Bella S. Guerrouahen

Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance

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.

Microparticles mediated cross-talk between tumoral and endothelial cells promote the constitution of a pro-metastatic vascular niche through Arf6 up regulation

The tumor stroma plays an essential role in tumor growth, resistance to therapy and occurrence of metastatic phenotype. Tumor vessels have been considered as passive conducts for nutrients but several studies have demonstrated secretion of pro-tumoral factors by endothelial cells. The failure of anti-angiogenic therapies to meet expectations raised by pre-clinical studies prompt us to better study the cross-talk between endothelial and cancer cells. Here, we hypothesized that tumor cells and the endothelium secrete bio-active microparticles (MPs) participating to a functional cross-talk. We characterized the cancer cells MPs, using breast and ovarian cancer cell lines (MCF7, MDA-MB231, SKOV3, OVCAR3 and a primary cell lines, APOCC). Our data show that MPs from mesenchymal-like cell lines (MDA-MB231, SKOV3 and APOCC) were able to promote an activation of endothelial cells through Akt phosphorylation, compared to MPs from epithelial-like cell lines (OVCAR3 and MCF7). The MPs from mesenchymal-like cells contained increased angiogenic molecules including PDGF, IL8 and angiogenin. The endothelial activation was associated to increased Arf6 expression and MPs secretion. Endothelial activation functionalized an MP dependent pro-tumoral vascular niche promoting cancer cells proliferation, invasiveness, stem cell phenotype and chemoresistance. MPs from cancer and endothelial cells displayed phenotypic heterogeneity, and participated to a functional cross-talk where endothelial activation by cancer MPs resulted in increased secretion of EC-MPs sustaining tumor cells. Such cross-talk may play a role in perfusion independent role of the endothelium.

Osteoblastic and Vascular Endothelial Niches, Their Control on Normal Hematopoietic Stem Cells, and Their Consequences on the Development of Leukemia

Stem cell self-renewal is regulated by intrinsic mechanisms and extrinsic signals mediated via specialized microenvironments called “niches.” The best-characterized stem cell is the hematopoietic stem cell (HSC). Self-renewal and differentiation ability of HSC are regulated by two major elements: endosteal and vascular regulatory elements. The osteoblastic niche localized at the inner surface of the bone cavity might serve as a reservoir for long-term HSC storage in a quiescent state. Whereas the vascular niche, which consists of sinusoidal endothelial cell lining blood vessel, provides an environment for short-term HSC proliferation and differentiation. Both niches act together to maintain hematopoietic homeostasis. In this paper, we provide some principles applying to the hematopoietic niches, which will be useful in the study and understanding of other stem cell niches. We will discuss altered microenvironment signaling leading to myeloid lineage disease. And finally, we will review some data on the development of acute myeloid leukemia from a subpopulation called leukemia-initiating cells (LIC), and we will discuss on the emerging evidences supporting the influence of the microenvironment on chemotherapy resistance.

Metabolic signatures differentiate ovarian from colon cancer cell lines

BackgroundIn this era of precision medicine, the deep and comprehensive characterization of tumor phenotypes will lead to therapeutic strategies beyond classical factors such as primary sites or anatomical staging. Recently, “-omics” approached have enlightened our knowledge of tumor biology. Such approaches have been extensively implemented in order to provide biomarkers for monitoring of the disease as well as to improve readouts of therapeutic impact. The application of metabolomics to the study of cancer is especially beneficial, since it reflects the biochemical consequences of many cancer type-specific pathophysiological processes. Here, we characterize metabolic profiles of colon and ovarian cancer cell lines to provide broader insight into differentiating metabolic processes for prospective drug development and clinical screening.MethodsWe applied non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography and gas chromatography for the metabolic phenotyping of four cancer cell lines: two from colon cancer (HCT15, HCT116) and two from ovarian cancer (OVCAR3, SKOV3). We used the MetaP server for statistical data analysis.ResultsA total of 225 metabolites were detected in all four cell lines; 67 of these molecules significantly discriminated colon cancer from ovarian cancer cells. Metabolic signatures revealed in our study suggest elevated tricarboxylic acid cycle and lipid metabolism in ovarian cancer cell lines, as well as increased β-oxidation and urea cycle metabolism in colon cancer cell lines.ConclusionsOur study provides a panel of distinct metabolic fingerprints between colon and ovarian cancer cell lines. These may serve as potential drug targets, and now can be evaluated further in primary cells, biofluids, and tissue samples for biomarker purposes.

Breast cancer cells promote a notch-dependent mesenchymal phenotype in endothelial cells participating to a pro-tumoral niche

BackgroundEndothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness.MethodsXenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition.ResultsOur results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECsMes) in vitro. The ECsMes acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECsMes were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECsMes were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFβ and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFβ down-regulated Smad1/5 phosphorylation and Jag1KD tumor cells were unable to initiate the process.ConclusionsOverall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFβ pathways in the initiation of mesenchymal phenotype may propose new stromal targets.

P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods

Background. P-gp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Fluorescence techniques have been widely applied to measure the P-gp activity. In this paper, there is a comparison between the advantages of two fluorescence approaches of commonly available and affordable instruments: the microplate reader (MPR) and the flow cytometer to detect the P-gp efflux activity using calcein-AM. Results. The selectivity, sensibility, and reproducibility of the two methods have been defined. Our results showed that the MPR is more powerful for the detection of small inhibition, whereas the flow cytometry method is more reliable at higher concentrations of the inhibitors. We showed that to determine precisely the inhibition efficacy the flow cytometry is better; hence, to get the correct E max and EC50 values, we cannot only rely on the MPR. Conclusion. Both techniques can potentially be used extensively in the pharmaceutical industry for high-throughput drug screening and in biology laboratories for academic research, monitoring the P-gp efflux in specific assays.

Nesting of colon and ovarian cancer cells in the endothelial niche is associated with alterations in glycan and lipid metabolism

The metabolic phenotype of a cancer cell is determined by its genetic makeup and microenvironment, which dynamically modulates the tumor landscape. The endothelial cells provide both a promoting and protective microenvironment – a niche for cancer cells. Although metabolic alterations associated with cancer and its progression have been fairly defined, there is a significant gap in our understanding of cancer metabolism in context of its microenvironment. We deployed an in vitro co-culture system based on direct contact of cancer cells with endothelial cells (E4+EC), mimicking the tumor microenvironment. Metabolism of colon (HTC15 and HTC116) and ovarian (OVCAR3 and SKOV3) cancer cell lines was profiled with non-targeted metabolic approaches at different time points in the first 48 hours after co-culture was established. We found significant, coherent and non-cell line specific changes in fatty acids, glycerophospholipids and carbohydrates over time, induced by endothelial cell contact. The metabolic patterns pinpoint alterations in hexosamine biosynthetic pathway, glycosylation and lipid metabolism as crucial for cancer – endothelial cells interaction. We demonstrated that “Warburg effect” is not modulated in the initial stage of nesting of cancer cell in the endothelial niche. Our study provides novel insight into cancer cell metabolism in the context of the endothelial microenvironment.

Akt-Activated Endothelium Constitutes the Niche for Residual Disease and Resistance to Bevacizumab in Ovarian Cancer

Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Despite optimal cytoreduction and adequate adjuvant therapies, initial tumor response is often followed by relapse suggesting the existence of a tumor niche. Targeted therapies have been evaluated in ovarian cancer to overcome resistant disease. Among them, antiangiogenic therapies inhibit new blood vessel growth, induce endothelial cell apoptosis, and block the incorporation of hematopoietic and endothelial progenitor cells into new blood vessels. Despite in vitro and in vivo successes, antivascular therapy with bevacizumab targeting VEGF-A has limited efficacy in ovarian cancer. The precise molecular mechanisms underlying clinical resistance to anti-VEGF therapies are not yet well understood. Among them, tumor and stromal heterogeneity might determine the treatment outcomes. The present study investigates whether abnormalities in the tumor endothelium may contribute to treatment resistance to bevacizumab and promote a residual microscopic disease. Here, we showed that ovarian cancer cells activate Akt phosphorylation in endothelial cells inducing resistance to bevacizumab leading to an autocrine loop based on FGF2 secretion. Altogether, our results point out the role of an activated endothelium in the resistance to bevacizumab and in the constitution of a niche for a residual disease. Mol Cancer Ther; 13(12); 3123–36. ©2014 AACR.

Abstract 2998: Akt-activated endothelium constitute the niche for residual disease and resistance to bevacizumab in ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Despite optimal cytoreduction and adequate adjuvant therapies, initial tumor response is often followed by relapse suggesting the existence of a tumor niche. Targeted therapies have been evaluated in ovarian cancer to overcome resistant disease. Among them anti-angiogenic therapies inhibit new blood vessel growth, induce endothelial cell apoptosis, and block the incorporation of haematopoietic and endothelial progenitor cells into new blood vessels. Despite in vitro and in vivo successes antivascular therapy with bevacizumab targeting VEGF has limited efficacy in ovarian cancer. The precise molecular mechanisms underlying clinical resistance to anti-VEGF therapies are not yet well understood. Among them tumor and stromal heterogeneity might determine the treatment outcomes. The present study investigates whether abnormalities in the tumor endothelium may contribute to treatment resistance to bevacizumab and promote a residual microscopic disease. Here we showed that ovarian cancer cells (OCC) activate akt phosphorylation in endothelial cells inducing resistance to bevacizumab leading to an autocrine loop based on FGF2 secretion. Altogether our results point out the role of an activated endothelium in the resistance to bevacizumab and in the constitution of a niche for a residual disease. Citation Format: Bella Samia Guerrouahen, Jennifer Pasquier, Nadine Abou Kaoud, Marie-Claude Beauchamp, Mahtab Maleki, Pegah Ghiabi, Raphael Lis, Ahmed Saleh, Walter H. Gotlieb, Shahin Rafii, Arash Rafii. Akt-activated endothelium constitute the niche for residual disease and resistance to bevacizumab in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2998. doi:10.1158/1538-7445.AM2014-2998

Abstract B69: Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance

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.