Extracellular Vesicle - MDM2-DNA as a Potential Liquid Biopsy Biomarker for Disease Identification in Retroperitoneal Liposarcoma.

OBJECTIVE: We aimed to assess the levels of MDM2-DNA within extracellular vesicles (EVs) isolated from the serum of retroperitoneal liposarcoma (RLS) patients versus healthy donors, as well as within the same patients at the time of surgery versus post-operative surveillance visits. To determine whether EV-MDM2 may serve as a possible first-ever biomarker of liposarcoma recurrence. BACKGROUND: A hallmark of well-differentiated and de-differentiated (WD/DD) retroperitoneal liposarcoma is elevated MDM2 due to genome amplification, with recurrence rates of >50% even after complete resection. Imaging technologies frequently cannot resolve recurrent WD/DD-RLS versus postoperative scarring. Early detection of recurrent lesions, for which biomarkers are lacking, would guide surveillance and treatment decisions. METHODS: WD/DD-RLS serum samples were collected both at the time of surgery and during follow-up visits from 42 patients, along with sera from healthy donors (n=14). EVs were isolated, DNA purified and MDM2-DNA levels determined through q-PCR analysis. Non-parametric tests were employed to compare EV-MDM2 DNA levels from patients versus control group, as well as the time of surgery versus post-surgery conditions. RESULTS: EV-MDM2 levels were significantly higher in WD/DD-RLS than controls (P= 0.00085). Moreover, EV-MDM2 levels were remarkably decreased in WD/DD-RLS patients after resection (P=0.00036), reaching values comparable to control group (P=0.124). During post-operative surveillance, significant increases of EV-MDM2 was observed in some patients, correlating with CT scan evidence of recurrent or persistent post-resection disease. CONCLUSIONS: Serum EV-MDM2 may serve as a potential biomarker of early recurrent or post-operatively persistent WD/DD-RLS, a disease currently lacking such determinants.

Enhancer Coamplification and Hijacking Promote Oncogene Expression in Liposarcoma.

SIGNIFICANCE: Comprehensive profiling of the enhancer landscape and 3D genome structure in liposarcoma identifies extensive enhancer-oncogene coamplification and enhancer hijacking events, deepening the understanding of how oncogenes are regulated in cancer.

Three dimensional models of dedifferentiated liposarcoma cell lines: scaffold-based and scaffold-free approaches.

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

Preclinical efficacy of the Wnt/ß-catenin pathway inhibitor BC2059 for the treatment of desmoid tumors.

Mutation in the CTNNB1 gene, leading to a deregulation of the WTN/ß-catenin pathway, is a common feature of desmoid tumors (DTs). Many ß-catenin inhibitors have recently been tested in clinical studies; however, BC2059 (also referred as Tegavivint), a selective inhibitor of nuclear ß-catenin that works through binding TBL-1, is the only one being evaluated in a clinical study, specifically for treatment of desmoid tumor patients. Preclinical studies on BC2059 have shown activity in multiple myeloma, acute myeloid leukemia and osteosarcoma. Our preclinical studies provide data on the efficacy of BC2059 in desmoid cell lines, which could help provide insight regarding antitumor activity of this therapy in desmoid tumor patients. In vitro activity of BC2059 was evaluated using desmoid tumor cell lines. Ex vivo activity of BC2059 was assessed using an explant tissue culture model. Pharmacological inhibition of the nuclear ß-catenin activity using BC2059 markedly inhibited cell viability, migration and invasion of mutated DT cells, but with lower effect on wild-type DTs. The decrease in cell viability of mutated DT cells caused by BC2059 was due to apoptosis. Treatment with BC2059 led to a reduction of ß-catenin-associated TBL1 in all mutated DT cells, resulting in a reduction of nuclear ß-catenin. mRNA and protein levels of AXIN2, a ß-catenin target gene, were also found to be downregulated after BC2059 treatment. Taken together, our results demonstrate that nuclear ß-catenin inhibition using BC2059 may be a novel therapeutic strategy for desmoid tumor treatment, especially in patients with CTNNB1 mutation.

In situ hybridization to detect DNA amplification in extracellular vesicles.

EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

Cancer-Derived Extracellular Vesicles: Their Role in Sarcoma.

Soft tissue sarcomas (STS) are rare malignancies with limited responses to anticancer therapy. Extracellular vesicles (EVs) are a heterogeneous group of bi-lipid layer sacs secreted by cells into extracellular space. Investigations of tumor-derived EVs have revealed their functional capabilities, including cell-to-cell communication and their impact on tumorigenesis, progression, and metastasis; however information on the roles of EVs in sarcoma is currently limited. In this review we investigate the role of various EV cargos in sarcoma and the mechanisms by which those cargos can affect the recipient cell phenotype and the aggressivity of the tumor itself. The study of EVs in sarcoma may help establish novel therapeutic approaches that target specific sarcoma subtypes or biologies, thereby improving sarcoma therapeutics in the future.

Targetable Pathways in the Treatment of Retroperitoneal Liposarcoma.

Liposarcoma (LPS) is the most prevalent soft tissue sarcoma histological subtype. When it occurs in the abdomen the overall survival rate is as low as 10% at 10 years and is fraught with high rates of recurrence, particularly for the more aggressive dedifferentiated subtype. Surgery remains the mainstay of treatment. Systemic therapies for the treatment of metastatic or unresectable disease have low response rates. Deep understanding of well-differentiated and de-differentiated LPS (WDLPS and DDLPS, respectively) oncologic drivers is necessary for the development of new efficacious targeted therapies for the management of this disease. This review discusses the current treatments under evaluation for retroperitoneal DDLPS and the potential targetable pathways in DDLPS.

Clinical biomarkers in soft tissue sarcoma A comprehensive review of current soft tissue sarcoma biomarkers.

Soft tissue sarcomas (STS) are a heterogeneous group of tumors that arise from mesenchymal tissue. Investigation at the molecular level has been challenging due to the rarity of STS and the number of histologic subtypes. However, recent research has provided new insight into potential genomic, proteomic, and immunological biomarkers of STS. The identification of biomarkers can improve diagnosis, prognosis, and prediction of recurrence and treatment response. This review provides an understanding of biomarkers, discussing the current status of biomarker research in STS.

Cross-flow microfiltration for isolation, selective capture and release of liposarcoma extracellular vesicles.

We present a resource-efficient approach to fabricate and operate a micro-nanofluidic device that uses cross-flow filtration to isolate and capture liposarcoma derived extracellular vesicles (EVs). The isolated extracellular vesicles were captured using EV-specific protein markers to obtain vesicle enriched media, which was then eluted for further analysis. Therefore, the micro-nanofluidic device integrates the unit operations of size-based separation with CD63 antibody immunoaffinity-based capture of extracellular vesicles in the same device to evaluate EV-cargo content for liposarcoma. The eluted media collected showed ∼76% extracellular vesicle recovery from the liposarcoma cell conditioned media and ∼32% extracellular vesicle recovery from dedifferentiated liposarcoma patient serum when compared against state-of-art extracellular vesicle isolation and subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma-relevant extracellular vesicle cargo obtained in 30 min presenting a significant advance over existing state-of-art.

ß-catenin S45F mutation results in apoptotic resistance.

Wnt/ß-catenin signaling is one of the key cascades regulating embryogenesis and tissue homeostasis; it has also been intimately associated with carcinogenesis. This pathway is deregulated in several tumors, including colorectal cancer, breast cancer, and desmoid tumors. It has been shown that CTNNB1 exon 3 mutations are associated with an aggressive phenotype in several of these tumor types and may be associated with therapeutic tolerance. Desmoid tumors typically have a stable genome with ß-catenin mutations as a main feature, making these tumors an ideal model to study the changes associated with different types of ß-catenin mutations. Here, we show that the apoptosis mechanism is deregulated in ß-catenin S45F mutants, resulting in decreased induction of apoptosis in these cells. Our findings also demonstrate that RUNX3 plays a pivotal role in the inhibition of apoptosis found in the ß-catenin S45F mutants. Restoration of RUNX3 overcomes this inhibition in the S45F mutants, highlighting it as a potential therapeutic target for malignancies harboring this specific CTNNB1 mutation. While the regulatory effect of RUNX3 in ß-catenin is already known, our results suggest the possibility of a feedback loop involving these two genes, with the CTNNB1 S45F mutation downregulating expression of RUNX3, thus providing additional possible novel therapeutic targets for tumors having deregulated Wnt/ß-catenin signaling induced by this mutation.

Extracellular vesicle cross-talk in the liposarcoma microenvironment.

Liposarcoma (LPS) is the most prevalent soft tissue sarcoma; among the four different LPS subtypes, dedifferentiated liposarcoma (DDLPS) is especially worrisome given its propensity for local and distant recurrence, with an overall survival rate of only 10% at 10 years. Our understanding of the molecular drivers of this disease is rudimentary at best; knowledge about how DDLPS interacts with cells in the tumor microenvironment (TME) is also lacking. Extracellular vesicle (EVs) have been studied in a number of different systems concerning their ability to influence the TME transferring bioactive molecules. In this review, we outline the role of the TME in the DDLPS progression and recurrence, focusing on the interplay between EVs released from the tumor and their target recipient cells in the TME. Success in the understanding of this process will be critical to an enhanced understanding of the underlying biologic drivers at play, potentially leading to new therapeutic strategies of benefit to patients with this disease.

Cracking the riddle of dedifferentiated liposarcoma: is EV-MDM2 a key?

Dedifferentiated liposarcoma (DDLPS) is molecularly characterized by wt p53 and MDM2 gene amplification causing MDM2 protein over-production, the key oncogenic process in DDLPS. Commonly located in fat-bearing retroperitoneal areas, almost 60% of DDLPS patients undergo multifocal recurrence, typically amenable to palliative treatment only, and occasionally develop distant metastasis. These factors lead to an abysmal 10% 10 year overall survival rate. Tumor cell-derived extracellular vesicles (EVs) can facilitate loco-regional malignancy dissemination by depositing molecular factors that participate in the development of pre-metastatic niches for tumor cell implantation and growth. High number of MDM2 DNA molecules was identified within EVs from DDLPS patient serum (ROC vs normal; 0.95) as well as from DDLPS cell lines. This MDM2 DNA could be transferred to preadipocytes (P-a), a major and ubiquitous cellular component of the DDLPS tumor microenvironment (TME), with subsequent P-a production of matrix metalloproteinase 2 (MMP2), a critical component in the metastatic cascade. From here the hypothesis that the DDLPS microenvironment (specifically P-a cells) may participate in DDLPS recurrence events. Since multifocal loco-regional DDLPS spreading is the main cause of the remarkably high lethality of this disease, a better understanding of the underlying oncogenic processes and their regulatory mechanisms is essential to improve the outcome of this devastating disease.

Adipose Tumor Microenvironment.

The term "adipose tissue" represents a multicellular and multifunctional organ involved in lipid storage, in hormone and temperature regulation, and in the protection of bones and vital organs from impact-based damage. Emerging evidence now suggests a more malignant role of adipose tissue in promoting cancer onset and progression via the release of secreted factors such as interleukin-6 (IL6) and extracellular vesicles (EVs). These adipose-source factors subsequently affect various aspects of tumorigenesis and/or cancer progression by either directly enhancing the tumor cell oncogenic phenotype or indirectly by the stimulating adjacent normal cells to adopt a more pro-cancer phenotype. Due to the recent growing interest in the role of IL6 and EVs released by adipose tissue in cancer promotion and progression, we are focusing on the protumorigenic impact of fat tissue via IL6 and EV secretion.

Multi-Layer Micro-Nanofluidic Device for Isolation and Capture of Extracellular Vesicles Derived from Liposarcoma Cell Conditioned Media.

We report on isolation, capture, and subsequent elution for analysis of extracellular vesicles derived from human liposarcoma cell conditioned media, using a multi-layer micro-nanofluidic device operated with tangential flow separation. Our device integrates size-based separation followed by immunoaffinity-based capture of extracellular vesicles in the same device. For liposarcomas, this is the first report on isolating, capturing, and then eluting the extracellular vesicles using a micro-nanofluidic device. The results show a significantly higher yield of the eluted extracellular vesicles (~84%) compared to the current methods of ultracentrifugation (~6%) and ExoQuick-based separations (~16%).

MDM2 Derived from Dedifferentiated Liposarcoma Extracellular Vesicles Induces MMP2 Production from Preadipocytes.

Dedifferentiated liposarcoma (DDLPS) is frequently diagnosed late, and patients typically respond poorly to treatments. DDLPS is molecularly characterized by wild-type p53 and amplification of the MDM2 gene, which results in overexpression of MDM2 protein, a key oncogenic process in DDLPS. In this study, we demonstrate that extracellular vesicles derived from patients with DDLPS or from DDLPS cell lines are carriers of MDM2 DNA that can be transferred to preadipocytes, a major and ubiquitous cellular component of the DDLPS tumor microenvironment, leading to impaired p53 activity in preadipocytes and increased proliferation, migration, and production of matrix metalloproteinase 2; treatment with MDM2 inhibitors repressed these effects. Overall, these findings indicate that MDM2 plays a crucial role in DDLPS by enabling cross-talk between tumor cells and the surrounding microenvironment and that targeting vesicular MDM2 could represent a therapeutic option for treating DDLPS. SIGNIFICANCE: Extracellular vesicles derived from dedifferentiated liposarcoma cells induce oncogenic properties in preadipocytes.

Autophagy inhibition overcomes sorafenib resistance in S45F-mutated desmoid tumors.

BACKGROUND: Desmoid tumors (DTs) are rare and understudied fibroblastic lesions that are frequently recurrent and locally invasive. DT patients often experience chronic pain, organ dysfunction, decrease in quality of life, and even death. METHODS: Sorafenib has emerged as a promising therapeutic strategy, which has led to the first randomized phase 3 clinical trial devoted to DTs. Concurrently, we conducted a comprehensive analysis of sorafenib efficacy in a large panel of desmoid cell strains to probe for response mechanism. RESULTS: We found distinctive groups of higher- and lower-responder cells. Clustering the lower-responder group, we observed that CTNNB1 mutation was determinant of outcome. Our results revealed that a lower dose of sorafenib was able to inhibit cell viability, migration, and invasion of wild-type and T41A-mutated DTs. Apoptosis induction was observed in those cells after treatment with sorafenib. On the other hand, the lower dose of sorafenib was not able to inhibit cell viability, migration, or invasion or to induce apoptosis in the S45F-mutated DTs. The investigation of autophagy showed the dependency of S45F-mutated DTs on this pathway as a part of cell survival mechanism. Significantly, when autophagy was inhibited genetically or pharmacologically in the S45F mutant cell strains, sensitivity to sorafenib was restored. CONCLUSIONS: Our findings suggest that the response to sorafenib differs when comparing S45F-mutated DTs and T41A-mutated or wild-type DTs. Furthermore, the combination of hydroxychloroquine and sorafenib enhances the antiproliferative and proapoptotic effects in S45F-mutated DT cells, suggesting that profiling ß-catenin status could guide clinical management of desmoid patients who are considering sorafenib treatment.

The TLR7/8/9 Antagonist IMO-8503 Inhibits Cancer-Induced Cachexia.

: Muscle wasting is a feature of the cachexia syndrome, which contributes significantly to the mortality of patients with cancer. We have previously demonstrated that miR-21 is secreted through extracellular vesicles (EV) by lung and pancreatic cancer cells and promotes JNK-dependent cell death through its binding to the TLR7 receptor in murine myoblasts. Here, we evaluate the ability of IMO-8503, a TLR7, 8, and 9 antagonist, to inhibit cancer-induced cachexia. Using EVs isolated from lung and pancreatic cancer cells and from patient plasma samples, we demonstrate that IMO-8503 inhibits cell death induced by circulating miRNAs with no significant toxicity. Intraperitoneal administration of the antagonist in a murine model for Lewis lung carcinoma (LLC-induced cachexia) strongly impaired several cachexia-related features, such as the expression of Pax7 as well as caspase-3 and PARP cleavage in skeletal muscles, and significantly prevented the loss of lean mass in tumor-bearing mice. IMO-8503 also impaired circulating miRNA-induced cell death in human primary myoblasts. Taken together, our findings strongly indicate that IMO-8503 serves as a potential therapy for the treatment of cancer cachexia. SIGNIFICANCE: Cancer-associated cachexia is a significant problem for patients with cancer that remain poorly understood, understudied, and inadequately treated; these findings report a potential new therapeutic for the treatment of TLR7-mediated cancer cachexia.

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates.

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.

Concurrent Aerobic and Resistance Training Has Anti-Inflammatory Effects and Increases Both Plasma and Leukocyte Levels of IGF-1 in Late Middle-Aged Type 2 Diabetic Patients.

Type 2 diabetes (T2D) is an age-related chronic disease associated with metabolic dysregulation, chronic inflammation, and activation of peripheral blood mononuclear cells (PBMC). The aim of this study was to assess the effects of a concurrent exercise training program on inflammatory status and metabolic parameters of T2D patients. Sixteen male patients (age range 55-70) were randomly assigned to an intervention group (n = 8), which underwent a concurrent aerobic and resistance training program (3 times a week; 16 weeks), or to a control group, which followed physicians' usual diabetes care advices. Training intervention significantly improved patients' body composition, blood pressure, total cholesterol, and overall fitness level. After training, plasma levels of adipokines leptin (-33.9%) and RBP4 (-21.3%), and proinflammatory markers IL-6 (-25.3%), TNF-α (-19.8%) and MCP-1 (-15.3%) decreased, whereas anabolic hormone IGF-1 level increased (+16.4%). All improvements were significantly greater than those of control patients. Plasma proteomic profile of exercised patients showed a reduction of immunoglobulin K light chain and fibrinogen as well. Training also induced a modulation of IL-6, IGF-1, and IGFBP-3 mRNAs in the PBMCs. These findings confirm that concurrent aerobic and resistance training improves T2D-related metabolic abnormalities and has the potential to reduce the deleterious health effects of diabetes-related inflammation.