VEGF-R2/CAV-1 Interaction Induced by Resveratrol/Eicosapentaenoic Acid/Docosahexaenoic Acid-Enriched Formulation through Functional Detergent-Resistant Membranes Is Associated with Decreased VEGF-A Release in ARPE-19 Cells.

SCOPE: Omega-3 fatty acids (O3FAs) and resveratrol (RSV) are known to be beneficial for certain eye diseases, such as age-related macular degeneration (AMD). Neovascular AMD is characterized by abnormal blood vessel formation due to the excessive synthesis of vascular endothelial growth factor (VEGF) by retinal pigment epithelium (RPE) cells. The study investigates whether a formulation based on eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and RSV is capable of counteracting VEGF-A secretion, and elucidates the molecular mechanism. METHODS AND RESULTS: The study finds, using ELISA, that O3FAs/RSV reduces VEGF-A secretion in human RPE cells. This phenomenon is related to the increased interaction between VEGF-receptor 2 (VEGF-R2) and caveolin-1 (CAV-1), a protein of detergent-resistant membranes (DRMs), as determined by co-immunoprecipitation and proximity ligation assay. Using microscale thermophoresis, the study confirms that O3FAs/RSV causes a high-affinity interaction. Isolation and analysis of DRMs reveal that this interaction is concomitant with VEGF-R2 relocalization in DRMs. The depletion of DRMs by a cholesterol-chelating agent blocks the VEGF-R2/CAV-1 interaction and EPA/DHA/RSV-mediated impairment of VEGF production. CONCLUSION: This specific interaction can provide a new strategy for countering VEGF-A production in human RPE cells and, consequently, reducing neovascularization in AMD. Further preclinical studies involving O3FAs and polyphenols are warranted.

Development of an Immuno-SPECT/Fluorescent Bimodal Tracer Targeting Human or Murine PD-L1 on Preclinical Models.

Detection of biomarkers to diagnose, treat, and predict the efficacy of cancer therapies is a major clinical challenge. Currently, biomarkers such as PD-L1 are commonly detected from biopsies, but this approach does not take into account the spatiotemporal heterogeneity of their expression in tumors. A solution consists in conjugating monoclonal antibodies (mAbs) targeting these biomarkers with multimodal imaging probes. In this study, a bimodal [111In]-DOTA-aza-BODIPY probe emitting in the near-infrared (NIR) was grafted onto mAbs targeting murine or human PD-L1 either in a site-specific or random manner. In vitro, these bimodal mAbs showed a good stability and affinity for PD-L1. In vivo, they targeted specifically PD-L1 and were detected by both fluorescence and SPECT imaging. A significant benefit of site-specific conjugation on glycans was observed compared to random conjugation on lysine. The potential of this bimodal agent was also highlighted, thanks to a proof of concept of fluorescence-guided surgery in a human PD-L1+ tumor model.

Emergence of Lipid Droplets in the Mechanisms of Carcinogenesis and Therapeutic Responses.

Cancer shares common risk factors with cardiovascular diseases such as dyslipidemia, obesity and inflammation. In both cases, dysregulations of lipid metabolism occur, and lipid vesicles emerge as important factors that can influence carcinogenesis. In this review, the role of different lipids known to be involved in cancer and its response to treatments is detailed. In particular, lipid droplets (LDs), initially described for their role in lipid storage, exert multiple functions, from the physiological prevention of LD coalescence and regulation of endoplasmic reticulum homeostasis to pathological involvement in tumor progression and aggressiveness. Analysis of LDs highlights the importance of phosphatidylcholine metabolism and the diversity of lipid synthesis enzymes. In many cancers, the phosphatidylcholine pathways are disrupted, modifying the expression of genes coding for metabolic enzymes. Tumor microenvironment conditions, such as hypoxia, different types of stress or inflammatory conditions, are also important determinants of LD behavior in cancer cells. Therefore, LDs represent therapeutic targets in cancer, and many lipid mediators have emerged as potential biomarkers for cancer onset, progression, and/or resistance.

Red Wine Extract Prevents Oxidative Stress and Inflammation in ARPE-19 Retinal Cells.

Age-related macular degeneration (AMD) is one of the most commonly occurring ocular diseases worldwide. This degenerative condition affects the retina and leads to the loss of central vision. The current treatments are focused on the late stage of the disease, but recent studies have highlighted the importance and benefits of preventive treatments and how good dietary habits can reduce the risk of progression to an advanced form of the disease. In this context, we studied whether resveratrol (RSV) or a polyphenolic cocktail, red wine extract (RWE), are able to prevent the initiating events of AMD (i.e., oxidative stress and inflammation) in human ARPE-19 retinal pigment epithelial (RPE) cells and macrophages. This study highlights that RWE and RSV can prevent hydrogen peroxide (H2O2) or 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress and can subsequently prevent DNA damage via the inhibition of the ATM (ataxia telangiectasia-mutated)/Chk2 (checkpoint kinase 2) or Chk1 signaling pathways, respectively. Moreover, ELISA assays show that RWE and RSV can prevent the secretion of proinflammatory cytokines in RPE cells and in human macrophages. Interestingly, RWE exhibits a greater protective impact compared to RSV alone, even though RSV was more concentrated when used alone than in the red wine extract. Our results suggest that RWE and RSV may have potential interest as preventive nutritional supplementations against AMD.

GTN Enhances Antitumor Effects of Doxorubicin in TNBC by Targeting the Immunosuppressive Activity of PMN-MDSC.

(1) Background: Immunosuppression is a key barrier to effective anti-cancer therapies, particularly in triple-negative breast cancer (TNBC), an aggressive and difficult to treat form of breast cancer. We investigated here whether the combination of doxorubicin, a standard chemotherapy in TNBC with glyceryltrinitrate (GTN), a nitric oxide (NO) donor, could overcome chemotherapy resistance and highlight the mechanisms involved in a mouse model of TNBC. (2) Methods: Balb/C-bearing subcutaneous 4T1 (TNBC) tumors were treated with doxorubicin (8 mg/Kg) and GTN (5 mg/kg) and monitored for tumor growth and tumor-infiltrating immune cells. The effect of treatments on MDSCs reprogramming was investigated ex vivo and in vitro. (3) Results: GTN improved the anti-tumor efficacy of doxorubicin in TNBC tumors. This combination increases the intra-tumor recruitment and activation of CD8+ lymphocytes and dampens the immunosuppressive function of PMN-MDSCs PD-L1low. Mechanistically, in PMN-MDSC, the doxorubicin/GTN combination reduced STAT5 phosphorylation, while GTN +/- doxorubicin induced a ROS-dependent cleavage of STAT5 associated with a decrease in FATP2. (4) Conclusion: We have identified a new combination enhancing the immune-mediated anticancer therapy in a TNBC mouse model through the reprograming of PMN-MDSCs towards a less immunosuppressive phenotype. These findings prompt the testing of GTN combined with chemotherapies as an adjuvant in TNBC patients experiencing treatment failure.

Tannic Acid, A Hydrolysable Tannin, Prevents Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition to Counteract Colorectal Tumor Growth.

Despite the medico-surgical progress that has been made in the management of patients with colorectal cancer (CRC), the prognosis at five years remains poor. This resistance of cancer cells partly results from their phenotypic characteristics in connection with the epithelial-mesenchymal transition (EMT). In the present study, we have explored the ability of a polyphenol, tannic acid (TA), to counteract CRC cell proliferation and invasion through an action on the EMT. We highlight that TA decreases human SW480 and SW620 CRC cell and murine CT26 CRC cell viability, and TA inhibits their adhesion in the presence of important factors comprising the extracellular matrix, particularly in the presence of collagen type I and IV, and fibronectin. Moreover, these properties were associated with TA's ability to disrupt CRC cell migration and invasion, which are induced by transforming growth factor-ß (TGF-ß), as evidence in the video microscopy experiments showing that TA blocks the TGF-ß1-induced migration of SW480 and CT26 cells. At the molecular level, TA promotes a reversal of the epithelial-mesenchymal transition by repressing the mesenchymal markers (i.e., Slug, Snail, ZEB1, and N-cadherin) and re-expressing the epithelial markers (i.e., E-cadherin and ß-catenin). These effects could result from a disruption of the non-canonical signaling pathway that is induced by TGF-ß1, where TA strongly decreases the phosphorylation of extracellular-signal regulated kinase ERK1/2, P38 and the AKT proteins that are well known to contribute to the EMT, the cell motility, and the acquisition of invasive properties by tumor cells. Very interestingly, a preclinical study of mice with subcutaneous murine tumor colon CT26 cells has shown that TA was able to significantly delay the growth of tumors without hepato- and nephrotoxicities.

Resvega, a Nutraceutical Preparation, Affects NFκB Pathway and Prolongs the Anti-VEGF Effect of Bevacizumab in Undifferentiated ARPE-19 Retina Cells.

Age-related macular degeneration (AMD) is an irreversible chronic degenerative pathology that affects the retina. Despite therapeutic advances thanks to the use of anti-vascular endothelial growth factor (VEGF) agents, resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, called Resvega®, was able to disrupt VEGF-A secretion in human ARPE-19 retina cells. We found that Resvega® inhibits VEGF-A secretion through decreases in both the PI3K-AKT-mTOR and NFκB signaling pathways. In NFκB signaling pathways, Resvega® inhibits the phosphorylation of the inhibitor of NFκB, IκB, which can bind NFκB dimers and sequester them in the cytoplasm. Thus, the NFκB subunits cannot migrate to the nucleus where they normally bind and stimulate the transcription of target genes such as VEGF-A. The IκB kinase complex (IKK) is also affected by Resvega® since the nutraceutical formulation decreases both IKKα and IKKß subunits and the IKKγ subunit which is required for the stimulation of IKK. Very interestingly, we highlight that Resvega® could prolong the anti-angiogenic effect of Avastin®, which is an anti-VEGF agent typically used in clinical practice. Our results suggest that Resvega® may have potential interest as nutritional supplementation against AMD.

NF-κB signaling controls H3K9me3 levels at intronic LINE-1 and hematopoietic stem cell genes in cis.

Ionizing radiations (IR) alter hematopoietic stem cell (HSC) function on the long term, but the mechanisms underlying these effects are still poorly understood. We recently showed that IR induces the derepression of L1Md, the mouse young subfamilies of LINE-1/L1 retroelements. L1 contributes to gene regulatory networks. However, how L1Md are derepressed and impact HSC gene expression are not known. Here, we show that IR triggers genome-wide H3K9me3 decrease that occurs mainly at L1Md. Loss of H3K9me3 at intronic L1Md harboring NF-κB binding sites motifs but not at promoters is associated with the repression of HSC-specific genes. This is correlated with reduced NFKB1 repressor expression. TNF-α treatment rescued all these effects and prevented IR-induced HSC loss of function in vivo. This TNF-α/NF-κB/H3K9me3/L1Md axis might be important to maintain HSCs while allowing expression of immune genes during myeloid regeneration or damage-induced bone marrow ablation.

Genetic Background of Polycythemia Vera.

Polycythemia vera belongs to myeloproliferative neoplasms, essentially by affecting the erythroblastic lineage. JAK2 alterations have emerged as major driver mutations triggering PV-phenotype with the V617F mutation detected in nearly 98% of cases. That's why JAK2 targeting therapeutic strategies have rapidly emerged to counter the aggravation of the disease. Over decades of research, to go further in the understanding of the disease and its evolution, a wide panel of genetic alterations affecting multiple genes has been highlighted. These are mainly involved in alternative splicing, epigenetic, miRNA regulation, intracellular signaling, and transcription factors expression. If JAK2 mutation, irrespective of the nature of the alteration, is known to be a crucial event for the disease to initiate, additional mutations seem to be markers of progression and poor prognosis. These discoveries have helped to characterize the complex genomic landscape of PV, resulting in potentially new adapted therapeutic strategies for patients concerning all the genetic interferences.

PD-1/PD-L1 Checkpoints and Resveratrol: A Controversial New Way for a Therapeutic Strategy.

Immune checkpoints refer to a range of immunoregulatory molecules that modulate the immune response. For example, proteins expressed at the surface of T-cells (including PD-1 and CTLA-4) and their ligands (PD-L1 and B7-1/B7-2, respectively), expressed by cancer cells and antigen-presenting cells, are needed to prevent excessive immune responses. However, they dampen anti-tumor immunity by limiting T-cell activity, making them promising therapeutic targets in cancer. Although immunotherapies using checkpoint blocking/neutralizing antibodies targeting PD-L1 or PD-1 have proven their superiority over conventional chemotherapies or targeted therapies by enhancing T-cell-mediated anti-tumor immunity, some limitations have emerged. These include a relatively low rate of "responders" (<50%; irrespective of cancer type), the high cost of injections, and a rare risk of hyper-progression. For clinicians, the current challenge is thus to improve the existing therapies, potentially through combinatory approaches. Polyphenols such as resveratrol (RSV), a trihydroxystilbene found in various plants and an adjuvant in numerous nutraceuticals, have been proposed as potential therapeutic targets. Beyond its well-known pleiotropic effects, RSV affects PD-L1 and PD-1 expression as well as PD-L1 subcellular localization and post-translational modifications, which we review here. We also summarize the consequences of PD-1/PD-L1 signaling, the modalities of their blockade in the context of cancer, and the current status and limitations of these immunotherapies. Finally, we discuss their potential use in combination with chemotherapies, and, using RSV as a model, we propose polyphenols as adjuvants to enhance the efficacy of anti-PD-1/anti-PD-L1 immunotherapies.

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

Nanofitins targeting heat shock protein 110: An innovative immunotherapeutic modality in cancer.

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds.

High-fat diet intensifies MLL-AF9-induced acute myeloid leukemia through activation of the FLT3 signaling in mouse primitive hematopoietic cells.

Using a MLL-AF9 knock-in mouse model, we discovered that consumption of a high-fat diet (HFD) accelerates the risk of developing acute myeloid leukemia (AML). This regimen increases the clusterization of FLT3 within lipid rafts on the cell surface of primitive hematopoietic cells, which overactivates this receptor as well as the downstream JAK/STAT signaling known to enhance the transformation of MLL-AF9 knock-in cells. Treatment of mice on a HFD with Quizartinib, a potent inhibitor of FLT3 phosphorylation, inhibits the JAK3/STAT3, signaling and finally antagonizes the accelerated development of AML that occurred following the HFD regimen. We can therefore conclude that, on a mouse model of AML, a HFD enforces the FLT3 signaling pathway on primitive hematopoietic cells and, in turn, improves the oncogenic transformation of MLL-AF9 knock-in cells and the leukemia initiation.

Heat Shock Proteins and PD-1/PD-L1 as Potential Therapeutic Targets in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are a group of clonal disorders that affect hematopoietic stem/progenitor cells. These disorders are often caused by oncogenic driver mutations associated with persistent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. While JAK inhibitors, such as ruxolitinib, reduce MPN-related symptoms in myelofibrosis, they do not influence the underlying cause of the disease and are not curative. Due to these limitations, there is a need for alternative therapeutic strategies and targets. Heat shock proteins (HSPs) are cytoprotective stress-response chaperones involved in protein homeostasis and in many critical pathways, including inflammation. Over the last decade, several research teams have unraveled the mechanistic connection between STAT signaling and several HSPs, showing that HSPs are potential therapeutic targets for MPN. These HSPs include HSP70, HSP90 (chaperoning JAK2) and both HSP110 and HSP27, which are key factors modulating STAT3 phosphorylation status. Like the HSPs, the PD-1/PD-L1 signaling pathway has been widely studied in cancer, but the importance of PD-L1-mediated immune escape in MPN was only recently reported. In this review, we summarize the role of HSPs and PD-1/PD-L1 signaling, the modalities of their experimental blockade, and the effect in MPN. Finally, we discuss the potential of these emerging targeted approaches in MPN therapy.

Selecting the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy.

Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.

Chaperoning STAT3/5 by Heat Shock Proteins: Interest of Their Targeting in Cancer Therapy.

While cells from multicellular organisms are dependent upon exogenous signals for their survival, growth, and proliferation, commitment to a specific cell fate requires the correct folding and maturation of proteins, as well as the degradation of misfolded or aggregated proteins within the cell. This general control of protein quality involves the expression and the activity of molecular chaperones such as heat shock proteins (HSPs). HSPs, through their interaction with the STAT3/STAT5 transcription factor pathway, can be crucial both for the tumorigenic properties of cancer cells (cell proliferation, survival) and for the microenvironmental immune cell compartment (differentiation, activation, cytokine secretion) that contributes to immunosuppression, which, in turn, potentially promotes tumor progression. Understanding the contribution of chaperones such as HSP27, HSP70, HSP90, and HSP110 to the STAT3/5 signaling pathway has raised the possibility of targeting such HSPs to specifically restrain STAT3/5 oncogenic functions. In this review, we present how HSPs control STAT3 and STAT5 activation, and vice versa, how the STAT signaling pathways modulate HSP expression. We also discuss whether targeting HSPs is a valid therapeutic option and which HSP would be the best candidate for such a strategy.

T-cell acute lymphoblastic leukemia displays autocrine production of Interleukin-7.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by an accumulation of immature T cells. Although patient outcomes have improved, novel targeted therapies are needed to reduce the intensity of chemotherapy and improve the prognosis of high-risk patients. Interleukin-7 (IL-7) modulates the survival and proliferation of normal and malignant T cells. Targeting the IL-7 signaling pathway is thus a potentially effective therapeutic strategy. To achieve such aim, it is essential to first understand how the IL-7 signaling pathway is activated. Although IL-7 production has been observed from multiple stromal tissues, T-ALL autocrine IL-7 secretion has not yet been described. Interestingly, using T-ALL cell lines, primary and patient-derived xenotransplanted (PDX) T-ALL cells, we demonstrate that T-ALL cells produce IL-7 whereas normal T cells do not. Finally, using knock down of IL7 gene in T-ALL cells, we describe to what extent IL-7 autocrine secretion is involved in the T-ALL cells propagation in bone marrow and how it affects the number of leukemia-initiating cells in PDX mice. Together, these results demonstrate how the autocrine production of the IL-7 cytokine mediated by T-ALL cells can be involved in the oncogenic development of T-ALL and offer novel insights into T-ALL spreading.

High-fat diet disturbs lipid raft/TGF-ß signaling-mediated maintenance of hematopoietic stem cells in mouse bone marrow.

Despite recent in vivo data demonstrating that high-fat diet (HFD)-induced obesity leads to major perturbations in murine hematopoietic stem cells (HSC), the direct role of a HFD is not yet completely understood. Here, we investigate the direct impact of a short-term HFD on HSC and hematopoiesis in C57BL/6J mice compared with standard diet-fed mice. We detect a loss of half of the most primitive HSC in the bone marrow (BM) cells of HFD-fed mice, which exhibit lower hematopoietic reconstitution potential after transplantation. Impaired maintenance of HSC is due to reduced dormancy after HFD feeding. We discover that a HFD disrupts the TGF-ß receptor within lipid rafts, associated to impaired Smad2/3-dependent TGF-ß signaling, as the main molecular mechanism of action. Finally, injecting HFD-fed mice with recombinant TGF-ß1 avoids the loss of HSC and alteration of the BM's ability to recover, underscoring the fact that a HFD affects TGF-ß signaling on HSC.

Exosomes and Other Extracellular Vesicles in HPV Transmission and Carcinogenesis.

Extracellular vesicles (EVs), including exosomes (Exos), microvesicles (MVs) and apoptotic bodies (ABs) are released in biofluids by virtually all living cells. Tumor-derived Exos and MVs are garnering increasing attention because of their ability to participate in cellular communication or transfer of bioactive molecules (mRNAs, microRNAs, DNA and proteins) between neighboring cancerous or normal cells, and to contribute to human cancer progression. Malignant traits can also be transferred from apoptotic cancer cells to phagocytizing cells, either professional or non-professional. In this review, we focus on Exos and ABs and their relationship with human papillomavirus (HPV)-associated tumor development. The potential implication of EVs as theranostic biomarkers is also addressed.

Efferocytosis of apoptotic human papillomavirus-positive cervical cancer cells by human primary fibroblasts.

BACKGROUND INFORMATION: Efficient clearance of apoptotic cells, named efferocytosis, is a fundamental physiological process for tissue development and homeostasis. The contribution of non-professional phagocytes like fibroblasts to efferocytosis has been established, although the underlying mechanisms are not well understood. We recently demonstrated that horizontal DNA transfer can occur through the uptake of apoptotic human papillomavirus-positive cancer cells by human primary fibroblasts leading to their transformation. The aim of this present study was to analyse the cellular and molecular mechanisms that drive the phagocytic activity of human primary fibroblasts in the context of apoptotic cervical cancer cell removal. RESULTS: Here we provide evidence that human primary fibroblasts engulf late more efficiently than early apoptotic cells, but their phagocytic ability remains limited compared to professional phagocytes such as human monocyte-derived macrophages. The engulfment occurs in a time-, temperature- and calcium-dependent manner. Remodelling of actin-fibers contributes to the biogenesis of apoptotic cell containing macroendocytic vacuoles. Both morphological analyses and pharmacological approaches confirmed the involvement of actin-driven phagocytosis and likely macropinocytotic mechanisms in apoptotic target internalization. The uptake of apoptotic cells requires phosphatidylserine recognition, which is mainly mediated by phosphatidylserine-receptor brain-specific angiogenesis inhibitor 1. Confocal microscopy analyses with organelle-specific markers revealed that internalised apoptotic material traffics into late phagolysosomes and specific features of microtubule-associated protein 1 light chain 3-associated phagocytosis were observed. CONCLUSIONS: Our in vitro data show that fibroblasts contribute to apoptotic tumour cell removal by phagocytosis and likely macropinocytotic mechanisms. Efferocytosis by fibroblasts involves phosphatidylserine receptor brain-specific angiogenesis inhibitor 1, which participates in subsequent uptake orchestration via actin cytoskeleton remodelling. SIGNIFICANCE: Our results highlight the cellular and molecular mechanisms of fibroblast-mediated clearance of apoptotic tumour cells. Consequences regarding alternative mechanism of carcinogenesis or tumour progression should be addressed.