-1 (1.4-fold) in H2170-ER compared to H2170-P cells, as can be seen in Immunoblotting studies were performed to assess the modulation of VEGFR-2 and NP-1 protein expression in wild-type EGFR NSCLC cells (H2170-P/ER and H358-P/ER), TKI-sensitive EGFR L858R-mutant NSCLC cells (H3255), and TKI-resistant EGFR L858R- and T790M-mutant NSCLC cells (H1975). Each cell line was analyzed without treatment, after treatment with Erlotinib, after treatment with epidermal growth factor (EGF), and after treatment with a combination of Erlotinib and EGF A.The expressions of VEGFR-2 and NP-1 from Western blots were quantified and analyzed. The results demonstrated an increase in VEGFR-2 (3.6-fold) and NP-1 (2.8-fold) expression in the presence of 10 µM Erlotinib in H2170-ER cells compared to the H2170-P cell line subjected to the same treatment B. AdditiAfter observing upregulation of gene and protein expression of VEGFR-2 and NP-1 in ER cells, the levels of expression of these proteins were measured on cell surfaces. Flow cytometry assays were performed to measure the expression of VEGFR-2 and NP-1 in H2170-P/ER, H358-P/ER, H3255, and H1975 cell lines. In H2170-ER cells as compared to H2170-P cells, there was an increase in mean fluorescent intensity for VEGFR-2 (2.1-fold) and NP-1 (3.8-fold) A. In H35Immunofluorescence (IF) was performed to confirm and further study the specific localization of VEGFR-2 and NP-1 in parental (H2170-P and H358-P), Erlotinib-resistant (H2170-ER and H358-ER), EGFR single-mutated (H3255), and EGFR double-mutated (H1975) cell lines. In H2170-ER cells, VEGFR-2 was expressed mainly on the cell surface and in the cytoplasm, while in H2170-P cells, VEGFR-2 was expressed mainly in the cytoplasm A. In H21In H358-ER cells, VEGFR-2 was expressed on the cell surface, in the cytoplasm, and in the nucleus, while in H358-P cells, VEGFR-2 was minimally expressed on the cell surface, in the nucleus, and in the cytoplasm B. NP-1 wIn H1975 cells, VEGFR-2 was expressed on the cell surface, in the cytoplasm, and in the nucleus, while VEGFR-2 was minimally expressed in the nucleus and on the membrane of H3255 cells C. NP-1 wAn enzyme-linked immunosorbent assay (ELISA) was performed to study the role of VEGF secretion in angiogenesis and EGFR-TKI resistance. Human VEGF concentration was quantified and analyzed for all cell lines and used to calculate levels of VEGF secretion. The results demonstrated that VEGF concentration (pg/mL) increased from 24 h to 48 h in all cell lines studied . We obseTo further study the effect of VEGF in EGFR-TKI resistance, we investigated the use of a humanized monoclonal VEGF antibody, bevacizumab (Avastin), which binds to and neutralizes VEGF. An MTT cell viability assay was performed to assess the effects of Avastin, Erlotinib, and both in combination in Erlotinib-resistant and EGFR-mutated NSCLC cells lines. Cells being resistant to 10 µM of Erlotinib, this was the concentration that was used, and since Avastin showed a similar inhibitory effect on cells in a concentration range of 200 ng/mL to 1000 ng/mL, we used 200 ng/mL of Avastin. The 10 µM Erlotinib treatment decreased cell viability to between 62 and 82% compared to the diluent, while the 200 ng/mL Avastin treatment decreased cell viability to between 84 and 89% compared to the diluent . FurtherAfter studying the effects of treatment against VEGF, we studied the inhibition of VEGFR-2 in Erlotinib-resistant and EGFR-mutated NSCLC cell lines to further elucidate its role in EGFR-TKI resistance. An MTT cell viability assay was performed with 5 µM Erlotinib treatment, 12 µM ZM 323–881 HCl (ZM) (VEGFR-2 inhibitor) treatment, and a combination treatment of Erlotinib and ZM. We tried different concentrations of ZM and selected a concentration of ZM which resulted in less than 50% inhibition. We used Erlotinib at a concentration of 5 uM, since the resistant cell lines studied showed 25–26% inhibition with 5 μM Erlotinib. We wanted to study a combination effect, so we kept the concentration of Erlotinib at half the dose we used for other aspects of the studies. As seen in p < 0.05) (Immunohistochemistry (IHC) was performed to analyze the expression of VEGFR-2 in tumor tissues from NSCLC patients. Forty-eight NSCLC tumor tissues were assessed for VEGFR-2 expression through IHC with VEGFR-2 staining A. Kaplan < 0.05) B.In this study, we recorded upregulation of the gene expression of VEGF, VEGFR-2, and NP-1 in Erlotinib-resistant cell lines as compared to parental cells and in EGFR double-mutant cells as compared to their single-mutant counterparts . In all VEGFR-2 and NP-1 protein expressions were also increased in EGFR double-mutant H1975 cells compared to H2170-ER and H358-ER cell lines, a result attributed to the T790M mutation in H1975 cells B. UnlikeThese mutations cause altered expression of EGFR, which results in the activation of downstream signaling pathways, such as the MAPK, FAK/Paxillin, and PI3K/AKT/mTOR pathways, which, in turn, promotes tumor cell proliferation, survival, migration, and angiogenesis .AccordinStudies have shown that NP-1 interacts with VEGFR-2 in a cis position, which, in turn, increases the binding affinity of VEGF to VEGFR-2 . In thisOur results also demonstrated that total VEGF secretion increased over time . VEGF seAfter determining that VEGF inhibition is not useful for overcoming EGFR-TKI resistance, we inhibited VEGFR-2 using a selective VEGFR-2 inhibitor, ZM 323–881 HCl (ZM). Based on our results, treatment with ZM alone and treatment with a combination of Erlotinib and ZM both significantly decreased cell viability in Erlotinib-resistant and EGFR-mutated cell lines . These rDuring NSCLC tumor screening with VEGFR-2 and NP-1 by IHC assays, we studied the expression levels of these angiogenic biomarkers in late- and early-stage NSCLC patients. VEGFR-2 is a transmembrane tyrosine kinase receptor, and studies have shown that VEGFR-2 is internalized with the help of NP-1 . Our floThis study investigated three angiogenic biomarkers, VEGF, VEGFR-2, and NP-1, that may play a role in EGFR-TKI resistance. In EGFR-TKI-resistant cells compared to TKI-sensitive cell lines, an upregulation of VEGFR-2 and NP-1 gene and protein expression, as well as increased cell surface expression, was observed. In addition, we observed that combinatorial treatment of Erlotinib and a selective VEGFR-2 inhibitor may serve as a more effective treatment option than inhibition of VEGF with a humanized monoclonal antibody Avastin. TKIs such as Erlotinib and Gefitinib have no effect on lung adenocarcinoma patients with KRAS mutations; however, combination therapy with Erlotinib and VEGFR-2 inhibitor can help achieve better prognoses for these patients. Our studies suggested that high VEGFR-2 expression is associated with poor prognosis in NSCLC patients, and we also studied single- and double-mutant NSCLC cell lines to provide a new outlook on ways to overcome EGFR-TKI resistance in NSCLC patients.


Soluble α-Klotho (s-Klotho) is a circulating protein with pleiotropic effects that mainly induce protective effects. Our study investigates the associations between s-Klotho and several established inflammatory biomarkers, with the aim of examining whether s-Klotho levels are representative of inflammatory states.A total of 11,128 eligible participants from NHANES 2007–2016 were included in our study. Levels of four inflammatory biomarkers, uric acid (UA), C-reactive protein (CRP), white blood cell (WBC) count, and mean platelet volume (MPV), were examined for their relationship with s-Klotho levels. Sub-analyses sorted the total population by gender and into four quartiles. Linear regression models were used to evaluate the strengths of associations.p<.001), and this relationship was stronger in women than in men . In addition, all four biomarkers manifested stronger associations with s-Klotho in higher quartiles, and the highest absolute values of β coefficients appeared in Q4 vs. Q1.All four inflammatory biomarkers were significantly associated with s-Klotho levels. UA, CRP, and WBC count showed an inverse association, while MPV showed a direct one. Of the four markers, UA was most strongly correlated with s-Klotho levels  levels are significantly associated with the inflammatory markers uric acid, C-reactive protein, white blood cell count, and mean platelet volume.S-Klotho is involved in inflammatory processes and plays a protective role.S-Klotho may serve as an inverse indicator of inflammation.s-Klotho is significantly associated with well-recognized inflammatory biomarkers. A decrease in s-Klotho levels implies a general inflammatory status; therefore, s-Klotho serves as a potential biomarker that is inversely correlated with inflammatory conditions. Further applications in clinical practice will provide us with a better understanding of its role. In addienotypes ,6, endotenotypes .2+ transient receptor potential vanilloid type 5 in nephrons, which contributes to the maintenance of normal renal function [Emerging evidence illustrates a protective role of s-Klotho in various disorders, prompting researchers to investigate its underlying mechanisms. A study demonstrated that s-Klotho inhibits the insulin/insulin-like growth factor-1 signalling pathway, which promotes resistance to oxidative stress, thereby extending the lifespans of mice . Other sfunction ,13.Because many studies have focussed on the benefits of s-Klotho in individual organs, there is less known about s-Klotho as a circulating factor and how it affects systemic health. This study explored the relationship between established inflammatory biomarkers and s-Klotho levels, aiming to determine whether s-Klotho modulates inflammation generally and its relationship with separate biomarkers.The National Health and Nutrition Examination Survey (NHANES) 2007–2016  databaseWe collected information from NHANES 2007–2016 because the main biomarker of interest, s-Klotho, is available from these years. There were 40,617 participants between 2007 and 2016, and we initially included those with complete data about s-Klotho levels. Subsequently, we excluded those with missing data on inflammatory biomarkers, including serum levels of uric acid (UA) and C-reactive protein (CRP), white blood cell (WBC) count, and mean platelet volume (MPV), leaving 11,128 subjects eligible for further analyses. We performed analyses in the total cohort and in men and women separately to evaluate gender differences. In addition, we divided the values of inflammatory biomarkers into quartiles for detailed comparisons. A flowchart showing the design and structure of our study is presented in All details about laboratory methodology and protocols are provided in the NHANES Laboratory/Medical Technologists Procedures Manual . s-KlothCRP concentration data included CRP values from 1999–2010 and high-sensitivity CRP from 2015–2018. The former values were determined using latex-enhanced nephelometry, and the latter was based on the highly sensitive near-infrared particle immunoassay rate methodology. WBC counts and MPVs are included in complete blood count tests. They were both analysed on the Beckman Coulter UniCel DxH 800 . Albumin levels were measured using the DxC800 as a bichromatic digital endpoint method. High-density lipoprotein cholesterol levels were examined enzymatically with a Roche/Hitachi Modular P Chemistry Analyser  according to the manual.2 (weight in kilograms/height in meters). The following categories for race were used: Mexican–American, other Hispanic, non-Hispanic White, non-Hispanic Black, and other races. A history of smoking was defined as having smoked at least 100 cigarettes in life. Histories of medical conditions were confirmed by asking the participants if they had ever been told by a doctor that they had one of the following conditions: coronary heart disease, angina, diabetes, and hypertension.Demographic information was acquired by trained examiners in household interviews. The formula for body mass index was kg/mp values<.05 were considered statistically significant. Linear regression analyses were utilized, and β coefficients were calculated to estimate the strengths of relationships. Three models were provided for each analysis to eliminate the effects of nuisance variables: Model 1 = unadjusted; Model 2 = adjusted for age, gender, body mass index, and race/ethnicity; Model 3 = Model 2 + adjusted for history of angina, coronary heart disease, diabetes mellitus, hypertension, and smoking status.SPSS  was used for statistical analyses. Qualitative data were expressed in percentages (%), while quantitative variables were expressed as means and standard deviations. All 3 cells/μl for WBC count, 862.29 ± 306.37 pg/ml for s-Klotho, 5.54 ± 1.43 mg/dl for UA, 0.46 ± 0.89 mg/dl for CRP, and 8.11 ± 0.95 fl for MPV.This study included 11,128 participants, including 5402 (48.5%) men and 5726 (51.5%) women. Demographic and laboratory profiles are shown in p<.001). The remaining variables had the following β coefficients in descending order: MPV ; CRP ; and WBC count . Further analyses showed gender differences for each biomarker. In women, UA and WBC count were more strongly correlated with s-Klotho levels  ). In men, CRP and MPV were more strongly correlated with s-Klotho levels  ).As shown in p<.001; Q3 vs. Q1, β coefficient= −65.72, 95% CI= −82.32, −49.13, p<.001; Q4 vs. Q1, β coefficient= −107.13, 95% CI= −123.78, −90.48, p<.001).To determine whether the associations between s-Klotho and the four inflammatory biomarkers changed on a gradient, we stratified levels of the four biomarkers into quartiles. In Our study indicates that several established and widely used inflammatory biomarkers are associated with s-Klotho levels. We demonstrated that s-Klotho functions as an inverse indicator of inflammation because of its inverse association with UA, CRP, and WBC count, and its direct association with MPV. Among these, UA was most strongly correlated with s-Klotho. These findings suggest that s-Klotho may serve as a predictive biomarker for inflammation.p=.022) [Few studies have mentioned the relationship between s-Klotho and inflammatory cytokines. A case–control study with a designated experimental group of 110 patients with histories of cardiovascular disease (CVD) showed that s-Klotho levels were significantly lower in the CVD group and inversely correlated with CRP and TNFα/IL10 . Anotherp=.022) . Howevervia the ROS–MAPK–NF-κB signalling pathway in rat vascular smooth muscle cells, which contributes to the development of CVD [Hyperuricaemia has long been associated with gout , but rect of CVD . In contt of CVD . The medt of CVD . Neverthp = .13). In our study, the inverse association remained significant in all models, including both the unadjusted and fully adjusted ones. This indicated that even after taking the following factors into consideration—age [The inverse relationship between CRP and s-Klotho has been described in CVD patients in a study performed in Santa Cruz de Tenerife, Spain . Howevertion—age , historytion—age —which alElevated WBC count, or leukocytosis, is a clinical sign often linked with infection or inflammation . MPV is et al. reported that under hyperglycaemic conditions, the shedding of Klotho is expedited, causing a decrease in membrane-bound Klotho and an increase in s-Klotho. They also demonstrated the addition of Klotho to cell medium improved kidney function via effects such as increased glycolytic capacity and lowered albumin permeability. Collectively, their findings supported the idea that Klotho ameliorates inflammation, although how s-Klotho predicts inflammatory status in diabetic nephropathy differed from our results. In view of this, the interpretation of serum s-Klotho levels in specific diseases requires further investigation.Despite the general negative relationship between s-Klotho levels and inflammatory biomarkers, under certain circumstances, the association may be positive. A recent study measuring serum and urine s-Klotho levels in rats found significantly higher serum levels of s-Klotho in diabetic subjects . InitialLevels of biomarkers discussed in the present study were altered significantly during the inflammatory process; however, each was regulated in by a unique mechanism. Elevation in circulating WBCs indicates the initiation of host defences against various kinds of injuries, and the five types of WBCs, neutrophils, lymphocytes, eosinophils, basophils, and monocytes, share general anti-inflammatory properties, although each mediates specific immune responses . IncreasNumerous studies that have investigated the functions of Klotho have demonstrated its distinctive role in vascular homeostasis. In CVD, Klotho suppresses IL-6 production in endothelial cells , inhibitComparing the four biomarkers led to the noteworthy finding that UA levels had the strongest relationship with s-Klotho levels, which may be attributable to their shared site of production—the kidney. Serum UA level is determined mainly by reabsorption and excretion in the kidney , and s-KThe pros and cons of incorporating s-Klotho testing into routine clinical practice are worth discussing. Compared with UA, CRP, WBC count, and MPV, which are already commonly used laboratory markers, the importance of Klotho has only been determined in the past decade. While the other four markers are altered in most inflammatory conditions, s-Klotho stands out because it is regulated in particular organs, such as the kidneys, and in cardiovascular-involved diseases, which permits it to provide distinct diagnostic values. However, Klotho is more costly to determine than established biomarkers, which may limit its application in general practice at the present time.The findings of this study must be viewed considering some limitations. First, our study revealed a positive relationship between s-Klotho and MPV, illustrating that decreased MPV is linked to inflammation. However, studies have shown that either increases or decreases in MPV can indicate various inflammatory statuses and diseases . Interprs-Klotho is significantly associated with well-recognized inflammatory biomarkers. Negative associations were demonstrated with UA, CRP, and WBC count, while a positive association was demonstrated with MPV. UA had the strongest relationship with s-Klotho, suggesting that s-Klotho could serve as an alternative inverse indicator for evaluation of inflammatory states. Further studies are required to assess the applicability of s-Klotho in clinical practice.