Use of non-small cell lung cancer multicellular tumor spheroids to study the impact of chemotherapy.

BACKGROUND: Lung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies. METHODS: To overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy. RESULTS: We showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level. CONCLUSIONS: This study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

Therapeutic strategies for non-small cell lung cancer: Experimental models and emerging biomarkers to monitor drug efficacies.

While new targeted therapies have considerably changed the treatment and prognosis of non-small cell lung cancer (NSCLC), they are frequently unsuccessful due to primary or acquired resistances. Chemoresistance is a complex process that combines cancer cell intrinsic mechanisms including molecular and genetic abnormalities, aberrant interactions within the tumor microenvironment, and the pharmacokinetic characteristics of each molecule. From a pharmacological point of view, two levers could improve the response to treatment: (i) developing tools to predict the response to chemo- and targeted therapies and (ii) gaining a better understanding of the influence of the tumor microenvironment. Both personalized medicine approaches require the identification of relevant experimental models and biomarkers to understand and fight against chemoresistance mechanisms. After describing the main therapies in NSCLC, the scope of this review will be to identify and to discuss relevant in vitro and ex vivo experimental models that are able to mimic tumors. In addition, the interests of these models in the predictive responses to proposed therapies will be discussed. Finally, this review will evaluate the involvement of novel secreted biomarkers such as tumor DNA or micro RNA in predicting responses to anti-tumor therapies.

Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast.

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.

Protocols for endothelial cell isolation from mouse tissues: brain, choroid, lung, and muscle.

Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).

Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis.

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Tumor vessel co-option probed by single-cell analysis.

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.

Protocols for endothelial cell isolation from mouse tissues: small intestine, colon, heart, and liver.

Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).

Comparative meta-analysis of cystic fibrosis cell models suggests partial endothelial-to-mesenchymal transition.

The mesenchymal conversion of epithelial cells (EMT) has been suggested as a potential contributor in cystic fibrosis (CF) disease progression. Endothelial cells (EndCs), the cells lining blood vessels, express functional CFTR and CFTR impairment promotes endothelial activation and dysfunction. However, if the mesenchymal switch also exists in CF EndCs remains uncharacterized. To understand whether the endothelial-to-mesenchymal transition (EndMT) could occur in CF, we have conducted a transcriptomic meta-analysis of primary CFTR-impaired and patient-derived EndCs, and further compared our results to data from CF epithelial cells (EpCs) where EMT has been demonstrated. As compared to EpCs, we show that CFTR-impaired EndCs display a limited signature of EndMT, and that expression of the mesenchymal inducer Twist1 remained unchanged. Nonetheless, the use of CFTR modulators reduced the expression of mesenchymal markers from CF patient-derived EndCs, suggesting an additional therapeutic added-value next to the known effect on CFTR ion transport.

Vasculogenic mimicry, a complex and devious process favoring tumorigenesis - Interest in making it a therapeutic target.

Tumor cell vasculogenic mimicry (VM), also dubbed vascular mimicry, describes the plasticity of aggressive cancer cells forming de novo vascular networks and is associated with the malignant phenotype and poor clinical outcome. VM is described in a plethora of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas and melanomas. The presence of VM is associated with a high tumor grade, short survival, invasion and metastasis. A variety of molecular mechanisms and signal pathways participates in VM induction and formation. Due to VM's contribution on tumor progression, more VM-related strategies are being utilized for anticancer treatment. After describing the main features of VM, this review will outline the importance of the tumor microenvironment during this process, and highlight the predominant molecular targets and signaling pathways involved. These data will make it possible to discuss the importance of VM-associated mediators in antitumor therapy and how it could allow to better understand the resistance to anticancer therapy.

Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype.

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

Critical Roles of Tumor Extracellular Vesicles in the Microenvironment of Thoracic Cancers.

Extracellular vesicles (EVs), such as exosomes, are critical mediators of intercellular communication between tumor cells and other cells located in the microenvironment but also in more distant sites. Exosomes are small EVs that can carry a variety of molecules, such as lipids, proteins, and non-coding RNA, especially microRNAs (miRNAs). In thoracic cancers, including lung cancers and malignant pleural mesothelioma, EVs contribute to the immune-suppressive tumor microenvironment and to tumor growth and metastasis. In this review, we discuss the recent understanding of how exosomes behave in thoracic cancers and how and why they are promising liquid biomarkers for diagnosis, prognosis, and therapy, with a special focus on exosomal miRNAs.

Basic and Therapeutic Aspects of Angiogenesis Updated.

All organisms growing beyond the oxygen diffusion limit critically depend on a functional vasculature for survival. Yet blood vessels are far more than passive, uniform conduits for oxygen and nutrient supply. A remarkable organotypic heterogeneity is brought about by tissue-specific differentiated endothelial cells (lining the blood vessels' lumen) and allows blood vessels to deal with organ-specific demands for homeostasis. On the flip side, when blood vessels go awry, they promote life-threatening diseases characterized by endothelial cells inappropriately adopting an angiogenic state (eg, tumor vascularization) or becoming dysfunctional (eg, diabetic microvasculopathies), calling respectively for antiangiogenic therapies and proangiogenic/vascular regenerative strategies. In solid tumors, despite initial enthusiasm, growth factor-based (mostly anti-VEGF [vascular endothelial growth factor]) antiangiogenic therapies do not sufficiently live up to the expectations in terms of efficiency and patient survival, in part, due to intrinsic and acquired therapy resistance. Tumors cunningly deploy alternative growth factors than the ones targeted by the antiangiogenic therapies to reinstigate angiogenesis or revert to other ways of securing blood flow, independently of the targeted growth factors. In trying to alleviate tissue ischemia and to repair dysfunctional or damaged endothelium, local in-tissue administration of (genes encoding) proangiogenic factors or endothelial (stem) cells harnessing regenerative potential have been explored. Notwithstanding evaluation in clinical trials, these approaches are often hampered by dosing issues and limited half-life or local retention of the administered agents. Here, without intending to provide an all-encompassing historical overview, we focus on some recent advances in understanding endothelial cell behavior in health and disease and identify novel molecular players and concepts that could eventually be considered for therapeutic targeting.

BIOMEX: an interactive workflow for (single cell) omics data interpretation and visualization.

The amount of biological data, generated with (single cell) omics technologies, is rapidly increasing, thereby exacerbating bottlenecks in the data analysis and interpretation of omics experiments. Data mining platforms that facilitate non-bioinformatician experimental scientists to analyze a wide range of experimental designs and data types can alleviate such bottlenecks, aiding in the exploration of (newly generated or publicly available) omics datasets. Here, we present BIOMEX, a browser-based software, designed to facilitate the Biological Interpretation Of Multi-omics EXperiments by bench scientists. BIOMEX integrates state-of-the-art statistical tools and field-tested algorithms into a flexible but well-defined workflow that accommodates metabolomics, transcriptomics, proteomics, mass cytometry and single cell data from different platforms and organisms. The BIOMEX workflow is accompanied by a manual and video tutorials that provide the necessary background to navigate the interface and get acquainted with the employed methods. BIOMEX guides the user through omics-tailored analyses, such as data pretreatment and normalization, dimensionality reduction, differential and enrichment analysis, pathway mapping, clustering, marker analysis, trajectory inference, meta-analysis and others. BIOMEX is fully interactive, allowing users to easily change parameters and generate customized plots exportable as high-quality publication-ready figures. BIOMEX is open source and freely available at https://www.vibcancer.be/software-tools/biomex.

Single-Cell RNA Sequencing Maps Endothelial Metabolic Plasticity in Pathological Angiogenesis.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets.

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

An Integrated Gene Expression Landscape Profiling Approach to Identify Lung Tumor Endothelial Cell Heterogeneity and Angiogenic Candidates.

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.

BRAF, A gatekeeper controlling endothelial permeability.

The RAF/MEK/ERK signal transduction pathway is commonly deregulated in cancer and is activated by various stimuli regulating a variety of cell responses. In wild-type endothelial cells, upon permeability stimuli, ROKα, RAF1, BRAF, and RAP1 become activated, inducing a cascade of reactions resulting in F-actin remodeling and increased cell permeability. Here, Dorard et al. showed that BRAF ablated cells had more RAF1/ROKα dimerization and relocalization to VE-cadherin occurred, ultimately leading to less F-actin content and reduced vessel permeability.