Although the high offset Tri-Lock bone preservation stem (BPS) was used widely, few studies explored the clinical and radiological results. The purpose of this study was to determine the clinical and radiological results of high offset Tri-Lock BPS in unilateral primary total hip arthroplasty (THA) at a minimum follow-up of 3 years.55 patients who underwent cementless THA with high offset Tri-lock BPS from 2017 to 2018 were followed for a minimum follow-up of 3 years. Patients were assessed clinically for complications, Harris hip score (HHS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Oxford Hip Score (OHS). Femoral offset (FO), acetabular offset (AO), hip offset (HO), HO difference, and leg length discrepancy (LLD) were measured on the anteroposterior (AP) pelvic radiograph. Standard pelvic AP and lateral radiographs were used to evaluate for evidence of bone ingrowth, stem subsidence, stem alignment, radiolucent line around the stem, osteolysis, loosening, ectopic ossification, and femoral stress shielding.No patients reported complications during hospitalization nor the follow-up period. At a mean follow-up of 42.5 months, the mean HHS, WOMAC, and OHS scores showed a significant improvement from preoperative to the latest follow-up. No patients reported thigh pain. No revision nor sign of radiographic loosening had been detected. The high offset Tri-Lock BPS significantly improved the FO and HO postoperatively. HO difference and LLD were balanced postoperatively. No sign of stem subsidence, radiolucent line, osteolysis, loosening, ectopic ossification, nor severe stress shielding (more than grade 3–4) were observed at the latest follow-up.The high offset Tri-Lock BPS demonstrated excellent clinical and radiographic outcomes at a minimum follow-up of 3 years. HO difference and LLD between legs decreased significantly and achieved balance postoperatively. Long-term follow-up is required for a definitive conclusion. Total hip arthroplasty (THA) is an effective method to treat end-stage hip disease for relieving the pain and improving joint activity and the quality of life , 2. The Recently, shorter bone preservation stems (BPS) were widely used in THA because of specific advantages . The novTri-Lock BPS manages soft tissue laxity without affecting leg length by choosing different offset stems, including standard and high offset stems . FemoralAlthough the high offset BPS was used widely, few studies explored the clinical and radiological results. Given that any design of an implant demands a careful follow-up, we conduct this specific study on outcomes of high offset Tri-Lock BPS in unilateral primary THA. The purpose of this study was to determine the clinical and radiological results of high offset Tri-Lock BPS in unilateral primary THA at a minimum follow-up of 3 years.The inclusion criterion was as follows: (1) patients who underwent primary THAs in our institution without contraindication; (2) patients using high offset Tri-lock BPS  in the THA surgery; (3) patients had enough radiological and clinical data; (4) patients had a contralateral native hip;The exclusion criterion was as follows: (1) patients with one-stage bilateral THA; (2) patients who were diagnosed as dysplasia of the hip (DDH) Crowe type III or IV; (3) patients who had disqualified or incomplete radiological data; (4) patients who were lost to follow up; (5) patients had a contralateral hip with prosthesis or deformity.This retrospective study was approved by the clinical trials and biomedical ethics committee of West China Hospital and written informed consents were obtained from all the participants. From April 2017 to April 2018, 80 patients (87 hips) were identified who underwent cementless THAs with high offset Tri-lock BPS in our institution by five experienced senior surgeons. Of all the 80 patients, 7 patients (14 hips) were one-stage bilateral THAs, 6 patients (6 hips) had disqualified radiological data, 5 patients (5 hips) were lost to follow up, 6 patients (6 hips) had a contralateral hip with prosthesis or deformity, and 1 patient died for cholangiocarcinoma, which was unrelated to the THA surgery. Thus, 55 hips in 55 patients comprised the study population. Patients' demographic and characteristic data were collected from the electronic medical data (Table Preoperative templating in the standard anteroposterior (AP) pelvic radiograph was used to identify appropriate acetabular size and location. Appropriate femoral stem size and neck length were confirmed to fit the geometry of the femur and acquired proper hip offset (HO) and leg length discrepancy (LLD) then. Tri-lock BPS dual offset options provided surgeons with standard and high offset stems to restore femoral offset without affecting leg length. We only included patients who use high offset Tri-lock BPS during the THA in our study. All patients were treated with general anesthesia. Pinnacle acetabular cup systems  were used in the surgeries, including 32/36 mm ceramic heads and appropriate liner. A polyethylene/ceramic liner accommodating a ceramic head was used in all the patients. All the surgeries were performed through the posterolateral (PL) approach or direct anterior approach (DAA). Conventional prophylactic intravenous antibiotics and thromboprophylaxis were used postoperatively. Furthermore, patients were encouraged to take active exercise in bed after surgery. On the second day postoperatively, partial weight training with the help of a walking aid was conducted after confirming the AP pelvic and lateral X-rays. We educated patients to walk with a walking stick two weeks postoperatively. They gradually change to full weight-bearing training four weeks postoperatively. Patients were followed up at two weeks, four weeks, three months, six months, 12 months, and annually postoperatively.Electronic medical records were evaluated to obtain all the in-hospital complications. Complications such as wound infection, periprosthetic infection (PJI), deep venous thrombosis (DVT), pulmonary embolism (PE), periprosthetic fracture (PFF), dislocation, and persistent thigh pain were recorded during the follow-up. Reoperation and revision for any reason were recorded during the follow-up period. Harris hip score (HHS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Oxford Hip Score (OHS) were used to assess the clinical function of the patients preoperatively and at the latest follow-up. OHS, WOMAC and HHS were classic and common functional hip scores and widely used to report clinical outcomes following THA , 28.All the radiographs were collected and measured by two independent reviewers who were unrelated to the surgeries and blinded to the clinical outcomes. Another measurement was performed by the same two observers one week after the initial measurement, blinded to the previous results. Radiographic assessment was measured on the AP pelvic radiograph with the lower limb in 15° internal rotation using Syngo  preoperatively, second day postoperatively, and at the latest follow-up. The preoperative geometry of proximal femur was classified by the classification system of Dorr et al. Fig.  37]. Th. Th37]. The data were analyzed using SPSS 26.0 . A two-tailed paired t-test was used to assess the differences between the FO, HO, HO difference, and LLD preoperatively and on the second day postoperatively. A two-tailed paired t-test was used to assess the HHS, WOMAC, OHS scores. Intraclass correlation coefficients (ICC) were used to measure intraobserver and interobserver reliability with a two-way random model for absolute agreement. P-value < 0.05 was defined as statistical significance.2(range 17.9–33.8). Twenty patients had left side surgeries, while 35 patients had right. The most common diagnosis was osteonecrosis of the femoral head (ONFH), which was found in 29 (52.7%) patients. According to the Dorr classification, ten patients (18.2%) were classified as Dorr A, 43 patients (78.2%) as Dorr B, and two patients (3.6%) as Dorr C. And the mean follow-up was 42.5 months (range 36–48) (Table The mean age of the study population was 49.8 years (range 25–73). Our study included 13 females (23.6%) and 42 males (76.4%). Patients' mean body mass index (BMI) was 23.8 kg/mThirty patients (54.5%) underwent THA through a PL approach, while 25 patients (45.5%) through a DAA approach. The mean stem size was 3.3# (range 0#-8#). Nineteen patients (34.5%) received 32 mm Ceramic femoral heads, while 36 patients (65.5%) received 36 mm ones. The bearing surface was ceramic-on-ceramic in 50 patients (90.9%) and ceramic-on-polyethylene in 5 patients (9.1%). Fifty-three patients (96.4%) had a neutral stem alignment, one patient (1.8%) had valgus, and one (1.8%) had varus . At the latest follow-up, all the patients (100%) had excellent or good function results on HHS. The WOMAC total score decreased from 50.04 ± 9.40 preoperatively to 3.27 ± 3.36 at the latest follow-up (p < 0.01). Besides, the OHS decreased from 36.15 ± 8.80 preoperatively to 15.33 ± 3.12 at the latest follow-up (p < 0.01) (Table The HHS showed a significant improvement from 48.13 ± 9.66 preoperatively to 96.84 ± 5.60 at the latest follow-up (p < 0.01). The HO improved from 81.0 ± 8.6 mm to 83.4 ± 8.5 mm (p < 0.01). The HO difference between legs decreased from 2.8 ± 7.0 mm preoperatively to 0.4 ± 5.7 mm postoperatively (p < 0.01). Besides, the LLD decreased from 7.4 ± 8.4 mm to 4.3 ± 3.3 mm (p < 0.01). On the second day postoperatively, 53 patients (96.4%) limited LLD within 10 mm, and all the patients limited LLD within 20 mm . The mean FO significantly improved from 43.2 ± 9.1 mm to 48.2 ± 7.4 mm  had a neutral stem alignment. Only one patient (1.8%) had a valgus stem, and another (1.8%) had a varus stem. Those two patients also achieved good clinical and radiographic outcomes at the latest follow-up. There were no radiographic signs of stem subsidence, radiolucent line, osteolysis, loosening, or ectopic ossification. Stress shielding was observed in 55 hips (100%). Among them, 32 patients (58.2%) were classified in grade 1 and 23 patients (41.8%) were classified in grade 2  stress shielding in our study. Our results were consistent with previous studies. Zhen et al.  found thHO and leg length reconstruction postoperatively is crucial for an additive effect on clinical outcomes . RestoraThe strengths of this study were its completeness of the clinical and radiological results of high offset Tri-Lock BPS at a minimum follow-up of 3 years. Moreover, we have quantified the improvement of offset and LLD firstly by using this specific stem.There are also some limitations to our study. First, the sample size of this study was relatively small and lacked a control group. Second, this was a retrospective study and the risk of selection bias cannot be avoided. Third, all the patients were followed for not more than 4 years. Further studies are required to detect long-term clinical and radiological results of high offset Tri-Lock BPS.The high offset Tri-Lock BPS demonstrated excellent clinical and radiographic outcomes at a minimum follow-up of 3 years. There were no radiographic signs of stem subsidence, radiolucent line, osteolysis, loosening, or ectopic ossification. HO difference and LLD between legs decreased significantly and achieved balance postoperatively. Extended follow-up is required to assess the durability of this stem.


Since cancer cachexia is a disease condition presenting with weight loss, skeletal muscle atrophy, and loss of adipose tissue in patients with advanced cancer, we hypothesized that NR intake could ameliorate sarcopenia. In this study, we investigated whether preemptive administration of NR ameliorated C26 adenocarcinoma–induced cancer cachexia and explored anti-cachexic mechanisms focused on the changes in muscle atrophy, cachexic inflammation, and catabolic catastrophe. Dietary intake of the NR-containing pellet diet significantly attenuated cancer cachexia in a mouse model. Starting with significant inhibition of cachexic factors, tumor necrosis factor alpha, and interleukin-6, NR significantly inhibited muscle-specific ubiquitin-proteasome ligases, such as atrogin-1, muscle RING-finger protein-1 (MuRF-1), mitofusin-2, and peroxisome proliferator–activated receptor gamma coactivator-1-alpha (PCG-1α). Significant inhibition of epididymal fat lipolysis was noted with significant inhibition of adipose triglyceride lipase (ATGL) gene. Furthermore, NR administration significantly increased the levels of crucial enzymes involved in the biosynthesis of NAD+ and nicotinamide phosphoribosyl transferase and significantly inhibited the NAD+-sensitive deacetylase sirtuin 1 (SIRT1). Preemptive intake of NR in patients vulnerable to cachexia can be a preemptive option to ameliorate cancer cachexia.Nicotinamide riboside (NR), vitamin B3, is a substrate for nicotinamide adenine dinucleotide (NAD Since complete treatment of cancer as well as the associated and complicated cachexia, except for the early detection of cancer, is not easy, and recovery from the underlying condition causing cancer is impregnable, troublesome cancer cachexia is an unmet medical need. Therefore, as a scientific approach to cancer cachexia, many attempts have been made to inhibit cancer cachexia by targeting the inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6. Downstream of these cytokine-associated cachexic factors, muscle atrophy–associated genes, such as +) metabolism in health and disease is of increased interest because nicotinamide can protect tissues, and NAD+ metabolism has been implicated in a variety of disease states in addition to extending the lifespan  cells, were obtained from Cell Lines Service GmbH  and maintained according to the CLS’s instructions. C26 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum  and 1% antibiotic anti-mycotic solution  at 37°C in a humidified atmosphere composed 5% COWe administered a mouse diet supplemented with NR at 200 or 400 mg/kg daily for 3 weeks as pellet diet.Balb/c mice  were randomly divided into four groups: normal group (n = 10), cachectic C26 adenocarcinoma–bearing group (n = 10), cachectic C26 adenocarcinoma–bearing group with 200 mg/kg containing pellet diets (n = 10), and cachectic C26 adenocarcinoma–bearing group with 400 mg/kg containing pellet diets (n = 10). Cachectic C26 adenocarcinoma–bearing groups were shaved on the legs side and injected in their right flank with 1 × 107 C26 cells. According to the method of , cells were counted using a hemocytometer, suspended in 100 μl of sterilized phosphate-buffered saline and then C26 cells were injected into the mice. For 3 weeks, we fed a normal pellet diet or pellet diet with NR with daily measurement of intake. We restricted feeds to 100 g per group to measure reduced food intake due to anorexia. Following this experimental regimen, mice were monitored every 3 days, including body weight and food intake. Leg muscles were isolated and subjected to further histologic examination, enzyme-linked immunosorbent assay (ELISA), Western blotting, and reverse transcription polymerase chain reaction (RT-PCR). Animal studies were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of CHA University CHA Bio Complex after IRB approval (IACAC 2019-0601).Six-week-old male On day 21 of the experiments, the mice were sacrificed. After the sacrifice of the animals, blood was collected for ELISA assay. After centrifugation at 3,000 rpm, 4°C for 30 min, the IL-6 and TNF-α levels in the supernatant were measured by ELISA, and concentration of IL-6/TNF-α is expressed as pg/mg protein. The process was performed strictly as prescribed in IL-6 or TNF-α ELISA kit manuscript . All samples were measured at individual levels, and each sample was analyzed in triplicate manner, taking a mean of the three determinations.Total RNA was isolated from leg muscle tissues using TRIzol reagent , and 1–5 mg of each total RNA was transcribed to cDNA using cDNA synthesis kit . The PCR mixture contained 2× PCR MasterMix , autoclaved water, primer (10 pmole/lL), and cDNA in final volume of 20 μl. PCR was performed over 30 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s. Oligonucleotide primers were purchased from Bioneer . Oligonucleotide primers for IL-6, Atrogin-1, MuRF-1, Mfn-2, and GAPDH are shown in Extracted tissues were washed twice with PBS and then lysed in ice-cold cell lysis buffer  containing 1 mM phenylmethylsulfonyl fluoride . After 20 min of incubation, samples were centrifuged at 10,000 × g for 10 min. Supernatants were then collected. Proteins in lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, which were incubated with primary antibodies, washed, incubated with peroxidase-conjugated secondary antibodies, rewashed, and then visualized using an enhanced chemiluminescence (ECL) system  and the relative amounts of proteins associated with specific antibody were quantified using Lumi Vision Imager software (TAITEC). Primary antibodies for Western blotting against NAMPT1, SIRT1, Pax7, ubiquitin, p-p38, ATGL (adipose triglyceride lipase), PARP-1, caspase-8, Bax, and Cyt c were from Cell Signaling Technology . Antibodies against MuRF-1 (muscle ring-finger protein-1), Mfn-2 (mitofusin-2), p-AMPKα 1/2, PGC-1α , p-JNK, p-ERK, and β-actin were from Santa Cruz Biotechnology  Antibody against Atrogin-1 was from ECM biosciences . Anti-mouse and anti-rabbit horseradish peroxidase–conjugated secondary antibodies were from Cell Signaling .p < 0.05.Statistical analysis results are expressed as the mean standard deviation (SD). Statistical analyses were conducted with GraphPad Prism  and SPSS software . Statistical significance between groups was determined by a multivariate test, Kruskal–Wallis test. Statistical significance was accepted at n = 10/group) were included as follows: normal control, cancer cachexia control, and cancer cachexia administered 200 mg/kg NR and 400 mg/kg NR as pellets in their daily diet. The experiments continued until 3 weeks after C26 cell administration because 20% weight loss was noted in the cachexia control group . Although there were no significant differences in the transplanted C26 cells between groups, weight changes were significantly different between the control group and the groups administered the NR-containing pellet diet . However, groups 3 and 4 that were administered 200 mg or 400 mg/kg NR mixed in pellet diet presented significantly decreased TNF-α and IL-6 levels (p < 0.01).TNF-α and IL-6 are well-known cytokines that provoke cancer cachexia in the C26 adenocarcinoma model, where we measured the serum levels of both. As seen in + pool utilizing nicotinamide riboside kinase 1 (NRK1) to synthesize NAD+ from NAD+ precursors , cancer cachexia was significantly associated with the activation of either extracellular-signal regulated kinase 1/2 (ERK1/2) or c-Jun N-terminal kinase (JNK) . ERK1/2 ecursors , was sigecursors . In contatrogin-1 and MuRF-1, were significantly increased in group 2, signifying that muscle-specific ubiquitin-proteasome ligase was enhanced during muscle atrophy in cancer cachexia , whereas epididymal fat was significantly preserved in the NR group. Adipose triacylglycerol lipase was significantly increased in group 2, leading to significant lipolysis in cancer cachexia, but was significantly decreased in the NR group  and NR as NAD+ precursors appears efficacious in elevating muscle NAD+ (+ availability. NAMPT catalyzes the conversion of nicotinamide to NMN, which is associated with most of the NAD+ formation.Since NADcle NAD+ . Therefo+ intermediates, including NR and NMN, have been studied over the past several years (+ intermediates has shown preventive and therapeutic effects against aging and sarcopenia. Although not measured in this investigation, NRK1 is a rate-limiting enzyme for the use of exogenous NR for NAD+ synthesis (+ synthesis, we measured the changes in SIRT1 instead of NRK because NAD+ is a vital redox cofactor and a substrate required for the activity of various enzyme families, including sirtuins and PARP.As the pathogenesis of age- or cachexia-associated muscle declines, two key NADal years  because ynthesis . Since m+ also play important roles in the regulation of NAD+-consuming enzymes, including sirtuins, PARPs, and CD38/157 ectoenzymes, and NAD+ biosynthesis mediated by NAMPT and SIRT1 function together to regulate metabolism, NAD+ levels decline during cachexia, causing defects in mitochondrial functions and resulting in sarcopenia (+ by supplementing NR could dramatically ameliorate cachexia-associated sarcopenia and counteract cancer cachexia (Since NR and NADrcopenia . As seenrcopenia , NAMPT ecachexia .via SIRT1 (In ia SIRT1 . Pharmacia SIRT1 . As an eia SIRT1 .+ homeostasis in experimental cancer cachexia (+ deficiency, alterations in NAD+ biosynthesis, and decreased muscle protein synthesis. Our findings supported these results such as NAD+ depletion, muscle protein degradation, and the elevation of inflammatory cytokine in TB-mice. However, the induction of enzymes of the NAD+ biosynthesis pathway such as NAMPT and the reduction of SIRT1, a conserved protein NAD+-dependent deacetylase, in TB-mice are inconsistent with our study. Hulmi et al. collected samples 11 days after the C26 cell inoculation, but we collected samples 21 days after the C26 cell inoculation. In our study, the elevation of SIRT1 might be due to compensatory mechanism against muscle atrophy. Further research should also examine by time dependent manner.Recently, Hulmi et al. reported about disturbed muscle NADcachexia . Muscle The basic action of NR in mitigating cancer cachexia with NR administration, as shown in “Preemptive administration” is usually employed for pain control , includiLipolysis is also a prominent event implicated in cancer cachexia. In conclusion, we found that preemptive dietary intake of NR in preclinical models of cancer cachexia can be a potential regimen to inhibit the occurrence of a catabolic catastrophe, cachexic inflammation, and muscle atrophy. A well-designed randomized clinical trial should be developed to study whether dietary intake of NR can cover the unmet medical needs in cancer cachexia.atrogin-1/MuRF-1 levels, and lipolysis. However, preemptive administration of NR led to significant rescue from cancer cachexia. Considering the fact that more than 30% of patients with chronic illness die due to cachexia and more than 50% of patients with cancer because of cachexia, but not due to chemotherapeutic drugs or agents, our investigation revealed that preemptive and dietary intake of NR in patients at high risk of developing cancer cachexia can be a potential regimen to inhibit the occurrence of a catabolic catastrophe, cachexia inflammation, and muscle atrophy. However, well-designed RCTs should be developed for the unmet medical needs in cancer cachexia.In this study, we documented that dietary intake of NR, a vitamin B3, can be a feasible preemptive intervention in patients with advanced cancer who are vulnerable to cachexia. As summarized in


A total of 18 morpho-physiological and ion accumulation traits were dissected using multivariate analysis, and salt tolerance index (STI) values of the examined traits were evaluated for grouping of cultivars into salt-tolerant and -sensitive groups. Salt stress significantly declined all measured traits except root–shoot ratio (RSR), while the cultivars responded differently. The cultivars were grouped into three clusters and the cultivars in Cluster-1 such as Prabhat, UniGreen NK41, Bisco 51, UniGreen UB100, Bharati 981 and Star Beej 7Star exhibited salt tolerance to a greater extent, accounting for higher STI in comparison to other cultivars grouped in Cluster-2 and Cluster-3. The high heritability  and genetic advance  were recorded in 13 measured traits, indicating considerable genetic variations present in these traits. Therefore, using multivariate analysis based on the measured traits, six hybrid maize cultivars were selected as salt-tolerant and some traits such as Total Fresh Weight (TFW), Total Dry Weight (TDW), Total Na+, Total K+ contents and K+–Na+ Ratio could be effectively used for the selection criteria evaluating salt-tolerant maize genotypes at the early seedling stage. Increasing soil salinity due to global warming severely restricts crop growth and yield. To select and recommend salt-tolerant cultivars, extensive genotypic screening and examination of plants’ morpho-physiological responses to salt stress are required. In this study, 18 prescreened maize hybrid cultivars were examined at the early growth stage under a hydroponic system using multivariate analysis to demonstrate the genotypic and phenotypic variations of the selected cultivars under salt stress. The seedlings of all maize cultivars were evaluated with two salt levels: control (without NaCl) and salt stress (12 dS m High crop [−1 . Around crop [−1  because crop [−1 . Moreovecrop [−1 . Thus, ecrop [−1 . Secondlp plants ,12,13. Hp plants ,15. For p plants ,17 whichop yield . High Nauctivity ,20. Besin leaves ,21, resun leaves ,23. To cn leaves , which rn leaves .Zea mays L.) is an important C4 plant from the Poaceae family and is moderately sensitive to salt stress [Maize  hybrid cultivars were tested for salt tolerance in a hydroponic trial 2·4H2O (2 mM), KH2PO4 (0.2 mM), K2SO4 (1 mM), CaCl2·2H2O (2 mM), MgSO4·7H2O (0.5 mM), Fe-EDTA (200 µM), H3BO3 (1 µM), CuSO4·5H2O (0.3 µM), MnSO4·6H2O (2 µM), (NH4)6Mo7O24 (0.01 µM), ZnSO4·7H2O (0.5 µM). Salinity was imposed after four days of adding nutrients in the tanks and kept for 14 days to observe salt tolerance. The seedlings of all cultivars were evaluated under two growing conditions: control (0 dS m−1) and salt stress (12 dS m−1). The salt level was obtained by dissolving laboratory-grade NaCl into the nutrient solution until the electrical conductivity (EC) reached 12 dS m−1 with the help of an EC meter, while the control medium only received the nutrient solution. The experiment was followed by a completely randomized design (CRD) with nine replications. Each tank was accommodated by three hills of nine cultivars (27 hills). Each growth condition consisted of six tanks (replicated thrice), and, thus, 12 tanks consisting of 324 plants (18 × 2 × 9) were used. A photoperiod of 16 h was maintained, providing artificial lighting using fluorescent lamps (40 W) at the initial stage. Later, high-pressure sodium lamps  were used, and approximately 350 µmol m−2 s−1 of Photosynthetic Photon Flux Density (PPFD) was provided for the seedling growth. An optimum growing temperature (30 ± 2 °C day and 25 ± 1 °C night) was maintained during the experiment. The nutrient solution was replaced once a week and aerated continuously using individual air pump for each tank.The hydroponic system was maintained in rectangular plastic tanks . Perforated cork sheets were used as trays having 27 holes (3 × 9) per tray in each tank . PregermThe 28-day-old seedlings of all genotypes grown under control and salt conditions were harvested and the morphological data were recorded. Roots and shoots were separated, and root length (RL) and shoot length (SL) were measured (cm) using a one-meter ruler. Fresh weight of roots and shoots  were obtained using a digital balance (g). Then the roots and shoots were oven-dried at 70 °C for 72 h and the root dry weight (RDW) and shoot dry weight (SDW) in grams were determined. The total fresh (TFW) and dry weight (TDW) were calculated by the summation of RFW and SFW, and RDW and SDW, respectively. Root–Shoot ratio (RSR) was calculated as root dry weight over shoot dry weight. The morphological data were taken from three biological replicates of each treatment.A) was measured in the first bottom true leaf of the plants in all growth conditions. Both SPAD and A measurements were recorded in three leaves  of each treatment. The A measurements were performed by a portable photosynthetic system  at a PPFD of 300 µmol m−2 s−1 and ambient temperature in the growth room. The CO2 level was maintained as 400 ppm during A determination.The leaf greenness  of the 28-day-old plants of each cultivar in all growth conditions was measured using a handheld portable chlorophyll meter . The reading was taken at the top, middle and bottom of the leaves and average values of those three readings were used as a single replicate value. The rate of photosynthesis  in three replicates of each treatment were measured by a flame photometer .The root and shoot dry samples of 21-day-old maize seedlings of all cultivars were collected to analyze the Kfication . Approxip < 0.05. The multiple comparisons of treatment means and STI values among cultivars were performed by the Tukey HSD test of the R program. The standardized STI values were used to construct a two-way hierarchical clustering heatmap using the package ComplexHeatmap in R. The functions ggpair and fviz_pca of R were used to generate correlation–matrix scatter plot and principal component analysis (PCA) biplot. The genotypic (σ2g), genotype × salinity (σ2gs), residual (σ2e) and phenotypic (σ2p) variances were computed from the respective mean squares as described by [g = mean square of genotype, MSgs = mean square due to genotype by salinity interactions, MSe = error mean square, l = number of salt levels, r = number of replications.The open-source statistical software R  version ribed by ,48,49.(2bs) was estimated using the method described in [The broad sense heritability (hribed in .(2) h2bThe heritability was categorized as low (0–30%), moderate (30–60%) and high (>60%) following . The genp = phenotypic standard deviation, k = selection differential at 5% selection intensity. The value of k is 2.06. GAM was classified and rated based on the scales given by [The genetic advance (GA) and genetic advance as percent of the mean (GAM) were estimated following the formula suggested by ,53.(4)Ggiven by  as low (+ concentration (ShootK) while the difference in root–shoot ratio (RSR) between the stress treatments was statistically similar  and lower in Cluster-2 (85%). Considering the STI of SFW, RFW, RDW, TDW and K+–Na+ ratio, the clusters were ranked as Cluster-1 > Cluster-2 > Cluster-3 (+ was observed in Cluster-3 (408%) followed by Cluster-2 (354%) and Cluster-1 (345%). The STI of K+–Na+ ratios were 21%, 16% and 15% in Cluster-1, Cluster-2 and Cluster-3, respectively  in control plants ranged from 7.0 to 7.9 µmol CO2 m−2s−1, while A ranged from 5.2 to 6.0 µmol CO2 m−2s−1 in the salt–stressed plants in the three clusters (+–Na+ Ratio under the control condition was observed in Cluster-3 (12.5), which was statistically similar to Cluster-1 (12.4). In contrast, under salt stress, the K+–Na+ Ratio was calculated as 2.6, 1.8 and 1.9 in Cluster-1, Cluster-2 and Cluster-3, respectively  were contributed majorly by the PC1 and PC2, and these traits were also considerably linked to the categorization of the cultivars into salt−tolerant and salt−sensitive groups (Clusters 1–3).The principal component analysis (PCA) was conducted using the experimental dataset including 18 maize cultivars and 18 different variables to reduce the dimensionality of the data and to reveal the potential relationships among the measured traits . The PCAL and RL a,c. The L and RL a,d. The L and RL a. The PCeatments a. The treatments  such as + and K+ accumulation traits, but PC2 was strongly associated with biomass-related features  values ranged from 0.32 to 0.96 (A) showed a positive correlation with almost all the traits except SDW, TDW and Shoot Na+. The K+–Na+ Ratio maintained positive and significant correlations with Root K+ (0.5), Shoot K+ (0.42) and Total K+ (0.67), whereas a significant negative correlation between the K+–Na+ Ratio and each of Root Na+ (−0.56), Shoot Na+ (−0.35) and Total Na+ (−0.7) was observed  and Total Na+ (0.21) (+ positively and significantly associated with SL (0.33), Root K+ (0.37), Root Na+ (0.46) and Total K+ (0.64). Apart from this, a significant negative correlation between Total Na+ and SPAD (−0.4) was observed .Similar to the control condition, the morphological traits under the stress condition showed a significantly positive correlation with  to 0.97 b. The RS to 0.97 b. The A ch other b. The Sh+ (0.21) b. Total observed b. K+–Na+2g) and phenotypic variance (σ2p) were ranged from 0.024–39.54 and from 0.03–142.4, respectively  for all traits except SPAD, Root Na+, Shoot Na+, Total Na+ and K+–Na+ ratio, which suggests that genetic variance had the predominant role in determining these traits (2bs) in all measured traits varied from 25.4% for Total Na+ to 96.4% for SDW  were significantly declined due to salt stress. However, the cultivars in Cluster-1 showed less decline in chlorophyll concentrations, although the rate of photosynthesis in both salt-resistant and salt-sensitive clusters was identified to be declined in a similar magnitude  score and used as an indicator for selecting salt-tolerant cultivars. Cluster analysis is practiced by examining large datasets with multiple variables, and this analysis allows grouping of the cultivars with similar traits related to salt tolerance. The 18 maize cultivars showed considerable variations in STI for all measured traits in the present study, and, therefore, the cultivars were grouped into salt–sensitive and salt–tolerant groups by a two-way heatmap clustering pattern using standardized STI values . The clu+ traits . Clusterultivars . Total Kclusters .+, Total Na+, K+–Na+ ratio, Shoot K+, Total K+, Root Na+, SDW, TDW, TFW, RFW, SFW, RL and SL contributed more to describing the variation across cultivars  estimates the relative magnitude of genetic or environmental variation in a population, and a high h2bs indicates less environmental influence in the observed variations. The high h2bs (>60%) was found in all morphological traits, A, Root K+, Shoot K+ and Total K+, indicating that considerable genetic variation is present in these traits  is, therefore, most useful for effective and reliable selection providing additive gene action. Johnson et al. [2bs, GCV and GAM (>20%) in this study indicate that the variation in these examined traits is largely due to the genetic factors, and these traits can be used as reliable screening criteria for the evaluation of salt-tolerant maize genotypes.The study of genetic variability and genetic advance provides clear information regarding the extent of variability in a plant population. It gives a relative efficiency measure in genotype selection based on phenotype in a highly variable population . In the aracters . Thus, te traits . Howevern et al.  suggesten et al. ,88. The + and higher K+ accumulation, and maintaining an increased K+–Na+ ratio. Integration of high heritability  and genetic advance  was recorded in 13 measured traits indicating the variations among these traits were largely due to genetic factors. The highlighted salt-tolerant maize cultivars could be used for cultivation in the coastal saline soils with further field trials. Some traits such as Total Na+, Total K+ contents, K+–Na+ Ratio, TFW, SDW and TDW could be effectively used for the selection in salt-tolerant maize cultivar development at the early seedling stage. The study could uphold the understanding of the methods to utilize the existing plant genetic resources for the improvement of salt-tolerant maize cultivars.Establishing salt–tolerant cultivars is the key approach to mitigating soil salinity under global climate change. A comprehensive understanding of the genotypes–traits relationship is needed for selection. A total of 18 hybrid maize cultivars were screened based on diverse morpho–physiological and biochemical traits in this study. The results revealed a considerable genotypic variability in response to salt stress in the maize seedlings. The cultivars Prabhat, UniGreen NK41, Bisco 51, UniGreen UB100, Bharati 981 and Star Beej 7Star exhibited greater salt tolerance characterizing higher plant biomass, lower Na


Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings.We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro  and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity .We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity., it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomasThe online version contains supplementary material available at 10.1186/s13046-021-02213-0. Chloride intracellular channels CLIC1–6) are non-canonical self-assembling anion channels involved in physiological cell functions as well as in cancer development –6 are no, 3 incluCLIC1 exists as either soluble cytosolic protein or transmembrane isoform (tmCLIC1), assembled in multimeric ion channel conformation  and its − current, via tmCLIC1, regulates reactive oxygen species (ROS) accumulation, and causes pH changes to promote cell cycle progression [GBM is a molecularly complex and heterogeneous primary brain tumor exhibiting high lethality despite aggressive multimodal treatment  , due to gression . Notablygression , 7, showgression  and tumogression .The improved knowledge of the molecular mechanisms of cancer growth favored the exploitation of drug repurposing, identifying antineoplastic activity of compounds developed for other diseases via a target-driven selection which allows the targeting of new vulnerabilities of cancer cells . MoreoveThe prototype of non-oncology drugs repurposed as anticancer agent is metformin, a first-line anti-hyperglycemic drug for type-2 diabetes . MetformIn this context, the evidence that metformin inhibition of GSC proliferation and migration is dependent on the blockade of CLIC1-mediated ion currents , paved tHowever, translational studies and retrospective analyses in diabetic metformin users, although confirming safety of the treatment , reporteIn the present study, patient-derived GSCs from different GBMs, characterized for CLIC1 expression, were used as monolayer, 3D organoids, and in vivo after zebrafish xenograft, to investigate the role of CLIC1 in GSC growth. In particular, we have tested the effects of CLIC1 blockade by novel biguanide derivatives focusing on GSC proliferation, migration, and invasion. We highlight the relevance of the selection of tumors which express this biomarker to obtain positive responses to treatments with the novel compounds we developed, and move toward a patient tailored therapy for GBM.n = 8) and grade III (n = 1) according to WHO criteria, are reported in Table Glioma post-surgical specimens were obtained from the Neurosurgery Department of IRCCS Ospedale Policlinico San Martino  after patients’ informed consent and Institutional Ethical Committee approval (CER Liguria register number 360/2019). All patients underwent surgery for the first time and never received chemo- or radio-therapy. Clinical, histopathological and molecular features of patients and tumors, classified as glioma grade IV  and sequenced on Illumina platform with a 150 cycles paired end flowcells. Up to 80 M reads were obtained for each sample. Reads were aligned with STAR 2.7.5c  and counPatient-derived GBM primary cell cultures were isolated and characterized as previously detailed  and mainTo induce differentiation, GSCs were shifted for 2 weeks in DMEM-F12 medium supplemented with 10% FBS 2 mM L-glutamine, 1% penicillin-streptomycin .n = 4) were collected from full-term women, immediately after caesarean section at the Obstetrics and Gynecology Department , after informed consent and Institutional Ethic Committee approval (register number 2/2010). Uc-mesenchymal stem cells (ucMSCs) were isolated as reported [Human umbilical cords  and firefly luciferase (5′-CGTACGCGGAATACTTCGA-3′) sequences were cloned into the XhoI/HpaI sites of the pLentiLox 3.7 lentiviral vector [Metformin, methotrexate, 1-phenylbiguanide hydrochloride Q46) and 1-(4-chlorophenyl)-biguanide hydrochloride (Q42) are commercially available (Sigma-Aldrich). Other linear biguanides  were synthesized by refluxing an ethanolic solution of the proper aniline derivative hydrochloride with dicyandiamide, according to previously published protocols: Q48 and Q49 , Q50 [46 and 1- dil. 1:750 (WB)Anti-Sox2  dil. 1:100 (IF), 1:1000 (WB)Anti-GFAP  dil. 1:1000 (WB), 1:100 (IF)Anti-β-III-Tubulin  dil. 1:100 (IF)Anti-Olig2  dil. 1:100 (IF)Anti-α-tubulin (Sigma-Aldrich) dil. 1:7500 (WB).Anti phospho ERK1/2  dil. 1:1000 (WB)The following primary antibodies were used:2O2 for 20 min; ii) 1% BSA diluted in phosphate saline buffer 0.01 M (PBS) containing 0.1% Triton X-100 for 20 min (BPBS); iii) the primary antibody, anti-CLIC1 (in BPBS) overnight, at room temperature (RT). Antigen-antibody binding was visualized using EnVision anti-mouse secondary antibody  and with 3,3′-Dia-minobenzidine as chromogen . Tissue sections were counterstained with hematoxylin and mounted with permanent mounting medium (Eukitt). The specificity of the primary antibody was assessed by incubating adjacent sections from the same GBM with BPBS in place of CLIC1 antibody.Tissue sections were sequentially incubated with: i) 3% HMTT -2,5-diphenyltetrazolium bromide, Sigma-Aldrich) tetrazolium reduction assay was used to test cell viability. GSCs were plated in 96-well plates at the concentration of 3000 cells/well. After 48 h treatment with test compounds, MTT substrate  was added to cells and incubated for 2 h. The quantity of formazan crystals formed was measured, after being dissolved in DMSO (Sigma-Aldrich), by recording absorbance at 570 nm using a BioTek ELx800 plate reading spectrophotometer .Trypan blue exclusion assay was used to evaluate cell viability reduction induced by test compounds. GSCs  were seeded in 6-well plates, and after 24 h were treated with drugs for further 48-72 h. Viable cells were counted in the presence of Trypan blue 0.4% w/v  using the TC-20™ automated cell counter (Bio-Rad Laboratories) and reported as % of viable cells divided by the total number of counted cells .GSCs were seeded in complete medium without Matrigel™, in 48-well plates at 1000 cells/well. After 24 h, cells were exposed to drugs and spherogenesis was monitored for 7 days. The number of spheres/well was quantified using a digital camera mounted on a transmitted light microscope to image each individual well, and visually calculated by two independent operators .Migration assay was performed using Falcon™ FluoroBlok™ HTS96 transwell Support System (ThermoFisher Scientific) with a light-tight PET membrane with 8 μm pores, that blocks light transmission between 400 and 700 nm. Cells  were labeled with Vybrant™ CFDA-SE Cell Tracer (Invitrogen-ThermoFisher Scientific) , plated To test the effects of novel biguanides on GSC invasiveness, cells were seeded without Matrigel™ in complete medium for 1 week to generate spheres. Then, spheres were harvested and embedded in 80% Matrigel™ and 20% complete medium in the presence of Q48 (100 μM), Q54 (100 μM), Q46 (100 μM) and metformin (10 mM). After Matrigel™ polymerization (45 min), complete medium with Q48, Q54, Q46 or metformin was added to the dish (T0) and Matrigel™ invasion was allowed for 15 h (T15). Within this time frame compounds do not impair cell proliferation. Photos were taken using a digital camera ICC50 HD  mounted on a transmitted light microscope DM IL (Leica). Analysis was performed by ImageJ software, calculating at least 2 diameters for each sphere. The differences between the average diameter of T15 and T0 of each condition were calculated and expressed as percentage of vehicle-treated controls.Patch electrodes  were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller  and fire-polished to a tip diameter of 1–1.5 μm and an electrical resistance of 5–7 MΩ. Patch-clamp electrophysiology was performed in perforated-patch whole cell configurations, as reported . In patcbath solution (mM): 140 NaCl, 5 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, 5 D-Glucose, pH 7.4;pipette solution (mM): 135 KCl, 10 HEPES, 10 NaCl, 1 MgCl2, 2 CaCl2, 5 D-Glucose, pH 7.4.Patch clamp solutions were the following:GSCs, grown in chamberslides, were fixed with 4% paraformaldehyde (PFA), permeabilized in PBS/0.1% Triton X-100, blocked with normal goat serum and immunostained with primary antibodies, followed by AlexaFluor-568 and -488 fluorochrome-conjugated secondary antibodies (Invitrogen-ThermoFisher Scientific), as reported . Nuclei Cells were lysed in a buffer containing 1% Igepal, 20 mM Tris–HCl, pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM NaF , and the “Complete protease inhibitor mixture”  . Nuclei The Purity™ anti-mouse/anti-rabbit detection system was used  and chemiluminescent detection  was performed to visualize and quantify protein bands.Microscale thermophoresis (MST) was used to assess Q48 and Q54 interaction with CLIC1 protein . MST mea2 at 37 °C. Organoid morphology was monitored until dense aggregates are formed (about 7–10 days). Organoids were fixed in PFA for 1.5 h, at RT, washed with PBS and dehydrated overnight at 4 °C with 30% sucrose solution. To assess whole organoid structure, samples were permeabilized with 0.1% Triton X-100 for 20 min and blocked in 10% normal goat serum for 2 h, at RT, followed by overnight primary antibody incubation. Then, organoids were washed 4 times in PBS for 1.5 h each and incubated overnight with secondary antibodies AlexaFluor-568 and -488 (ThermoFisher Scientific). After further 4 washes in PBS 1x for 1.5 h each, fluorescent images were captured with Stellaris 8 TAU STED confocal microscope (Leica) and 3D images were generated using ImageJ software. Alternatively, organoids were frozen in OCT compound (Sigma-Aldrich), sectioned at 20 μM, and placed on microscope slides previously coated with gelatin  and chromium(III) potassium sulfate dodecahydrate . Slices were probed overnight with primary antibodies and nuclei were stained with DAPI following a standard immunofluorescence protocol, and then imaged with DM 2500 microscope equipped with DFC 350 FX digital camera (Leica).3D GSC organoids were obtained as previously described . BrieflyCell proliferation within treated organoids was assessed after 7 days in the absence or presence of 10 mM metformin, and 100 μM Q46, Q48, and Q54. Proliferating cells were detected using the Edu DetectPro Imaging kit 488  , followiTo extract RNA, organoids were treated with Cell Recovery solution  following the manufacturer’s protocol to allow Matrigel™ depolymerization. Total RNA was isolated from recovered cells using the PureLink RNA Mini Kit (Invitrogen). iScript™ Reverse Transcription Supermix was used for cDNA synthesis and cDNA products were analyzed using the SsoFast EvaGreen Supermix on a CFX96 Touch Real-Time PCR Detection System .Ribosomal protein large P0 (RPLP0) and 28S housekeeping genes, were used to normalize levels of target genes in each sample.Primers (human sequences):CD44: forward = 5′-TGAATATAACCTGCCGCTTTG-3′; reverse = 5′-GCTTTCTCCATCTGGGCCAT-3′MAP2: forward = 5′-GAAAGACCAAGAGCCTACCACAG-3′; reverse = 5′-TCGGTCATGGCTTTCTCCAG-3′RPLP0: forward = 5′-TGTGGGCTCCAAGCAGATGCA-3′; reverse = 5′-GCAGCAGTTTCTCCAGAGCTGGG-3′28S rRNA: forward = 5′-GTAACCCGTTGAACCCCATT-3′; reverse = 5′-CCATCCAATCGGTAGTAGCG-3′Danio rerio) adults were raised and maintained following standard methods, according to National  and European (2010/63/EU and 86/609/EEC) animal welfare laws. A breeding stock of mature zebrafish was used for egg production: males and females (ratio 2:1) were kept in a tank under the following conditions: 26 °C ± 1 °C, 14 h/10 h light/dark cycle [Zebrafish  using an Eppendorf FemtoJet microinjector, under the observation by stereoscope . After transplantation, embryos were incubated for 4 h at 32 °C and checked for the presence of fluorescent cells in the correct site. Then embryos were incubated at 32 °C in fresh medium for the following days. For the treatments, screened embryos were transferred in a 48-well plate with 1 mM compound concentration prior to the incubation at 32 °C. Five days post-fertilization , images of the tumors were captured and the relative integrated density, obtained by the product of the fluorescence intensity and the area of the tumor mass, was calculated as the ratio between the final and the initial tumor integrated density using ImageJ software.Test compounds were prepared immediately prior to use by diluting the stocks with the culture medium. Embryos were transferred to 6-well plates (20 embryos/well) in 3 ml of medium containing different concentrations of compounds or vehicle for control groups. Plates containing experimental embryos were placed in the laboratory with controlled light and temperature conditions.The potential in vivo toxicity of the novel biguanide derivatives was assessed evaluating the effects on zebrafish embryo survival in comparison with those of metformin , 60. Emb2 window was cut in the eggshell above the CAM. Metformin and Q54 were incubated from day 9 to 19 of embryo development, being administered every 2 days. At the end of the treatment the percentage of alive/dead embryos was recorded ad analyzed as percentage of untreated eggs. Experiments were performed using an average of 20 eggs for each experimental group. According to French legislation, no ethical approval is needed for scientific experiments using oviparous embryos .All activities related to the in ovo experiments, including the preparation of the final concentration of compounds, the incubation of eggs, the administration of test compounds, and the toxicity test, were carried out by the Inovotion lab team . All activities related to the in ovo experiments were conducted as previously described . BrieflyDHFR activity was performed using the Abcam colorimetric kit (ab239705) following manufacturer’s instructions. Spectrophotometric reading was performed at 30 °C, each 5 s, for 7 min, using a ClarioStar Plus microplate reader (BMG LabTech).50 values, calculated using nonlinear regression curve fit analysis, were done using Prism 5.02 . Statistical significance between groups was assessed by t-test  or one-way ANOVA followed by post-hoc Dunnett’s test, or Tukey multiple comparison test for zebrafish xenograft experiments. Selectivity Index was calculated as the ratio of IC50 of non-malignant astrocytes to IC50 of GSC cultures, as reported [p value ≤0.05.All experiments, repeated at least three times and performed in triplicate or quadruplicate, were expressed as mean ± S.D./S.E.M. Statistical analyses and ICreported . SurvivaCLIC1 expression was initially analyzed in human GBM sections by immunohistochemistry. CLIC1 expression in GBM cells is diffuse and uniform within tumor tissue Fig. A. An intTo measure CLIC1 expression in GSCs, transcript quantification by RNA-Seq was carried out in a panel of 14 cultures. Overall, average gene expression across GSCs showed high levels of CLIC1 mRNA, although reflecting the expected heterogeneity among distinct patient-derived cultures Fig. A. RNA-SeThe high percentage of GSC cultures expressing CLIC1, and the known ability of biguanides to act as CLIC1 blockers, prompted us to screen the efficacy of novel biguanide derivatives. In particular, metformin and cycloguanil core structures were selected as valuable chemotypes, to be optimized for the development of novel anticancer agents . To adeqThe likely pharmacokinetic behavior of the ionizable form of these compounds has been predicted calculating pKa and lipophilicity LogD descriptors Table . A small50s for both linear and cyclic biguanides are listed in Table 1 within Fig. Antiproliferative efficacy of the novel biguanide-based compounds, in comparison with metformin used as reference drug, was tested on CLIC1-expressing GSC cultures, according to data reported in Fig. 50 9.78 mM, Table 1) in agreement with previous reports [50 values within the μM range, three  in the low mM range, and only two (Q46 and Q52) did not reach an adequate inhibitory activity to allow the calculation. In particular, the best performing molecules, i.e. exhibiting the highest potency, were the linear compound Q48 (mean IC50 = 0.082 mM) and the cyclized analogue Q54 (mean IC50 = 0.43 mM).Metformin inhibited cell proliferation confirming high efficacy and low potency  caused a highly significant inhibition of GSC viability, showing efficacy ranging between − 71 and − 83%. Q46 (− 27%), Q51 (− 61%) and Q52 (− 56%) were the least effective compounds (Table 1). Metformin also showed high efficacy (− 82%) but required 30 mM concentration to yield the maximal effect, which is 10-fold higher than that of the novel derivatives. Importantly, all tested GSC cultures showed similar sensitivity to all biguanides Fig. , and theMetformin antiproliferative activity in GSCs is mostly dependent on CLIC1 inhibition, whilst loses efficacy in cells in which CLIC1 activity is not required for cell-cycle progression, such as non-stem GBM cells (differentiated cells obtained from GSC cultures shifted in FBS-containing medium) and primary cultures of ucMSCs , 27, 38.50 range, which induced a significant inhibition of proliferation in GSCs isolated from the same GBM   Fig. .These results clearly indicate that Q42, Q49 and Q50 effectiveness was not (or not only) mediated by CLIC1 inhibition, and suggest that other targets, relevant for non-tumor stem cell survival are involved, which may possibly cause systemic toxicity, when used in patients.On these bases, we excluded the compounds with high off-target activity  or displaying modest efficacy  and selected, for more in-depth investigations, the two compounds  ensuring the best performance in terms of higher potency, efficacy, and lower toxicity towards ucMSCs; moreover, metformin and Q46 were also included in the following experiments as positive and negative controls, respectively.50 values of each drug: metformin (1-10 mM), Q48 (0.03-1 mM) and Q54 (0.1-10 mM). Lethal and sub-lethal endpoints were microscopically inspected and recorded. Treated embryos did not show visible developmental defects or phenotypic changes within 5-day exposure to these compounds. Kaplan-Meier’s plot revealed limited toxicity only for concentrations significantly higher than the respective antitumor IC50s , demonstrating a non-significant impact on embryo survival. Similarly, Q54 had no toxic effects at concentrations up to 10 mM, with survival curves non-different from controls (log rank test for trend p = 0.64). Importantly, toxicity induced by Q48 was comparable to that induced by metformin, which decreased the survival of embryos (52%), while the effects of Q54 were even lower, but they did not differ from one another (log rank test for trend p = 0.38).One of the best metformin features, pushing its repositioning as anticancer drug, is the combination of antiproliferative activity and low systemic toxicity, as demonstrated by its widespread use as hypoglycemic agent . Thus be50s Fig. . In detaSimilar results were obtained using chick embryos, which did not display signs of toxicity after 10-day incubation with concentrations of metformin and Q54 up to 3 mM Fig. . Overall50 obtained in normal astrocytes and GSCs allowed us to define the selectivity index [Antiproliferative efficacy of Q48, Q54, and Q46 (10-100 μM) was directly measured on GSC growth rate, comparing viable cell number after 48-72 h treatment of GBM3, GBM23 and GBM19. By means of Trypan blue exclusion assay Fig. A. Comparty index  of each 50 and above, reduced CLIC1 activity in a concentration-dependent manner, although only Q54 experienced an efficacy comparable to metformin (about − 80% of CLIC1 current) for concentrations corresponding to the antiproliferative IC50 , and a complete CLIC1 inhibition occurred only at the concentration of 0.5 mM. Thus, differently from Q54, whose antiproliferative activity was strictly correlated to a complete CLIC1 inhibition, Q48 exerted a maximal inhibition of GSC proliferation at concentrations in which CLIC1 current is not completely blocked, suggesting that other molecular targets may contribute to its antiproliferative effect. Finally, as expected from cell viability data, Q46 did not affect CLIC1 activity at concentrations up to 1 mM.Then we checked the direct effects of these biguanide derivatives on CLIC1 activity, evaluating CLIC1-mediated ion current in perforated patch clamp experiments. GBM3 GSCs were sequentially perfused with vehicle (controls) or different concentrations of Q46, Q48, Q54 and metformin, followed by the CLIC1 inhibitor IAA94, in current time-course experiments. Box plots in Fig. 50 above reported (Table 1). Data reported in Fig. The direct interaction of Q48 and Q54 with recombinant CLIC1 protein was tested through MST experiments by labelling CLIC1 with fluorescent dyes covalently bound to the primary amines (lysine residues) of the protein. Thermophoretic-induced fluorescence change of the labelled-target was detected upon addition of increasing concentrations of the two compounds. All data were collected right after sample preparation. A dose response curve was built for each compound, providing affinity parameters of Kd = 15.6 ± 1.9 μM and 1.9 ± 0.5 mM, for Q48 and Q54, respectively Fig. C, in linThe specificity of CLIC1 as target for biguanide antiproliferative activity was further supported in GBM19 GSCs in which CLIC1 expression was down-regulated by RNA interference  a highly significant inhibition of spherogenesis was observed . In comparison, metformin impaired sphere-forming GBM3 and GBM19  when used at 10 mM, reflecting its antiproliferative IC50  for 7 days nearly abolished sphere formation, reducing both number and size , migration slowed-down in all GSC cultures, with a maximal inhibition observed in GBM3 , as well as metformin, were able to inhibit GSC invasion from spheroids into Matrigel™ Fig. C, with sIn vitro 3D GBM cultures (organoids) better mimic tumor tissue structure, cell-to-cell and cell-to matrix interactions occurring in vivo, and more accurately predict drug efficacy than 2D cultures . IsolateInitially, we characterized the phenotypical features of cells composing 3D cultures in comparison with 2D ones. GSC-derived organoids developed a tissue-like layered cellular organization Fig. Importantly, proliferating cells (Edu green-labeled cells) were mainly located in the external layers, showing a GSC-like phenotype, as for Sox2 expression, while differentiated internal cells, expressing GFAP, were mainly, non-proliferating Fig. C.50 values from dose-responses curves by MTT assay. Q48 and Q54 confirmed a higher potency than metformin, with the calculated IC50 values only slightly different from what observed in 2D. Q48 displayed a 5-fold reduction in IC50 (from 0.082 to 0.43 mM), while no significant differences occurred for Q54 (0.43 vs. 0.4 mM). However, as expected, drug efficacy  detected in 3D cultures was significantly reduced in comparison to the maximal antiproliferative effect detected in 2D (p < 0.01): − 39, − 54%, and − 55% vs. -81, − 78%, and − 82% (see Table 1), for Q48, Q54 and metformin, respectively.Organoids were treated for 7 days with the novel biguanides, and assayed for proliferation activity in comparison with vehicle-treated 3D cultures Fig. A. Q54 anp = 0.0002) reduction of tumor growth, confirming its in vitro antitumor efficacy, while Q46 was unable to affect GSC growth in vivo as it did in vitro. Conversely, Q48, which was highly effective in vitro, slightly reduced GBM3 tumor mass without reaching statistical significance.To test the in vivo efficacy of the novel compounds, we evaluated tumor growth after ZsGreen labeled GBM3 GSC xenotransplant in zebrafish hindbrain Fig. B. Q48, Q+ cells by IF , Q54 (300 μM), and metformin (10 mM) for 48 h, and evaluated Sox2 expression by Western blotting  and spontaneously low CLIC1-expressing (GBM39) GSCs for up to 11 days. Growth curves evidenced that, consistently with the downregulation of CLIC1, GBM39 in vitro proliferation was much slower than GBM3 and GBM23 Fig. B.p = 0.34 and 0.14, respectively; Fig. However, we have to point out that, not only low CLIC1-expressing GSCs were isolated from patients which developed aggressive GBMs, but also that GBM39 cells retain tumorigenic potential in vivo, similar to high CLIC1-expressing GSCs  and metformin (from 9.8 mM to 117.1 mM in GBM39 and 68.7 mM in GBM44). Q54 showed, besides lower efficacy, slightly higher potency in GBM39, as compared to high CLIC1 expressing GBMs (0.12 mM vs. 0.43 mM), while IC50 values for Q48 and Q54 in GBM44 cannot be calculated due to the lack of dose-dependent effects  were treated with Q48, Q54 and metformin for 48 h in concentration-response experiments, and cell survival monitored by MTT assay. GBM39 and GBM44 cells display reduced sensitivity to biguanides, as compared to high CLIC1-expressing GSCs. Indeed, at the maximal concentrations tested, 3 mM for Q48 and Q54, and 30 mM for metformin, cell viability was reduced by only − 50, − 55%, and − 49% in GBM39 and − 17, − 40%, and − 50% in GBM44, respectively, as compared to − 81, − 78%, and − 82% as average effect in CLIC1-expressing GBMs Fig. E. Moreovts Table .R2 = 0.99). Q46 was ineffective in both GBM3 and GBM39, confirming its inability to affect GSC proliferation in both 2D and 3D cultures.Low CLIC1-expressing GSCs were able to generate 3D organoids, although showing a lower proliferating cell fraction, confirming that tumor formation may be, at least partially, independent from CLIC1 activity. GSC organoid treatment with Q48, but not Q54, caused a comparable inhibition in GBM3, GBM50, and GBM39, that thus was independent from CLIC1 expression levels Fig. A and B. p < 0.05) was reached only after 72 h of treatment for Q48 and metformin, although with low efficacy . Conversely, no effects were observed using Q54  granting potencies in the μM range, while the polar 4-OCH3 group (Q48) produced a negative effect, abolishing the activity. In silico analysis of the pharmacokinetic profile of both linear aryl-biguanides  and cycloguanil-like derivatives (Q51-Q54) predicted a slightly better ability to cross the BBB than metformin, but not enough to avoid a carrier-mediated organic cation transport. Noteworthy, in mice, metformin accumulation in CNS was demonstrated, reaching up to 20% of the plasma concentration [Here we tested a small library of novel biguanide derivatives for the ability to affect GSC proliferation, self-renewal and invasiveness with higher potency than metformin, which is active only at concentrations within mM range , 28, 71.ntration . Thus, a3) on the phenyl ring, displayed high antiproliferative activity, while the substitution with the apolar and electron-donor 4-CH3 (Q49) was responsible for a net decrease of efficacy. In the dihydrotriazine (cyclic biguanides) subset, Q53 and Q52 were, respectively, the 3-Cl and 2-Cl isomers of cycloguanil (4-Cl) which previously demonstrated a mean IC50 = 0.18 mM against seven GBMs [50 = 0.082 and 0.43 mM, respectively, vs. 9.78 mM of metformin), and selectivity as CLIC1 blockers , and were identified as best lead compounds.Initially, we evaluated the potency of the novel compounds to inhibit GSC proliferation, and the blockade of CLIC1 current as specific molecular signaling mediating this effect, using cells not requiring CLIC1 for proliferation (i.e. non-stem GBM cells and ucMSCs ). From tven GBMs . Notably50, thus confirming their low toxicity observed in vitro in non-tumor cell cultures, including ucMSCs and astrocytes. Antitumor activity of Q48 and Q54 also involved the inhibition of GSC spherogenesis, an in vitro index of self-renewal, migration, and invasiveness, with efficacy similar to that of metformin, but a 100-fold higher potency.Safety of Q48 and Q54 after systemic administration was showed in vivo, demonstrating the absence of zebrafish and chick embryo toxicity after prolonged treatment  using higher concentrations (within the mM range) than antiproliferative IC50 observed in the antitumor activity, and act as CLIC1 blockers in GSC whole-cell electrophysiology experiments. The correlation between the inhibition of CLIC1-mediated ion current by Q48, Q54, and the antiproliferative effects was further supported by the observation that Q46 that did not affect GSC proliferation was also inactive as CLIC1 blocker. While these results clearly correlate biguanide ability to bind to CLIC1 and to block CLIC1-mediated current with their antiproliferative activity, it is worth noting that, differently from Q54, the Q48 concentration able to completely hamper CLIC1 activity was higher than antiproliferative IC50 (0.5 vs. 0.082 mM), thus implying the possible involvement of adjunctive mechanisms of action.The main molecular target of Q48 and Q54 biguanide derivatives was identified in CLIC1, since these compounds directly bind to a recombinant form of the channel, with Kd similar to the IC, Q54 significantly reduced the growth of GBM3 GSCs, xenotransplanted in zebrafish hindbrain, while Q46 was ineffective, as already shown in vitro. Conversely, Q48 which was highly active in vitro, caused a modest inhibition of GSC growth in vivo, which did not reach the statistical significance. Presently, we do not have a definitive explanation for the low activity of Q48 in vivo, but a possibility could reside in the animal model used (zebrafish) which requires testing molecules to be added to the fishes’ culture water. In particular, being Q48 much less hydrosoluble than Q54, it might not be able to reach effective concentrations in animal brain.Then we tested biguanide derivatives activity on GSC proliferation in 3D organoids, and tumor growth in vivo, using the zebrafish model . When gr+ cells). Moreover, also other relevant stem-like features strictly related to GBM relapse [Interestingly, in both 2D and 3D cultures, Q48 and Q54 reduced proliferating cell number mainly acting on the GSC component  without evident systemic side effects [50 for the antiproliferative activity in vitro, even after prolonged treatments (10 days in ovo and 5 days in zebrafish embryos). Although novel biguanides Q48 and Q54 are preclinical drug candidates, therefore still lack safety and pharmacokinetic data in humans, preliminary toxicological assessment reasonably predicts their low toxicity and the potential translational relevance if used at the efficacious concentrations identified in this study.One of the main issues for clinical translation of preclinical studies using metformin and derivatives is in vivo safety of the corresponding antitumor concentrations active in vitro. The effective antiproliferative concentration we identified in the in vitro model in which short-term treatments (48-72 h) are used, is in the mM range, accordingly to most studies reporting antitumor activity of metformin in vitro. Plasma concentrations corresponding to the standard treatment in diabetes patients (1.5-2 g/day = ≈30 mg/kg) correspond to about 10–30 μM . However effects . In line effects . Thus shAltogether these data provide relevant pieces of information: (i) CLIC1 inhibition reduces GSC proliferation, self-renewal, migration, and invasiveness; (ii) the antiproliferative activity of biguanides (including the novel derivatives characterized in this study) is mainly mediated by CLIC1 inhibition, as shown by the minimal inhibition of viability observed in ucMSCs, which do not require CLIC1 activity to proliferate, or in GSCs interfered for CLIC1 expression; (iii) metformin antitumor activity can be improved, as demonstrated by the novel compounds tested, in particular as far as potency, without losing the safety of the antidiabetic drug, as shown by the low toxicity in normal astrocytes and ucMSCs, or when systemically administered to whole organisms; (iv) antitumor effects exerted in 3D GSC organoids and in vivo in zebrafish are highly suggestive of efficacy also in more complex systems. These latter data also provide evidence that, although unable to freely diffuse through membranes, these compounds are able to be vehiculated inside tissues, likely by transport systems. However, similarly to metformin, the physical-chemical properties of the novel biguanide derivatives allow their ionization at physiological pH in tissues, a molecular condition required for an effective interaction with CLIC1. In fact, in the only model available, basing on single-amino acid mutations in the putative pore region, it was proposed that biguanides might affect channel activity by interacting with negative charged amino acids . FurtherCurrent oncological drug development, in the view of precision medicine, is focused on the identification of molecular drug targets to improve pharmacological activity and reduce systemic toxic effects. Thus, analysis of drug target gene expression in individual patients is a mandatory requirement. CLIC1, highly expressed in most GBMs, acts as a main regulator of growth rate  patients likely receiving benefit from biguanide treatment.50 and the concentration required to completely block CLIC1-mediated current (0.082 mM vs. 0.5 mM).However, the study of low CLIC1-expressing GBMs allowed us to check the specificity of the novel biguanides as CLIC1 blockers. In 2D cultures of CLIC1 low-expressing GSCs, a significant reduction in potency and efficacy of Q48 and Q54 (and metformin) was detected. It is worth noting that the novel biguanides retained, although impaired, antiproliferative activity, likely because GSCs from these GBMs still express low levels of CLIC1, which represent the expected molecular target of these compounds. Similarly, lack of biguanide efficacy was also observed in differentiated GBM cells and ucMSCs, in which no CLIC1 activity can be recorded , or in GWe show that, similarly to metformin, Q48 and Q54 affect GSC proliferation, self-renewal, migration, and invasion, mainly acting via CLIC1 inhibition. Antitumor effects were observed in both 2D and 3D models, and, for Q54, in vivo in the zebrafish model. Importantly, our data, considering the low toxicity for normal cells and the significant higher potency than metformin, support the possible clinical translation of Q48 and Q54, or of derivatives obtained from these lead compounds. Moreover, we propose that CLIC1 is a major determinant of cancer stem cell activity, at least in GBM, controlling not only cell proliferation but also invasiveness and, more in general, tumorigenesis, and that antitumor activity of biguanide-based compounds requires its inhibition, among all the different intracellular mechanisms that these drugs can affect. Thus CLIC1 expression and activity represents a relevant biomarker which have to be considered to predict GBM responsiveness to these novel biguanide derivatives. In fact, we also identified a subset of GBM displaying a CLIC1-independent growth, in which CLIC1 activity is likely bypassed by the activity of different intracellular signaling. In these tumors, biguanides are less effective although adjunctive mechanisms, including the ability to inhibit DHFR activity, may cooperate to induce antiproliferative effects. However, this effect may be hampered according to the compound analyzed due to the uneven intracellular permeation of these compounds within GSCs, being prone to ionization due to their strongly basic nature. On the other hand, their hydrosolubility is essential for an effective interaction between these molecules and CLIC1 channel pore. Hence, we believe that Q48 and Q54 may represent relevant leads to develop novel, powerful and selective treatments for GBM and other CLIC1-dependent tumors, in a precision medicine oriented approach.Additional file 1: Figure S1. Immunohistochemical distribution of CLIC1 in GBM. Figure S2. A) CLIC1 expression in GSCs isolated from 14 human GBMs, evaluated by RNA-seq, and expressed as counts per million reads mapped (CPM). Only GBM 39 (arrow) displayed low CLIC1 mRNA content. The Table reports the corresponding codes of the GSC cultures used in this study and the RNA-seq data deposited at NCBI Geo data set. B) Upper panel: CLIC1 expression evaluated by WB, in total cell lysates from selected GSC cultures. Membranes were re-probed with α-tubulin antibody after stripping and used as a reference for protein loading. Lower panel: Histograms report CLIC1/α-tubulin ratio of densitometric values and expressed in arbitrary units (A.U.) as mean ± S.D. Figure S3. Dose-response curves of novel biguanide derivatives and metformin on individual GSC cultures. The average response is reported in the Fig. Figure S4. Dose-response curves of novel biguanide derivatives and metformin on individual non-stem differentiated GBM cell cultures (GBM D). The average response is reported in the Fig. Figure S5. Dose-response curves of metformin and novel biguanide derivatives on individual ucMSC cultures. The average response is reported in the Fig. Figure S6. Kaplan-Mayer curves of Q48, Q54, and metformin (MET) depicting the effect of supramaximal concentration of these compounds on zebrafish embryos survival. Limited toxicity, not different from controls, was observed for all the compounds up to 5 days of treatment. Experiments were repeated twice, n = 20 per experimental group. Q48: log rank test for trend p = 0.62; Q54: log rank test for trend p = 0.64; metformin: log rank test for trend p = 0.38. Figure S7. Effect of different doses of metformin and Q54 on chick embryo survival after 10 days of incubation. Experiments were performed by Inovotion . No toxicity was observed for these compounds up to 3 mM. Experiments were repeated twice, n = 18 per experimental group. Figure S8. A. Dose-response curves of Q46, Q48, Q54 and metformin on rat astrocyte cultures. Limited toxicity is observed for all the novel compounds. Only metformin reduced astrocyte viability (− 50%) at the highest concentration tested (30 mM). Data are expressed as average of experiments preformed in quadruplicate and repeated twice. B. Table reports IC50 values in non-malignant rat astrocytes and GSCs and the calculated selectivity indices for each compound. According to the “selectivity criteria” all biguanides are considered selective compounds against GSCs (selectivity index > 10)  and CLIC1 (siCLIC1). B. Cell proliferation of GBM19 siLuc and GBM19 siCLIC1, treated with Q48 and Q54 (100 μM) for 48 h and evaluated by counting live cells. Data represent the mean ± S.E.M. ***p < 0.001 vs. respective control (CTR). Figure S10. Upper panels: Western blot depicting phospho-ERK1/2 (pERK) levels in GSC cultures in control conditions (CTR), after 5 min stimulation with bFGF (40 ng/ml) and after treatment of bFGF-stimulated cells with with Q48 (100 μM), Q54 and Q46 (300 μM), and metformin (10 mM) for 24 h. α-tubulin was used as loading reference. M.W. = molecular weight markers. Lower panels: densitometric analysis of pERK normalized for α-tubulin levels, * = p < 0.05; ** = p < 0.01. Figure S11. Comparison of CD44 (A) and MAP2 (B) mRNA expression in GBM3 GSCs cultivated as 2D monolayer or grown as 3D organoids, for 15, 21, and 30 days. Data are obtained by quantitative RT-PCR experiments. Figure S12. Kaplan-Meier analysis of the relationship between overall survival and CLIC1 expression. The statistical difference between the curves is measured by log-rank test. The prognostic effect of CLIC1 mRNA level in GBM according to The Cancer Genome Atlas (TGCA) databasea nalyzed using the GEPIA  software (A) and Chinese Glioma Genome Atlas (CGCA) (B). TPM: transcripts of per million; n(high): samples with expression level higher than the median of TPM; n(low): samples with expression level lower than the median of TPM. Figure S13. Expression stem cell-related markers  in GSCs isolated from 14 human GBMs, evaluated by RNA-seq, and expressed as counts per million reads mapped (CPM). RNA-seq have been data deposited at NCBI Geo data set . Low CLIC1-expressing cells (GBM39) do not display differences as compared to high expressing GBMs. Figure S14. A) Dihydrofolate reductase (DHFR) expression in 14 human GSC cultures evaluated by RNA-seq, and expressed as counts per million reads mapped (CPM). Comparable expression was detected in cells derived from all the GBM analyzed. RNA-seq have been data deposited at NCBI Geo data set . B) Effect of Q48, Q54, Q46 and metformin (MET) on DHFR activity, incubating the compounds with purified enzyme. Methotrexate (MTX) was used as positive control. Q48, Q54, and MET, but not Q46, inhibited DHFR activity with an efficacy comparable to MTX. * p < 0.05. C) Effect of Q48, Q54, and metformin, on DHFR activity in living cells. Q48 and metformin, but not Q54, caused a moderate inhibition of enzyme activity in a time-dependent manner. * p < 0.05. Table S1. Patients’ and tumors’ characteristics. Table S2. Elemental analysis of biguanide derivatives. Table S3. Acidity constant (pKa) and distribution constant (LogD) at pH 7 of the novel biguanide derivatives and reference drugs (metformin and cycloguanil). Table S4. Statistical analysis of the effects of novel biguanide derivatives and metformin on GSCs. Table S5. Statistical analysis of the effects of novel biguanide derivatives and metformin on differentiated non-stem GBM cells. Table S6. Statistical analysis of the effects of novel biguanide derivatives and metformin on ucMSCs. Table S7. Statistical analysis of the effects of novel biguanides on low CLIC1-expressing GSCs (GBM39 and GBM44) and the average results from high CLIC1-expressing GSCs .