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 (ΔΨ m ) and optical redox ratio. We demonstrated that physical constraints by the stromal cells play a major role in ΔΨ m 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 ΔΨ m pattern. In addition, we showed a positive correlation between ΔΨ m 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.

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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.

T lymphocytes are the central coordinator and executor of many immune functions. The activation and function of T lymphocytes are mediated through the engagement of cell surface receptors and regulated by a myriad of intracellular signaling network. Bioengineering tools, including imaging modalities and fluorescent probes, have been developed and employed to elucidate the cellular events throughout the functional lifespan of T cells. A better understanding of these events can broaden our knowledge in the immune systems biology, as well as accelerate the development of effective diagnostics and immunotherapies. Here we review the commonly used and recently developed techniques and probes for monitoring T lymphocyte intracellular events, following the order of intracellular events in T cells from activation, signaling, metabolism to apoptosis. The techniques introduced here can be broadly applied to other immune cells and cell systems.