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Influence of bevacizumab (Beva), erlotinib (Erlo) and their combination (Beva + Erlo) on the tumor growth of CRC xenografts.

Purpose: There is extensive cross-talk between VEGF- and EGFR-pathway signaling in colorectal cancer. However, combinations of VEGF- and EGFR-targeted monoclonal antibodies (mAb) show disappointing activity, in particular for patients with mutant RAS. Previous results show that tyrosine kinase inhibitors (TKI) can be active in colorectal cancer models resistant to mAbs. This prompted us to examine whether the activity of bevacizumab can be increased by combination with erlotinib.Experimental Design: The antitumor activity of bevacizumab, erlotinib, and their combination was determined in colorectal cancer models with different RAS status and bevacizumab sensitivity. EGFR/VEGF pathway activation was characterized by immunohistochemistry, Western blot, and ELISA assays. The influence of cetuximab and erlotinib on EGF-mediated migration and the EGFR–EGF ligand feedback loop was established in colorectal cancer cell lines with different RAS status.Results: The addition of erlotinib increased bevacizumab activity in all models independent of RAS status. Bevacizumab exposure was accompanied by marked EGFR activation in tumor cells as well as in tumor-associated endothelial cells (TECs) and resulted in strong accumulation of intracellular EGFR, which could be attenuated by erlotinib. In cellular models, erlotinib was able to attenuate EGF-mediated functions in all cell lines independent of RAS status while cetuximab only showed activity in RAS wild-type cells.Conclusions: These results should provide a molecular framework to better understand the increased activity of the bevacizumab–erlotinib combination, compared with bevacizumab alone, in the GERCOR DREAM phase III clinical trial. Differential activity of mAbs and TKIs targeting the same signaling pathway is likely applicable for other tumor types. Clin Cancer Res; 24(11); 2548–58. ©2018 AACR.

The influence of 72 hrs exposure to vehicle, erlotinib (Erlo, 10 µM) or cetuximab (Cetux, 1 µg/mL) on the viability of the indicated CRC cell lines as measured by the TUNEL assay.

The expression of total VEGFR1 and VEGFR2 was determined by ELISA analysis of tumor extracts (3 tumors per treatment).

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PDF file - 317K, List of EMT- and autophagy-related differentially expressed genes in SNAI1- and SNAI1-6SA-expressing MCF-7 cells

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PDF file - 204K, Cancer stem cell properties of EMTed and autophagic cell lines

PDF file - 10396K, Human breast cancer biopsies were stained for SNAI2 and LC3B expression by immunohistochemistry on serial tumor sections

PDF file - 72K, SNAI1-6SA cells were sorted by flow cytometry (MoFlo� Astrios� - Beckman Coulter, Inc) based on their ALDH activity

PDF file - 160K, Data mining of microarray data showing the connections between EMT and autophagy-related genes

International audience; Poly (ADP-ribose) polymerase inhibitors (PARPi) are targeted therapies that inhibit PARP proteins which are involved in a variety of cell functions. PARPi may act as modulators of angiogenesis; however, the relationship between PARPi and the vasculogenic mimicry (VM) in breast cancer remains unclear. To determine whether PARPi regulate the vascular channel formation, we assessed whether the treatment with olaparib, talazoparib and veliparib inhibits the vascular channel formation by breast cancer cell lines. Here, we found that PARPi act as potent inhibitors of the VM formation in triple negative breast cancer cells, independently of the BRCA status. Mechanistically, we find that PARPi trigger and inhibit the NF-κB signaling, leading to the inhibition of the VM. We further show that PARPi decrease the expression of the angiogenic factor PTX3. Moreover, PTX3 rescued the PARPi-inhibited VM inhibition. In conclusion, our results indicate that PARPi, by targeting the VM, may provide a new therapeutic approach for triple negative breast cancer.

Adipose tissue is a major component of breast stroma. This study focused on delineating the effects of adipose stem cells (ASCs) derived from breast of healthy women and cancer patients with normal or tumor breast cells.The ASCs were induced to differentiate into adipocytes, and the subsequent adipocyte conditioned media (ACM) were evaluated for their fatty acid profile, adipokine secretion and influence on proliferation, migration and invasion on tumoral (MCF-7 and SUM159) and normal (HMEC) human breast cell lines.An enrichment of arachidonic acid was observed in ACM from tumor tissues. Adipose tissues from tumor free secrete twice as much leptin than those from proximal or distal to the tumor. All ACMs display proliferative activity and favor invasiveness of SUM159 cells compared to MCF-7 and HMEC. All ACMs induced lipid droplets accumulation in MCF-7 cells and increased CD36 expression in tumor cells.We conclude that among secreted factors analyzed, only arachidonic acid and leptin levels did discriminate ASCs from tumor-bearing and tumor-free breasts emphasizing the importance that other cell types could contribute to the adipose tissue secretome in a tumor context.

A fluorescent monomer-free one-step strategy is developed for the synthesis of fluorescent acrylamide gels, using inexpensive and commercially available rhodamine B as the hydrogen donor in a type II photoinitiation system. The obtained hydrogels are fluorescent and have limited fluorophore leaching over time, due to the covalent bond formed between the polymer network and rhodamine dye.

Breast cancer (BC) is the first worldwide most frequent cancer in both sexes and the most commonly diagnosed in females. Although BC mortality has been thoroughly declining over the past decades, there are still considerable differences between women diagnosed with early BC and when metastatic BC is diagnosed. BC treatment choice is widely dependent on precise histological and molecular characterization. However, recurrence or distant metastasis still occurs even with the most recent efficient therapies. Thus, a better understanding of the different factors underlying tumor escape is mainly mandatory. Among the leading candidates is the continuous interplay between tumor cells and their microenvironment, where extracellular vesicles play a significant role. Among extracellular vesicles, smaller ones, also called exosomes, can carry biomolecules, such as lipids, proteins, and nucleic acids, and generate signal transmission through an intercellular transfer of their content. This mechanism allows tumor cells to recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. By reciprocity, stromal cells can also use exosomes to profoundly modify tumor cell behavior. This review intends to cover the most recent literature on the role of extracellular vesicle production in normal and cancerous breast tissues. Specific attention is paid to the use of extracellular vesicles for early BC diagnosis, follow-up, and prognosis because exosomes are actually under the spotlight of researchers as a high-potential source of liquid biopsies. Extracellular vesicles in BC treatment as new targets for therapy or efficient nanovectors to drive drug delivery are also summarized.

Recent Advancements in Hematology: Knowledge, Methods and Dissemination is a series of editorials which is published on a biannual basis by the editorial board of the journal Bloods [...]

International audience; Vasculogenic mimicry (VM) formed by aggressive tumor cells to create vascular networks connected with the endothelial cells, plays an important role in breast cancer progression. WISP2 has been considered as a tumor suppressor protein; however, the relationship between WISP2 and VM formation remains unclear. We used the in vitro tube formation assay and in vivo immunohistochemical analysis in a mouse model, and human breast tumors were used to evaluate the effect of WISP2 on VM formation. Here we report that WISP2 acts as a potent inhibitor of VM formation in breast cancer. Enforced expression of WISP2 decreased network formation while knockdown of WISP2 increased VM. Mechanistically, WISP2 increased retention of oncogenic activators YAP/TAZ in cytoplasm, leading to decreased expression of the angiogenic factor CYR61. Studies using an in vivo mouse model and human breast tumors confirmed the in vitro cell lines data. In conclusion, our results indicate that WISP2 may play a critical role in VM and highlight the critical role of WISP2 as a tumor suppressor.

Molecularly imprinted polymers (MIPs) are really interesting for nanomedicine. To be suitable for such application, they need to be small, stable in aqueous media and sometimes fluorescent for bioimaging. We report herein, the facile synthesis of fluorescent, small (below 200 nm), water-soluble and water-stable MIP capable of specific and selective recognition of their target epitope (small part of a protein). To synthesize these materials, we used dithiocarbamate-based photoiniferter polymerization in water. The use of a rhodamine-based monomer makes the resulting polymers fluorescent. Isothermal titration calorimetry (ITC) is used to determine the affinity as well as the selectivity of the MIP for its imprinted epitope, according to the significant differences observed when comparing the binding enthalpy of the original epitope with that of other peptides. The toxicity of the nanoparticles is also tested in two breast cancer cell lines to show the possible use of these particle for future in vivo applications. The materials demonstrated a high specificity and selectivity for the imprinted epitope, with a Kd value comparable with the affinity values of antibodies. The synthesized MIP are not toxic, which makes them suitable for nanomedicine.

International audience; Molecularly imprinted polymers (MIPs) have been widely used in nanomedicine in the last few years. However, their potential is limited by their intrinsic properties resulting, for instance, in lack of control in drug release processes or complex detection for in vivo imaging. Recent attempts in creating hybrid nanomaterials combining MIPs with inorganic nanomaterials succeeded in providing a wide range of new interesting properties suitable for nanomedicine. Through this review, we aim to illustrate how hybrid molecularly imprinted polymers may improve patient care with enhanced imaging, treatments, and a combination of both.

International audience; The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through “RNA-Sequencing” (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca2+ observed after ATP exposure in all CRC cell lines, Ca2+ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether, our study provides novel insights into the characterization of purinergic signaling in CRC.

Molecularly imprinted polymers (MIPs) have been widely used in nanomedicine during the last few years. However, their potential is limited by their intrinsic properties resulting, for instance, in lack of control in drug release processes or complex detection for in vivo imaging. Recent attempts in creating hybrid nanomaterials combining MIPs with inorganic nanomaterials succeeded in providing a wide range of new interesting properties suitable for nanomedicine. Through this review, we aim to illustrate how hybrids molecularly imprinted polymers may improve patient care with enhanced imaging, treatments and combination of both.

Background One year after the declaration of the coronavirus disease 2019 (COVID-19) pandemic by the World Health Organization (WHO) and despite the implementation of mandatory physical barriers and social distancing, humanity remains challenged by a long-lasting and devastating public health crisis. Management Non-pharmacological interventions (NPIs) are efficient mitigation strategies. The success of these NPIs is dependent on the approval and commitment of the population. The launch of a mass vaccination program in many countries in late December 2020 with mRNA vaccines, adenovirus-based vaccines, and inactivated virus vaccines has generated hope for the end of the pandemic. Current Issues The continuous appearance of new pathogenic viral strains and the ability of vaccines to prevent infection and transmission raise important concerns as we try to achieve community immunity against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and its variants. The need of a second and even third generation of vaccines has already been acknowledged by the WHO and governments. Perspectives There is a critical and urgent need for a balanced and integrated strategy for the management of the COVID-19 outbreaks organized on three axes: (1) Prevention of the SARS-CoV-2 infection, (2) Detection and early diagnosis of patients at risk of disease worsening, and (3) Anticipation of medical care (PDA). Conclusion The “PDA strategy” integrated into state policy for the support and expansion of health systems and introduction of digital organizations (i.e., telemedicine, e-Health, artificial intelligence, and machine-learning technology) is of major importance for the preservation of citizens' health and life world-wide.

International audience; A high expression of the phosphoprotein osteopontin (OPN) has been associated with cancer progression in several tumor types, including breast cancer, hepatocarcinoma, ovarian cancer, and colorectal cancer (CRC). Interestingly, OPN is overexpressed in CRC and is associated with a poor prognosis linked to invasion and metastasis. Here, we review the regulation and functions of OPN with an emphasis on CRC. We examine how epigenetic and genetic regulators interact with the key signaling pathways involved in this disease. Then, we describe the role of OPN in cancer progression, including proliferation, survival, migration, invasion, and angiogenesis. Furthermore, we outline the interest of using OPN as a clinical biomarker, and discuss if and how osteopontin can be implemented as a routine assay in clinical laboratories for monitoring CRC patients. Finally, we discuss the use of OPN an attractive, but challenging, therapeutic target.

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.

Simple Summary The mortality of colorectal cancer is principally related to metastatic disease at the time of diagnosis or to the growth of initially undetectable micro-metastasis. Current therapeutic strategies are efficient in patients with locally advanced cancer, but are rarely able to cure patients with metastatic disease. Therapeutic failure is mainly associated with drug resistance and an aggressive phenotype. The identification of new biomarkers for micro-metastasis and tumor progression remains an unmet clinical need that should allow for improved patient stratification for optimal treatment and may lead to the identification of novel therapeutic targets. Osteopontin (OPN), a multifunctional protein, has emerged as a potentially valuable biomarker in several cancer types. This review principally describes the molecular mechanisms of OPN that are associated with colorectal cancer (CRC) progression and metastasis, as well as the use of OPN as a clinical biomarker. This review identifies a role for OPN as a biomarker ready for extended clinical application and discusses its use as a therapeutic target. Abstract A high expression of the phosphoprotein osteopontin (OPN) has been associated with cancer progression in several tumor types, including breast cancer, hepatocarcinoma, ovarian cancer, and colorectal cancer (CRC). Interestingly, OPN is overexpressed in CRC and is associated with a poor prognosis linked to invasion and metastasis. Here, we review the regulation and functions of OPN with an emphasis on CRC. We examine how epigenetic and genetic regulators interact with the key signaling pathways involved in this disease. Then, we describe the role of OPN in cancer progression, including proliferation, survival, migration, invasion, and angiogenesis. Furthermore, we outline the interest of using OPN as a clinical biomarker, and discuss if and how osteopontin can be implemented as a routine assay in clinical laboratories for monitoring CRC patients. Finally, we discuss the use of OPN an attractive, but challenging, therapeutic target.

International audience; Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocytederived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs). Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB, and TARC) were increased in MM-plasmas and separately inhibited the osteoblastic differentiation of HD-ASCs. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Finally, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM-and HD-ASCs with a skewing towards adipocyte differentiation.

Simple Summary Endometrial cancer (EC) is the 2nd most common gynecologic cancer worldwide. MicroRNAs (miRNAs) are small noncoding RNAs that contribute to epigenetic regulation. The objective of this systematic review is to summarize our current knowledge on the role of miRNAs in the epigenetic deregulation of tumor-related genes in EC. It includes all miRNAs reported to be involved in EC including their roles in DNA methylation and RNA-associated silencing. This systematic review should be useful for development of novel strategies to improve diagnosis and risk assessment as well as for new treatments aimed at miRNAs, their target genes or DNA methylation. Abstract The objective of this systematic review is to summarize our current knowledge on the influence of miRNAs in the epigenetic deregulation of tumor-related genes in endometrial cancer (EC). We conducted a literature search on the role of miRNAs in the epigenetic regulation of EC applying the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The following terms were used: microRNA, miRNA, miR, endometrial cancer, endometrium, epigenetic, epimutation, hypermethylation, lynch, deacetylase, DICER, novel biomarker, histone, chromatin. The miRNAs were classified and are presented according to their function (tumor suppressor or onco-miRNA), their targets (when known), their expression levels in EC tissue vs the normal surrounding tissue, and the degree of DNA methylation in miRNA loci and CpG sites. Data were collected from 201 articles, including 190 original articles, published between November 1, 2008 and September 30, 2020 identifying 313 different miRNAs implicated in epigenetic regulation of EC. Overall, we identified a total of 148 miRNAs with decreased expression in EC, 140 miRNAs with increased expression in EC, and 22 miRNAs with discordant expression levels. The literature implicated different epigenetic phenomena including altered miRNA expression levels (miR-182, -230), changes in the methylation of miRNA loci (miR-34b, -129-2, -130a/b, -152, -200b, -625) and increased/decreased methylation of target genes (miR-30d,-191). This work provides an overview of all miRNAs reported to be involved in epigenetic regulation in EC including DNA methylation and RNA-associated silencing. These findings may contribute to novel strategies in diagnosis, risk assessment, and treatments aimed at miRNAs, their target genes or DNA methylation.

There is an urgent need for specific antiviral drugs directed against SARS-CoV-2 both to prevent the most severe forms of COVID-19 and to reduce viral excretion and subsequent virus dissemination; in the present pandemic context, drug repurposing is a priority. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus in order to inhibit its association with viral RNA could be a strategy to impeding viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, belonging to the NSAID family, previously demonstrated against Influenza A virus, were evaluated against SARS-CoV-2. Naproxen binding to the nucleoprotein of SARS-CoV2 was shown by molecular modeling. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2 induced-damage. The benefit of naproxen addition to the standard of care is tested in an on-going clinical study.

International audience; High-throughput screening assays have been designed to identify compounds capable of inhibiting phenotypes involved in cancer aggressiveness. However, most studies used commercially available chemical libraries. This prompted us to explore natural products isolated from marine-derived fungi as a new source of molecules. In this study, we established a chemical library from 99 strains corresponding to 45 molecular operational taxonomic units and evaluated their anticancer activity against the MCF7 epithelial cancer cell line and its invasive stem cell-like MCF7-Sh-WISP2 counterpart. We identified the marine fungal Paradendryphiella salina PC 362H strain, isolated from the brown alga Pelvetia caniculata (PC), as one of the most promising fungi which produce active compounds. Further chemical and biological characterizations of the culture of the Paradendryphiella salina PC 362H strain identified (-)-hyalodendrin as the active secondary metabolite responsible for the cytotoxic activity of the crude extract. The antitumor activity of (-)-hyalodendrin was not only limited to the MCF7 cell lines, but also prominent on cancer cells with invasive phenotypes including colorectal cancer cells resistant to chemotherapy. Further investigations showed that treatment of MCF7-Sh-WISP2 cells with (-)-hyalodendrin induced changes in the phosphorylation status of p53 and altered expression of HSP60, HSP70 and PRAS40 proteins. Altogether, our study reveals that this uninvestigated marine fungal crude extract possesses a strong therapeutic potential against tumor cells with aggressive phenotypes and confirms that members of the epidithiodioxopiperazines are interesting fungal toxins with anticancer activities.

Purpose: There is extensive cross-talk between VEGF- and EGFR-pathway signaling in colorectal cancer. However, combinations of VEGF- and EGFR-targeted monoclonal antibodies (mAb) show disappointing activity, in particular for patients with mutant RAS. Previous results show that tyrosine kinase inhibitors (TKI) can be active in colorectal cancer models resistant to mAbs. This prompted us to examine whether the activity of bevacizumab can be increased by combination with erlotinib. Experimental Design: The antitumor activity of bevacizumab, erlotinib, and their combination was determined in colorectal cancer models with different RAS status and bevacizumab sensitivity. EGFR/VEGF pathway activation was characterized by immunohistochemistry, Western blot, and ELISA assays. The influence of cetuximab and erlotinib on EGF-mediated migration and the EGFR–EGF ligand feedback loop was established in colorectal cancer cell lines with different RAS status. Results: The addition of erlotinib increased bevacizumab activity in all models independent of RAS status. Bevacizumab exposure was accompanied by marked EGFR activation in tumor cells as well as in tumor-associated endothelial cells (TECs) and resulted in strong accumulation of intracellular EGFR, which could be attenuated by erlotinib. In cellular models, erlotinib was able to attenuate EGF-mediated functions in all cell lines independent of RAS status while cetuximab only showed activity in RAS wild-type cells. Conclusions: These results should provide a molecular framework to better understand the increased activity of the bevacizumab–erlotinib combination, compared with bevacizumab alone, in the GERCOR DREAM phase III clinical trial. Differential activity of mAbs and TKIs targeting the same signaling pathway is likely applicable for other tumor types. Clin Cancer Res; 24(11); 2548–58. ©2018 AACR.

Background: COVID-19 has been associated with hypercoagulability, endothelial cell injury and frequent thrombotic complications resulting both from direct effects of the virus on the endothelium and from the 'cytokine storm' resulting from the host's immune response. Since the COVID-19 vaccines have been shown to effectively prevent symptomatic infection including hospital admissions and severe disease, the risk of COVID-19-related thrombosis should be expected to (almost) disappear in vaccinated individuals. However, some rare cases of venous thrombosis have been reported in individuals vaccinated with mRNA vaccines. Thus, there is a sharp contrast between the clinical or experimental data reported in the literature on COVID-19 and on the rare thrombotic events observed after the vaccination with these vaccines. This phenomenon raised some scepticism of even some fear about the safety of these vaccines which could compromise the adhesion of the citizens in the vaccination program. Aims: We conducted a prospective observational study, to explore the impact of vaccination with the BNT162b2 (Pfizer/BioNTech) on blood hypercoagulability and endothelial cell activation and to investigate if this is modified by the presence of active cancer. Methods: In total 229 subjects were prospectively included in the study from April to June 2021. Subjects were stratified in three predefined groups: 127 vaccinated patients with active cancer (VOnco group), 72 vaccinated health care workers (VHcw group) and 30 non vaccinated health individuals (Control group). Blood samples were obtained 2 days after the administration of the first dose of BNT162b2 vaccine and collected in Vacutainer® tubes (0.109 mol/L trisodium citrate). Platelet poor plasma (PPP) was prepared by double centrifugation at 2000 g for 20 minutes at room temperature and plasma aliquots were stored at -80°C until assayed. Samples of PPP were assessed for thrombin generation (TG) with PPP-Reagent® (Thrombogram-Thrombinoscope assay with PPP-Reagent®TF 5pM), E-selectin, D-dimers, (D-Di), Tissue Factor (TFa), procoagulant phospholipid-dependent clotting time (Procag-PPL) and von Willebrand factor (vWF), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), and platelet factor 4 (PF4). All assays were from Diagnostica Stago (France). The upper and lower normal limits (UNL and LNL) for each biomarker were calculated by the mean±2SD for the control group. Results: All vaccinated subjects showed significantly increased levels of PF4 (71% >UNL, pUNL, pUNL, pUNL, pUNL, pUNL, pUNL, p Conclusion: The ROADMAP-COVID-19-Vaccine study shows that administration of the first dose of the BNT162b2 vaccine induced significant platelet activation documented by shorter Procoag-PPL associated with increased levels of PF4. Plasma hypercoagulability was less frequent in vaccinated individuals whereas there was no evidence of significant endothelial cells activation after vaccination. Interestingly, the presence of active cancer was not associated with an enhancement of platelet activation, hypercoagulability, or endothelial cell activation after the vaccination. Probably, the generated antibodies against the spike protein or lead to platelet activation in a FcyRIIa dependent manner that results in PF4 release. The implication of the mild inflammatory reaction triggered by the vaccination could be another possible pathway leading to platelet activation. Nevertheless, vaccination does not provoke endothelial activation even in patients with cancer. The findings of the ROADMAP-COVID-19-Vaccine study support the concept administration of mRNA based vaccines does not directly cause a systematic hypercoagulability. Disclosures Gligorov: Roche-Genentech: Research Funding; Novartis: Research Funding; Onxeo: Research Funding; Daichi: Research Funding; MSD: Research Funding; Eisai: Research Funding; Genomic Heatlh: Research Funding; Ipsen: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding. Terpos: Novartis: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria, Research Funding. Dimopoulos: Amgen: Honoraria; BMS: Honoraria; Janssen: Honoraria; Beigene: Honoraria; Takeda: Honoraria.

Background Cancer patients with venous thromboembolism (VTE) or at risk of VTE are treated with antithrombotic agents. Cancer cells express procoagulant properties and induce hypercoagulability in the microenvironment, that could impact the efficiency of the antithrombotic agents. Aims In the present study, we investigated the interaction between antithrombotic agents with pancreatic cancer cells, as well as with their microenvironment. The impact of apixaban, fondaparinux, enoxaparin and tinzaparin on the procoagulant properties of pancreatic cancer cells BXPC3 was examinated. Reciprocally, we also investigated the impact of BXPC3 on the potency of these antithrombotic agents. Methods BXPC3 cells (400 cells/μl) were exposed for 48 hours to apixaban (2 µg/ml), fondaparinux (2 µg/ml), enoxaparin, tinzaparin (2 anti-Xa IU/ml) or NaCL (control). Then, conditioned media (CM) and BXPC3 cells were harvested, separated and put in contact with normal platelet-poor plasma (PPP). Subsequently, thrombin generation (TG) was assessed using Thrombogram-Thrombinoscope® assay (Diagnostica Stago). Cells' viability was also assessed with the MTT assay. Gene expression for Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Thrombospondin 1 (THSB1) was assessed with RT-qPCR at the cells exposed or not to the antithrombotic agents. Expression of TF protein and activity of cancer cells was assessed using ELISA method. Residual anti-Xa activity in CM was measured using specific amidolytic assays for each antithrombotic agent. Results Apixaban, fondaparinux, enoxaparin, and tinzaparin significantly reduced cell viability by 25%, 12%, 14%, and 11% respectively. In the control experiment non treated BXPC3 cells enhanced TG. Pre-treatment of BXPC3 with the antithrombotic agents did not significantly modify their capacity to trigger and enhance TG. Among the studied agents only apixaban resulted in significant decrease of TF mRNA expression. However, protein expression of TF was not significantly modified by any of the antithrombotic agents. VEGF's mRNA expression was significantly decreased by fondaparinux and enoxaparin. THBS1's mRNA expression was significantly increased by apixaban. The concentrations of the anti-Xa activity of fondaparinux, enoxaparin and tinzaparin in the CM obtained at 48h after exposure of cells were reduced by 27%, 48% and 26% respectively as compared to those initially added in the culture medium. In contrast, the concentration of apixaban in the CM did not significantly change. The CM obtained by cells exposed to apixaban, fondaparinux, enoxaparin and tinzaparin inhibited TG by 70%, 30%, 40% and 90% respectively. Conclusion. Antithrombotic agents reduced the viability of BXPC3 cells. Among the studied agents, apixaban had the most pronounced effect on cells' viability. The antithrombotic agents had a potential downregulating effect on the proangiogenetic properties of BXPC3 via the decrease of VEGF gene expression (fondaparinux and enoxaparin) and enhancement of THBS1 gene expression (apixaban). Nevertheless, preincubation of BXPC3 with the antithrombotic agents did not alter the expression of TF protein and their effect on thrombin generation. Moreover, BXPCE exerted a "degradation" effect on LMWH and fondaparinux. Apixaban appeared to escape from this effect of the cancer cells. A significant inhibitory effect on thrombin generation was exerted by the residual concentrations of the antithrombotic agents in the microenvironment of cancer cells. The ensemble of these data highlight for the first time that the presence of antithrombotic agents in cancer cell microenvironment alters the biology of cancer cells and offer a constant antithrombotic effect in the microenvironment. Disclosures No relevant conflicts of interest to declare.

Introduction: Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We therefore examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Materials and Methods: We studied 11 MM-ASCs, 5 HD-ASCs and 3 HD-BM-MSCs. The plasmas were from myeloma patients with bone lesions at diagnosis (n=12), in complete remission (n=8) and from 5 pools of 100 healthy donors (HD-plasmas). HD-ASCs were differentiated into osteoblasts and adipocytes and HD-BM-MSCs into osteoblasts with the three types of plasmas as well as newly discovered cytokines. Results: Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Alizarin red coloration and alkaline phosphatase activity were strongly decreased along with a decreased RUNX2 and osteocalcin expression. However, adipocyte differentiation was unaltered. The osteoblastic differentiation deficiency was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas which was confirmed by ELISA (Figure). They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, myeloma patients in remission had a cytokine plasma level almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomittantly, we observed that MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. When myeloma patients were in remission, the cytokines levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition by the cytokines were also observed on HD-BM-MSCs suggesting that this could also be the case on myeloma-BM-MSCs. Legend to figure: Cytokine expression in MM, CR and HD-plasmas (A) Representative images of cytokine array blots probed with the plasma samples. The red boxes identify the cytokines significantly dysregulated in MM- as compared to HD- or CR-plasmas and further analyzed by ELISA. The blue boxes identified the cytokines similarly expressed in MM-/CR-/HD-plasmas. (B) Cytokine concentrations in the HD-plasmas (n=5), MM-plasmas (n=11) and CR-plasmas (n=8) were measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant). Figure 1 Figure 1. Disclosures Delhommeau: Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Garderet: Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Takeda: Consultancy.

Background Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We aimed to identify the cause of this defect. Materials and Methods We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas), in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of MM-ASCs, healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Alizarin red coloration, alkaline phosphatase activity, RUNX2 and osteocalcin expression determined osteoblastic differentiation while oil-red O staining, PPARγ, C/EBPα and CD36 expression reflected the adipogenic capacity. Cytokine arrays were used along with Elisa assays for identification and quantification of relevant cytokines. RNA sequencing was performed on MM and HD-ASCs. Results Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas but adipocyte differentiation was unaltered. This defect was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas. They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, in CR-plasmas, the cytokine plasma levels were almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomitantly, MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. In CR-plasmas, the cytokine levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. Conclusion We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition, by these new circulating cytokines, was also observed on HD-BM-MSCs, suggesting that this could also be the case on myeloma-BM-MSCs.

Introduction: Endometrial cancer (EC) is the most important gynecological cancer in terms of incidence. microRNAs (miRs), which are post-transcriptional regulators implicated in a variety of cellular functions including carcinogenesis, are particularly attractive candidates as biomarkers. Indeed, several studies have shown that the miR expression pattern appears to be associated with prognostic factors in EC. Our objective is to review the current knowledge of the role of miRs in carcinogenesis and tumor progression and their association with the prognosis of endometrial cancer. Materials and Method: We performed a literature search for miR expression in EC using MEDLINE, PubMed (the Internet portal of the National Library of Medicine) and The Cochrane Library, Cochrane databases “Cochrane Reviews” and “Clinical Trials” using the following keywords: microRNA, endometrial cancer, prognosis, diagnosis, lymph node, survival, plasma, FFPE (formalin-fixed, paraffin-embedded). The miRs were classified and presented according to their expression levels in cancer tissue in relation to different prognostic factors. Results: Data were collected from 74 original articles and 8 literature reviews which described the expression levels of 261 miRs in ECs, including 133 onco-miRs, 110 miR onco-suppressors, and 18 miRs with discordant functions. The review identified 30 articles studying the expression pattern of miR in neoplastic endometrial tissue compared to benign and/or hyperplastic tissues, 12 articles detailing the expression profile of miRs as a function of lymph node status, and 14 articles that detailed the expression pattern of miRs in endometrial tumor tissue according to overall survival or in the absence of recurrence. Conclusions: The findings presented here suggest that miR analysis merits a role as a prognostic factor in the management of patients with endometrial cancer.