Dedifferentiated liposarcoma (DDLPS) is an aggressive mesenchymal malignancy coexisting with a low grade well-differentiated component. Pathways implicated in liposarcoma growth and dedifferentiation are promoted by heparan sulfate (HS) proteoglycans (HSPG) and their modifying enzymes including heparanase. HSPGs serve as co-receptors enhancing tyrosine kinase signaling and tumor aggressiveness. Targeting these interactions bears promise in attenuating liposarcoma growth. We employed an investigational HS mimetic, the non-anticoagulant heparin CX-01 (dociparstat), to assess its HS competition impact on deregulated adipogenic differentiation and growth of human DDLPS cell lines and patient-derived xenografts (PDXs). Remarkably, CX-01 reduced colony formation and invasive capacities of DDLPS cell lines, inducing cytoskeleton remodeling, lipid accumulation and reactivation of adipogenic program. Mechanistic studies into the anti-DDLPS activity of CX-01 unveiled Syndecan 1 (SDC1)/heparanase system and receptor tyrosine kinase-AKT signaling as targets of cell growth inhibition and induction of differentiation. CX-01 treatment of mice harboring DDLPS PDXs attenuated tumor growth, enhanced lipid content and consistently altered the transcriptome, modulating pathways associated with tumor dedifferentiation (adipogenesis and fatty acid metabolism) and tumor-microenvironment interaction (TGFβ signaling, inflammatory response). In two independent cohorts of DDLPS patients, genes downregulated in CX-01-treated PDXs (SDC1, TIMP1, FN1, COL5A1, and MMP14), were found preeminently expressed in the dedifferentiated, compared to the well-differentiated tumor component and normal fat. This suggests a role for these genes in disease progression. Collectively, this study demonstrates the remarkable potential of HS competition to simultaneously block multiple anti-adipogenic players representing metabolic vulnerabilities, and to promote a differentiated tumor phenotype markedly less aggressive.

Background. Epithelioid sarcoma (EpS) is an ultra-rare, aggressive soft tissue sarcoma (STS) driven by loss of the tumor suppressor INI1, encoded by SMARCB1 , which triggers hyperactivation of the chromatin-modifying enzyme EZH2. However, objective responses to the EZH2 inhibitor (EZH2i) tazemetostat (TZM) remain limited to approximately 15% of patients. Combination strategies incorporating EZH2i with doxorubicin (DOX), the first-line standard for advanced STSs, may enhance the activity of either agent alone, warranting investigation of determinants of response and resistance. Methods. Two INI1-deficient in-house generated proximal-type EpS (P-EpS) patient-derived xenograft (PDX) models, EpS-1 and EpS-2, and their matched cell lines were established. EpS-1 and EpS-2 models harbored a homozygous SMARCB1 deletion and underwent comprehensive genomic, epigenomic, transcriptomic, protein-level, and histological characterization. Both models were used to evaluate TZM and DOX, alone and in combination, and to investigate determinants of response and resistance. Results. TZM + DOX greatly inhibited tumor growth in EpS-1 in vivo, though regrowth occurred approximately three weeks after treatment cessation. EpS-2 displayed intrinsic resistance to all treatments. RNA sequencing of post-treatment PDXs revealed divergent transcriptional responses: in EpS-1 but not in EpS-2 TZM + DOX downregulated gene sets linked to chromatin organization and histone deacetylases (HDACs). Consistently, H3K27 acetylation (H3K27ac) accumulated exclusively in EpS-1, suggesting HDAC activity limits EZH2i-induced chromatin remodeling in resistant EpS. In EpS-2, co-treatment with TZM, DOX, and the pan-HDAC inhibitor vorinostat (SAHA) restored H3K27ac, induced apoptosis, and showed tendency toward synergism. Conclusions. This study identifies distinct resistance profiles to EZH2i-based therapy in P-EpS PDXs. HDAC activity limits chromatin remodeling induced by EZH2i (± DOX), and its inhibition with SAHA restores sensitivity, supporting HDAC inhibition as a rational strategy to overcome EZH2i resistance.

Background Metastatic recurrence represents the major clinical challenge in early-stage lung cancer after curative surgery. Here, we investigated the role of circulating extracellular vesicles and particles (EVPs) in promoting formation of pre-metastatic niches (PMNs).Methods Plasma-derived EVPs were obtained by ultracentrifugation from pre-surgery blood samples of patients with poor prognosis. Heavy-smokers cancer free individuals were used as control. EVP were characterized following MISEV guidelines. Functional experiments were carried out in vitro in 2D and 3D-bioprinted models as well as in vivo.Results EVPs from patients with early relapse show distinct molecular profiles, characterized by elevated levels of miR-29a and complement protein C4a. These EVPs preferentially target endothelial cells inducing a pro-inflammatory condition with upregulation of VCAM1 and CXCL1. In turn, endothelial modulation stimulated fibroblast activation and promoted neutrophils recruitment supporting PMNs formation. Mechanistically, we demonstrate that miR-29a and C4A act synergistically through SPARC down-modulation promoting cancer cell colonization. Preconditioning of mouse lungs using EVPs from patients with poor prognosis increased metastatic growth of human tumor cells, which was inhibited by miR-29a blockade.Conclusions Circulating EVPs could be novel prognostic biomarkers and key players in PMN formation offering new targets to reduce relapses in lung cancer.

PURPOSE:The Complexity INdex in SARComas (CINSARC) predicts the metastatic risk in patients with soft-tissue sarcoma. The aims of this study were to provide the first independent validation of CINSARC in patients with retroperitoneal sarcoma (RPS) and evaluate whether CINSARC could enhance the performance of Sarculator. EXPERIMENTAL DESIGN:A retrospective cohort included patients with primary localized RPS resected with curative intent (2011-2015) at a single institution. The STRASS cohort comprised patients from the surgery-only arm of the EORTC-STBSG-62092 (STRASS) trial who had undergone CINSARC categorization. Patients were classified as CINSARC low-risk (C1) versus high-risk (C2). Primary study endpoints were overall survival (OS) and disease-free survival (DFS). Sarculator performance was assessed in terms of discrimination (the Harrell C-index) and calibration (calibration plots and the Brier score) before and after adding CINSARC. RESULTS:The study cohorts included 104 and 69 patients, respectively, with similar OS. In a pooled cohort, in multivariable analysis for OS considering Sarculator and CINSARC, only Sarculator was significantly associated with OS [HR, 1.93; 95% confidence interval (CI), 1.35-2.74; P < 0.001]. In multivariable analysis for DFS, both Sarculator (HR, 1.51; 95% CI, 1.09-2.09; P = 0.013) and CINSARC (HR, 2.01; 95% CI, 1.26-3.23; P = 0.004) were significantly associated with DFS. However, the addition of CINSARC did not improve Sarculator's discrimination or calibration for either OS or DFS. CONCLUSIONS:This study validates CINSARC as a prognostic predictor for OS and DFS in patients with primary RPS. CINSARC did not improve the performance of Sarculator, suggesting that its addition to the Sarculator may not provide added clinical benefit.

Supplementary Table 2: univariate and multivariable analyses for OS in specific subgroups

Introduction: Hormone receptor-positive, HER2-negative metastatic breast cancer (HR+/HER2- mBC) is an almost invariably incurable disease. Endocrine therapy plus CDK4/6 inhibitors (ET+CDK4/6i) represents the standard-of-care for 1st line treatment. However, primary or acquired resistance leads to tumor progression in most patients, and there is therefore an unmet need to unravel mechanisms of resistance and actionable targets in patients progressing upon CDK4/6i. To date, genomic studies pinpointed several putative resistance biomarkers. including alterations in ESR1, PIK3CA, FGFR1, TP53, RB1, KRAS, HRAS, NRAS, AURKA, AKT1, FGFR2, and CCNE2 genes. However, genomics can only partially explain resistance to CDK4/6i in HR+/HER2- mBC patients, while a combined analysis of genomic, transcriptomic and epigenetic profiles could potentially provide a more accurate characterization of primary and acquired resistance mechanisms. In this scenario, in June 2022 we launched the UNDERSTAND trial, an observational, open-label study aimed at identifying markers of resistance and tracing cancer cell evolution by sequential multi-omic analysis, in prospectively collected tissue and plasma samples from 100 HR+/HER2- mBC patients undergoing 1st line ET+CDK4/6i. Methods: The trial plans to collect tumor tissue at baseline (T0, prior to ET+CDK4/6i) and at disease progression (T1) in patients with accessible metastatic sites (cohort A) to longitudinally obtain genomic (FFPE), methylomic (fresh/frozen), bulk transcriptomic (fresh/frozen), single cell RNAseq (fresh tissue) and spatial transcriptomic (FFPE) data. If tumor biopsy would not feasible (bone-only disease, refusal of the biopsy procedure, lack of accessible tumor sites), blood samples will be prospectively collected at baseline, after 15 days of treatment, 3-months, 1-year or at tumor progression, for circulating methylome and ctDNA analysis (cohort B). Blood samples will be collected in cohort A patients as well. A dedicated electronic clinical record was set up with REDCap to collect demographic, clinical and sample information. Results: Between January 2022 and July 2024, we enrolled 56 patients with HR+/HER2- advanced breast cancer. Multiple plasma samples were longitudinally collected (cohort A + B). We were able to collect baseline tumor tissue in 20 patients, including 3 relapses (cohort A). T0 samples were genomically characterized with the Illumina TSO500 NGS panel. scRNAseq data were obtained from 14 samples (ChromiumX platform, 10X Genomics), including 7 nodal, 4 liver and 3 local relapses. After stringent quality controls, we identified and annotated 90.000 high quality (mean: 6400 cells per sample), including 43000 neoplastic cells, as well as T cells, B cells, plasma cells, mast cells, myeloid cells, endothelial cells, perycites, fibroblasts and liver cells. Intra- and inter-tumor heterogeneity was investigated, and the PAM50 classifier was used, highlighting a site-specific variability of molecular subtype proportion. Exploratory case-control analysis on patients with early (<6 months) and late (>12 months) relapses confirmed the role of PIK3CA and MAPK pathway activation in therapy resistance, identifying neoplastic cells as the primary contributors. Additionally, other pathways, such as WNT, NF-κB, and JAK/STAT, were implicated in resistance, suggesting areas for further investigation. For the upcoming SABCS, we plan to analyze and present the complete results of translational analyses on matched T0/T1 tissue samples from 30 patients, including genomic profiling, bulk and scRNA sequencing data. Conclusions: Our highly integrated infrastructure allows us to collect and integrate unprecedented longitudinal multiomic molecular data from HR+/HER2- mBC patients. A combination of genomic, transcriptomic, and epigenetic analyses will provide a more accurate characterization of primary and acquired resistance mechanisms to CDK4/6i. Citation Format: Andrea Vingiani, A. Belfiore, T. Torelli, D. Stetco, F. Ligorio, E. Minna, A. Bertolotti, S. Brich, A. Piccolo, E. Conca, E. Tamborini, F. Perrone, A. Busico, I. Capone, C. Sposetti, G. Scaperrotta, C. Depretto, R. Lanocita, D. Lorenzini L. Provenzano, G. Mazzoli, G. Fotia, I. Maugeri, C. Ferraris, S. Folli, G. Bianchi, F. Ravera, M. Dameri, L. Agnelli, L. Ferrando, G. Zoppoli, L. Magnani, C. Vernieri, G. Pruneri. The UNDERSTAND trial: a multi-omic platform for investigating CDK4/6 inhibitors resistance mechanisms in HR+ advanced breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS13-05.

Background Dedifferentiated liposarcoma (DDLPS) is an aggressive mesenchymal malignancy sharing driver oncogenes with the low-grade well differentiated liposarcoma. DDLPS, which includes a well differentiated component in most cases, has limited therapeutic options in advanced stages. Exploiting tumor cell plasticity to promote differentiation represents an attractive approach to counteract DDLPS aggressiveness. Heparan sulfate (HS) proteoglycans support pathways implicated in liposarcoma growth and dedifferentiation such as growth factor-mediated and epigenetic regulatory signaling. We used the HS mimetic non-anticoagulant heparin derivative CX-01 (dociparstat) to investigate the effect of HS competition on deregulated adipogenic differentiation and growth of DDLPS models. Methods The antitumor activity of CX-01 was assessed in-house generated DDLPS cell lines and patient-derived xenografts (PDXs). In vitro, reactivation of adipogenic program, cell phenotypic changes and accumulation of neutral lipids induced by CX-01 were determined through biochemical analyses, immunofluorescent microscopy and cytofluorimetric/colorimetric methods. The antiadipogenic and growth-promoting activities of pathways targeted by CX-01 were validated through pharmacological/molecular approaches. RNA-Seq transcriptomic analysis was performed on control and CX-01-treated PDX samples. Selected differentially expressed genes (DEGs) were further analyzed in two independent clinical DDLPS databases. Results CX-01 reduced the proliferative and invasive abilities of DDLPS cell lines inducing cytoskeleton remodeling, lipid accumulation and adipocytic markers. Mechanistic insights demonstrated that CX-01 targets, such as receptor tyrosine kinase-AKT pathways involving c-Met and FGFR, the HS proteoglycan syndecan 1 (SDC1) and heparanase, promoted DDLPS cell growth and impaired differentiation. CX-01 treatment of mice harboring DDLPS PDXs delayed tumor growth and modified the transcriptome affecting pathways associated with the tumor dedifferentiated phenotype (adipogenesis, fatty acid metabolism) and tumor-microenvironment interaction (TGFβ signaling, inflammatory response). SDC1 and other DEGs, including TIMP1, FN1, COL5A1, and MMP14, downmodulated after treatment with CX-01, were found preeminently expressed in the dedifferentiated compared to the well-differentiated tumor component in two independent cohorts of patients with primary DDLPS. Conclusions These findings demonstrated the potential of the approach based on HS competition to simultaneously inhibit multiple anti-adipogenic players, which singly represent actionable vulnerabilities, and modify the DDLPS transcriptomic profile promoting a more differentiated phenotype compatible with a less aggressive behavior.

Abstract Background: Preclinical studies showed that severely calorie restricted fasting-mimicking diets (FMDs) enhance the antitumor efficacy of chemotherapy (CT) or immunotherapy (IO) in murine triple negative breast cancer (TNBC) models. These effects are mediated by a combination of blood glucose reduction and positive immunomodulatory effects. Moreover, combining fasting and metformin produced synergistic anticancer effects in a broad range of tumor models. The BREAKFAST trial was designed to investigate if FMD, plus/minus metformin, could increase the antitumor activity of neoadjuvant CT in patients with stage I-III TNBC. Methods: BREAKFAST (NCT04248998) is a randomized, non-comparative, phase II, pilot trial that enrolled stage I-III (cT>1 cm) TNBC patients (pts) candidate to receive 4 cycles of neoadjuvant CT with doxorubicin-cyclophosphamide every 3 weeks, followed by 12 cycles of weekly paclitaxel. Pts were randomized 1:1 to receive: CT plus 5-day FMD every 3 weeks, up to 8 cycles (arm A); CT plus FMD plus daily metformin (1700 mg) (arm B). The primary study endpoint was the rate of pCR in either experimental arm. Secondary/exploratory endpoints included safety, compliance, and biomarker analyses based on blood metabolomics and tumor transcriptomics analyses at different timepoints. Results: Between June 2020 and February 2022 we enrolled 30 pts. Then, the study was interrupted after the introduction of chemo-immunotherapy (CT-IO) as a standard neoadjuvant therapy for early stage TNBC pts. Among 30 pts, 13 were treated with CT plus FMD, while 17 pts received CT plus FMD plus metformin. Overall, pCR rate was 56.6%, i.e., significantly higher than pCR rates reported with anthracycline-taxane CT alone in previous phase II/III trials (26-39%), with no significant differences among treatment arms (p=0.49). The FMD acutely reduced blood glucose, insulin and LDH levels, which reflects a reduction in systemic and/or tumor glucose metabolism. Of note, precocious LDH reduction was more pronounced in patients undergoing pCR. RNA-seq analysis of tumor samples revealed a significant downmodulation of glycolysis and TCA cycle pathways after one treatment cycle, paralleled by an increase of intratumor activated T cells, memory T cells and NK cells, as estimated by deconvolution analyses of tumor transcriptomic data. Of note, these changes were observed only in patients achieving pCR. While intratumor metabolic changes were similar in the two treatment arms, the modulation of intratumor immunity was more pronounced in patients not receiving metformin. Conclusion: Preoperative CT plus cyclic FMD (plus/minus metformin) results in excellent pCR rates in localized TNBC patients. Early on-treatment downregulation of systemic and intratumor metabolic parameters related to glucose metabolism predicts pCR, and this is independent of metformin use. Based on results of this study, we recently initiated a large, multicentric trial, namely the BREAKFAST-2 (NCT05763992) study, which will investigate if adding cyclic FMD to neoadjuvant CT-IO increases pCR rates in ~ 145 pts with stage II-III TNBC. Citation Format: Francesca Ligorio, Giovanni Fucà, Andrea Vingiani, Fabio Iannelli, Riccardo Lobefaro, Leonardo Provenzano, Lucrezia Zanenga, Cristina Ferraris, Antonino Belfiore, Silvia Brich, Alessia Bertolotti, Gianfranco Scaperrotta, Catherine Depretto, Antonia Martinetti, Elisa Sottotetti, Paola Antonia Corsetto, Giulia Valeria Bianchi, Giuseppe Capri, Secondo Folli, Saverio Minucci, Marco Foiani, Massimiliano Pagani, Giancarlo Pruneri, Filippo De Braud, Claudio Vernieri. Precocious modulation of metabolic and immunological parameters predicts tumor response to fasting-mimicking diet plus chemotherapy in patients with early stage TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-07.

Supplementary Figure 4. Transcriptomic profiles of EHE clinical tumor and PDX.

Supplementary Figure 6. Time-course analysis of siRNA-mediated down regulation of GDF-15 in EHE cell line.

Supplementary Figure 1. Representative growth curves of the EHE PDX at different passages in mice.

Abstract Background: The recommended cut-off of < 1% positive cells to define estrogen receptor (ER) negative status in breast cancer (BC) is highly debated. Few data exist on transcriptomic features of ER-low BC, suggesting no major differences vs ER-neg. Among HER2-negative BCs, we compared gene-expression profiling (including PAM50 intrinsic subtyping) according to ER status (ER-neg: 0% vs. ER-low: 1-9%) and assessed its impact on Tumor-infiltrating lymphocytes (TILs) levels. Methods: Across two cancer centers, stage I-III HER2-negative BCs FFPE samples were reviewed for ER status. All ER-low (ER 1-9%) samples were identified and a cohort of ER-neg BC samples, matched for age and stage, was also retrieved. A small cohort of pts with intermediate ER expression (ER-int: 10-50%) was included as a control cohort. Expression of 776 BC–related genes was evaluated by nCounter® (Breast Cancer 360TM Panel) and intrinsic molecular subtyping was determined using the PAM50 subtype predictor. A False Discovery Rate (FDR) corrected unpaired two-class SAM was used to identify genes differentially expressed in different subgroups. TILs were evaluated on archival H&E slides following guidelines. Results: Of 116 stage I-III HER2-neg BC pts included, 39 had ER-neg BC, 65 had ER-low BC, and 12 had ER-int disease. PAM50 intrinsic subtype distribution was similar in ER-neg and ER-low BCs, (with an enrichment in basal-like tumors), while both subgroups differed significantly from ER-int samples (Table). As compared to ER-neg tumors, ER-low BCs showed significantly higher expression of GATA3 gene and lower expression of EDN1 and PROM1 genes by SAM analysis (FDR< 5%); however, when the analysis was limited to PAM50 basal-like tumors (N=77) we only identified two genes that were downregulated in ER-low BCs (EDN1, PROM1) as compared to ER-neg BCs. On the contrary, significant differences in transcriptomic regulation were observed between ER-low and ER-int BCs, with ER-low BCs showing significantly higher expression of 53 genes and lower expression of 398 genes, as compared to ER-int (SAM analysis FDR< 5%). We previously reported similar TIL levels in ER-low and ER-neg tumors. In both ER-low and ER-int BCs, PAM50 basal-like subtype was associated with significantly higher TILs: median TIL levels 20 (range 0-80) and 6 (range 1-40) for PAM50 Basal-like BCs versus other PAM50 subtypes respectively in ER-low samples (p< 0.001); median TIL levels 53 (range 25-80) and 5 (range 0-10) for PAM50 Basal-like BCs versus other PAM50 subtypes respectively in ER-int samples (p=0.036). No significant difference in TIL levels according to PAM50 subtyping were observed in ER-neg tumors. No significant difference in mRNA levels for PD-1 (PDCD1) and PD-L1 (CD274) genes was observed between ER-low and ER-neg tumors. Conclusions: Among patients with HER2-neg BC, gene-expression profiling is similar in ER-neg (i.e., < 1%) vs. ER-low tumors with a high prevalence of PAM50 Basal-like tumors and few differentially expressed genes. In both ER-low and ER-int HER2-neg BCs, PAM50 Basal-like subtype is also associated with higher TIL levels. Our results strongly indicate that HER2-neg/ER-low BCs and HER2-neg/ER-neg BCs are similar biological entities, thus further supporting the use of similar treatments in patients with early-stage ER-low and triple-negative BC, including immune checkpoint inhibitors. PAM50 intrinsic subtype distribution according to ER levels Citation Format: Gaia Griguolo, Claudio Vernieri, Davide Massa, Lorenzo Nicolè, Claudia Pinato, Federica Miglietta, Andrea Vingiani, Silvia Brich, Riccardo Lobefaro, Francesca Schiavi, Matteo Fassan, Giancarlo Pruneri, Valentina Guarneri, Maria Vittoria Dieci. Transcriptomic profiling of ER-negative and ER-low, HER2-negative breast cancer and its implications on immune regulation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-03-09.

BACKGROUND:The cutoff of <1% positive cells to define estrogen receptor (ER) negativity by immunohistochemistry (IHC) in breast cancer (BC) is debated. We explored the tumor immune microenvironment and gene-expression profile of patients with early-stage HER2-negative ER-low (ER 1%-9%) BC, comparing them to ER-negative (ER <1%) and ER-intermediate (ER 10%-50%) tumors. METHODS:Among 921 patients with early-stage I-III, ER ≤50%, HER2-negative BCs, tumors were classified as ER-negative (n = 712), ER-low (n = 128), or ER-intermediate (n = 81). Tumor-infiltrating lymphocytes (TILs) were evaluated. CD8+, FOXP3+ cells, and PD-L1 status were assessed by IHC and quantified by digital pathology. We analyzed 776 BC-related genes in 116 samples. All tests were 2-sided at a <.05 significance level. RESULTS:ER-low and ER-negative tumors exhibited similar median TILs, statistically significantly higher than ER-intermediate tumors. CD8/FOXP3 ratio and PD-L1 positivity rates were comparable between ER-low and ER-negative groups. These groups showed similar enrichment in basal-like intrinsic subtypes and comparable expression of immune-related genes. ER-low and ER-intermediate tumors showed significant transcriptomic differences. High TILs (≥30%) were associated with improved relapse-free survival (RFS) in ER-low (5-year RFS 78.6% vs 66.2%, log-rank P = .033, hazard ratio [HR] 0.37 [95% CI = 0.15 to 0.96]) and ER-negative patients (5-year RFS 85.2% vs 69.8%, log-rank P < .001, HR 0.41 [95% CI = 0.27 to 0.60]). CONCLUSIONS:ER-low and ER-negative tumors are similar biological and molecular entities, supporting their comparable clinical outcomes and treatment responses, including to immunotherapy. Our findings contribute to the growing evidence calling for a reevaluation of ER-positive BC classification and management, aligning ER-low and ER-negative tumors more closely.

Introduction The synthesis of the periprosthetic capsule during implant-based breast reconstruction is the result of a coordinate cascade of inflammatory events ending in a fibrous tissue deposition around the expander or implant. Although the development of small volumes of fluid is one of the complications of prosthetic-based breast reconstruction, the characterization of the periprosthetic effusions coupled with the micro-textured devices, that have been recently introduced after the recall of macro-textured ones, is still lacking. The investigation of these periprosthetic effusions and paired capsules in terms of immunological content were the primary and secondary aims of the present study, respectively. Methods For this, 68 women, 41 of whom had periprosthetic effusions at the time of expander replacement with implant, were recruited. For each case, capsule and healthy dermal tissues were taken and for women with periprosthetic effusion, peripheral blood was also collected. Periprosthetic effusions and peripheral blood were characterized by cytometry while capsules and dermal tissues by immunohistochemistry and Nanostring analysis. Results The results showed an increase of Th1, Th2 lymphocytes and a HLA-DR+ bright CD16+ cells (likely representing monocytes-derived macrophages) in periprosthetic effusions in respect to peripheral blood. These pro-inflammatory cells were counterbalanced by the gain of suppressive CD4 Treg cells. In the corresponding capsules, immunohistochemistry revealed the absence of Th1 cells and the presence of tissutal FOXP3 Treg. No significant difference in expression of inflammatory-related genes between capsules and dermal tissues was present. Conclusions These results suggest the presence of a Treg-controlled inflammation in both periprosthetic effusions and capsules.

GDF-15 was released by tumor cells in patient-derived models of EHE. A, Assessment of released cytokines in the culture medium of EHE cells using the Human XL Cytokine Array. Detection of GDF-15 by ELISA in culture medium of EHE cell lines and cell lines of PLPS and DDLPS-1 and -2. B, Data were normalized as amount (pg) of released GDF-15 to total released (mg) proteins. RT-qPCR and ELISA results are reported as mean ± SD of three independent experiments. C, Detection of GDF-15 by ELISA in the plasma of healthy mice and mice carrying EHE PDX or PLPS PDX. D, siRNA-mediated downregulation of GDF-15 in the EHE cell line as detected at the mRNA level by RT-qPCR (left), protein level by Western blotting (middle), and as cytokine released in cell culture medium by ELISA 3 days after transfection (right). ELISA data were normalized as amount (pg) of released GDF-15 to total released (mg) proteins and reported as mean ± SD of three independent experiments. E, GSEA (RRID: SCR_003199) was employed on Hallmark (H) collection of the Molecular Signature Database, showing a limited number of modulated pathways following GDF-15 knockdown. DDLPS, dedifferentiated liposarcoma; PLPS, pleomorphic liposarcoma.