Impact of rising seawater temperature on a phagocytic cell population during V. parahaemolyticus infection in the sea anemone E. pallida

Climate change is increasing ocean temperatures and consequently impacts marine life (e.g., bacterial communities). In this context, studying host–pathogen interactions in marine organisms is becoming increasingly important, not only for ecological conservation, but also to reduce economic loss due to mass mortalities in cultured species. In this study, we used Exaiptasia pallida (E. pallida), an anemone, as an emerging marine model to better understand the effect of rising temperatures on the infection induced by the pathogenic marine bacterium Vibrio parahaemolyticus. The effect of temperature on E. pallida was examined at 6, 24, or 30 h after bath inoculation with 108 CFU of V. parahaemolyticus expressing GFP (Vp-GFP) at 27°C (husbandry temperature) or 31°C (heat stress). Morphological observations of E. pallida and their Hsps expression demonstrated heat stress induced increasing damage to anemones. The kinetics of the infections revealed that Vp-GFP were localized on the surface of the ectoderm and in the mucus during the first hours of infection and in the mesenterial filaments thereafter. To better identify the E. pallida cells targeted by Vp-GFP infection, we used spectral flow cytometry. E. pallida cell types were identified based on their autofluorescent properties. corresponding to different cell types (algae and cnidocytes). We identified an AF10 population whose autofluorescent spectrum was identical to that of human monocytes/macrophage, suggesting that this spectral print could be the hallmark of phagocytic cells called “amebocytes’’. AF10 autofluorescent cells had a high capacity to phagocytize Vp-GFP, suggesting their possible role in fighting infection. This was confirmed by microscopy using sorted AF10 and GFP-positive cells (AF10+/GFP+). The number of AF10+/GFP+ cells were reduced at 31°C, demonstrating that increased temperature not only damages tissue but also affects the immune response of E. pallida. In conclusion, our study provides a springboard for more comprehensive studies of immune defense in marine organisms and paves the way for future studies of the dynamics, activation patterns, and functional responses of immune cells when encountering pathogens

Nanoblades allow high-level genome editing in murine and human organoids

Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called “organoids” is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the “nanoblade (NB)” technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%–50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression

Metformin targets the GTPase Rac1 to inhibit prostate cancer cell migration

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Extracellular matrix stiffness determines the phenotypic behavior of dedifferentiated melanoma cells through a DDR1/2-dependent YAP mechanotransduction pathway

Abstract Extracellular matrix (ECM) stiffening, resulting from increased collagen deposition and cross-linking, is a key biophysical factor of the tumor microenvironment. Cutaneous melanoma is a deadly metastatic cancer. Its aggressiveness stems from high intratumoral heterogeneity, resulting from the plasticity of melanoma cells, which transit from a melanocytic state to dedifferentiated therapy-resistant and invasive phenotypes, characterized by mesenchymal and/or neural crest stem cell-like features. Phenotypic plasticity is regulated by stroma-derived soluble factors, but the functional impact of ECM stiffening on melanoma cell phenotypes remains ill defined. Here, we found that melanoma cell subpopulations display difference in mechanical responsiveness. Compared to melanocytic cells, mesenchymal dedifferentiated cells showed increased proliferation, migration and resistance to MAP kinase-targeted therapy when seeded on stiff collagen. By contrast, a soft ECM impaired their proliferation and migration and sensitized them to targeted therapy. In addition, extracellular mechanical signals are required to sustain melanoma cell identity and dedifferentiation features. Further analyses indicated that the mechanosensitivity nature of dedifferentiated cells relies on the expression and activation of collagen receptors DDR1 and DDR2 that control actomyosin cytoskeleton reorganization and YAP mechanotransduction pathway. Inhibiting both DDR in dedifferentiated melanoma cells abrogated their mechano-induced behavior and drug-resistant phenotype, while forcing their expression in melanocytic cells induced mechanical responsiveness and a less differentiated phenotype. Our results reveal that phenotypic reprogramming endows dedifferentiated melanoma cells with increased sensitivity and addiction to ECM stiffness. We propose that mechano-addiction mediated by DDR collagen receptors may represent a novel vulnerability for aggressive dedifferentiated cancer cells that can be exploited for therapeutic benefits

Targeting Discoidin Domain Receptors DDR1 and DDR2 overcomes matrix‐mediated tumor cell adaptation and tolerance to BRAF‐targeted therapy in melanoma

International audienceResistance to BRAF/MEK inhibitor therapy in BRAFV600 -mutated advanced melanoma remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MMDR) in response to BRAFV600 pathway inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MMDR is mediated by drug-induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors. Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF-targeted therapy. In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling, and delays tumor relapse. Mechanistically, DDR-dependent MMDR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. These findings reveal a novel role for a collagen-rich matrix and DDR in tumor cell adaptation and resistance. They also provide important insights into environment-mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma

A Feed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma

International audienceAberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg