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.

Multi-drug resistance (MDR) is a phenomenon by which tumor cells exhibit resistance to a variety of chemically unrelated chemotherapeutic drugs. The classical form of multidrug resistance is connected to overexpression of membrane P-glycoprotein (P-gp), which acts as an energy dependent drug efflux pump. P-glycoprotein expression is known to be controlled by genetic and epigenetic mechanisms. Until now processes of P-gp gene up-regulation and resistant cell selection were considered sufficient to explain the emergence of MDR phenotype within a cell population. Recently, however, "non-genetic" acquisitions of MDR by cell-to-cell P-gp transfers have been pointed out. In the present study we show that intercellular transfers of functional P-gp occur by two different but complementary modalities through donor-recipient cells interactions in the absence of drug selection pressure. P-glycoprotein and drug efflux activity transfers were followed over 7 days by confocal microscopy and flow cytometry in drug-sensitive parental MCF-7 breast cancer cells co-cultured with P-gp overexpressing resistant variants. An early process of remote transfer was established based on the release and binding of P-gp-containing microparticles. Microparticle-mediated transfers were detected after only 4 h of incubation. We also identify an alternative mode of transfer by contact, consisting of cell-to-cell P-gp trafficking by tunneling nanotubes bridging neighboring cells. Our findings supply new mechanistic evidences for the extragenetic emergence of MDR in cancer cells and indicate that new treatment strategies designed to overcome MDR may include inhibition of both microparticles and Tunneling nanotube-mediated intercellular P-gp transfers.

Cancer is a proliferation disease affecting a genetically unstable cell population, in which molecular alterations can be somatically inherited by genetic, epigenetic or extragenetic transmission processes, leading to a cooperation of neoplastic cells within tumoural tissue. The efflux protein P-glycoprotein (P-gp) is overexpressed in many cancer cells and has known capacity to confer multidrug resistance to cytotoxic therapies. Recently, cell-to-cell P-gp transfers have been shown. Herein, we combine experimental evidence and a mathematical model to examine the consequences of an intercellular P-gp trafficking in the extragenetic transfer of multidrug resistance from resistant to sensitive cell subpopulations.