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supplementary materials & methods

Supplementary Figure S8 shows that EVT801 doesn’t modify blood pressure in rat while sorafenib increases it

Supplementary Table S2 shows EVT801 metabolite potency and selectivity profile

This file contains the additional information for Material and method, including method for cell transduction, cell treatment and WB, RT-qPCR analysis, viablity assays, proliferation assays, DEVD cleavage assay, flow cytometry, soft agar assay, organoid and soft agar assay quantification and ImageJ plugin, scriot for analaysis of unpaired heavy/light phosphopeptides, method for SILAC labelling, SILAC sample preparation, data-dependent acquisition LC-MS/MS, phosphoproteomic data analysis, mice housing, details on statistics, as well as Legends of supplementary figures and tables, table with sequences of shRNAs, used antibodies, RT-qPCR primers.

Supplementary Table 3 contains the location of isoform selective phosphorylations in the protein.

Supplementary Table 2 contains the list of isoform selective peptides in each condition, with quantification and global pathway analysis.

Supplementary Figures file contains merged Figure S1 to S8 including description of cell lines, Human samples, PI3K inhibitor stability, SILAC setup and controls, STRING analysis, quantifications of WB and cellular assays, prediction of kinase motifs found regulated, effects of combination of treatment in vitro, correlation between cell survival and phosphopeptide intensity, effects of combination treatment in Human samples and in vivo.

Supplementary Table 1 contains the list of files deposited on PRIDE Proteome X Change under the number PXD008410.

Supplementary Figure from Immune Infiltrate and Tumor Microenvironment Transcriptional Programs Stratify Pediatric Osteosarcoma into Prognostic Groups at Diagnosis

Figure S2. Expression of endogenous VEGF-D in mice tissues

Figure S5. VEGF-D 5'UTR mRNA exhibits two structural forms that allow the binding of different proteins

Supplementary Table from Immune Infiltrate and Tumor Microenvironment Transcriptional Programs Stratify Pediatric Osteosarcoma into Prognostic Groups at Diagnosis

The outcomes of adolescents/young adults with osteosarcoma have not improved in decades. The chaotic karyotype of this rare tumor has precluded the identification of prognostic biomarkers and patient stratification. We reasoned that transcriptomic studies should overcome this genetic complexity. RNA sequencing (RNA-seq) of 79 osteosarcoma diagnostic biopsies identified stable independent components that recapitulate the tumor and microenvironment cell composition. Unsupervised classification of the independent components stratified this cohort into favorable (G1) and unfavorable (G2) prognostic tumors in terms of overall survival. Multivariate survival analysis ranked this stratification as the most influential variable. Functional characterization associated G1 tumors with innate immunity and G2 tumors with angiogenic, osteoclastic, and adipogenic activities as well as PPARγ pathway upregulation. A focused gene signature that predicted G1/G2 tumors from RNA-seq data was developed and validated within an independent cohort of 82 osteosarcomas. This signature was further validated with a custom NanoString panel in 96 additional osteosarcomas. This study thus proposes new biomarkers to detect high-risk patients and new therapeutic options for osteosarcoma.Significance:These findings indicate that the osteosarcoma microenvironment composition is a major feature to identify hard-to-treat patient tumors at diagnosis and define the biological pathways and potential actionable targets associated with these tumors.

Figure S7. Tumor growth after NSAID treatment

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

Figure S8. Schematic representation of VEGF-D synthesis in tumor cells

Supplementary figure legend

Figure S6. Polysome profiling of 4T1 cells

Figure S4. Lymphangiogenesis and tumor gowth are not affected by 4T1 or 67NR lentiviral transduction

Figure S3. Podoplanin staining of lymphatic vessels

The outcomes of adolescents/young adults with osteosarcoma have not improved in decades. The chaotic karyotype of this rare tumor has precluded the identification of prognostic biomarkers and patient stratification. We reasoned that transcriptomic studies should overcome this genetic complexity. RNA sequencing (RNA-seq) of 79 osteosarcoma diagnostic biopsies identified stable independent components that recapitulate the tumor and microenvironment cell composition. Unsupervised classification of the independent components stratified this cohort into favorable (G1) and unfavorable (G2) prognostic tumors in terms of overall survival. Multivariate survival analysis ranked this stratification as the most influential variable. Functional characterization associated G1 tumors with innate immunity and G2 tumors with angiogenic, osteoclastic, and adipogenic activities as well as PPARγ pathway upregulation. A focused gene signature that predicted G1/G2 tumors from RNA-seq data was developed and validated within an independent cohort of 82 osteosarcomas. This signature was further validated with a custom NanoString panel in 96 additional osteosarcomas. This study thus proposes new biomarkers to detect high-risk patients and new therapeutic options for osteosarcoma. Significance: These findings indicate that the osteosarcoma microenvironment composition is a major feature to identify hard-to-treat patient tumors at diagnosis and define the biological pathways and potential actionable targets associated with these tumors.

Background—The purpose of this study was to investigate the bone resorption, as well as the vascular and immune microenvironment, of jaw osteosarcomas (JO) and to correlate these features with patient clinical outcomes. Methods—We studied 50 JO biopsy samples by immunohistochemical analysis of tissue microarrays (TMAs). We investigated the bone remodeling markers RANK/RANKL/OPG, the endothelial glycoprotein CD146, and biomarkers of the immune environment (CD163 and CD68 of macrophages, CD4+ and CD8+ of tumor-infiltrating lymphocytes (TILs), and an immune checkpoint PD-1/PD-L1). The biomarkers were analyzed for their influence on progression (recurrence and metastasis), overall survival (OS), and disease-free survival (DFS). Results—A strong and significant correlation has been found between CD163 staining and lower OS and DFS. The level of CD4+ and CD8+ staining was low and non-significantly associated with survival outcomes. High levels of RANK and RANKL were found in the tumor samples and correlated with lower DFS. Conclusion—Our findings suggest that CD163+ TAMs represent markers of poor prognosis in JO. Targeting TAMs could represent a valuable therapeutic strategy in JO.

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (

International audience; Acral soft tissue tumors are common neoplasms, a subset of which pose a diagnostic challenge. We report 10 cases of a previously unrecognized acral benign soft tissue tumor. These tumors arose on the fingers and toes and involved bone in half of cases. Histologically, the tumors were lobulated and displayed an abundant stroma made of variable fibrous, chondroid and myxoid material reminiscent of cartilaginous or myoepithelial differentiation. Tumor cells harbored small round to reniform nuclei with clear chromatin and inconspicuous nucleoli along with scant eosinophilic cytoplasm. The cells were mostly arranged haphazardly in the stroma but also in small clusters. No mitotic activity was detected. No specific feature was identified in recurrent cases. By immunohistochemistry, the cells consistently stained for CD34 (10/10), ERG (9/10), and SOX9 (7/10). Whole RNA sequencing identified a previously undescribed recurrent in frame THBS1-ADGRF5 gene fusion in all cases. The transcript was confirmed by RT-PCR and was not found in the control group of mimickers including soft tissue chondromas. We propose the name of Acral FibroChondroMyxoid Tumors for this new entity.

The Sphingosine kinase-1/Sphingosine 1-Phosphate (SphK1/S1P) signaling pathway is overexpressed in various cancers, and is instrumental for the adaptation to hypoxia in a number of solid tumor models, but no data are available in osteosarcoma. Here we report that SphK1 and the S1P1 receptor are involved in HIF-1α accumulation in hypoxic osteosarcoma cells. FTY720 (Fingolimod), which targets SphK1 and S1P1, prevented HIF-1α accumulation, and also inhibited cell proliferation in both normoxia and hypoxia unlike conventional chemotherapy. In human biopsies, a significant increase of SphK1 activity was observed in cancer compared with normal bones. In all sets of TMA samples (130 cases of osteosarcoma), immunohistochemical analysis showed the hypoxic marker GLUT-1, SphK1 and S1P1 were expressed in tumors. SphK1 correlated with the GLUT-1 suggesting that SphK1 is overexpressed and correlates with intratumoral hypoxia. No correlation was found between GLUT-1 or SphK1 and response to chemotherapy, but a statistical difference was found with increased S1P1 expression in patients with poor response in long bone osteosarcomas. Importantly, multivariate analyses showed that GLUT-1 was associated with an increased risk of death in flat bone, whereas SphK1 and S1P1 were associated with an increased risk of death in long bones.

Cytotoxic therapy for breast cancer inhibits the growth of primary tumors, but promotes metastasis to the sentinel lymph nodes through the lymphatic system. However, the effect of first-line chemotherapy on the lymphatic endothelium has been poorly investigated. In this study, we determined that paclitaxel, the anti-cancer drug approved for the treatment of metastatic or locally advanced breast cancer, induces lymphatic endothelial cell (LEC) autophagy to increase metastases. While paclitaxel treatment was largely efficacious in inhibiting LEC adhesion, it had no effect on cell survival. Paclitaxel inhibited LEC migration and branch point formation by inducing an autophagy mechanism independent of Akt phosphorylation. In vivo, paclitaxel mediated a higher permeability of lymphatic endothelium to tumor cells and this effect was reversed by chloroquine, an autophagy-lysosome inhibitor. Despite a strong effect on reducing tumor size, paclitaxel significantly increased metastasis to the sentinel lymph nodes. This effect was restricted to a lymphatic dissemination, as chemotherapy did not affect the blood endothelium. Taken together, our findings suggest that the lymphatic system resists to chemotherapy through an autophagy mechanism to promote malignant progression and metastatic lesions. This study paves the way for new combinative therapies aimed at reducing the number of metastases.

Bone tissue can be involved by primitive or metastatic tumors and requires a specific processing both at the department of pathology and during multidisciplinary meetings. The development of fine-needle percutaneous biopsies and of molecular techniques in bone tumor pathology requires a specific management. Moreover, decalcification of samples is crucial but can be deleterious if not controlled or not appropriate. The aim of this review is to provide recommendations for management and decalcification of bone tumor samples.

Resume Contexte L’utilisation d’un compte rendu standardise est indispensable pour repondre aux exigences de la certification des centres de references « sarcome osseux » par l’Institut national du cancer (INCa). Les classifications de reference, de la Musculoskeletal Tumor Society (MSTS), de l’American Joint Committee on Cancer (classification R du TNM/AJCC/IUCC) et du college americain des pathologistes, manquant de precision n’integrant pas l’effet de la chimiotherapie sur les cellules tumorales dans la mesure des marges chirurgicales, menant a une interpretation equivoque des marges par les equipes qui traitent les sarcomes osseux. Cette analyse de la litterature est destinee a evaluer les limites des classifications existantes pour etablir un compte rendu standardise de la prise en charge des pieces de resection osseuse des patients traites par chimiotherapie neoadjuvante et atteints d’osteosarcome ou de sarcome d’Ewing, en repondant aux questions : (1) quelle est la valeur pronostique des marges et de la reponse a la chimiotherapie dans les classifications ? ; (2) quelles sont les modifications histologiques induites par la chimiotherapie et leur impact sur l’interpretation des marges ? Methode Une analyse de la litterature ciblee sur la valeur pronostique des marges de resections chirurgicales, en utilisant le moteur de recherche PubMed a ete realisee en septembre 2018. Les recommandations du Groupe francais des pathologistes osseux, et internationales, de prise en charge des pieces de resection osseuses ont ete considerees pour choisir les items du compte rendu standardise. Sur 523 articles, 8 repondent aux criteres d’eligibilite. Resultats La mesure de la plus petite distance entre la tumeur et le trait chirurgical avec un seuil > 2 mm semblent les plus performants pour evaluer le risque de recidive locale. Une bonne reponse a la chimiotherapie et la localisation au squelette appendiculaire diminuent la probabilite de recidives locales. Aucune des classifications n’integre les modifications microscopiques induites par la chimiotherapie dans l’interpretation des marges chirurgicales. Discussion Pour uniformiser les pratiques, les membres du GROUPOS ont etabli collegialement un compte rendu standardise des pieces de resection des sarcomes osseux qui tient compte des modifications histopathologiques de la tumeur apres chimiotherapie. Le systeme R du TNM a ete adapte et une distance > 2 mm entre la tumeur et le plan de dissection a ete valide pour definir une marge saine R0. Le statut R1, ≤ 2 mm, a ete subdivise en 3 sous-groupes (a, b, c) pour integrer la mesure des marges par rapport a la cicatrice post-chimiotherapie : R1a, resection dans la « cicatrice » ; R1b, resection dans du tissu normal a ≤ 2 mm de la cicatrice et/ou de cellules viables residuelles ; R1c, resection intralesionnelle au contact de cellules viables ou dans des territoires de necrose de coagulation. Les membres du GROUPOS ont construit ce compte rendu standardise pour etablir un langage unique et ainsi ameliorer la prise en charge des sarcomes osseux dans les centres specialises. Des donnees fiables pourront etre etablies pour mener des etudes multicentriques nationales et internationales. Niveau de preuve IV.

The PI3K pathway is highly active in human cancers. The four class I isoforms of PI3K are activated by distinct mechanisms leading to a common downstream signaling. Their downstream redundancy is thought to be responsible for treatment failures of PI3K inhibitors. We challenged this concept, by mapping the differential phosphoproteome evolution in response to PI3K inhibitors with different isoform selectivity patterns in pancreatic cancer, a disease currently without effective therapy. In this cancer, the PI3K signal was shown to control cell proliferation. We compared the effects of LY294002 that inhibit with equal potency all class I isoenzymes and downstream mTOR with the action of inhibitors with higher isoform-selectivity towards PI3Kα, PI3Kβ or PI3Kγ (namely A66, TGX-221 and AS-252424). A bioinformatics global pathway analysis of phosphoproteomics data allowed us to identify common and specific signals activated by PI3K inhibitors supported by the biological data. AS-252424 was the most effective treatment and induced apoptotic pathway activation as well as the highest changes in global phosphorylation-regulated cell signal. However, AS-252424 treatment induced re-activation of Akt, therefore decreasing the treatment outcome on cell survival. Reversely, AS-252424 and A66 combination treatment prevented p-Akt reactivation and led to synergistic action in cell lines and patient organoids. The combination of clinically approved α-selective BYL-719 with γ-selective IPI-549 was more efficient than single molecule treatment on xenograft growth. Mapping unique adaptive signaling responses to isoform-selective PI3K inhibition will help to design better combinative treatments that prevent the induction of selective compensatory signals.Graphical abstractSignificanceHalf of all human cancers show increased PI3K activity but the mutational pattern of the PI3K pathway is not sufficient to predict sensitivity to PI3K inhibitors. By identifying for the first time specific signaling induced by PI3K inhibitors with different isoform-selectivity patterns, we provide insight in how to handle heterogeneity of PI3K expression in tumor samples for the choice of available PI3K-targetting drugs. Our work provides a roadmap to target the PI3K pathway, balancing the use of isoform-selective PI3K inhibitors with personalized information extending beyond the specific mutational status.

Background Since 2010, nationwide networks of reference centers for sarcomas (RREPS/NETSARC/RESOS) collected and prospectively reviewed all cases of sarcomas and connective tumors of intermediate malignancy (TIM) in France. Methods The nationwide incidence of sarcoma or TIM (2013–2016) was measured using the 2013 WHO classification and confirmed by a second independent review by expert pathologists. Simple clinical characteristics, yearly variations and correlation of incidence with published clinical trials are presented and analyzed. Results Over 150 different histological subtypes are reported from the 25172 patients with sarcomas (n = 18712, 74,3%) or TIM (n = 6460, 25.7%), with n = 5838, n = 6153, n = 6654, and n = 6527 yearly cases from 2013 to 2016. Over these 4 years, the yearly incidence of sarcomas and TIM was therefore 70.7 and 24.4 respectively, with a combined incidence of 95.1/106/year, higher than previously reported. GIST, liposarcoma, leiomyosarcomas, undifferentiated sarcomas represented 13%, 13%, 11% and 11% of tumors. Only GIST, as a single entity had a yearly incidence above 10/106/year. There were respectively 30, 64 and 66 different histological subtypes of sarcomas or TIM with an incidence ranging from 10 to 1/106, 1–0.1/106, or < 0.1/106/year respectively. The 2 latter incidence groups represented 21% of the patients with 130 histotypes. Published phase III and phase II clinical trials (p−6) are significantly higher with sarcomas subtypes with an incidence above 1/106 per. Conclusions This nationwide registry of sarcoma patients, with exhaustive histology review by sarcoma experts, shows that the incidence of sarcoma and TIM is higher than reported, and that tumors with a very low incidence (16/year) are less likely to be included in clinical trials.

Background: Some authors have suggested that the semimembranosus tendon is involved in the pathophysiology of ramp lesions. This led us to conduct a gross and microscopic analysis of the posterior horn of the medial meniscus and the structures inserted on it. Hypothesis: (1) The semimembranosus tendon has a tendinous branch inserting into the posterior horn of the medial meniscus, and (2) the meniscotibial ligament is inserted on the posteroinferior edge of the medial meniscus. Study Design: Descriptive laboratory study. Methods: In total, 14 fresh cadaveric knees were dissected. From each cadaveric donor, a stable anatomic specimen was harvested en bloc, including the medial femoral condyle, medial tibial plateau, whole medial meniscus, cruciate ligaments, joint capsule, and distal insertion of the semimembranosus tendon. The harvested blocks were cut along the sagittal plane to isolate the distal insertion of the semimembranosus tendon on the posterior joint capsule and the posterior horn of the medial meniscus in a single slice. Histological slides were made from these samples and analyzed under a microscope. Results: In all knees, gross examination revealed a direct branch of the semimembranosus and a tendinous capsular branch ending behind the posterior horn of the medial meniscus. This capsular branch protruded over the joint capsule, over the meniscotibial ligament below and the meniscocapsular ligament above, but never ended directly in the meniscal tissue. The capsular branch was 14.3 ± 4.4 mm long (mean ± SD). The direct tendon inserted 11 ± 2.8 mm below the articular surface of the tibial plateau. The meniscotibial ligament inserted on the posteroinferior edge of the medial meniscus, and the meniscocapsular ligament insertion was on its posterosuperior edge. Highly vascularized adipose tissue was found, delimited by the posterior horn of the medial meniscus, meniscotibial ligament, meniscocapsular ligament, and capsular branch of the semimembranosus tendon. Conclusion: In all knees, our study found a capsular branch of the semimembranosus tendon inserted behind the medial meniscus. The meniscotibial ligament was inserted on the posteroinferior edge of the medial meniscus. Histological analysis of this area revealed that this ligament inserted differently from the insertion previously described in the literature. Clinical Relevance: This laboratory study provides insight into the pathophysiology of ramp lesions frequently associated with anterior cruciate ligament injury. To restore anatomy, it is mandatory to reestablish meniscotibial ligament continuity in ramp repairs.

Background: Osteosarcomas (OTS) represent the most common primary bone cancer diagnosed in adolescents and young adults. Despite remarkable advances, there are no objective molecular or imaging markers able to predict an OTS outcome at diagnosis. Focusing on biomarkers contributing broadly to treatment resistance, we examine the interplay between the tumor-associated macrophages and intra-tumor hypoxia. Methods: Radiological and immunohistochemical (IHC) data were correlated with the outcome in a retrospective and monocentric cohort of 30 pediatric OTS. We studied hypoxic (pS6, phospho-mTor, HIF-1α and carbonic anhydrase IX (CAIX)) and macrophagic (CD68 and CD163) biomarkers. Results: The imaging analyses were based on MRI manual volumetric measures on axial post-contrast T1 weighted images, where, for each tumor, we determined the necrotic volume and its ratio to the entire tumor volume. When they were above 50 cm3 and 20%, respectively, they correlated with a worse overall survival (p = 0.0072 and p = 0.0136, respectively) and event-free survival (p = 0.0059 and p = 0.0143, respectively). IHC assessments enable a significant statistical link between HIF-1α/CAIX hyper-expressions, CD68+ cells and a worse outcome, whereas activation of mTor pathway was linked to a better survival rate and CD163+ cells. Conclusions: This study evidenced the links between hypoxia and immunity in OTS, as their poor outcome may be related to a larger necrotic volume on diagnostic MRI and, in biopsies, to a specific IHC profile.