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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.

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Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.

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Taxanes and epothilones are chemotherapeutic agents that ultimately lead to cell death through inhibition of normal microtubular function. This review summarizes the literature demonstrating their current use and potential promise as therapeutic agents in the treatment of epithelial ovarian cancer (EOC), as well as putative mechanisms of resistance. Historically, taxanes have become the standard of care in the front-line and recurrent treatment of epithelial ovarian cancer. In the past few years, epothilones (i.e., ixabepilone) have become of interest as they may retain activity in taxane-treated patients since they harbor several features that may overcome mechanisms of taxane resistance. Clinical data now support the use of ixabepilone in the treatment of platinum-resistant or refractory ovarian cancer. Clinical data strongly support the use of microtubule-interfering drugs alone or in combination in the treatment of epithelial ovarian cancer. Ongoing clinical trials will shed further light into the potential of making these drugs part of current standard practice.

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Circulating tumor cells (CTCs) are associated with a higher risk of metastasis in tumor patients. The adhesion and arrest of CTCs at a secondary site is an essential prerequisite for the occurrence of tumor metastasis. CTC reattachment has shown to be dependent on microtentacle (McTN) formation in vivo. However, the specific molecular mechanism of McTN formation in suspended cancer cells remains largely unclear. Here, we demonstrated that the activation of Notch-1 signaling triggers McTN formation to facilitate cell reattachment in suspended cell culture conditions. Moreover, molecular mechanistic studies revealed that McTN formation is governed by the balance between microtubule-driven outgrowth and actomyosin-driven cell contractility. The activation of Notch-1 downregulates the acetylation level of microtubules via the Cdc42/HDAC6 pathway, which contributes to microtubule polymerization. Simultaneously, Notch-1 signaling-induced Cdc42 activation also reduced phosphorylation of myosin regulatory light chain, leading to cell contractility attenuation. Altogether, these results defined a novel mechanism by which Notch-1 signaling disturbs the balance between the expansion of microtubules and contraction of the cortical actin, which promotes McTN formation and cell reattachment. Our findings provide a new perspective on the effective therapeutic target to prevent CTC reattachment.

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BACKGROUND: Circulating tumor cells (CTCs) are the major players in the metastatic process. A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by α-tubulin (TUB), detyrosinated α-tubulin (GLU), and vimentin (VIM). In the current study, we evaluated the expression of those cytoskeletal proteins in CTCs. METHODS: Forty patients with breast cancer (BC) (16 early and 24 metastatic) were enrolled in the study. CTCs were isolated using the ISET platform and stained with the following combinations of antibodies: pancytokeratin (CK)/VIM/TUB and CK/VIM/GLU. Samples were analyzed with the ARIOL platform and confocal laser scanning microscopy. RESULTS: Fluorescence quantification revealed that the ratios CK/TUB, CK/VIM, and CK/GLU were statistically increased in MCF7 compared with more aggressive cell lines (SKBR3 and MDA-MB-231). In addition, all of these ratios were statistically increased in MCF7 cells compared with metastatic BC patients' CTCs (p = 0.0001, p = 0.0001, and p = 0.003, respectively). Interestingly, intercellular connections among CTCs and between CTCs and blood cells through cytoskeleton bridges were revealed, whereas microtentacles were increased in patients with CTC clusters. These intercellular connections were supported by TUB, VIM, and GLU. Quantification of the examined molecules revealed that the median intensity of TUB, GLU, and VIM was significantly increased in patients with metastatic BC compared with those with early disease (TUB, 62.27 vs 11.5, p = 0.0001; GLU, 6.99 vs 5.29, p = 0.029; and VIM, 8.24 vs 5.38, p = 0.0001, respectively). CONCLUSIONS: CTCs from patients with BC aggregate to each other and to blood cells through cytoskeletal protrusions, supported by VIM, TUB, and GLU. Quantification of these molecules could potentially identify CTCs related to more aggressive disease.

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Microtentacles are mostly microtubule-based cell protrusions that are formed by detached tumor cells. Here, we report that the formation of tumor cell microtentacles depends on the presence and dynamics of guanine nucleotide-binding proteins of the septin family, which are part of the cytoskeleton. In matrix-attached breast, lung, prostate and pancreas cancer cells, septins are associated with the cytosolic actin cytoskeleton. Detachment of cells causes redistribution of septins to the membrane, where microtentacle formation occurs. Forchlorfenuron, which inhibits septin functions, blocks microtentacle formation. The small GTPase Cdc42 and its effector proteins Borgs regulate septins and are essential for microtentacle formation. Dominant active and inactive Cdc42 inhibit microtentacle formation indicating that the free cycling of Cdc42 between its active and inactive state is essential for septin regulation and microtentacle formation. Cell attachment and aggregation models suggest that septins play an essential role in the metastatic behavior of tumor cells.

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Microtentacles are thin, flexible cell protrusions that have recently been described and whose presence enhances efficient attachment of circulating cells. They are found on circulating tumor cells and can be induced on a wide range of breast cancer cell lines, where they are promoted by factors that either stabilize microtubules or destabilize the actin cytoskeleton. Evidence suggests that they are relevant to the metastatic spread of cancer, so understanding their structure and formation may lead to useful therapies. Microtentacles are formed by microtubules and contain vimentin intermediate filaments, but beyond this, there is little information about their ultrastructure. We have used electron microscopy of high pressure frozen sections and tomography of cryo-prepared intact cells, along with super resolution fluorescence microscopy, to provide the first ultrastructural insights into microtubule and intermediate filament arrangement within microtentacles. By scanning electron microscopy it was seen that microtentacles form within minutes of addition of drugs that stabilize microtubules and destabilize actin filaments. Mature microtentacles were found to be well below one micrometer in diameter, tapering gradually to below 100nm at the distal ends. They also contained frequent branches and bulges suggestive of heterogeneous internal structure. Super resolution fluorescence microscopy and examination of sectioned samples showed that the microtubules and intermediate filaments can occupy different areas within the microtentacles, rather than interacting intimately as had been expected. Cryo-electron tomography of thin regions of microtentacles revealed densely packed microtubules and absence of intermediate filaments. The number of microtubules ranged from several dozen in some areas to just a few in the thinnest regions, with none of the regular arrangement found in axonemes. Improved understanding of the mechanism of microtentacle formation, as well as the resultant structure, will be valuable in developing therapies against metastasis, if the hypothesized role of microtentacles in metastasis is confirmed. This work provides a significant step in this direction.

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During metastasis, tumor cells dynamically change their cytoskeleton to traverse through a variety of non-adherent microenvironments, including the vasculature or lymphatics. Due to the challenges of imaging drift in non-adhered tumor cells, the dynamic cytoskeletal phenotypes are poorly understood. We present a new approach to analyze the dynamic cytoskeletal phenotypes of non-adhered cells that support microtentacles (McTNs), which are cell surface projections implicated in metastatic reattachment. Combining a recently-developed cell tethering method with a novel image analysis framework allowed McTN attribute extraction. Full cell outlines, number of McTNs, and distance of McTN tips from the cell body boundary were calculated by integrating a rotating anisotropic filtering method for identifying thin features with retinal segmentation and active contour algorithms. Tethered cells behave like free-floating cells; however tethering reduces cell drift and improves the accuracy of McTN measurements. Tethering cells does not significantly alter McTN number, but rather allows better visualization of existing McTNs. In drug treatment experiments, stabilizing tubulin with paclitaxel significantly increases McTN length, while destabilizing tubulin with colchicine significantly decreases McTN length. Finally, we quantify McTN dynamics by computing the time delay autocorrelations of 2 composite phenotype metrics (cumulative McTN tip distance, cell perimeter:cell body ratio). Our automated analysis demonstrates that treatment with paclitaxel increases total McTN amount and colchicine reduces total McTN amount, while paclitaxel also reduces McTN dynamics. This analysis method enables rapid quantitative measurement of tumor cell drug responses within non-adherent microenvironments, using the small numbers of tumor cells that would be available from patient samples.

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Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis.

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The presence of tumor cells in the circulation is associated with a higher risk of metastasis in patients with breast cancer. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. AMP-activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We report that AMPK can regulate the cytoskeleton of breast cancer cells in both attached and suspended conditions. We tested the effects of AMPK on microtubule stability and the actin-severing protein, cofilin. AMPK inhibition with compound c increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation with A-769662 decreased microtubule stability and cofilin activation with concurrent decreases in McTN formation and cell re-attachment. This data shows for the first time that AMPK shifts the balance of cytoskeletal forces in suspended breast cancer cells, which affect their ability to form McTNs and re-attach. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of breast tumor cells.

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The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.

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