Intracellular and microenvironmental regulation of mitochondrial membrane potential in cancer cells.

Cancer cells have an abnormally high mitochondrial membrane potential (ΔΨm ), which is associated with enhanced invasive properties in vitro and increased metastases in vivo. The mechanisms underlying the abnormal ΔΨm in cancer cells remain unclear. Research on different cell types has shown that ΔΨm is regulated by various intracellular mechanisms such as by mitochondrial inner and outer membrane ion transporters, cytoskeletal elements, and biochemical signaling pathways. On the other hand, the role of extrinsic, tumor microenvironment (TME) derived cues in regulating ΔΨm is not well defined. In this review, we first summarize the existing literature on intercellular mechanisms of ΔΨm regulation, with a focus on cancer cells. We then offer our perspective on the different ways through which the microenvironmental cues such as hypoxia and mechanical stresses may regulate cancer cell ΔΨm . This article is categorized under: Cancer > Environmental Factors Cancer > Biomedical Engineering Cancer > Molecular and Cellular Physiology.

Recapitulating Tumor Hypoxia in a Cleanroom-Free, Liquid-Pinning-Based Microfluidic Tumor Model.

In tumors, the metabolic demand of cancer cells often outpaces oxygen supply, resulting in a gradient of tumor hypoxia accompanied with heterogeneous resistance to cancer therapeutics. Models recapitulating tumor hypoxia are therefore essential for developing more effective cancer therapeutics. Existing in vitro models often fail to capture the spatial heterogeneity of tumor hypoxia or involve high-cost, complex fabrication/handling techniques. Here, we designed a highly tunable microfluidic device that induces hypoxia through natural cell metabolism and oxygen diffusion barriers. We adopted a cleanroom-free, micromilling-replica-molding strategy and a microfluidic liquid-pinning approach to streamline the fabrication and tumor model establishment. We also implemented a thin-film oxygen diffusion barrier design, which was optimized through COMSOL simulation, to support both two-dimensional (2-D) and three-dimensional (3-D) hypoxic models. We demonstrated that liquid-pinning enables an easy, injection-based micropatterning of cancer cells of a wide range of parameters, showing the high tunability of our design. Human breast cancer and prostate cancer cells were seeded and stained after 24 h of 2-D and 3-D culture to validate the natural induction of hypoxia. We further demonstrated the feasibility of the parallel microfluidic channel design to evaluate dual therapeutic conditions in the same device. Overall, our new microfluidic tumor model serves as a user-friendly, cost-effective, and highly scalable platform that provides spatiotemporal analysis of the hypoxic tumor microenvironments suitable for high-content biological studies and therapeutic discoveries.

E-Cadherin Regulates Mitochondrial Membrane Potential in Cancer Cells.

Epithelial cancer cells often have unusually higher mitochondrial membrane potential (ΔΨm) than their normal counterparts, which has been associated with increased invasiveness in vitro and higher metastatic potential in vivo. However, the mechanisms by which ΔΨm in cancer cells is regulated in tumor microenvironment (TME) remain unclear. In this study, we used an in vitro micropatterning platform to recapitulate biophysical confinement cues in the TME and investigated the mechanisms by which these regulate cancer cell ΔΨm. We found that micropatterning resulted in a spatial distribution of ΔΨm, which correlated with the level of E-cadherin mediated intercellular adhesion. There was a stark contrast in the spatial distribution of ΔΨm in the micropattern of E-cadherin-negative breast cancer cells (MDA-MB-231) compared to that of the high E-cadherin expressing (MCF-7) cancer cells. Disruption and knockout of E-cadherin adhesions rescued the low ΔΨm found at the center of MCF-7 micropatterns with high E-cadherin expression, while E-cadherin overexpression in MDA-MB-231 and MCF-7 cells lowered their ΔΨm at the micropattern center. These results show that E-cadherin plays an important role in regulating the ΔΨm of cancer cells in the context of biophysical cues in TME.

Spatial Regulation of Mitochondrial Heterogeneity by Stromal Confinement in Micropatterned Tumor Models.

Heterogeneity of mitochondrial activities in cancer cells exists across different disease stages and even in the same patient, with increased mitochondrial activities associated with invasive cancer phenotypes and circulating tumor cells. Here, we use a micropatterned tumor-stromal assay (µTSA) comprised of MCF-7 breast cancer cells and bone marrow stromal cells (BMSCs) as a model to investigate the role of stromal constraints in altering the mitochondrial activities of cancer cells within the tumor microenvironment (TME). Using microdissection and RNA sequencing, we revealed a differentially regulated pattern of gene expression related to mitochondrial activities and metastatic potential at the tumor-stromal interface. Gene expression was confirmed by immunostaining of mitochondrial mass, and live microscopic imaging of mitochondrial membrane potential (ΔΨm) and optical redox ratio. We demonstrated that physical constraints by the stromal cells play a major role in ΔΨm heterogeneity, which was positively associated with nuclear translocation of the YAP/TAZ transcriptional co-activators. Importantly, inhibiting actin polymerization and Rho-associated protein kinase disrupted the differential ΔΨm pattern. In addition, we showed a positive correlation between ΔΨm level and metastatic burden in vivo in mice injected with MDA-MB-231 breast cancer cells. This study supports a new regulatory role for the TME in mitochondrial heterogeneity and metastatic potential.