Background: Soft-tissue sarcomas (STSs) are rare and heterogeneous malignancies with generally poor and unpredictable prognosis. Tertiary lymphoid structures (TLSs) have been identified as favorable prognostic indicators in several cancer types, yet their role in STS remains poorly defined. This study investigates the prognostic relevance of TLS presence, maturity, location and density in resected STSs. Methods: We retrospectively analyzed 219 cases of primary STS surgically resected at the Bergonié Institute (France) between 1990 and 2020. TLSs were assessed for presence, spatial distribution, semi-quantitative density and degree of maturity using CD20 and CD23 immunohistochemistry, categorizing tumors as fully mature TLS-positive (fmTLS+) or -negative (fmTLS-). RNA sequencing was performed on 126 formalin-fixed paraffin-embedded samples to characterize immune microenvironment profiles. Survival outcomes-including overall survival (OS), time to locoregional progression (TTLRP), and time to distant progression (TTDP)-were analyzed using Kaplan-Meier estimates and Cox proportional hazards models. Results: The presence of fmTLS was significantly associated with improved 5-year OS (p = 0.012) and cause-specific survival (p = 0.006). Unexpectedly, fmTLS+ tumors showed a higher rate of local recurrence (22.9% vs. 8.1%, p = 0.002). On multivariate analysis, high-density fmTLS+ tumors conferred a 2.68-fold increased risk of locoregional progression (95% CI: 1.28-5.59, p = 0.009). Transcriptomic profiling confirmed a significant correlation between fmTLS+ status and a high-immune phenotype (Φ = 0.30, p < 0.001). Conclusions: STSs with fmTLS are associated with improved OS but increased risk of local recurrence. These findings support fmTLS as a dual prognostic biomarker and highlight the need for tailored surveillance and adjuvant strategies in fmTLS+ patients.

Purpose: Undifferentiated pleomorphic sarcoma (UPS) is an aggressive subtype of soft tissue sarcoma with poor outcomes, particularly in metastatic cases. The mechanisms driving metastasis in UPS remain poorly understood, limiting therapeutic advances.Experimental Design: A multi-omics approach was used to analyze paired primary and metastatic UPS tumor samples. Spatial transcriptomics, bulk RNA sequencing, and deconvolution analyses were performed to identify molecular pathways and immune microenvironment alterations associated with metastasis. Functional assays using CRISPR-Cas9 knockout (KO) UPS cell lines, alongside in vivo models, were used for functional validation experiments.Results: Transcriptomic analyses on 13 patients with UPS revealed significant upregulation of hypoxia, epithelial-mesenchymal transition, and immune-suppressive pathways in metastatic UPS. ADORA2B was identified as a key driver of these processes, with elevated expression correlating with poor disease-free survival in patients with UPS. Functional studies confirmed that ADORA2B promotes proliferation, migration, invasion, and matrix remodeling via metalloprotease regulation. In vivo, ADORA2B KO reduced primary tumor growth and metastatic dissemination in UPS models.Conclusions: This study identifies ADORA2B as a critical regulator of metastatic progression in UPS, implicating it as a promising therapeutic target. Ongoing clinical trials targeting adenosine pathways further support the translational potential of ADORA2B inhibition to disrupt metastasis and improve outcomes for patients with UPS.

In this review, we discuss classic and recently described fibroblastic tumors, emphasizing practical aspects of their diagnostic approach in routine practice. The fibroblastic lineage is a fuzzy term that refers to a bland cytomorphology of small cells with small elongated, monotonous nuclei and inconspicuous cytoplasm, arranged in short fascicles frequently associated with a collagenous stroma. These morphological features are hardly specific; for many of these entities, there is limited evidence to definitively assert a fibroblastic lineage. To illustrate the complexity of assigning a definitive fibroblastic lineage to a neoplasm, recent studies have speculated on the fibroblastic lineage of atypical fibroxanthomas based on their proteomic profiles and expression analysis. The list of fibroblastic tumors keeps evolving. We will mention the various clinical, morphological, phenotypic, and molecular aspects of fibroblastic tumors that occur exclusively in children—lipofibromatosis and calcifying aponeurotic fibroma—as well as fibroblastic tumors found at any age or sometimes exclusively in adults: superficial CD34-positive pleomorphic fibroblastic tumors and PRDM10-rearranged tumors; EWSR1::SMAD3-rearranged fibroblastic tumors; nodular necrotizing fibroblastic sarcoma associated with MAML2 fusions; sclerosing epithelioid fibrosarcoma; KMT2A-rearranged tumor; MUC4-positive fibroblastoma; PRRX1-rearranged fibroblastic tumors and acral fibrochondromyxoid tumors.

The advent of widespread genomic testing of uterine mesenchymal tumors has led to novel insights into the biology of these diverse tumors, and many genomically defined entities have been described in recent years. During a larger study of endometrial stromal sarcomas and unclassified uterine sarcomas, we identified 3 tumors harboring KDM2B gene fusions. Patients were 32, 61, and 67 years old, and all initially underwent incomplete sampling via laparoscopic myomectomy (n = 1), laparoscopic biopsy (n = 1), or hysteroscopic myomectomy (n = 1). One patient’s tumor was densely adherent to the pelvic sidewall; she was treated with chemotherapy and died of widely metastatic disease at 29 weeks. Another underwent a subsequent recent hysterectomy with the tumor confined to the uterus and minimal follow-up to date. The final patient refused further treatment and was alive at 28 weeks, although the status of the disease progression was unknown. On microscopic examination, 2 tumors showed infiltrative borders, whereas interface with the myometrium was not present in the third. The tumors were variably cellular with alternating hypercellular and hypocellular zones in a myxoid to loosely collagenous stroma. The hypercellular areas contained round to ovoid cells in diffuse (n = 3) and sex cord-like arrangements, including cords (n = 3), nests (n = 2), and tubules (n = 1); 2 also contained occasional spindled cells arranged in vague fascicles. These cells showed moderate atypia with open chromatin, numerous mitoses (8, 24, and 25 per 10 high-power fields), and frequent apoptosis. The hypocellular areas contained sparse, ovoid-to-spindled cells with minimal atypia. All tumors were diffusely positive for cyclin D1, whereas BCL6 corepressor was diffusely positive in 1 and negative in 2; desmin and caldesmon were negative in all 3 neoplasms. All harbored KDM2B gene fusions; partner genes included EPC1, EP400, and CITED1. MDM2 amplification was also noted in 2. Clustering analysis based on RNA expression profiling revealed tight clustering of all 3 tumors within the broad group of high-grade endometrial stromal sarcomas. Based on the overall clinicopathologic and genomic features, we suggest that these tumors may represent a novel subtype of uterine sarcoma and may be best classified as high-grade endometrial stromal sarcoma, although additional confirmatory studies are needed.

Pseudoendocrine sarcoma (PS) is a recently described neoplasm of uncertain differentiation, characterized by recurrent CTNNB1 mutations, frequent paravertebral location, and a neuroendocrine-like histomorphology. In this study, we report the clinicopathologic, immunohistochemical, transcriptomic, and epigenetic findings of 12 PS cases. The tumors affected 7 men and 5 women with a median age of 66 years and were located in the paraspinal/paravertebral region (n = 11) and the thigh (n = 1). Median tumor size was 82 mm (range, 32-170 mm). Histologically, the tumors comprised sheets and nests of epithelioid-to-ovoid cells with uniform nuclei and speckled chromatin, frequently associated with extracellular hyaline globules and fibrovascular cords/septa. Uncommon findings included microcalcifications, myxoid stroma, pseudopapillary, pseudoglandular, microcystic or corded architecture, and lumen and rosette-like structures. Necrosis was absent, and mitotic activity was low. On immunohistochemistry, the tumors showed aberrant nuclear staining for beta-catenin (8/8) and expression of CD56 (7/7), S100 (8/8), desmin (2/6), and androgen receptor (1/4). Pankeratin (AE1/AE3), progesterone receptor, synaptophysin, chromogranin, and INSM1 were negative. All tested cases harbored CTNNB1 mutations. Using a customized cohort, methylation profiling revealed that PS formed a common cluster with solid pseudopapillary neoplasm of the pancreas (SPNP), distinct from all methylation classes from the Heidelberg sarcoma classifier and a subset of paragangliomas. Transcriptomic analysis showed that PS formed an independent cluster from a control group of tumors (including SPNP). Differential gene expression analysis showed enrichment in genes of the Wnt signaling pathway (HALLMARK gene sets) and biological processes related to sensory perception, among others (gene ontology - biological process [GO-BP]). Additionally, upregulated genes were related to various fetal cell types from the cell type signature data set (MSigDB), particularly of neuronal and epithelial lineage. Immunohistochemical assessment of potential markers identified through gene expression analysis revealed focal-to-diffuse expression of GLUT1 (6/6) and focal/multifocal expression of Brachyury (4/9) and HuD (3/7). Follow-up information, available for 10 cases (median duration of 18 months; range, 7-69 months), showed local recurrences and metastatic spread in 2 patients each. Evidence of response to radiotherapy was documented in one tumor. Altogether, this study expands knowledge on PS and suggests biological links with SPNP, including a potential shared cell of origin.

This review focuses on uterine mesenchymal tumors that are defined on a molecular level by a single and unique genetic alteration, that is somehow necessary and sufficient to allow tumor growth and progression. Although diverse from a clinical, morphological and immunohistochemical point of view, the different entities we are going to talk about share both a simple genomic profile with a low number of chromosomal alterations observed by CGH Array (few deletions, gains or amplifications...) and a low mutational burden observed by sequencing technics. Some of these entities are already well known and described in the literature when found outside of the uterus and gynecological tract. It remains intriguing that uterine mesenchymal pathology has been lagging behind when compared to its extrauterine counterpart. How can we explain that when it comes to inflammatory myofibroblastic tumors, abundant numbers of articles have been published since the 70's, but it was only in the early 2000s that the first relevant descriptions of this tumor in the uterus emerged? Certainly, the increased accuracy, availability, and use of molecular biology technics and in particular RNA sequencing in the area of uterine pathology can partly explain the reduction of the gap between soft tissue and uterine pathology we currently observe. Other reasons explaining this gap may be the high prevalence of smooth muscle tumors in the uterus and the abounding diversity of their morphological aspects, which may have partly eclipsed the array of differential diagnoses. Last but not least, one can hypothesize that the relative "simplicity"of hysterectomy procedures, referring to their safety and accessibility, has cured most of the lesions and partly clouded our knowledge regarding the biological potential and natural history of these newly described entities. As a consequence of this situation, our reader will often encounter the wording "uncertain malignant potential", as for some of these rare entities, evidence to establish reliable prognostic variables is still insufficient. We hope this review to be a useful tool to guide pathologists through the diversity and complexity of uterine mesenchymal tumors. As a scientific and medical community, sharing this knowledge will help us to collectively raise our vigilance and awareness by expanding the array of our differential diagnoses. We hope this will lead to more cases being accurately diagnosed, and ultimately, to a deeper knowledge regarding the biological potential and clinical evolution of these tumors. From a therapeutical point of view, the consequences of an accurate diagnosis for the patient are already appreciable through the use of targeted therapy. Examples include: ALK inhibitors in inflammatory myofibroblastic tumor, tyrosine-kinase inhibitors in COL1A::PDGFB rearranged sarcomas or mTOR inhibitors in PEComa. (c) 2024 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Metropolitan areas are known to be anthropogenic “hot spots” of Greenhouse Gas (GHG) fluxes. To track the effectiveness of climate mitigation policies and emission reduction objectives, large metropolitan areas like Munich and Paris regions are currently being instrumented with dense atmospheric GHG networks, further assimilated in inversion systems with high-resolution inventories, also complementing the data collected by remote sensing instruments on the ground and in space. To study medium-sized cities, where a large fraction of the global population lives, spaceborne measurements often fail to quantify fossil fuel emissions since the atmospheric signatures are below the detection threshold of current instruments. For the past two years (2022 and 2023), two large-scale campaigns of the MAGIC initiative led by CNRS and CNES (https://magic.aeris-data.fr) have been taking place in Reims, France, a city with a population of 300,000 inhabitants (207 hab./km2) located to the East of Paris (approx. 100 km away). During these two intensive measurement campaigns, a wide range of ground-based instruments have been deployed around the city to measure CO2 concentrations, in addition to instrumented balloons and aircraft. The goal of these campaigns was to evaluate CO2 emissions from the area and to assess the detection capabilities of current satellite instruments. In our study, we simulated the atmospheric CO2 mixing ratios using the Weather Research Forecast model coupled to a chemistry transport model (WRF-Chem) at 4 horizontal resolutions (9 km, 3 km, 1 km, and 333 m). Typically, mesoscale models are used for resolutions coarser than 1 km while microscale Large-Eddy Simulation models (LES) are used for resolutions finer than 100m. In between, i.e. the grey-zone, turbulent motions are not resolved explicitly but high resolutions might offer a better representation of fine plume structures. Here, we present the results of a multi-scale multi-instrument comparison between the model and the observations to characterize the model performances and the ability of the model to reproduce the observed variations in concentrations. We found that the detectability of the various CO2 plumes remains challenging. First, the strength of the anthropogenic signals from the city remains low compared to gradients from nearby sources, whether industrial or metropolitan, hence making the city plume hard to study. We also showed that improvements in the modelling of CO2 plumes were not significant between the 1 and 0.3 km horizontal resolution scales, thus suggesting that LES models might be better suited for such studies.

Selective KRAS(G12C) inhibitors have been developed to covalently lock the oncogene in the inactive GDP-bound state. Two of these molecules, sotorasib and adagrasib, are approved for the treatment of adult patients with KRAS(G12C)-mutated previously treated advanced non-small cell lung cancer. Drug treatment imposes selective pressures leading to the outgrowth of drug-resistant variants. Mass sequencing from patients' biopsies identified a number of acquired KRAS mutations -both in cis and in trans- in resistant tumors. We demonstrate here that disease progression in vivo can also occur due to adaptive mechanisms and increased KRAS-GTP loading. Using the preclinical tool tri-complex KRAS(G12C)-selective covalent inhibitor, RMC-4998 (also known as RM-029), that targets the active GTP-bound (ON) state of the oncogene, we provide a proof-of-concept that the clinical stage KRAS(G12C)(ON) inhibitor RMC-6291 alone or in combination with KRAS(G12C)(OFF) drugs can be an alternative potential therapeutic strategy to circumvent resistance due to increased KRAS-GTP loading.

Undifferentiated pleomorphic sarcoma (UPS) is the most frequent and the most aggressive sarcoma subtype for which therapeutic options are limited. The identification of new therapeutic strategies is therefore an important medical need. Epigenetic modifiers has been extensively investigated in recent years leading to the development of novel therapeutic agents. Dual BET/EP300 inhibitors have shown synergistic antitumor activity and have recently entered clinical development. To date, no data related to potential of BET/EP300 inhibition as a treatment in UPS have been reported. To investigate the therapeutic potential of BET/EP300 inhibition, we evaluated the antitumor activity of three compounds in vitro via MTT, apoptosis and cell cycle assays. The most potent inhibitor was evaluated in vivo in two animal models and the mechanisms of action were investigated by RNA sequencing, Western blotting and immunofluorescence staining. A CRISPR knockout screen was performed to identify resistance mechanisms. Among the three compounds tested, the dual inhibitor NEO2734 was the most potent, decreased the viability of UPS cells in vitro through a regulation of E2F targets and cell cycle and decreased the tumor growth in vivo. Moreover, we identified GPX4 as a gene involved in resistance and showed synergy between BET inhibition and ferroptosis induction.The present study demonstrated that dual BET/EP300 inhibitors have a relevant antitumor activity in a subgroup of UPS characterized by expression of MYC-targets pathway and identified a potent combination therapeutic strategy that deserves further investigation in the clinical setting.

Our objective was to capture subgroups of soft-tissue sarcoma (STS) using handcraft and deep radiomics approaches to understand their relationship with histopathology, gene-expression profiles, and metastatic relapse-free survival (MFS). We included all consecutive adults with newly diagnosed locally advanced STS (N = 225, 120 men, median age: 62 years) managed at our sarcoma reference center between 2008 and 2020, with contrast-enhanced baseline MRI. After MRI postprocessing, segmentation, and reproducibility assessment, 175 handcrafted radiomics features (h-RFs) were calculated. Convolutional autoencoder neural network (CAE) and half-supervised CAE (HSCAE) were trained in repeated cross-validation on representative contrast-enhanced slices to extract 1024 deep radiomics features (d-RFs). Gene-expression levels were calculated following RNA sequencing (RNAseq) of 110 untreated samples from the same cohort. Unsupervised classifications based on h-RFs, CAE, HSCAE, and RNAseq were built. The h-RFs, CAE, and HSCAE grouping were not associated with the transcriptomics groups but with prognostic radiological features known to correlate with lower survivals and higher grade and SARCULATOR groups (a validated prognostic clinical-histological nomogram). HSCAE and h-RF groups were also associated with MFS in multivariable Cox regressions. Combining HSCAE and transcriptomics groups significantly improved the prognostic performances compared to each group alone, according to the concordance index. The combined radiomic-transcriptomic group with worse MFS was characterized by the up-regulation of 707 genes and 292 genesets related to inflammation, hypoxia, apoptosis, and cell differentiation. Overall, subgroups of STS identified on pre-treatment MRI using handcrafted and deep radiomics were associated with meaningful clinical, histological, and radiological characteristics, and could strengthen the prognostic value of transcriptomics signatures.

Cette revue se concentre sur les entités mésenchymateuses utérines présentant sur le plan moléculaire une altération unique, nécessaire et suffisante à la tumorigénèse et à la progression tumorale. L’ensemble de ces entités ont en commun ce que l’on nomme un profil génomique simple, terme recoupant l’absence de remaniements chromosomiques significatifs objectivables en CGH Array (délétions, gains, amplifications….) et un faible nombre de mutations (« low mutational burden »).Certaines de ces entités diagnostiques sont connues et bien décrites depuis longtemps en dehors de la sphère gynécologique. On peut s’interroger sur les raisons pour lesquelles la pathologie utérine mésenchymateuse accuse un certain retard par rapport à la pathologie des tissus mous extra gynécologique. Comment expliquer que les tumeurs myofibroblastiques inflammatoires font l’objet de riches descriptions cliniques et morphologiques depuis les années 70 tandis que les premières séries utérines ad hoc n’apparaissent qu’au début des années 2000 ?Les progrès exponentiels et la démocratisation des techniques de biologie moléculaire et de séquençage ARN expliquent en partie ce différentiel dans la mise au jour d’entités diagnostiques nouvelles. Il semble également que la prépondérance effective des lésions musculaires lisses et leurs grandes richesses morphologiques ait en partie occulté l’éventail des diagnostics différentiels envisagés en pathologie utérine. On peut par ailleurs supposer que la facilité à la fois en termes de geste technique, d’accessibilité et de prise de décision thérapeutique à la procédure d’hystérectomie a en partie tronqué nos connaissances sur le potentiel évolutif et l’histoire naturelle de ces lésions, guéries par le geste chirurgical.Ainsi en parcourant cette revue, le lecteur pourra constater que pour nombre d’entités la question de la terminologie « bénin vs malin » n’est pas tranchée et que les outils morphologiques et/ou moléculaires permettant une stratification pronostique fiable sont en cours d’évaluation et amenés à évoluer. Le manque de recul clinique pour beaucoup de ces entités récentes appelle à la prudence, passant par l’utilisation de la terminologie « potentiel de malignité indéterminé ».Ce manque de recul clinique illustre le retard à combler en pathologie mésenchymateuse utérine, retard qui ne pourra être rattrapé qu’en généralisant auprès des pathologistes la richesse du spectre diagnostique des lésions utérines et en déconstruisant l’idée d’une hégémonie des lésions musculaires lisses.Nous espérons que cette revue ne soit pas qu’un simple inventaire des nouvelles entités diagnostiques et moléculaires, mais permette également d’accroitre la vigilance de tout pathologiste amené à prendre en charge une lésion utérine et à lui donner les outils nécessaires pour élargir son spectre de diagnostics différentiels. C’est en améliorant en tant que communautés scientifiques nos connaissances que nous pourrons accumuler les données cliniques nécessaires à une meilleure caractérisation de ces lésions et donc à une prise en charge adéquate de ces patientes.L’enjeu thérapeutique sur le court terme est déjà appréciable à travers l’utilisation de thérapies ciblées. Par exemple les inhibiteurs de ALK dans les tumeurs myofibroblastiques inflammatoires, les inhibiteurs de tyrosine kinase dans les sarcomes COL1A::PDGFB réarrangés, les inhibiteurs de la voie mTOR ou MET dans les PEComes…Face au bouleversement des paradigmes diagnostiques apporté par la biologie moléculaire et particulièrement par le RNAseq, l’étape suivante sera de reclasser les nouvelles entités selon l’évolution clinique, les données intégrées morphologiques, immunohistochimiques et moléculaires. Une avancée majeure consistera dans l’identification de biomarqueurs immunohistochimiques qui permettront une approche diagnostique plus immédiate, sans les temps d’attente des techniques moléculaires, permettant une diffusion ubiquitaire dans les laboratoires de pathologie.

Soft-tissue sarcoma (STS) are a heterogeneous group of rare tumors with different biological behavior that are fatal in more than 40% of cases, due to their metastatic evolution and inadequate treatment options. ATR inhibition already showed an activity, even if modest, in broad pre-clinical models of STS. By using genome-wide CRISPR/Cas9 library screening, we identified ATM signaling network genes as critical drivers for resistance to the specific ATR inhibitor AZD6738. The role of such genes in resistance to AZD6738 was confirmed by using CRISPR/Cas9 knockout models. More strikingly, the ATM inhibitor AZD0156 works synergistically with AZD6738 in vitro and abolishes STS growth in vivo in our models of most frequent histotypes (such as dedifferentiated liposarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma among others). Moreover, the combination of AZD6738 and AZD0156 induced significantly higher levels of DNA damage than either drug used as single agent alone. In summary, our results demonstrate that targeting ATM is an effective approach to overcome resistance to ATR inhibition in different STS subtypes, including the most frequent histologies.

Prognostic impact of PRMT5 gene expression in STSs. A, GEPIA dot plot showing PRMT5 expression in sarcomas and normal samples, with each dot representing a distinct tumor (n = 262) or normal sample (n = 2). B, Kaplan‒Meier curves of overall survival according to PRMT5 gene expression in 262 STSs from TCGA consortium (http://gepia.cancer-pku.cn/index.html). C, Kaplan‒Meier curves of metastasis-free survival in 255 patients with translocation-related sarcomas according to PRMT5 expression (green line: low expression; red line: high expression: n = 135). D, Kaplan‒Meier curves of metastasis-free survival in 389 patients with complex genomic sarcomas according to PRMT5 expression (green line: low expression, n = 159; red line: high expression: n = 180).

Antitumor effects of PRMT5 inhibition on STSs. A, Assessment of cell viability with PRMT5 inhibitor GSK3226595 (GSK595) in seven STS cell lines (with TP53 deleted or not) as determined by MTT assay (n = 3 or more). Cells were treated with a range of increasing concentrations of drug for 10 days and the IC50 was calculated with GraphPad Prism software. B, Western blot analysis showing of SDMA, p53, and p21 protein expression in four STS cell lines after 10 days of GSK595 treatment at the respective IC50. C, Quantitation of p53 and p21 bands in B using ImageJ software. GAPDH served as the loading control (n = 3). D, Proliferation assay after PRMT5 inhibition: Cells were untreated or treated with GSK595 at the IC50 and counted after 3, 6, and 10 days by flow cytometry. Fresh medium with drug treatment at the appropriate concentration was used to replace the medium every 3 days (n = 3; **, P < 0.01; ***, P < 0.001 two-way ANOVA and Bonferroni multiple comparisons test). E, Clonogenic assay: Representative images of stained cells (crystal violet) after 10 days of GSK595 treatment at the IC80 (n = 2). F, Efficacy of the PRMT5 inhibitor in vivo in IB115 xenograft. IB115 cells were used to establish xenograft tumors in NSG mice (n = 9 mice per group), and the tumors were treated with vehicle or GSK595 at 100 mg/kg orally twice daily (5 days-on/2 days-off; ***, P < 0.001 two-way ANOVA and Bonferroni multiple comparisons tests, arrow = start of treatment). Tumor growth, body weight, and final tumor weight were measured. G, Efficacy of the PRMT5 inhibitor in vivo in IB111 xenograft mice. IB111 cells were used to establish xenograft tumors in NSG mice, which were treated with the same concentrations as the IB115 cells. Tumor growth and body weight were measured, and the tumors were monitored until the end of the treatment period or until they reached 2,000 mm3 (day of sacrifice); the data were used to draw the survival curve.

PRMT5 inhibition impairs glycolysis in STSs. A, Western blot analysis showing key proteins involved in glycolysis. Sarcoma cell lines were treated for 10 days without or with GSK595 at the IC80 before protein extraction. B, Quantitation of Western blot bands (n = 2 or more). C and D, Relative glucose uptake and lactate production were measured by colorimetric assay after 10 days of treatment with GSK595 at the IC80. All values were normalized to the total amount of protein (n = 3 or more; *, P < 0.05; **, P < 0.01; ***, P < 0.001, multiple t test using the Holm‒Sidak method).