High throughput cell mechanotyping of cell response to cytoskeletal modulations using a microfluidic cell deformation system.

Cellular mechanical properties influence cellular functions across pathological and physiological systems. The observation of these mechanical properties is limited in part by methods with a low throughput of acquisition or with low accessibility. To overcome these limitations, we have designed, developed, validated, and optimized a microfluidic cellular deformation system (MCDS) capable of mechanotyping suspended cells on a population level at a high throughput rate of ∼300 cells pers second. The MCDS provides researchers with a viable method for efficiently quantifying cellular mechanical properties towards defining prognostic implications of mechanical changes in pathology or screening drugs to modulate cytoskeletal integrity.

Hydrogen Peroxide Induces α-Tubulin Detyrosination and Acetylation and Impacts Breast Cancer Metastatic Phenotypes.

Levels of hydrogen peroxide are highly elevated in the breast tumor microenvironment compared to normal tissue. Production of hydrogen peroxide is implicated in the mechanism of action of many anticancer therapies. Several lines of evidence suggest hydrogen peroxide mediates breast carcinogenesis and metastasis, though the molecular mechanism remains poorly understood. This study elucidates the effects of exposure to elevated hydrogen peroxide on non-tumorigenic MCF10A mammary epithelial cells, tumorigenic MCF7 cells, and metastatic MDA-MB-231 breast cancer cells. Hydrogen peroxide treatment resulted in a dose- and time-dependent induction of two α-tubulin post-translational modifications-de-tyrosination and acetylation-both of which are markers of poor patient prognosis in breast cancer. Hydrogen peroxide induced the formation of tubulin-based microtentacles in MCF10A and MDA-MB-231 cells, which were enriched in detyrosinated and acetylated α-tubulin. However, the hydrogen peroxide-induced microtentacles did not functionally promote metastatic phenotypes of cellular reattachment and homotypic cell clustering. These data establish for the first time that microtentacle formation can be separated from the functions to promote reattachment and clustering, which indicates that there are functional steps that remain to be identified. Moreover, signals in the primary tumor microenvironment may modulate α-tubulin post-translational modifications and induce microtentacles; however, the functional consequences appear to be context-dependent.

Metastatic breast cancer cells have reduced calcium and actin response after ATP-P2Y2 signaling.

The tumor microenvironment and wound healing after injury, both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it’s altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cellular junctions, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation could be blunted using a P2Y2 antagonist, which also prevented actin mobilization in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. STATEMENT OF SIGNIFICANCE: This work shows non-tumorigenic breast epithelial cells increase intracellular Ca 2+ after ATP-P2Y2 signaling and re-localize actin, while metastatic cells lack this response, due to decreased P2Y2 expression, which correlates with poorer survival.

Elevation of Cytoplasmic Calcium Suppresses Microtentacle Formation and Function in Breast Tumor Cells.

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling.

Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.