Extracting microtentacle dynamics of tumor cells in a non-adherent environment

Abstract

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.

Microtubule disruption reduces metastasis more effectively than primary tumor growth

“…For further metrics, outlines of the cell body and full cell outline including McTNs were extracted. For the cell body outline, a combination of built-in MATLAB functions for Otsu thresholding and morphological operators were used [ 22 ]. Any cell with a cell body’s centroid that migrated greater than 5 μm was excluded from this study.…”

Microtubule-Interfering Drugs: Current and Future Roles in Epithelial Ovarian Cancer Treatment

“…Furthermore, intra-abdominal pressure and fluid shear stresses from the accumulation and circulation of ascites likely induce epithelial to mesenchymal transition (EMT) and influence adhesion and migration in these floating tumor cells [ 90 ]. Single-cell tethering techniques to visualize microtentacles after drug exposure may even allow rapid identification of drug resistance without cell culture or xenografts [ 91 ].…”

Partial thermal imidization of polyelectrolyte multilayer cell tethering surfaces (TetherChip) enables efficient cell capture and microtentacle fixation for circulating tumor cell analysis

“…Further, the compatibility of thermal-crosslinking with downstream chemical cell fixation also reduces variables in this process to more tightly streamline a clinical workflow and produce a consistently archived sample. We have proven that with the TetherChip, CTCs can be spatially immobilized and therefore imaged in high-resolution and McTNs can be objectively measured with an automated software 42 for responses to chemotherapy drugs such as taxol and colchicine in less than one hour. With the nanoscale surfaces on TetherChip that provide maximal optical clarity, we can easily employ this platform for highcontent and high-resolution imaging.…”