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Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.

Ewing sarcoma (EWS), the second most common malignant bone tumor in children and adolescents, occurs abruptly without clear evidence of tumor history or progression. Previous association studies have identified some inherited variants associated with the risk of developing EWS but a common picture of the germline susceptibility to this tumor remains largely unclear. Here, we examine the association between thirty single nucleotide polymorphisms (SNPs) of the IGF2BP3, a gene that codes for an oncofetal RNA-binding protein demonstrated to be important for EWS patient’s risk stratification, and five SNPs of SENCR, a long non-coding RNA shown to regulate IGF2BP3. An association between polymorphisms and EWS susceptibility was observed for three IGF2BP3 SNPs - rs112316332, rs13242065, rs12700421 - and for four SENCR SNPs - rs10893909, rs11221437, rs12420823, rs4526784 -. In addition, IGF2BP3 rs34033684 and SENCR rs10893909 variants increased the risk for female respect to male subgroup when carried together, while IGF2BP3 rs13242065 or rs76983703 variants reduced the probability of a disease later onset (> 14 years). Moreover, the absence of IGF2BP3 rs10488282 variant and the presence of rs199653 or rs35875486 variant were significantly associated with a worse survival in EWS patients with localized disease at diagnosis. Overall, our data provide the first evidence linking genetic variants of IGF2BP3 and its modulator SENCR to the risk of EWS development and to disease progression, thus supporting the concept that heritable factors can influence susceptibility to EWS and may help to predict patient prognosis.

Abstract Background The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling. Methods The effects of ROCK2 depletion on metastasis were studied in NOD Scid gamma (NSG) mice injected with U-2OS cells in which ROCK2 expression had been stably silenced. Functional studies were performed in vitro in human U-2OS cells and in three novel cell lines derived from patient-derived xenografts (PDXs) by using standard methods to evaluate malignancy parameters and signaling transduction. The nuclear immunostaining of YAP and the evaluation of its downstream targets Cysteine Rich Angiogenic Inducer 6, Connective Tissue Growth Factor and Cyclin D1 by quantitative PCR were performed to analyze YAP activity. The effect of the expression and activity of ROCK2 and YAP on tumor progression was analyzed in 175 OS primary tumors. Results The silencing of ROCK2 markedly reduced tumor growth and completely abolished the metastatic ability of U-2OS cells. The depletion of ROCK2, either by pharmacological inhibition or silencing, induced a dose- and time-dependent reduction in the nuclear expression and transcriptional activity of YAP. The nuclear expression of YAP was observed in 80/175 (46%) tumor samples and was significantly correlated with worse patient prognosis and a higher likelihood of metastasis and death. The use of verteporfin, a molecule that specifically inhibits the TEAD–YAP association, remarkably impaired the growth and migration of OS cells in vitro. Moreover to inhibiting YAP activity, our findings indicate that verteporfin also affects the ROCK2 protein and its functions. Conclusions We describe the functional connection between ROCK2 and YAP in the regulation of OS cell migration and metastasis formation. These data provide support for the use of verteporfin as a possible therapeutic option to prevent OS cell dissemination.

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.

Abstract Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient’s tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches.

The ATP-binding cassette (ABC) transporter superfamily consists of several proteins with a wide repertoire of functions. Under physiological conditions, ABC transporters are involved in cellular trafficking of hormones, lipids, ions, xenobiotics, and several other molecules, including a broad spectrum of chemical substrates and chemotherapeutic drugs. In cancers, ABC transporters have been intensely studied over the past decades, mostly for their involvement in the multidrug resistance (MDR) phenotype. This review provides an overview of ABC transporters, both related and unrelated to MDR, which have been studied in osteosarcoma and Ewing’s sarcoma. Since different backbone drugs used in first-line or rescue chemotherapy for these two rare bone sarcomas are substrates of ABC transporters, this review particularly focused on studies that have provided findings that have been either translated to clinical practice or have indicated new candidate therapeutic targets; however, findings obtained from ABC transporters that were not directly involved in drug resistance were also discussed, in order to provide a more complete overview of the biological impacts of these molecules in osteosarcoma and Ewing’s sarcoma. Finally, therapeutic strategies and agents aimed to circumvent ABC-mediated chemoresistance were discussed to provide future perspectives about possible treatment improvements of these neoplasms.

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 3 (IGF2BP3) is an oncofetal protein that binds RNA, thereby influencing the fate of target transcripts. IGF2BP3 is synthesized de novo in cancer, where it promotes proliferation, drug resistance, and metastasis via both IGF2-dependent and IGF2-independent mechanisms. Ewing sarcoma (ES) is a rare bone and soft tissue tumor in which the IGF system plays a pivotal role. This study aimed to investigate the effect of IGF2BP3 on the regulation of the IGF system in ES. Among the components of the IGF axis, a direct significant correlation was identified between IGF2BP3 and IGF1R at mRNA and protein levels in two independent series of clinical specimens from patients with localized ES. After the formal demonstration of a direct association between IGF2BP3 and IGF1R mRNA using ribo-immunoprecipitation assay, we performed in vitro studies using A673 and TC-71 ES cell lines to demonstrate that IGF2BP3 loss promotes the downregulation of IGF1R and a decreased biological response to IGF1, represented by reduced migration and cell growth. Additionally, the compensatory activation of insulin receptor (IR) and its mitogenic ligand IGF2 is triggered in some but not all cell lines in response to IGF2BP3-mediated IGF1R loss. These findings have therapeutic implications because cells with a decreased expression of IGF2BP3/IGF1R axis but an increased expression of the IR/IGF2 loop display higher sensitivity to the dual inhibitor OSI-906 than do control cells. Therefore, studies on IGF2BP3, which was confirmed as a post-transcriptional regulator of IGF1R, provide a step forward in the identification of new mechanisms regulating the IGF system. In addition, our results demonstrate that the detection of IGF2BP3 expression should be combined with the assessment of the IGF1R/IR ratio to predict cell responses to anti-IGF1R/IR agents.

Recent studies have indicated that targeting glutathione-S-transferase (GST) isoenzymes may be a promising novel strategy to improve the efficacy of conventional chemotherapy in the three most common musculoskeletal tumours: osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma. By using a panel of 15 drug-sensitive and drug-resistant human osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma cell lines, the efficay of the GST-targeting agent 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has been assessed and related to GST isoenzymes expression (namely GSTP1, GSTA1, GSTM1, and MGST). NBDHEX showed a relevant in vitro activity on all cell lines, including the drug-resistant ones and those with higher GSTs levels. The in vitro activity of NBDHEX was mostly related to cytostatic effects, with a less evident apoptotic induction. NBDHEX positively interacted with doxorubicin, vincristine, cisplatin but showed antagonistic effects with methotrexate. In vivo studies confirmed the cytostatic efficay of NBDHEX and its positive interaction with vincristine in Ewing's sarcoma cells, and also indicated a positive effect against the metastatisation of osteosarcoma cells. The whole body of evidence found in this study indicated that targeting GSTs in osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma may be an interesting new therapeutic option, which can be considered for patients who are scarcely responsive to conventional regimens.