Current Advances in 3D Bioprinting for Cancer Modeling and Personalized Medicine.

Tumor cells evolve in a complex and heterogeneous environment composed of different cell types and an extracellular matrix. Current 2D culture methods are very limited in their ability to mimic the cancer cell environment. In recent years, various 3D models of cancer cells have been developed, notably in the form of spheroids/organoids, using scaffold or cancer-on-chip devices. However, these models have the disadvantage of not being able to precisely control the organization of multiple cell types in complex architecture and are sometimes not very reproducible in their production, and this is especially true for spheroids. Three-dimensional bioprinting can produce complex, multi-cellular, and reproducible constructs in which the matrix composition and rigidity can be adapted locally or globally to the tumor model studied. For these reasons, 3D bioprinting seems to be the technique of choice to mimic the tumor microenvironment in vivo as closely as possible. In this review, we discuss different 3D-bioprinting technologies, including bioinks and crosslinkers that can be used for in vitro cancer models and the techniques used to study cells grown in hydrogels; finally, we provide some applications of bioprinted cancer models.

Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non-transformed and cancer cells.

Mitochondrial metabolism must constantly adapt to stress conditions in order to maintain bioenergetic levels related to cellular functions. This absence of proper adaptation can be seen in a wide array of conditions, including cancer. Metabolic adaptation calls on mitochondrial function and draws on the mitochondrial reserve to meet increasing needs. Among mitochondrial respiratory parameters, the spare respiratory capacity (SRC) represents a particularly robust functional parameter to evaluate mitochondrial reserve. We provide an overview of potential SRC mechanisms and regulation with a focus on its particular significance in cancer cells.

Retrospective study of COVID-19 seroprevalence among tissue donors at the onset of the outbreak before implementation of strict lockdown measures in France.

Background The COVID-19 pandemic has altered organ and tissue donations as well as transplantation practices. SARS-CoV-2 serological tests could help in the selection of donors. We assessed COVID-19 seroprevalence in a population of tissue donors, at the onset of the outbreak in France, before systematic screening of donors for SARS-CoV-2 RNA. Methods 235 tissue donors at the Lille Tissue Bank between November 1, 2019 and March 16, 2020 were included. Archived serum samples were tested for SARS-CoV-2 antibodies using two FDA-approved kits. Results Most donors were at higher risks for severe COVID-19 illness including age over 65 years (142/235) and/or presence of co-morbidities (141/235). According to the COVID-19 risk assessment of transmission, 183 out of 235 tissue donors presented with a low risk level and 52 donors with an intermediate risk level of donor derived infection. Four out of the 235 (1.7%) tested specimens were positive for anti-SARS-CoV-2 antibodies: 2 donors with anti-N protein IgG and 2 other donors with anti-S protein total Ig. None of them had both type of antibodies. Conclusion Regarding the seroprevalence among tissue donors, we concluded that the transmission probability to recipient via tissue products was very low at the beginning of the outbreak.

A New Strategy to Preserve and Assess Oxygen Consumption in Murine Tissues.

Mitochondrial dysfunctions are implicated in several pathologies, such as metabolic, cardiovascular, respiratory, and neurological diseases, as well as in cancer and aging. These metabolic alterations are usually assessed in human or murine samples by mitochondrial respiratory chain enzymatic assays, by measuring the oxygen consumption of intact mitochondria isolated from tissues, or from cells obtained after physical or enzymatic disruption of the tissues. However, these methodologies do not maintain tissue multicellular organization and cell-cell interactions, known to influence mitochondrial metabolism. Here, we develop an optimal model to measure mitochondrial oxygen consumption in heart and lung tissue samples using the XF24 Extracellular Flux Analyzer (Seahorse) and discuss the advantages and limitations of this technological approach. Our results demonstrate that tissue organization, as well as mitochondrial ultrastructure and respiratory function, are preserved in heart and lung tissues freshly processed or after overnight conservation at 4 °C. Using this method, we confirmed the repeatedly reported obesity-associated mitochondrial dysfunction in the heart and extended it to the lungs. We set up and validated a new strategy to optimally assess mitochondrial function in murine tissues. As such, this method is of great potential interest for monitoring mitochondrial function in cohort samples.

First-line Screening of OXPHOS Deficiencies Using Microscale Oxygraphy in Human Skin Fibroblasts: A Preliminary Study.

The diagnosis of mitochondrial diseases is a real challenge because of the vast clinical and genetic heterogeneity. Classically, the clinical examination and genetic analysis must be completed by several biochemical assays to confirm the diagnosis of mitochondrial disease. Here, we tested the validity of microscale XF technology in measuring oxygen consumption in human skin fibroblasts isolated from 5 pediatric patients with heterogeneous mitochondrial disorders. We first set up the protocol conditions to allow the determination of respiratory parameters including respiration associated with ATP production, proton leak, maximal respiration, and spare respiratory capacity with reproducibility and repeatability. Maximum respiration and spare capacity were the only parameters decreased in patients irrespective of the type of OXPHOS deficiency. These results were confirmed by high-resolution oxygraphy, the reference method to measure cellular respiration. Given the fact that microscale XF technology allows fast, automated and standardized measurements, we propose to use microscale oxygraphy among the first-line methods to screen OXPHOS deficiencies.

Benefits of cryopreserved human amniotic membranes in association with conventional treatments in the management of full-thickness burns.

The use of split-thickness skin autografts (STSA) with dermal substitutes is the gold standard treatment for third-degree burn patients. In this article, we tested whether cryopreserved amniotic membranes could be beneficial to the current treatments for full-thickness burns. Swines were subjected to standardised full-thickness burn injuries, and then were randomly assigned to treatments: (a) STSA alone; (b) STSA associated with the dermal substitute, Matriderm; (c) STSA plus human amniotic membrane (HAM); and (d) STSA associated with Matriderm plus HAM. Clinical and histological assessments were performed over time. We also reported the clinical use of HAM in one patient. The addition of HAM to classic treatments reduced scar contraction. In the presence of HAM, skin wound healing displayed high elasticity and histological examination showed a dense network of long elastic fibres. The presence of HAM increased dermal neovascularization, but no effect was observed on the recruitment of inflammatory cells to the wound. Moreover, the use of HAM with classical treatments in one human patient revealed a clear benefit in terms of elasticity. These results give initial evidence to consider the clinical application of HAM to avoid post-burn contractures and therefore facilitate functional recovery after deep burn injury.

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors.

BACKGROUND: Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies. SCOPE OF THE REVIEW: To review the recent advances in elucidating the metabolic effects of BRAF and MEK inhibitors (clinical inhibitors of the MAPK/ERK pathway) in melanoma and discuss the underlying mechanisms involved in the way metabolism can influence melanoma cell death and resistance to BRAF and MEK inhibitors. We also underlined the therapeutic perspectives in terms of innovative drug combinations. MAJOR CONCLUSION: BRAF and MEK inhibitors inhibit aerobic glycolysis and induce high levels of metabolic stress leading to effective cell death by apoptosis in BRAF-mutated cancer cells. An increase in mitochondrial metabolism is required to survive to MAPK/ERK pathway inhibitors and the sub-population of cells that survives to these inhibitors are characterized by mitochondrial OXPHOS phenotype. Consequently, mitochondrial inhibition could be combined with oncogenic "drivers" inhibitors of the MAPK/ERK pathway for improving the efficacy of molecular-targeted therapy. GENERAL SIGNIFICANCE: Metabolism is a key component of the melanoma response to BRAF and/or MEK inhibitors. Mitochondrial targeting may offer novel therapeutic approaches to overwhelm the mitochondrial addiction that limits the efficacy of BRAF and/or MEK inhibitors. These therapeutic approaches might be quickly applicable to the clinical situation.

GILZ overexpression attenuates endoplasmic reticulum stress-mediated cell death via the activation of mitochondrial oxidative phosphorylation.

The Glucocorticoïd-induced leucine zipper (GILZ) protein has profound anti-inflammatory activities in haematopoietic cells. GILZ regulates numerous signal transduction pathways involved in proliferation and survival of normal and neoplastic cells. Here, we have demonstrated the potential of GILZ in alleviating apoptosis induced by ER stress inducers. Whereas the glucocorticoid, dexamethasone, protects from tunicamycin-induced cell death, silencing endogeneous GILZ in dexamethasone-treated cancer cells alter the capacity of glucocorticoids to protect from tunicamycin-mediated apoptosis. Under ER stress conditions, overexpression of GILZ significantly reduced activation of mitochondrial pathway of apoptosis by maintaining Bcl-xl level. GILZ protein affects the UPR signaling shifting the balance towards pro-survival signals as judged by down-regulation of CHOP, ATF4, XBP1s mRNA and increase in GRP78 protein level. Interestingly, GILZ sustains high mitochondrial OXPHOS during ER stress and cytoprotection mediated by GILZ is abolished in cells depleted of mitochondrial DNA, which are OXPHOS-deficient. These findings reveal a new role of GILZ, which acts as a cytoprotector against ER stress through a pathway involving mitochondrial OXPHOS.

Cigarette smoke alters the ability of human dendritic cells to promote anti-Streptococcus pneumoniae Th17 response.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation and impaired immune response to pathogens leading to bacteria-induced exacerbation of the disease. A defect in Th17 cytokines in response to Streptococcus pneumoniae, a bacteria associated with COPD exacerbations, has been recently reported. Dendritic cells (DC) are professional antigen presenting cells that drive T-cells differentiation and activation. In this study, we hypothesized that exposure to cigarette smoke, the main risk factor of COPD, might altered the pro-Th17 response to S. pneumoniae in COPD patients and human DC. METHODS: Pro-Th1 and -Th17 cytokine production by peripheral blood mononuclear cells (PBMC) from COPD patients was analyzed and compared to those from smokers and non-smokers healthy subjects. The effect of cigarette smoke extract (CSE) was analyzed on human monocyte-derived DC (MDDC) from controls exposed or not to S. pneumoniae. Bacteria endocytosis, maturation of MDDC and secretion of cytokines were assessed by flow cytometry and ELISA, respectively. Implication of the oxidative stress was analyzed by addition of antioxidants and mitochondria inhibitors. In parallel, MDDC were cocultured with autologous T-cells to analyze the consequence on Th1 and Th17 cytokine production. RESULTS: PBMC from COPD patients exhibited defective production of IL-1ß, IL-6, IL-12 and IL-23 to S. pneumoniae compared to healthy subjects and smokers. CSE significantly reduced S. pneumoniae-induced MDDC maturation, secretion of pro-Th1 and -Th17 cytokines and activation of Th1 and Th17 T-cell responses. CSE exposure was also associated with sustained CXCL8 secretion, bacteria endocytosis and mitochondrial oxidative stress. Antioxidants did not reverse these effects. Inhibitors of mitochondrial electron transport chain partly reproduced inhibition of S. pneumoniae-induced MDDC maturation but had no effect on cytokine secretion and T cell activation. CONCLUSIONS: We observed a defective pro-Th1 and -Th17 response to bacteria in COPD patients. CSE exposure was associated with an inhibition of DC capacity to activate antigen specific T-cell response, an effect that seems to be not only related to oxidative stress. These results suggest that new therapeutics boosting this response in DC may be helpful to improve treatment of COPD exacerbations.

Another facet to the anticancer response to lamellarin D: induction of cellular senescence through inhibition of topoisomerase I and intracellular Ros production.

Lamellarin D (LamD) is a marine alkaloid with broad spectrum antitumor activities. Multiple intracellular targets of LamD, which affect cancer cell growth and induce apoptosis, have been identified. These include nuclear topoisomerase I, relevant kinases (such as cyclin-dependent kinase 2) and the mitochondrial electron transport chain. While we have previously demonstrated that LamD at micromolar range deploys strong cytotoxicity by inducing mitochondrial apoptosis, mechanisms of its cytostatic effect have not yet been characterized. Here, we demonstrated that induction of cellular senescence (depicted by cell cycle arrest in G2 associated with ß-galactosidase activity) is a common response to subtoxic concentrations of LamD. Cellular senescence is observed in a large panel of cancer cells following in vitro or in vivo exposure to LamD. The onset of cellular senescence is dependent on the presence of intact topoisomerase I since topoisomerase I-mutated cells are resistant to senescence induced by LamD. LamD-induced senescence occurs without important loss of telomere integrity. Instead, incubation with LamD results in the production of intracellular reactive oxygen species (ROS), which are critical for senescence as demonstrated by the inhibitory effect of antioxidants. In addition, cancer cells lacking mitochondrial DNA also exhibit cellular senescence upon LamD exposure indicating that LamD can trigger senescence, unlike apoptosis, in the absence of functional mitochondria. Overall, our results identify senescence-associated growth arrest as a powerful effect of LamD and add compelling evidence for the pharmacological interest of lamellarins as potential anticancer agents.

Implementing Chicken Chorioallantoic Membrane (CAM) Assays for Validating Biomaterials in Tissue Engineering: Rationale and Methods.

Tissue engineering is a promising approach for generating or repairing living tissues. The development of innovative biomaterials for tissue engineering has the potential to address the unmet clinical needs in certain applications. However, before these biomaterials can be used in clinical settings, they must undergo preclinical testing to ensure safety and performance. The chicken chorioallantoic membrane (CAM) assay is a preferred screening tool for studying biocompatibility, angiogenesis, and inflammation induced by biomaterials owing to ethical and economic considerations. This CAM-based platform increased the throughput of biomaterial testing for tissue engineering before in vivo testing. In this paper, we discuss the advantages of the CAM model. We also provided a step-by-step guide for implementing the CAM model in a research laboratory, along with tips and tricks for successfully running CAM assays. Finally, we present examples of biomaterials screened using CAM assays. CAM assay is a powerful in vivo model for assessing the angiogenic potential of tissue-engineered scaffolds. This guide provides a framework for conducting the assay, but specific experimental conditions should be optimized based on the scaffold material and the research question.

Key role of glutamine metabolism in persistence of leukemic cells upon exposition to FLT3 tyrosine kinase inhibitors.

Acute myeloid leukemias are a group of hematological malignancies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FMS-like tyrosine kinase 3 (FLT3) gene has led to the development of tyrosine kinase inhibitors such as quizartinib. However, achieving complete remission in patients remains challenging because these new tyrosine kinase inhibitors (TKIs) are unable to completely eradicate all leukemic cells. Residual leukemic cells persist during quizartinib treatment, leading to the rapid emergence of drug-resistant leukemia. Given that mitochondrial oxidative metabolism promotes the survival of leukemic cells after exposure to multiple anticancer drugs, we characterized the metabolism of leukemic cells that persisted during quizartinib treatment and developed metabolic strategies to eradicate them. In our study, employing biochemical and metabolomics approaches, we confirmed that the survival of leukemic cells treated with FLT3 inhibitors critically depends on maintaining mitochondrial metabolism, specifically through glutamine oxidation. We uncovered a synergistic interaction between the FLT3 inhibitor quizartinib and L-asparaginase, operating through antimetabolic mechanisms. Utilizing various models of persistent leukemia, we demonstrated that leukemic cells resistant to quizartinib are susceptible to L-asparaginase. This combined therapeutic strategy shows promise in reducing the development of resistance to FLT3 inhibitors, offering a potential strategy to enhance treatment outcomes.

Clinical response under MEK inhibitor alone in metastatic melanoma with a novel fusion involving the RAF1 gene.

Currently, in the absence of BRAFV600 mutation, the management of advanced melanomas is based on immunotherapies, but only half of the patients are responders. RAF1 (also named CRAF) fusions occur in 1-2.1% of wild-type melanomas. Preclinical data suggest that the presence of RAF fusion may be sensitive to MEK inhibitors. We report the case of a patient with an advanced melanoma harboring an EFCC1-RAF1 fusion who showed a clinical benefit from and a partial response to a MEK inhibitor.

Engineering 3D-Printed Bioresorbable Scaffold to Improve Non-Vascularized Fat Grafting: A Proof-of-Concept Study.

Autologous fat grafting is the gold standard for treatment in patients with soft-tissue defects. However, the technique has a major limitation of unpredictable fat resorption due to insufficient blood supply in the initial phase after transplantation. To overcome this problem, we investigated the capability of a medical-grade poly L-lactide-co-poly ε-caprolactone (PLCL) scaffold to support adipose tissue and vascular regeneration. Deploying FDM 3D-printing, we produced a bioresorbable porous scaffold with interconnected pore networks to facilitate nutrient and oxygen diffusion. The compressive modulus of printed scaffold mimicked the mechanical properties of native adipose tissue. In vitro assays demonstrated that PLCL scaffolds or their degradation products supported differentiation of preadipocytes into viable mature adipocytes under appropriate induction. Interestingly, the chorioallantoic membrane assay revealed vascular invasion inside the porous scaffold, which represented a guiding structure for ingrowing blood vessels. Then, lipoaspirate-seeded scaffolds were transplanted subcutaneously into the dorsal region of immunocompetent rats (n = 16) for 1 or 2 months. The volume of adipose tissue was maintained inside the scaffold over time. Histomorphometric evaluation discovered small- and normal-sized perilipin+ adipocytes (no hypertrophy) classically organized into lobular structures inside the scaffold. Adipose tissue was surrounded by discrete layers of fibrous connective tissue associated with CD68+ macrophage patches around the scaffold filaments. Adipocyte viability, assessed via TUNEL staining, was sustained by the presence of a high number of CD31-positive vessels inside the scaffold, confirming the CAM results. Overall, our study provides proof that 3D-printed PLCL scaffolds can be used to improve fat graft volume preservation and vascularization, paving the way for new therapeutic options for soft-tissue defects.

Regulation by survivin of cancer cell death induced by F14512, a polyamine-containing inhibitor of DNA topoisomerase II.

F14512, an epipodophyllotoxin derivative equipped with a spermine moiety, is selectively taken up by the polyamine transport system over-active in tumor cells. F14512 was identified as a selective anticancer agent with a broad spectrum of antitumor activities and is currently undergoing phase I clinical trial in onco-hematology. However, the mechanism by which F14512 exerts its selective effects on neoplastic cells remains poorly understood. In this study, using mainly P388 leukemia cells, we showed that activation of the DNA damage response by F14512 did not induce immediate apoptosis but resulted in an early growth arrest. F14512-induced G2 arrest was accompanied by the appearance of a senescence-like phenotype (characterized by an increased ß-galactosidase staining) with up-regulation of the cyclin-dependent kinase inhibitor p16, and cyclin D1. The early senescence-based cell cycle block was characterized by a marked increase of the level of the IAP protein survivin, but not cIAP2, in P388 cells as well as in three other leukemia and melanoma cell types. The Thr(34)-phosphorylated form of survivin was observed within 4 h after F14512 exposure. Inhibition of survivin by siRNA resulted in a switch from senescence-like growth arrest to apoptosis. Compared with the parental drug etoposide, F14512-induced DNA damage signaling pathway resulted in greater senescence like-growth arrest and delayed apoptosis. Collectively, our data show that senescence arrest and subsequent apoptosis are powerful mechanisms mediating the chemotherapeutic effects of F14512 and identify survivin as the molecular determinant responsible for a qualitative shift in cell fate from senescence to apoptosis upon treatment with F14512.

Preclinical Study of Radiation on Fat Flap Regeneration under Tissue-engineering Chamber: Potential Consequences for Breast Reconstruction.

Use of a tissue-engineering chamber (TEC) for growth of fat flap is a promising approach for breast reconstruction. Here, we evaluated in a preclinical model the effects of radiation on adipose tissue growth either before or after 3D-printed bioresorbable TEC implantation. Methods: Twenty-eight female Wistar rats were distributed into three groups: TEC implantation as nonirradiated controls (G1), TEC insertion followed by irradiation 3 weeks later (G2), and irradiation 6 weeks before TEC insertion (G3). G2 and G3 received 33.3 Gy in nine sessions of 3.7 Gy. Growth of the fat flap was monitored via magnetic resonance imaging. At 6 months after implantation, fat flaps and TECs were harvested for analysis. Results: Irradiation did not alter the physicochemical features of poly(lactic-co-glycolic acid)-based TECs. Compared with G1, fat flap growth was significantly reduced by 1.6 times in irradiated G2 and G3 conditions. In G2 and G3, fat flaps consisted of mature viable adipocytes sustained by CD31+ vascular cells. However, 37% (3 of 8) of the G2 irradiated adipose tissues presented a disorganized architecture invaded by connective tissues with inflammatory CD68 + cells, and the presence of fibrosis was observed. Conclusions: Overall, this preclinical study does not reveal any major obstacle to the use of TEC in a radiotherapy context. Although irradiation reduces the growth of fat flap under the TEC by reducing adipogenesis and inducing inconsistent fibrosis, it does not impact flap survival and vascularization. These elements must be taken into account if radiotherapy is proposed before or after TEC-based breast reconstruction.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

Tips and Tricks and Clinical Outcome of Cryopreserved Human Amniotic Membrane Application for the Management of Medication-Related Osteonecrosis of the Jaw (MRONJ): A Pilot Study.

Medication-related osteonecrosis of the jaw (MRONJ) is a complication of certain pharmacological treatments such as bisphosphonates, denosumab, and angiogenesis inhibitors. There are currently no guidelines on its management, particularly in advanced stages. The human amniotic membrane (hAM) has low immunogenicity and exerts anti-inflammatory, antifibrotic, antimicrobial, antiviral, and analgesic effects. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with suitable mechanical properties (permeability, stability, elasticity, flexibility, and resorbability) to prevent the proliferation of fibrous tissue and promote early neovascularization at the surgical site. In oral surgery, hAM stimulates healing and facilitates the proliferation and differentiation of epithelial cells in the oral mucosa and therefore its regeneration. We proposed using cryopreserved hAM to eight patients suffering from cancer (11 lesions) with stage 2-3 MRONJ on a compassionate use basis. A collagen sponge was added in some cases to facilitate hAM grafting. One or three hAMs were applied and one patient had a reapplication. Three patients had complete closure of the surgical site with proper epithelialization at 2 weeks, and two of them maintained it until the last follow-up. At 1 week after surgery, three patients had partial wound dehiscence with partial healing 3 months later and two patients had complete wound dehiscence. hAM reapplication led to complete healing. All patients remained asymptomatic with excellent immediate significant pain relief, no infections, and a truly positive impact on the patients' quality of life. No adverse events occurred. At 6 months of follow-up, 80% of lesions had complete or partial wound healing (30 and 50%, respectively), while 62.5% of patients were in stage 3. Radiological evaluations found that 85.7% of patients had stable bone lesions (n = 5) or new bone formation (n = 1). One patient had a worsening MRONJ but remained asymptomatic. One patient did not attend his follow-up radiological examination. For the first time, this prospective pilot study extensively illustrates both the handling and surgical application of hAM in MRONJ, its possible association with a collagen sponge scaffold, its outcome at the site, the application of multiple hAM patches at the same time, and its reapplication.

TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.