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Identification of potential sEV-associated ESFT biomarkers. (A) Cellular localization of potential ESFT sEV biomarkers (Intraluminal, rectangles; plasma membrane, ovals). (B-C) Validation of ITGA5 (Integrin Subunit Alpha 5), JAK1 (Janus Kinase 1), CTNNB1 (Catenin Beta 1), EZR (Ezrin), ENO (Enolase), and NPMN1 (Nucleophosmin 1) as potential sEV-protein biomarkers for ESFT by western blot analysis.
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Purpose: Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and...
Contexts in source publication
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... has been shown to repress β-catenin-mediated transcription of Wnt target genes and had been noted to be differentially expressed between localized and metastatic ESFT [44]. All together a total of 10 proteins previously identified to be associated with sarcomas were further investigated (Supplementary Table 7 and Figure 4A). Our next step was to then confirm the presence and enrichment of these proteins within our cell lines and cell line-derived sEVs by western blot analysis ( Figure 4B and 4C). ...
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... together a total of 10 proteins previously identified to be associated with sarcomas were further investigated (Supplementary Table 7 and Figure 4A). Our next step was to then confirm the presence and enrichment of these proteins within our cell lines and cell line-derived sEVs by western blot analysis ( Figure 4B and 4C). This analysis corroborated that CD99 and NGFR were expressed within ESFT cell lines and enriched within ESFT-derived sEV. ...
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... protein levels were significantly lower or absent in the control Hs919. T cells and its associated sEVs ( Figure 4B). Likewise, HINT1, EZR, and ENO were enriched in a majority of ESFT sEV samples and minimally enriched or absent in control ( Figure 4B and 4C). ...
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... cells and its associated sEVs ( Figure 4B). Likewise, HINT1, EZR, and ENO were enriched in a majority of ESFT sEV samples and minimally enriched or absent in control ( Figure 4B and 4C). ITGA5 (Integrin Subunit Alpha 5), JAK1 (Janus Kinase 1), NPM1 (Nucleophosmin), and CTNNB1 (Catenin Beta 1) were either not detected in ESFT or not substantially enriched in ESFT samples as compared to the control ( Figure 4C). ...
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... HINT1, EZR, and ENO were enriched in a majority of ESFT sEV samples and minimally enriched or absent in control ( Figure 4B and 4C). ITGA5 (Integrin Subunit Alpha 5), JAK1 (Janus Kinase 1), NPM1 (Nucleophosmin), and CTNNB1 (Catenin Beta 1) were either not detected in ESFT or not substantially enriched in ESFT samples as compared to the control ( Figure 4C). Taken together, this analysis lead towards the identification of 5 proteins, (2 The color of the plotted protein represents the difference in mean expression. ...
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... using the COMBO immuno-isolation approach, but only 50% of the patients using CD9 antibody alone (Supplementary Figure 4B). These pre-clinical data are strongly supportive of the approach to use CD99 and NGFR for the enrichment for ESFT specific EVs from clinical patient plasma and that the proposed liquidbase biopsy can serve as a clinical tool for the diagnosis, monitoring of disease, and early detection of relapse ( Figure 6D). ...
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... has been shown to repress β-catenin-mediated transcription of Wnt target genes and had been noted to be differentially expressed between localized and metastatic ESFT [44]. All together a total of 10 proteins previously identified to be associated with sarcomas were further investigated (Supplementary Table 7 and Figure 4A). Our next step was to then confirm the presence and enrichment of these proteins within our cell lines and cell line-derived sEVs by western blot analysis ( Figure 4B and 4C). ...
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... together a total of 10 proteins previously identified to be associated with sarcomas were further investigated (Supplementary Table 7 and Figure 4A). Our next step was to then confirm the presence and enrichment of these proteins within our cell lines and cell line-derived sEVs by western blot analysis ( Figure 4B and 4C). This analysis corroborated that CD99 and NGFR were expressed within ESFT cell lines and enriched within ESFT-derived sEV. ...
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... protein levels were significantly lower or absent in the control Hs919. T cells and its associated sEVs ( Figure 4B). Likewise, HINT1, EZR, and ENO were enriched in a majority of ESFT sEV samples and minimally enriched or absent in control ( Figure 4B and 4C). ...
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... cells and its associated sEVs ( Figure 4B). Likewise, HINT1, EZR, and ENO were enriched in a majority of ESFT sEV samples and minimally enriched or absent in control ( Figure 4B and 4C). ITGA5 (Integrin Subunit Alpha 5), JAK1 (Janus Kinase 1), NPM1 (Nucleophosmin), and CTNNB1 (Catenin Beta 1) were either not detected in ESFT or not substantially enriched in ESFT samples as compared to the control ( Figure 4C). ...
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... HINT1, EZR, and ENO were enriched in a majority of ESFT sEV samples and minimally enriched or absent in control ( Figure 4B and 4C). ITGA5 (Integrin Subunit Alpha 5), JAK1 (Janus Kinase 1), NPM1 (Nucleophosmin), and CTNNB1 (Catenin Beta 1) were either not detected in ESFT or not substantially enriched in ESFT samples as compared to the control ( Figure 4C). Taken together, this analysis lead towards the identification of 5 proteins, (2 The color of the plotted protein represents the difference in mean expression. ...
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... using the COMBO immuno-isolation approach, but only 50% of the patients using CD9 antibody alone (Supplementary Figure 4B). These pre-clinical data are strongly supportive of the approach to use CD99 and NGFR for the enrichment for ESFT specific EVs from clinical patient plasma and that the proposed liquidbase biopsy can serve as a clinical tool for the diagnosis, monitoring of disease, and early detection of relapse ( Figure 6D). ...
Citations
... Clinical application of the proteomic content of EWS EVs is only starting to be revealed [47]. The presence of CD99 in EVs from EWS cells has been already established [18,20,53]. Data from the literature support the clinical utility for some of the EV-associated proteins identified in this study in EWS. ...
Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.
... Regarding diagnosis, EVs can be leveraged for several cancers, including rare types, such as pediatric Ewing sarcoma. Samuel et al. immunoenriched tumorderived EVs from pediatric patient plasma samples using EV membrane proteins, CD99/MIC2 and NGFR (nerve growth factor receptor), and then interrogated EV mRNA cargo for EWS-ETS transcripts, the fusion gene driving Ewing sarcoma (29). The EV count expressing EWS-ETS transcripts separated cases from controls with an AUC of 0.92 (95% CI, 0.80-1.00) ...
Abstract
Background
Cancer is a dynamic process and thus requires highly informative and reliable biomarkers to help guide patient care. Liquid-based biopsies have emerged as a clinical tool for tracking cancer dynamics. Extracellular vesicles (EVs), lipid bilayer delimited particles secreted by cells, are a new class of liquid-based biomarkers. EVs are rich in selectively sorted biomolecule cargos, which provide a spatiotemporal fingerprint of the cell of origin, including cancer cells.
Content
This review summarizes the performance characteristics of EV-based biomarkers at different stages of cancer progression, from early malignancy to recurrence, while emphasizing their potential as diagnostic, prognostic, and screening biomarkers. We discuss the characteristics of effective biomarkers, consider challenges associated with the EV biomarker field, and report guidelines based on the biomarker discovery pipeline.
Summary
Basic science and clinical trial studies have shown the potential of EVs as precision-based biomarkers for tracking cancer status, with promising applications for diagnosing disease, predicting response to therapy, and tracking disease burden. The multi-analyte cargos of EVs enhance the performance characteristics of biomarkers. Recent technological advances in ultrasensitive detection of EVs have shown promise with high specificity and sensitivity to differentiate early-cancer cases vs healthy individuals, potentially outperforming current gold-standard imaging-based cancer diagnosis. Ultimately, clinical translation will be dictated by how these new EV biomarker-based platforms perform in larger sample cohorts. Applying ultrasensitive, scalable, and reproducible EV detection platforms with better design considerations based upon the biomarker discovery pipeline should guide the field towards clinically useful liquid biopsy biomarkers.
... Ewing's sarcoma (ES) is a highly aggressive bone tumor, and the second most prevalent pediatric bone malignancy [1]. It belongs to a family known as Ewing's Sarcoma Family of Tumors (ESFT), comprising ES, extraosseous ES, peripheral primitive neuroectodermal tumor (PNET), and Askin tumor [2]. The exact origin of these tumors is a subject of much debate. ...
... The five-year survival rate for ES patients with initially localized tumors is around 70 to 75 % [1]. However, the presence of metastasis at diagnosis decreases the three-year survival rate to 20 % and contributes to poor prognosis [2]. Approximately 25 % of ES cases show metastatic tumors at preliminary diagnosis [10]. ...
... It usually occurs in the lungs, other bones, and bone marrow. Furthermore, the disease has a high recurrence rate, with about 30 to 40 % relapse in ES pediatric patients [2]. Prediction of the patients' disease clinical outcomes is mostly difficult. ...
... We selected a panel of confirmed cell lines that are representative of the most common EWS-ETS fusion types, namely, EWS-FLI1 type I (CHLA-9, CHLA-10, CHLA-32, TC-32, and TC-71), EWS-FLI1 type II (SK-ES-1), EWS-FLI1 type III (CHLA-258) fusion, and EWS-ERG fusion (COG-E-352) [30]. cell lines were obtained from the Children's Oncology Group (COG). ...
Simple Summary
Ewing sarcoma (EWS) is a rare pediatric sarcoma affecting children and adolescents, with median diagnosis around the age of 15. Despite an intensive therapeutic regimen, including chemotherapy, surgery/radiation patients with recurrent (10–15%) and metastatic disease (<30%) have poor overall survival rates. Moreover, standard chemotherapy is intense and is often associated with systemic toxicity and secondary malignancies. Hence, it is critical to find new treatments to improve outcomes in EWS patients. We identified a combination of mitotic inhibitors targeting KIF11 (SB-743921) and AURKA (VIC-1911) that are effective in inhibiting EWS tumor growth at physiologically relevant nanomolar doses. This drug combination inhibited EWS cell viability in vitro by promoting cell cycle arrest followed by cell death. In vivo, this treatment regimen led to significantly delayed tumor growth and improved overall survival in xenograft EWS mouse models. Overall, these preclinical data provide encouragement to consider a future clinical trial for patients with this deadly disease.
Abstract
Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1–3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45–52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan–Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).
... Finally, five studies explored RNA in circulating EVs and one study investigated lncRNA by qRT-PCR including a total of 257 patients [43,45,[105][106][107][108]. ...
... The use of ctRNA by RT-PCR only detected fusion transcripts in 32% (n=285) of patients included in eleven studies [33,[75][76][77]80,[82][83][84][85][86][87]. However, Samuel et al. investigating RNA in EVs detected cTM in 70% (n=10) of the patients [106]. Patients were distinguished from controls by CTC enumeration, ddPCR targeting ctDNA and differential expression profiling of miR-125b [54,65,96]. ...
Background:
Detection of circulating tumor-derived material (cTM) in the peripheral blood (PB) of cancer patients has been shown to be useful in early diagnosis, prediction of prognosis, and disease monitoring. However, it has not yet been thoroughly evaluated for pediatric sarcoma patients.
Methods:
We searched the PubMed and EMBASE databases for studies reporting the detection of circulating tumor cells, circulating tumor DNA, and circulating RNA in PB of pediatric sarcoma patients. Data on performance in identifying cTM and its applicability in diagnosis, and evaluation of tumor characteristics, prognostic factors, and treatment response was extracted from publications.
Results:
A total of 79 studies were assigned for the present systematic review, including detection of circulating tumor cells (116 patients), circulating tumor DNA (716 patients), and circulating RNA (2887 patients). Circulating tumor cells were detected in 76% of patients. Circulating DNA was detected in 63% by targeted NGS, 66% by shallow WGS, and 79% by digital droplet PCR. Circulating RNA was detected in 37% of patients.
Conclusion:
Of the cTM from Ewing's sarcoma and rhabdomyosarcoma ctDNA proved to be the best target for clinical application including diagnosis, tumor characterization, prognosis, and monitoring of disease progression and treatment response. For osteosarcoma the most promising targets are copy number alterations or patient specific micro RNAs, however, further investigations are needed to obtain consensus on clinical utility.
... ESFT includes Ewing sarcoma of the bone, extraosseous Ewing sarcoma (ES), and peripheral primitive neuroectodermal tumors (pPNET), and most ESFT patients present micrometastatic disease at diagnosis [69]. Over the past few decades, the diagnosis of ES has become more accurate due to detection of relevant hallmarks, including CD99/MIC2 in immunohistochemistry and the oncogenic fusions of the Ewing sarcoma RNA binding protein 1 gene (EWSR1) [70,71]. ES is characterized by highly recurrent translocations involving ETS transcription factors, with EWS-FLI1 and EWS-ERG being the most common [72,73]. ...
... Additionally, EZH2 mRNA into exosomes has been detected in plasma of ES type 1 patients, and not healthy donors or patients with other types of sarcoma tumors [80]. Thus, assaying circulating exosomes can help in diagnosis of ESFT and potentially in predicting response to therapy and recurrence [71]. ...
... Liquid-based immuno-enrichment for ESFT-specific exosomes has been performed using CD99 and NGFR, and the EWS-ETS fusion transcript has been detected with high specificity from as little as 250 µL of plasma samples of 10 metastatic and localized pediatric patients, with a significant diagnostic power (AUC = 0.92, p = 0.001 for sEV numeration) [71]. CD99 prevents cell differentiation in order to maintain the proliferative as well as the metastatic capabilities of tumor cells [81,82]. ...
The knowledge of exosome impact on sarcoma development and progression has been implemented in preclinical studies thanks to technological advances in exosome isolation. Moreover, the clinical relevance of liquid biopsy is well established in early diagnosis, prognosis prediction, tumor burden assessment, therapeutic responsiveness, and recurrence monitoring of tumors. In this review, we aimed to comprehensively summarize the existing literature pointing out the clinical relevance of detecting exosomes in liquid biopsy from sarcoma patients. Presently, the clinical utility of liquid biopsy based on exosomes in patients affected by sarcoma is under debate. The present manuscript collects evidence on the clinical impact of exosome detection in circulation of sarcoma patients. The majority of these data are not conclusive and the relevance of liquid biopsy-based approaches in some types of sarcoma is still insufficient. Nevertheless, the utility of circulating exosomes in precision medicine clearly emerged and further validation in larger and homogeneous cohorts of sarcoma patients is clearly needed, requiring collaborative projects between clinicians and translational researchers for these rare cancers.
... In fact, in a published study conducted in tandem with this analysis, PT 7 is the only ESFT patient out of 12 samples, to have a CD99 immunohistochemical score of "moderate" by a trained pathologist, while the 11 others tumor samples were ranked as "high." 26 Despite this finding, radiographic imaging including PET, along with both clinical and pathologic findings were all consistent with the diagnosis of localized osseous Ewing Sarcoma of the extremity as well demonstrated presence of EWSR1 breakage as determined by FISH ( Figure 6A-C). Given the high promiscuity of EWSR1 in fusion genes and fusion oncoproteins in a variety of malignancies outside of ESFT, we questioned whether this patient may have an alternative EWSR1 binding partner atypical of ESFT. ...
... Our previous work has identified the EWS-FLI1 oncoproteins in sEVs such as exosomes. 26 We suspect that EWS-FLI1 is important for miRNA enrichment in sEVs however a potential mechanism remains elusive. While this body of work is focused on biomarker development, future work should investigate potential roles of EWS-ETS in the enrichment of miRNAs into sEVs. ...
Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant ( P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson’s clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.
... These luminal or surface molecules act through paracrine or autocrine signaling and are involved in cancer development, immunoediting, metastasis, and drug resistance [14][15][16]. In clinical and translational oncology, tumor-derived EVs and exosomes can be used to infer cancer diagnosis, prognosis, and recurrence [10,[17][18][19][20][21]. Their potential as biomarkers for hepatocellular carcinoma (HCC), various pediatric solid tumors, pancreatic, ovarian, and bladder cancer has been shown in preliminary research [22][23][24][25]. ...
Cutaneous squamous cell carcinoma (cSCC) as one of the most prevalent cancers worldwide is associated with significant morbidity and mortality. Full-body skin exam and biopsy is the gold standard for cSCC diagnosis, but it is not always feasible given constraints on time and costs. Furthermore, biopsy fails to reflect the dynamic changes in tumor genomes, which challenges long-term medical treatment in patients with advanced diseases. Extracellular vesicle (EV) is an emerging biological entity in oncology with versatile clinical applications from screening to treatment. In this systematic review, pre-clinical and clinical studies on cSCC-derived EVs were summarized. Seven studies on the genomics, transcriptomics, and proteomics of cSCC-derived EVs were identified. The contents in cSCC-derived EVs may reflect the mutational landscape of the original cancer cells or be selectively enriched in EVs. Desmoglein 2 protein (Dsg2) is an important molecule in the biogenesis of cSCC-derived EVs. Ct-SLCO1B3 mRNA, and CYP24A1 circular RNA (circRNA) are enriched in cSCC-derived EVs, suggesting potentials in cSCC screening and diagnosis. p38 inhibited cSCC-associated long intergenic non-coding RNA (linc-PICSAR) and Dsg2 involved in EV-mediated tumor invasion and drug resistance served as prognostic and therapeutic predictors. We also proposed future directions to devise EV-based cSCC treatment based on these molecules and preliminary studies in other cancers.
... Proteomic investigations of circulating exosomes Ye et al. Journal of Nanobiotechnology (2022) 20:403 revealed that some proteins were helpful in indicating tumor progression and metastasis [82][83][84][85]. For example, the level of serum exosomal PD-L1 and N-cadherin was higher in osteosarcoma patients, especially in the ones with pulmonary metastasis, than that in healthy controls [86]. ...
The current diagnosis and treatment of sarcoma continue to show limited timeliness and efficacy. In order to enable the early detection and management of sarcoma, increasing attentions have been given to the tumor microenvironment (TME). TME is a dynamic network composed of multiple cells, extracellular matrix, vasculature, and exosomes. Exosomes are nano-sized extracellular vesicles derived from various cells in the TME. The major function of exosomes is to promote cancer progress and metastasis through mediating bidirectional cellular communications between sarcoma cells and TME cells. Due to the content specificity, cell tropism, and bioavailability, exosomes have been regarded as promising diagnostic and prognostic biomarkers, and therapeutic vehicles for sarcoma. This review summarizes recent studies on the roles of exosomes in TME of sarcoma, and explores the emerging clinical applications.
... Circulating miRNAs have also recently become the subject of study in Ewing sarcoma. As an example, a widely studied circulating miRNA related to Ewing sarcoma progression is miR-125b, which was found decreased in patients serum after surgical resection when compared to healthy controls [121] . In the same study, its downregulation in the group of patients analyzed was also correlated with poor response to chemotherapy [121] . ...
... As an example, a widely studied circulating miRNA related to Ewing sarcoma progression is miR-125b, which was found decreased in patients serum after surgical resection when compared to healthy controls [121] . In the same study, its downregulation in the group of patients analyzed was also correlated with poor response to chemotherapy [121] . Recently, the research focus is shifting towards Ewing sarcoma-derived EV cargo as a prognostic biomarker source, particularly to their protein content. ...
... As for the development of chemoresistance in Ewing sarcoma, a recent study showed that circulating miR-125b levels were decreased in patient serum, and this was associated with poor response to chemotherapy [121] . ...
Liquid biopsies are a powerful tool to non-invasively analyze tumor phenotype and progression as well as drug resistance. In the bone oncology field, liquid biopsies would be particularly important to develop, since standard biopsies can be very painful, dangerous (e.g., when found in proximity to the spinal cord), and hard to collect. In this review, we explore the recent advances in liquid biopsies in both primary (osteosarcoma and Ewing sarcoma) and secondary bone cancers (breast, prostate, and lung cancer-induced bone metastases), presenting their current role and highlighting their unexpressed potential, as well as the barriers limiting their possible adoption, including costs, scalability, reproducibility, and isolation methods. We discuss the use of circulating tumor cells, cell-free circulating tumor DNA, and extracellular vesicles for the purpose of improving diagnosis, prognosis, evaluation of therapy resistance, and driving therapy decisions in both primary and secondary bone malignancies.
