Notch signaling is an emerging regulator of liposarcoma (LPS), but its role in mediating communication with the tumor microenvironment (TME) is unclear. Here, we investigate how Notch activation (NICD overexpression) alters the proteomes of LPS-derived extracellular vesicles (EVs). We used quantitative mass spectrometry to profile the EV proteome in multiple contexts: cultured LPS cells, LPS tumor, circulating EVs of LPS-bearing mice, and human LPS samples. We found that Notch signaling increases the secretion of EV proteins that favor tumor progression and metastasis but suppresses immune responses in murine LPS cells. Overlapping murine and human LPS data identifies 18 proteins that are increased in LPS EVs of both species, including endotrophin as a biomarker of LPS. Functional analysis supports a role of LPS EVs in regulating gene expression and behaviors of endothelial cells in TME. Together, these data demonstrate that in addition to its known function in driving tumorigenesis, Notch signaling also regulates TME through EV secretion.

Introduction Aggressive surgical resection is the cornerstone of treatment for retroperitoneal sarcomas (RPS), but recurrent disease occurs in up to 50% of patients. Palbociclib, an oral CDK4/6 inhibitor, in adjuvant setting may delay the need for additional surgery. However, it is unclear which patients derive an oncologic benefit from Palbociclib and what duration is required to prevent recurrence. We sought to evaluate recurrence patterns in patients with high risk RPS who completed adjuvant Palbociclib. Methods Patients with no evidence of disease after resection of RPS and treated with adjuvant Palbociclib were identified from a prospectively-maintained institutional database. We performed bulk RNA sequencing of primary tumor samples. Results Of the thirty-four patients who received adjuvant Palbociclib, 8 met the inclusion criteria and 5 developed recurrence after stopping treatment. Most (n=7) patients completed at least 12 months of Palbociclib. The three patients without recurrence currently have disease-free interval of 16, 25, and 33 months. On RNA sequencing, the epithelial-mesenchymal transition pathway is significantly enriched in the recurrence cohort compared to the no-recurrence cohort. Conclusion Half of the patients in this cohort developed short interval recurrence after completing adjuvant Palbociclib. Studies with larger patient cohorts and genomic analysis of tumors may lend additional insight.

Abstract Dedifferentiated liposarcoma (DDLPS) is a rare and aggressive adipocytic malignancy with a nearly 85% local recurrence rate, exceptionally high compared to other tumors, and a 10-year survival rate of only 10%. No reliable biomarkers currently predict recurrence or therapeutic response. Current management relies primarily on radical surgery, often combined with non-specific chemotherapy, which yields poor outcomes and severely compromises patients’ quality of life. The aggressiveness of DDLPS and the lack of effective systemic therapies highlight an urgent need for novel molecular targets and treatment approaches. We identified the mitochondrial chaperone TRAP1 (TNF Receptor-Associated Protein 1) as a potential oncogenic driver and therapeutic target in DDLPS. TRAP1 is a master regulator of mitochondrial metabolism, oxidative stress, and apoptosis, yet its role in DDLPS remains unknown. Our preliminary data show that TRAP1 protein and mRNA levels are significantly overexpressed in DDLPS compared to low grade well-differentiated liposarcoma (WDLPS) and Normal Adjacent Tissue (NAT), suggesting a contribution to tumor progression. To explore its therapeutic potential, we silenced TRAP1 using siRNA and inhibited its activity with MitoQuinone (MitoQ), a TRAP1 inhibitor currently evaluated in non-cancer clinical trials. TRAP1 silencing significantly reduced DDLPS cell proliferation and increased cell death, as shown by MTS assays and Annexin V/PI assays. MitoQ treatment led to significant, dose- and time-dependent cytotoxicity in DDLPS cells compared to their WDLPS counterpart, and impaired spheroid growth in 3D models. Moreover, TRAP1 knockdown significantly decreased mitochondrial membrane potential compared to control conditions, and triggered G1/S cell-cycle arrest, indicating disruption of mitochondrial function. Ongoing studies are investigating how TRAP1 modulation affects ROS production, mitochondrial dynamics and energy metabolism using Seahorse metabolic flux analysis, to define its contribution to metabolic reprogramming and redox balance in DDLPS. Finally, to establish TRAP1’s clinical relevance, we will assess TRAP1 mRNA expression in a larger cohort of liposarcoma patient samples to correlate its levels with recurrence rates and survival outcomes. This work positions TRAP1 as a mitochondrial therapeutic target and potential biomarker, offering novel mechanistic insight and paving the way for innovative DDLPS treatment strategies. Citation Format: Roma Karna, Esin Ulker, Sydney Rentsch, Marina Capece, Sayumi Tahara, Qi Zhang, Patricia Sarchet, Giovanni Nigita, Paolo Fadda, Fernanda Costas Casal de Faria, Valerie Grignol, Nicholas C. Denko, Carlo M. Croce, Raphael E. Pollock, Federica Calore. TRAP1 represents a mitochondrial target and biomarker of dedifferentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 557.

Reliable, reproducible, and standardized characterization of extracellular vesicles (EVs) remains a central challenge due to methodological variability across analytical platforms and intrinsic EV heterogeneity within and across biofluids. Here, we evaluated EVs isolated using ultracentrifugation from dedifferentiated liposarcoma-conditioned media and commercially available healthy donor urine. Physical characterization and surface tetraspanin profiling of isolated EVs were performed using three widely used instruments: NanoFCM, CytoFLEX Nano, and ZetaView Evolution. All platforms consistently detected small EVs; however, absolute size distributions, particle concentrations, and tetraspanin expression levels varied, reflecting differences in optical configuration, detection principles, and fluorescence background sensitivity. Additionally, EV-associated DNA was quantified using two different methods, revealing further variability in both the DNA yield and quantity. Together, the observations highlight the method-dependent nature of EV analyses and underscore the importance of carefully considering instrument-specific metrics for interpreting EV data across biofluids.

Abstract Background Dedifferentiated liposarcoma (DDLPS) is characterized by abundant immune cell infiltration yet derives limited benefit from immune checkpoint blockade and stimulator of interferon genes (STING) agonist-based strategies, suggesting tumor-mediated suppression of antitumor immunity. Tumor-associated macrophages are the most abundant immune populations in DDLPS, but the factors regulating their function remain incompletely understood. Methods Extracellular vesicles (EVs) were isolated from two DDLPS cell lines and serum from 16 DDLPS patients and 13 healthy donors. EVs’ impact on cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) -induced macrophage activation was assessed by cytokine secretion, surface markers, functional assays and macrophage-T-cell coculture. Proteomics was performed in EV-treated and EV-untreated macrophages from three donors. Pathway and protein interaction analyses were integrated with The Cancer Genome Atlas (TCGA) DDLPS transcriptomic and survival data. Results We show that EVs released by DDLPS cells suppress macrophage responsiveness to classic STING agonist cGAMP. EVs derived from DDLPS attenuated cGAMP-induced expression of type I interferon-associated cytokines and chemokines, reduced IFN-β secretion, and impaired phosphorylation of STING, TBK1 and IRF3. Functionally, DDLPS EV exposure shifted macrophages toward an immunoregulatory phenotype, restrained phagocytic activity, and attenuated macrophage-dependent T-cell proliferation while promoting T-cell exhaustion. Proteomic profiling revealed extensive macrophage reprogramming characterized by suppression of STING-associated signaling, antigen processing and presentation associated pathways and proteins targeted by miR-16-5p. Consistent with these findings, STING expression was associated with prolonged overall survival in DDLPS, while reduced expression of miR-16-5p target proteins was associated with attenuated STING pathway activity and immunostimulatory macrophage signatures. Conclusions These findings identify EV-mediated suppression of macrophage STING signaling as a mechanism of immune dysfunction in DDLPS and provide a framework for understanding immune resistance in this disease.

Sequence Data Information in Lipo863B

Dedifferentiated liposarcoma (DDLPS) is a soft tissue sarcoma characterized by genomic amplification of proto-oncogene MDM2 alongside wildtype P53. Disease progression and poor survival rates in DDLPS are driven by MDM2 overexpression, which negatively regulates the tumor suppressor p53. While small molecules such as Nutlin and its derivatives have been employed to inhibit MDM2-p53 interaction, their significant toxicity presents a major limitation. This clinical challenge highlights the pressing need for alternative approaches to reactivate P53 through less toxic mechanisms. Alternatively spliced isoform of MDM2, MDM2-ALT1 inhibits full-length MDM2 (MDM2-FL) and stabilizes p53 expression. Additionally, MDM2-ALT1 can prevent the activity of MDMX, another negative regulator of p53, and the resulting p53 activation can sensitize tumor cells to p53-dependent chemotherapies. Thus, targeting MDM2 splicing to enhance MDM2-ALT1 production represents a promising non-toxic intervention strategy for DDLPS. Our laboratory has developed Splice Switching Oligonucleotides (SSOs) that can induce MDM2-ALT1 expression and subsequently reactivate p53 function. We hypothesize that in vivo delivery of SSOs to p53 wild-type tumor cells will induce MDM2-ALT1 expression and restore p53 activity to reverse the cancer phenotype. We have synthesized a 20-mer SSO targeting the SRSF2 binding site in MDM2 exon 11, which functions as a positive regulator of MDM2 splicing. These SSOs prevent SRSF2 binding and promote MDM2-ALT1 production. To enhance both in vitro and in vivo SSO delivery, we incorporated our SSO sequence in an AAV viral vector to be expressed as an anti-sense RNA and validated its ability to induce MDM2-ALT1 splicing in vitro using RT-PCR. The SSO sequence expressed by the AAV vector successfully induced MDM2-ALT1 expression. Next, we will measure the p53 activity and perform proliferation assay to validate that SSO expressed from the AAV viral vectors can reduce cellular proliferation through the activation of p53 tumor suppressive activity. Our goal is to facilitate target specific in vivo delivery of this SSO construct using AAV viruses or extracellular vesicles in Xenograft tumor models. This research presents a novel therapeutic strategy by designing SSOs that reactivate p53 tumor suppressor function and developing methods for target-specific delivery. This innovative strategy could potentially revolutionize treatment for DDLPS and other cancers where p53 function is suppressed by MDM2 overexpression. Rafia Rahat, Akila S. Venkataramany, Matias Montes, Ravi Dhital, Kevin Cassady, Raphael E. Pollock, Dawn S. Chandler. Therapeutic delivery of splice switching oligonucleotides to restore p53 function in liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5999.

Liposarcoma, a rare malignancy originating from adipose tissue, includes well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS). While WDLPS exhibits indolent behavior, DDLPS is a more aggressive, high-grade subtype, with a nearly 85% local recurrence rate, exceptionally high compared to other tumors, and a 10-year survival rate of only 10%. No reliable biomarkers currently predict prognosis or recurrence. Current treatment relies primarily on radical surgery, often combined with non-specific chemotherapy, yielding poor response rates and severely affecting patients’ quality of life. The aggressiveness of DDLPS and lack of effective systemic therapies highlight an urgent need for novel therapeutic targets and approaches. Mitochondrial dysfunction is increasingly recognized as a key driver in cancer progression. Cancer cells often exploit altered mitochondrial function to support rapid cell proliferation, resist apoptosis, and metastasize. TRAP1 (TNF Receptor-Associated Protein 1), a mitochondrial chaperone of the Hsp90 family, regulates mitochondrial metabolism, oxidative stress, and apoptosis, making it a promising target in various cancers. However, its role in DDLPS remains poorly defined. We propose to investigate TRAP1 as a novel potential target for DDLPS, leading to novel future therapeutic strategies. Our preliminary data demonstrate significantly elevated TRAP1 protein expression in DDLPS compared to WDLPS patient-derived cell lines, suggesting its involvement in DDLPS pathogenesis. To explore its therapeutic potential, we silenced TRAP1 using siRNA and inhibited its activity with MitoQuinone (MitoQ), a mitochondria-targeted antioxidant evaluated in non-cancer clinical trials. TRAP1 silencing significantly reduced DDLPS cell proliferation, as shown by MTS assays, and increased cell death, confirmed by Annexin V/PI staining. Similarly, MitoQ treatment led to significant, dose- and time-dependent cytotoxicity in DDLPS cells, validated using a 3D spheroid model. We are currently elucidating TRAP1’s role in regulating ROS production and mitochondrial function in DDLPS. Silencing TRAP1 disrupts mitochondrial homeostasis, potentially altering ROS balance and oxidative stress. Using MitoQ, we aim to clarify how TRAP1 modulation impacts ROS dynamics, mitochondrial membrane potential (ΔΨm), and ATP levels, potentially inducing energy imbalance and electron leakage. These analyses will deepen our understanding of TRAP1’s role in DDLPS pathogenesis and therapeutic response. Finally, to establish TRAP1’s clinical relevance, we will evaluate its mRNA expression in Normal Adjacent Tissue (NAT), WDLPS, and DDLPS patient samples, correlating levels with recurrence rates and survival outcomes. This research could reveal molecular mechanisms driving DDLPS tumorigenesis, identify TRAP1 as a potential therapeutic target, and validate it as a potential prognostic biomarker, ultimately improving DDLPS treatment strategies. Roma Karna, Marina Capece, Qi Zhang, Sayumi Tahara, Patricia Sarchet, Giovanni Nigita, Paolo Fadda, Sydney Rentsch, Fernanda Costas Casal de Faria, Valerie Grignol, Carlo Croce, Raphael Pollock, Federica Calore. Evaluating TRAP1 as a novel, potential therapeutic target in dedifferentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB264.

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 postoperative 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 dedifferentiated (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. Nonparametric tests were employed to compare EV- MDM2 DNA levels from patients versus the control group, as well as the time of surgery versus postsurgery 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 postoperative surveillance, significant increases of EV- MDM2 were observed in some patients, correlating with computed tomography scan evidence of recurrent or persistent postresection disease. CONCLUSIONS:Serum EV- MDM2 may serve as a potential biomarker of early recurrent or postoperatively persistent WD/DD-RLS, a disease currently lacking such determinants.

H3K27Ac ChIP-seq Patient Sample Statistics

Patient Sample Hi-C data Statistics

Effective therapies for retroperitoneal (RP) dedifferentiated liposarcoma (DDLPS) remain unavailable. Loco-regional recurrence occurs in >80% of cases; 5-year disease-specific survival is only 20%. DDLPS is especially prevalent in the retroperitoneum and abdomen; evaluation of the DDLPS microenvironment in these high-fat compartments appears pertinent. Adipose is a main supplier of interleukin-6 (IL6); excessive activation of IL6 signal transducer glycoprotein 130 (GP130) underlies the development of some diseases. The role of GP130 pathway activation remains unstudied in DDLPS, so we examined the role of microenvironment fat cell activation of the IL6/GP130 signaling cascade in DDLPS. All DDLPS tumors and cell lines studied expressed elevated levels of the GP130-encoding gene IL6ST and GP130 protein compared to normal tissue and cell line controls. IL6 increased DDLPS cell growth and migration, possibly through increased signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) activation, and upregulated mouse double minute 2 (MDM2). GP130 loss conveyed opposite effects; pharmacological blockade of GP130 by SC144 produced the MDM2 splice variant MDM2-ALT1, known to inhibit full length MDM2 (MDM2-FL). Although genomic MDM2 amplification is pathognomonic for DDLPS, mechanisms driving MDM2 expression, regulation, and function beyond the MDM2:p53 negative feedback loop are poorly understood. Our findings suggest a novel preadipocyte DDLPS-promoting role due to IL6 release, via upregulation of DDLPS MDM2 expression. Pharmacological GP130 blockade reduced the IL6-induced increase in DDLPS MDM2 mRNA and protein levels, possibly through enhanced expression of MDM2-ALT1, a possibly targetable pathway with potential as future DDLPS patient therapy.

Patient Sample WGS data statistics

Background: Surgery and radiation therapy remain the standard of care for patients with high-grade extremity soft tissue sarcoma that are >5 cm. Radiation therapy is time and labor-intensive for patients, and social determinants of health may affect adherence. The aim of this study was to define demographic, clinical, and treatment factors associated with the completion of radiation therapy and determine if preoperative radiation therapy improved adherence compared to postoperative radiation therapy. Methods: The cohort included patients in the National Cancer Database with high-grade extremity soft tissue sarcoma >5 cm without nodal or distant metastases who received limb-sparing surgery and radiation therapy with microscopically negative R0 margins. Multivariable logistic regression analyses identified factors associated with radiation therapy sequencing and adherence (defined as completion of 50 Gy preoperative radiation therapy or at least 60 Gy postoperative radiation therapy). A multivariable Cox Proportional Hazards model assessed overall survival. Results: Among 2,145 patients, 47.1% received preoperative radiation therapy (n = 1,010), and 52.9% (n = 1135) received postoperative radiation therapy. A greater proportion of patients treated with preoperative (77.2%) versus postoperative radiation therapy (64.9%, P < .0001) received the recommended dose. More patients with private insurance (49.8% vs 35.3% Medicaid vs 44.9% Medicare, P = .011) and patients treated at an academic medical center (52.6% vs 47.4%, P < .001) received preoperative radiation therapy. Patients who received preoperative radiation therapy had lower odds of receiving insufficient doses of radiation therapy (odds ratio 0.34 [95% CI 0.27-0.47]). Neither radiation therapy adherence nor sequencing were independent predictors of overall survival. Conclusions: Patients who received preoperative radiation therapy were more likely to complete therapy and receive an optimal dose than patients treated with postoperative radiation therapy. Preoperative radiation therapy improves adherence and should be widely considered in patients with high-grade extremity soft tissue sarcoma, particularly in patients at risk for not completing therapy. (c) 2023 Elsevier Inc. All rights reserved.

Analysis of single extracellular vesicles (EVs) has the potential to yield valuable label-free information on their morphological structure, biomarkers and therapeutic targets, though such analysis is hindered by the lack of reliable and quantitative measurements of the mechanical properties of these compliant nanoscale particles. The technical challenge in mechanical property measurements arises from the existing tools and methods that offer limited throughput, and the reported elastic moduli range over several orders of magnitude. Here, we report on a flow-based method complemented by transmission electron microscopy (TEM) imaging to provide a high throughput, whole EV deformation analysis for estimating the mechanical properties of liposarcoma-derived EVs as a function of their size. Our study includes extracting morphological data of EVs from a large dataset of 432 TEM images, with images containing single to multiple EVs, and implementing the thin-shell deformation theory. We estimated the elastic modulus, E = 0.16 +/- 0.02 MPa (mean +/- SE) for small EVs (sEVs; 30-150 nm) and E = 0.17 +/- 0.03 MPa (mean +/- SE) for large EVs (lEVs; >150 nm). To our knowledge, this is the first report on the mechanical property estimation of LPS-derived EVs and has the potential to establish a relationship between EV size and EV mechanical properties.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions.Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically.Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response.Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis.Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

Sarcomas comprise a wide range of diverse cancers of the bone and soft tissues, with ∼100 different types as classified by the World Health Organization.1,2 Sarcomas arise in any organ at any time, throughout the lifespan, and, depending on the specific type, can be treated with many known types of cancer therapies, including surgery, radiation, chemotherapy, targeted therapy, and immunotherapy. Care teams thus require multidisciplinary pediatric and adult expertise.