Intra-articular corticosteroids for the treatment of osteoarthritis: A systematic review and meta-analysis on the comparison of different molecules and doses.

Purpose: The purpose of this study was to quantify and compare the clinical relevance of the different intra-articular corticosteroids (CS) effects in vivo for osteoarthritis (OA) treatment. Methods: The search was conducted on PubMed, Cochrane, and Web of Science in October 2023. The PRISMA guidelines were used. Inclusion criteria: animal or human randomized controlled trials (RCTs), English language and no time limitation, on the comparison of different intra-articular CS for OA treatment. The articles' quality was assessed using the Cochrane RoB2 and GRADE guidelines for human RCTs, and SYRCLE's tool for animal RCTs. Results: Eighteen RCTs were selected (16 human and 2 animal studies), including 1577 patients (1837 joints) and 31 animals (51 joints). The CS used were triamcinolone (14 human and 2 animal studies), methylprednisolone (7 human and 1 animal study), betamethasone (3 human studies) and dexamethasone (1 human study). All studies addressed knee OA except for three human and one animal study. A meta-analysis was performed on the comparison of methylprednisolone and triamcinolone in humans with knee OA analysing VAS pain at very short- (≤2 weeks), short- (>2 and ≤4 weeks), mid- (>4 and ≤8 weeks), long- (>8 and ≤ 12 weeks), and very long-term (>12 and ≤24 weeks). Triamcinolone showed better post-injection values compared to methylprednisolone at very short-term (p = 0.028). No difference in terms of VAS improvement was observed at any follow-up. Conclusions: The available preclinical and clinical literature provides limited evidence on the comparison of different CS, hindering the possibility of determining the best CS approach in terms of molecule and dose for the intra-articular injection of OA joints. Level of Evidence: Level I.

A call for standardization for secretome and extracellular vesicles in osteoarthritis: results show disease-modifying potential, but protocols are too heterogeneous-a systematic review.

The currently available osteoarthritis (OA) treatments offer symptoms' relief without disease-modifying effects. Increasing evidence supports the role of human mesenchymal stem cells (MSCs) to drive beneficial effects provided by their secretome and extracellular vesicles (EVs), which includes trophic and biologically active factors. Aim of this study was to evaluate the in vitro literature to understand the potential of human secretome and EVs for OA treatment and identify trends, gaps, and potential translational challenges. A systematic review was performed on PubMed, Embase, and Web-of-Science, identifying 58 studies. The effects of secretome and EVs were analysed on osteoarthritic cells regarding anabolic, anti-apoptotic/anti-inflammatory and catabolic/pro-inflammatory/degenerative activity, chondroinduction, and immunomodulation. The results showed that MSC-derived EVs elicit an increase in proliferation and migration, reduction of cell death and inflammation, downregulation of catabolic pathways, regulation of immunomodulation, and promotion of anabolic processes in arthritic cells. However, a high heterogeneity in several technical or more applicative aspects emerged. In conclusion, the use of human secretome and EVs as strategy to address OA processes has overall positive effects and disease-modifying potential. However, it is crucial to reduce protocol variability and strive toward a higher standardization, which will be essential for the translation of this promising OA treatment from the in vitro research setting to the clinical practice.

Total knee arthroplasty survival rates are lower in younger men, as well as rheumatoid patients and after previous high-tibial osteotomy: A registry study.

PURPOSE: The purpose of this study was to investigate if patient's gender significantly affected the long-term outcome of patients undergoing total knee arthroplasty (TKA) and to provide a cross-gender comparison of a large patient sample from a single regional register. METHODS: The Registry of Prosthetic Orthopedic Implant of Emilia Romagna (RIPO) investigated all primary TKAs performed from July 2000 to December 2020 by collecting data of men and women separately. Primary bicompartmental and tricompartmental TKAs were included. The survival rates and the reasons for revision were assessed to check if any other factor could have influenced implant failure. RESULTS: In total, 66,032 TKAs were included and analysed, comprising 46,774 women and 19,258 men. The 15-year Kaplan-Meier survival percentage was 93.6% for women and 92.5% for men (p = 0.001). Men exhibited a higher revision risk following primary TKA (p = 0.012), particularly when the primary diagnosis was arthritis resulting from rheumatic disorders (p = 0.018) and arthritis following high-tibial osteotomy (p = 0.024). Failure risk was also higher for men below the age of 60 years (p = 0.038). CONCLUSION: The long-term outcome in TKA showed significant differences between men and women, with a significantly lower survival rate in men at 15 years, especially when they are under 60 years old or with a diagnosis of rheumatic disorders or arthritis following high-tibial osteotomy. It is necessary to design specific studies to have relevant data concerning gender differences in prosthetic surgery and to customise treatments to improve outcome and patient satisfaction. LEVEL OF EVIDENCE: Level III.

Corticosteroid injections for the treatment of osteoarthritis present a wide spectrum of effects ranging from detrimental to disease-modifying: A systematic review of preclinical evidence by the ESSKA Orthobiologic Initiative.

PURPOSE: Aim of this systematic review of preclinical evidence was to determine the effects of intra-articular corticosteroid (CS) injections in joints affected by osteoarthritis (OA). METHODS: A systematic review was performed on animal studies evaluating intra-articular CS injections for OA joints. The search was performed on PubMed, Cochrane, and Web of Science databases. A synthesis of the results was performed investigating CS effects by evaluating studies comparing CS with control groups. Morphological, histological, immunohistochemistry evaluations, clinical outcomes, biomarkers and imaging results were evaluated. The risk of bias was assessed according to the Systematic Review Centre for Laboratory Animal Experimentation's tool. RESULTS: Thirty-two articles analysing CS effects in OA animal models were included (1079 joints), 18 studies on small and 14 on large animals. CS injections showed overall positive effects in at least one of the outcomes in 68% of the studies, while 16% reported a deleterious effect. CS improved cartilage and synovial outcomes in 68% and 60% of the studies, but detrimental effects were documented in 11% and 20% of the studies, respectively. Clinical parameters evaluated in terms of pain, lameness or joint swelling improved in 63% of the studies but deteriorated in 13%. Evidence is limited on imaging and biomarkers results, as well as on the best CS type, dose, formulation and injection protocol. The risk of bias assessment revealed a 28% low and an 18% high risk of bias. CONCLUSION: Intra-articular CS injections induced a wide range of results on OA joints in experimental animal models, from disease-modifying and positive effects on pain and joint function at short-term evaluation to the lack of benefit or even negative effects. This underlines the need to identify more specific indications and treatment modalities to avoid possible detrimental effects while maximising the anti-inflammatory properties and the benefits of intra-articular CS in OA joints. LEVEL OF EVIDENCE: Level II.

Meniscal allograft transplantation: A matched-pair analysis reveals worse sport activity level but similar clinical improvement and survival in women compared to men.

PURPOSE: The aim of this study was to assess how gender might affect the clinical outcome and survival of meniscal allograft transplantation (MAT). METHODS: A total of 358 patients (23.2% women, 76.8% men) were treated with fresh-frozen nonirradiated allografts implantated arthroscopically using a single- or double-tunnel technique without bone plugs and peripheral suture to the capsule with 'all-inside' stitches. RESULTS: Patients were evaluated at baseline and 2-year follow-up with the Lysholm score, visual analogue scale (VAS) pain, the Knee Osteoarthritis Outcome Score (KOOS) subscales and Tegner score. Women presented higher body mass index (p < 0.0005), poorer baseline VAS (p = 0.012), Lysholm score (p = 0.005), KOOS symptom (p = 0.034) and KOOS pain (p = 0.030), Tegner score (preinjury and basal, p < 0.0001 and p = 0.002, respectively), a lower number of previous (p = 0.039) and concurrent (p = 0.001) anterior cruciate ligament reconstructions and a higher number of concurrent procedures (p = 0.032) and distal femoral osteotomies (p = 0.024). Worse results were documented in women at 2 years, with lower Lysholm score (p = 0.024) and Tegner score (p = 0.007) and a lower clinical survival rate (p = 0.03) (67.5% vs. 82.2%) in the overall patient cohort. However, the matched-pair analysis only confirmed a lower Tegner score value at 2 years (p = 0.016), while underlying the interplay of sex, age and concomitant cartilage lesions in determining the clinical outcome. The analysis of this large series of patients affected by postmeniscectomy syndrome and treated with MAT revealed gender differences. CONCLUSION: While both genders benefited from a significant improvement, the female population presents more often with older age, concomitant cartilage lesions and a lower activity level, all factors contributing towards a lower clinical success after MAT. LEVEL OF EVIDENCE: Level III, comparative study.

The influence of sex is a neglected focus in rotator cuff repair: A systematic review and meta-analysis.

PURPOSE: Rotator cuff (RC) disorders are the most common cause of shoulder disability. The aim of this study was to quantify the evidence on the sex-related differences in RC repair. METHODS: A systematic review of the literature was performed in January 2023 in PubMed, Wiley Cochrane Library and Web of Science on research articles on humans with RC tears treated surgically. A meta-analysis was performed to compare results in men and women. The Downs and Black score and the modified Coleman methodology score (MCMS) were used to assess the retrieved studies. RESULTS: A total of 39,909 patients were enroled in the 401 studies analysed (45% women, 55% men). A trend toward more sex-balanced recruitment was observed over time. Only 4% of the studies on 1.5% of the documented patients presented disaggregated outcome data and were quantitatively analysed. A tendency for lower range of motion values after surgery was found for external shoulder rotation in women, with 39.9° ± 6.9° versus 45.3° ± 4.1° in men (p = 0.066). According to Downs and Black scores, four studies were good and 12 fair, with a main MCMS score of 69/100. CONCLUSION: There is a lack of awareness on the importance to document women- and men-specific data. Only 4% of the articles disaggregated data, and it was possible to analyse only 1.5% of the literature population, a sample which cannot be considered representative of all patients. The lack of disaggregated data is alarming and calls for action to better study men and women differences to optimise the management of RC tears. This will be necessary to provide sex-specific information that could be of clinical relevance when managing RC repair. LEVEL OF EVIDENCE: Level IV.

Higher Eccentric Hamstring Muscle Fatigue After Participation in a Soccer Match in Young Female Athletes.

BACKGROUND: Hamstring (HS) strength deficits and imbalances have been identified as risk factors for sustaining anterior cruciate ligament (ACL) injuries and muscular strains, with HS injuries being the most prevalent muscle injuries in soccer athletes. The aim of this study was to investigate HS eccentric strength before and after a soccer match in both male and female soccer athletes. HYPOTHESIS: Soccer athletes have changes in eccentric HS strength after a soccer game. STUDY DESIGN: Cohort observational study. LEVEL OF EVIDENCE: Level 3. METHODS: HS eccentric strength (mean and absolute peak torque and total work) was measured in 64 healthy male and female competitive football athletes (14-25 years) with an automatic device during the execution of the Nordic hamstring exercise (NHE) test before and after a 90-minute soccer match. The anterior-knee laxity (AKL) was quantified with an arthrometer. RESULTS: Mean and absolute eccentric HS peak torque decreased by 24.5 N.m (-12.34%; P < 0.01) and 21.9 N.m (-10.08%; P < 0.01) in female athletes, whereas their male peers improved by 19.9 N.m (+9.01%; P = 0.01) and by 20.9 N.m (+8.51%; P = 0.02), respectively. HS total work in female athletes decreased by 831.1 J (P < 0.01) compared with the male athlete reduction of 235.3 J. Both the pre- versus postmatch intersex mean and absolute eccentric HS peak torque changes were significant (P < 0.01), as were the changes in HS total work (P < 0.01). The pre- versus postmatch AKL difference and the dominant versus nondominant limb comparison of the strength parameters were not significantly different. Younger female athletes (14-19 years old) presented a greater decrease in mean and absolute peak HS eccentric strength compared with those in older female athletes and men. CONCLUSION: HS eccentric strength and work differ based on athlete sex, as measured by the NHE test. Mean peak, absolute peak, and total work showed greater reductions in female athletes than those in their male peers. The subgroup of 14- to 19-year-old female athletes experienced the highest reduction in strength parameters.

Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair.

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100-150 µm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.

Sex does not influence the long-term outcome of matrix-assisted autologous chondrocyte transplantation.

PURPOSE: Regenerative techniques for articular cartilage lesions demonstrated heterogeneous clinical results. Several factors may influence the outcome, with sex being one of the most debated. This study aimed at quantifying the long-term influence of sex on the clinical outcome obtained with a regenerative procedure for knee chondral lesions. METHODS: Matrix-assisted autologous chondrocyte transplantation (MACT) was used to treat 235 knees which were prospectively evaluated with the International Knee Documentation Committee (IKDC), EuroQol visual analogue scale, and Tegner scores at 14-year mean follow-up. A multilevel analysis was performed with the IKDC subjective scores standardised according to the age/sex category of each patient and/or the selection of a match-paired subgroup to compare homogeneous men and women patients. RESULTS: At 14 years, men and women showed a failure rate of 10.7% and 28.8%, respectively (p < 0.0005). An overall improvement was observed in both sexes. Women had more patellar lesions and men more condylar lesions (p = 0.001), and the latter also presented a higher preinjury activity level (p < 0.0005). Men had significantly higher IKDC subjective scores at all follow-ups (at 14 years: 77.2 ± 18.9 vs. 62.8 ± 23.1; p < 0.0005). However, the analysis of homogeneous match-paired populations of men and women, with standardised IKDC subjective scores, showed no differences between men and women (at 14 years: -1.6 ± 1.7 vs. -1.9 ± 1.6). CONCLUSION: Men and women treated with MACT for knee chondral lesions presented a significant improvement and stable long-term results. When both sexes are compared with homogeneous match-paired groups, they have similar results over time. However, women present more often unfavourable lesion patterns, which proved more challenging in terms of long-term outcome after MACT. LEVEL OF EVIDENCE: Level II.

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

Adipose Tissue-Derived Products May Present Inflammatory Properties That Affect Chondrocytes and Synoviocytes from Patients with Knee Osteoarthritis.

Adipose tissue-derived cell-based injectable therapies have been demonstrated to have disease-modifying effects on joint tissues in preclinical studies on animal osteoarthritis (OA) models, but clinical results are heterogeneous and not always satisfactory. The aim of this study was to investigate the influence of adipose tissue properties on the therapeutic effects of the adipose-derived product in an in vitro OA setting. Micro-fragmented adipose tissue (MF-AT) samples were obtained from 21 OA patients (mean age 51.7 ± 11.8 years, mean BMI 25.7 ± 4.1 kg/m2). The analysis of the MF-AT supernatant was performed to analyze the release of inflammatory factors. The effects of MF-AT inflammatory factors were investigated on chondrocytes and synoviocytes gene expression levels. Patients' characteristics were analyzed to explore their influence on MF-AT inflammatory molecules and on the MF-AT effects on the gene expression of chondrocytes and synoviocytes. The study results demonstrated that adipose tissue-derived products may present inflammatory properties that influence the therapeutic potential for OA treatment, with products with a higher pro-inflammatory profile stimulating a higher expression of genes related to a more inflamed and catabolic phenotype. A higher pro-inflammatory cytokine pattern and a higher pro-inflammatory effect were found in adipose tissue-derived products obtained from OA patients with higher BMI.

Total Knee Arthroplasty (TKA): When Do the Risks of TKA Overcome the Benefits? Double Risk of Failure in Patients up to 65 Years Old.

OBJECTIVE: The aim of this study was to document the survival rate in the middle-aged patient group up to 65 years old and to compare it with other age groups of patients undergoing total knee arthroplasty (TKA) for knee osteoarthritis (OA). DESIGN: The Register of Orthopaedic Prosthetic Implants (RIPO) regional registry was used to analyze the results of patients <80 years old affected by primary OA and treated with TKA from 2000 to 2019. The database was investigated according to the age group: younger than 50 years, 50-65 years, or 66-79 years, with the aim to estimate revision surgeries and implant survivorship. RESULTS: A total of 45,488 TKAs for primary OA were included in the analysis (M: 11,388; F: 27,846). The percentage of patients <65 years old increased from 13.5% to 24.8% between 2000 and 2019 (P < 0.0001). The survival analysis showed an overall influence of age on the implant revision rate (P < 0.0001), with an estimated survival rate of 78.7%, 89.4%, and 94.8% at 15 years in the 3 groups, respectively. Compared with the older-aged group, the relative risk of failure was 3.1 (95% confidence interval [CI] = 2.2-4.3; P < 0.001) higher in patients <50 years old and 1.8 (95% CI = 1.6-2.0; P < 0.001) higher in patients 50-65 years old. CONCLUSIONS: TKA use in the middle-aged patient population up to 65 years old increased significantly over time. These patients present a double risk of failure with respect to older patients. This is particularly important considering the increasing life expectancy and the emergence of new joint preserving strategies, which could postpone the need for TKA to an older age.

Adipose Tissue-Derived Minimally Manipulated Products versus Platelet-Rich Plasma for the Treatment of Knee Osteoarthritis: A Systematic Review of Clinical Evidence and Meta-Analysis.

The use of minimally manipulated adipose tissue (MM-AT) products is gaining increasing interest for the treatment of knee osteoarthritis (OA). MM-AT represents an easy way to exploit adipose tissue properties, although clinical evidence is still limited, as well as their benefits with respect to more documented orthobiologics like platelet-rich plasma (PRP). A systematic review and meta-analysis were performed to evaluate the safety and efficacy of MM-AT products for knee OA management. The risk of bias of the included studies was evaluated using the Dawns and Black checklist for all the included studies and RoB-2.0 for randomized controlled trials (RCTs). Thirty-three clinical studies were included in the qualitative analysis: 13 prospective case series, 10 retrospective case series, 7 RCTs, 2 retrospective comparative studies, and 1 prospective comparative study. An overall clinical improvement and few minor adverse events were observed. Five RCTs comparing MM-AT and PRP injections were meta-analyzed, showing comparable results. The analysis also highlighted the limits of the literature, with only a few high-level trials and an overall low quality. Even though the current literature is still limited, the available evidence suggests the safety and overall positive results of the intra-articular injections of MM-AT products for knee OA treatment.

Tendinopathy: sex bias starts from the preclinical development of tendon treatments. A systematic review.

Tendinopathies are common overuse disorders that arise both in athletes and the general population. Available tendon treatments are used both for women and men without distinction. However, the existence of a sex-based difference in tendon biology is widely demonstrated. Since basic research represents the foundation for treatment development, an equal female-male representation should be pursued in preclinical studies. This systematic review quantified the current evidence by analyzing 150 studies on 8231 animals. Preclinical studies largely neglected the importance of sex, none analyzed sex-based differences, and only 4% of the studies reported disaggregated data suitable for the analysis of treatment results in males and females. There is an alarming female under-representation, in particular in the field of injective therapies. Despite the growing awareness on the importance of investigating treatments in both males and females, the investigated field proved resistant from properly designing studies including both sexes, and the lack of sex-representation remains critical.

Platelet-rich plasma injections induce disease-modifying effects in the treatment of osteoarthritis in animal models.

PURPOSE: The mechanisms of action and disease-modifying potential of platelet-rich plasma (PRP) injection for osteoarthritis (OA) treatment are still not fully established. The aim of this systematic review of preclinical evidence was to determine if PRP injections can induce disease-modifying effects in OA joints. METHODS: A systematic review was performed on animal studies evaluating intra-articular PRP injections as treatment for OA joints. A synthesis of the results was performed investigating the disease-modifying effects of PRP by evaluating studies that compared PRP with OA controls or other injectable products, different PRP formulations or injection intervals, and the combination of PRP with other products. The risk of bias was assessed according to the SYRCLE's tool. RESULTS: Forty-four articles were included, for a total of 1251 animals. The publication trend remarkably increased over time. PRP injections showed clinical effects in 80% and disease-modifying effects in 68% of the studies, attenuating cartilage damage progression and reducing synovial inflammation, coupled with changes in biomarker levels. Evidence is limited on the best PRP formulation, injection intervals, and synergistic effect with other injectables. The risk of bias was low in 40%, unclear in 56%, and high in 4% of items. CONCLUSION: Intra-articular PRP injections showed disease-modifying effects in most studies, both at the cartilage and synovial level. These findings in animal OA models can play a crucial role in understanding mechanism of action and structural effects of this biological approach. Nevertheless, the overall low quality of the published studies warrants further preclinical studies to confirm the positive findings, as well as high-level human trials to demonstrate if these results translate into disease-modifying effects when PRP is used in the clinical practice to treat OA. LEVEL OF EVIDENCE: Level II.

Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells.

The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.

Novel histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in sarcoma cancer stem cells.

Musculoskeletal sarcomas are aggressive malignancies of bone and soft tissues often affecting children and adolescents. Histone deacetylase inhibitors (HDACi) have been proposed to counteract cancer stem cells (CSCs) in solid neoplasms. When tested in human osteosarcoma, rhabdomyosarcoma, and Ewing's sarcoma stem cells, the new HDACi MC1742 (1) and MC2625 (2) increased acetyl-H3 and acetyl-tubulin levels and inhibited CSC growth by apoptosis induction. At nontoxic doses, 1 promoted osteogenic differentiation. Further investigation with 1 will be done in preclinical sarcoma models.

Impairment of lysosomal activity as a therapeutic modality targeting cancer stem cells of embryonal rhabdomyosarcoma cell line RD.

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewing's sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 µM, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease.

Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells.

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

V-ATPase as an effective therapeutic target for sarcomas.

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9­6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment.