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An area that has come to be of tremendous interest in tumor research in the last decade is the role of the microenvironment in the biology of neoplastic diseases. The tumor microenvironment (TME) comprises various cells that are collectively important for normal tissue homeostasis as well as tumor progression or regression. Seminal studies have demonstrated the role of the dialogue between cancer cells (at many sites) and the cellular component of the microenvironment in tumor progression, metastasis, and resistance to treatment. Using an appropriate system of microenvironment and tumor culture is the first step towards a better understanding of the complex interaction between cancer cells and their surroundings. Three-dimensional (3D) models have been widely described recently. However, while it is claimed that they can bridge the gap between in vitro and in vivo, it is sometimes hard to decipher their advantage or limitation compared to classical two-dimensional (2D) cultures, especially given the broad number of techniques used. We present here a comprehensive review of the different 3D methods developed recently, and, secondly, we discuss the pros and cons of 3D culture compared to 2D when studying interactions between cancer cells and their microenvironment.

The NAD + -dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD + boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD + booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD + metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.

The concept of cancer as a cell-autonomous disease has been challenged by the wealth of knowledge gathered in the past decades on the importance of tumor microenvironment (TM) in cancer progression and metastasis. The significance of endothelial cells (ECs) in this scenario was initially attributed to their role in vasculogenesis and angiogenesis that is critical for tumor initiation and growth. Nevertheless, the identification of endothelial-derived angiocrine factors illustrated an alternative non-angiogenic function of ECs contributing to both physiological and pathological tissue development. Gene expression profiling studies have demonstrated distinctive expression patterns in tumor-associated endothelial cells that imply a bilateral crosstalk between tumor and its endothelium. Recently, some of the molecular determinants of this reciprocal interaction have been identified which are considered as potential targets for developing novel anti-angiocrine therapeutic strategies.

Disparities in the genetic risk of cancer among various ancestry groups and populations remain poorly defined. This challenge is even more acute for Middle Eastern populations, where the paucity of genomic data could affect the clinical potential of cancer genetic risk profiling. We used data from the phase 1 cohort of the Qatar Genome Programme to investigate genetic variation in cancer-susceptibility genes in the Qatari population. The Qatar Genome Programme generated high-coverage genome sequencing on DNA samples collected from 6142 native Qataris, stratified into six distinct ancestry groups: general Arab, Persian, Arabian Peninsula, Admixture Arab, African, and South Asian. In this population-based, cohort study, we evaluated the performance of polygenic risk scores for the most common cancers in Qatar (breast, prostate, and colorectal cancers). Polygenic risk scores were trained in The Cancer Genome Atlas (TCGA) dataset, and their distributions were subsequently applied to the six different genetic ancestry groups of the Qatari population. Rare deleterious variants within 1218 cancer susceptibility genes were analysed, and their clinical pathogenicity was assessed by ClinVar and the CharGer computational tools. The cohort included in this study was recruited by the Qatar Biobank between Dec 11, 2012, and June 9, 2016. The initial dataset comprised 6218 cohort participants, and whole genome sequencing quality control filtering led to a final dataset of 6142 samples. Polygenic risk score analyses of the most common cancers in Qatar showed significant differences between the six ancestry groups (p<0·0001). Qataris with Arabian Peninsula ancestry showed the lowest polygenic risk score mean for colorectal cancer (−0·41), and those of African ancestry showed the highest average for prostate cancer (0·85). Cancer-gene rare variant analysis identified 76 Qataris (1·2% of 6142 individuals in the Qatar Genome Programme cohort) carrying ClinVar pathogenic or likely pathogenic variants in clinically actionable cancer genes. Variant analysis using CharGer identified 195 individuals carriers (3·17% of the cohort). Breast cancer pathogenic variants were over-represented in Qataris of Persian origin (22 [56·4%] of 39 BRCA1/BRCA2 variant carriers) and completely absent in those of Arabian Peninsula origin. We observed a high degree of heterogeneity for cancer predisposition genes and polygenic risk scores across ancestries in this population from Qatar. Stratification systems could be considered for the implementation of national cancer preventive medicine programmes. Qatar Foundation.

Our vision of cancer has changed during the past decades. Indeed tumors are now perceived as complex entities where tumoral and stromal components interact closely. Among the different elements of tumor stroma the cellular component play a primordial role. Bone Marrow derived mesenchymal cells (MSCs) are attracted to tumor sites and support tumor growth. Endothelial cells (ECs) play a major role in angiogenesis. While the literature documents many aspects of the cross talk between stromal and cancer cells, the role of direct hetero-cellular contact is not clearly established. Recently, Tunneling nanotubes (TnTs) have been shown to support cell-to-cell transfers of plasma membrane components, cytosolic molecules and organelles within cell lines. Herein, we have investigated the formation of heterocellular TnTs between stromal (MSCs and ECs) and cancer cells. We demonstrate that TnTs occur between different cancer cells, stromal cells and cancer-stromal cell lines. We showed that TnTs-like structure occurred in 3D anchorage independent spheroids and also in tumor explant cultures. In our culture condition, TnTs formation occurred after large membrane adhesion. We showed that intercellular transfers of cytoplasmic content occurred similarly between cancer cells and MSCs or ECs, but we highlighted that the exchange of mitochondria occurred preferentially between endothelial cells and cancer cells. We illustrated that the cancer cells acquiring mitochondria displayed chemoresistance. Our results illustrate the perfusion-independent role of the endothelium by showing a direct endothelial to cancer cell mitochondrial exchange associated to phenotypic modulation. This supports another role of the endothelium in the constitution of the metastatic niche.

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb , Gfi1 , Runx1 , and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0–8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8–20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. The authors reprogram in vitro endothelial cells from adult mice into engraftable haematopoietic stem cells that display single-cell and multilineage properties, are capable of long-term self-renewal and can reconstitute T cell adaptive immune function. Generating functional haematopoietic stem cells The transition pathways of endothelial cells into haematopoietic stem cells remain undefined. Shahin Rafii and colleagues reprogrammed in vitro mouse adult endothelial cells into mouse engraftable haematopoietic stem cells which display the range of functional properties expected from true haematopoietic stem cells. They use a sequential approach to express transcription factors known to participate in the induction of haematopoiesis from the embryonic endothelium, as well as an endothelial cell line that acts as the vascular niche to provide the required signals. The reprogramed cells display single-cell and multilineage properties, long-term self-renewal and reconstitute T cell adaptive immune function. Converting endothelial cells into functional and self-renewing haematopoietic stem cells could open up treatment opportunities for haematological disorders, the authors suggest. Elsewhere in this issue, George Daley and colleagues differentiated human pluripotent stem cells to the haemogenic endothelium by expressing a set of transcription factors that regulate hematopoiesis.

Tumor growth and metastatic dissemination rely on cellular plasticity. Among the different phenotypes acquired by cancer cells, epithelial to mesenchymal transition (EMT) has been extensively illustrated. Indeed, this transition allows an epithelial polarized cell to acquire a more mesenchymal phenotype with increased mobility and invasiveness. The role of EMT is quite clear during developmental stage. In the neoplastic context in many tumors EMT has been associated with a more aggressive tumor phenotype including local invasion and distant metastasis. EMT allows the cell to invade surrounding tissues and survive in the general circulation and through a stem cell phenotype grown in the host organ. The molecular pathways underlying EMT have also been clearly defined and their description is beyond the scope of this review. Here we will summarize and analyze the attempts made to block EMT in the therapeutic context. Indeed, till today, most of the studies are made in animal models. Few clinical trials are ongoing with no obvious benefits of EMT inhibitors yet. We point out the limitations of EMT targeting such tumor heterogeneity or the dynamics of EMT during disease progression.

Ovarian cancer (OC) is a heterogeneous disease characterized by its late diagnosis (FIGO stages III and IV) and the importance of abdominal metastases often observed at diagnosis. Detached ovarian cancer cells (OCCs) float in ascites and form multicellular spheroids. Here, we developed endothelial cell (EC)-based 3D spheroids to better represent in vivo conditions. When co-cultured in 3D conditions, ECs and OCCs formed organized tumor angiospheres with a core of ECs surrounded by proliferating OCCs. We established that Akt and Notch3/Jagged1 pathways played a role in angiosphere formation and peritoneum invasion. In patients’ ascites we found angiosphere-like structures and demonstrated in patients’ specimens that tumoral EC displayed Akt activation, which supports the importance of Akt activation in ECs in OC. Additionally, we demonstrated the importance of FGF2, Pentraxin 3 (PTX3), PD-ECGF and TIMP-1 in angiosphere organization. Finally, we confirmed the role of Notch3/Jagged1 in OCC–EC crosstalk relating to OCC proliferation and during peritoneal invasion. Our results support the use of multicellular spheroids to better model tumoral and stromal interaction. Such models could help decipher the complex pathways playing critical roles in metastasis spread and predict tumor response to chemotherapy or anti-angiogenic treatment.

Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both in vitro and in vivo approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44HighCD24Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The in vitro and in vivo results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.

Metastasis is the primary cause of cancer related deaths due to the limited number of efficient druggable targets. Signatures of dysregulated cancer metabolism could serve as a roadmap for the determination of new treatment strategies. However, the metabolic signatures of metastatic cells remain vastly elusive. Our aim was to determine metabolic dysregulations associated with high metastatic potential in breast cancer cell lines. We have selected 5 triple negative breast cancer (TNBC) cell lines including three with high metastatic potential (HMP) (MDA-MB-231, MDA-MB-436, MDA-MB-468) and two with low metastatic potential (LMP) (BT549, HCC1143). The normal epithelial breast cell line (hTERT-HME1) was also investigated. The untargeted metabolic profiling of cells and growth media was conducted and total of 479 metabolites were quantified. First we characterized metabolic features differentiating TNBC cell lines from normal cells as well as identified cell line specific metabolic fingerprints. Next, we determined 92 metabolites in cells and 22 in growth medium that display significant differences between LMP and HMP. The HMP cell lines had elevated level of molecules involved in glycolysis, TCA cycle and lipid metabolism. We identified metabolic advantages of cell lines with HMP beyond enhanced glycolysis by pinpointing the role of branched chain amino acids (BCAA) catabolism as well as molecules supporting coagulation and platelet activation as important contributors to the metastatic cascade. The landscape of metabolic dysregulations, characterized in our study, could serve as a roadmap for the identification of treatment strategies targeting cancer cells with enhanced metastatic potential.