Migratory Metrics of Wound Healing: A Quantification Approach for in vitro Scratch Assays

Abstract

Metastatic dissemination generates an aggressive disease facilitated by enhanced migratory and invasive properties. Experimental approaches employ several in vitro and in vivo assays toward quantification of these functionalities. In vitro assessments of cell motility often employ endpoint assays that rely on the global efficacy of wound closure and thwart quantification of migratory phenotypes observed during metastatic dissemination. Recent studies highlight the distinct signatures associated with individual vs. collective cell migration and necessitate the incorporation of these modalities into routine analyses. Advances in live cell imaging that permit real-time visualization of pathophysiological processes can be employed toward elucidating phenotypic plasticity associated with cell migration to overcome caveats inherent to end-point assays. Herein, we corroborate live cell imaging with the in vitro scratch assay toward quantification of migratory modalities in transformed cells. Our protocol describes a step-by-step approach for live cell setup of the scratch assay, and details analyses employed toward definition of three quantitative metrics viz., displacement, velocity and number of nearest neighbors. The current protocol (from scratch induction to data acquisition) is implemented for ~30 h and provides global/single-cell resolution of migratory phenotypes as opposed to the endpoint assays. Routine application of this protocol in cancer biology can aid the design of therapeutic regimes targeting specific migratory modalities and significantly contribute to the dissection of associated molecular networks.

Uncoupling Traditional Functionalities of Metastasis: The Parting of Ways with Real-Time Assays

“…In vitro migration and invasion assays present with similar shortcomings, wherein the heterogeneity and mechanics of these processes are excessively simplified and the modalities of translocation are overlooked. Wound closure assays widely imply cell migration, while disregarding the proliferative potential of the wound edge [99]. Similarly, experimental systems often employ scratch and gap closure assays interchangeably, with a complete disregard for the differential biological annotations represented by each method.…”

“…A recent study from our group, coupling live cell imaging with the in vitro scratch assay, not only discerned the distinct migratory modalities in ovarian carcinoma (CCM versus EMT), but also resolved subtle variations within CCM; this defined CCM as being mediated either through proliferation (passive CCM) or sheet-like migration (active CCM). We quantified the altered migratory modalities in response to extrinsic and intrinsic stimuli, further establishing an improved prototype of the scratch assay [99]. In a separate study, CCM was assessed in the context of durotaxis, wherein live cell imaging successfully quantified the extent and pattern of migration in response to ECM variations [133].…”

“…The extraction of relevant quantitative metrics must consider the biophysical properties of a system and examine the associative trends with the cellular processes under study. Examples highlighting the selection of quantitative metrics are summarized in Table S3, and include the micro-pillar array-based study, wherein six quantitative parameters extracted from individual cells generated a binary solidification model for describing the interconversion between collective versus individual modes of migration [135], fractal analysis that described the developing dermis as a lattice structure resulting from the dynamic stroma [158], derivation of migration modalities in ovarian cancer cell lines assisted by coupling live cell imaging with the in vitro scratch assay [99], and so on. Additional studies highlight the vast array of quantitative measures derived from real-time imaging datasets, and signify their contributions to an improved understanding of the biological processes [11,151,159,160,161,162].…”

Effects of Substrate-Coating Materials on the Wound-Healing Process

“…Then based on these images, wound-healing rates can be calculated. The most common method for creating a wound is to scratch out a cell-free area using a tip or needle [15,16,17]. This scratch wound-healing assay has been commercialized as the CellPlayer Migration Assay by Essen BioScience (Ann Arbor, MI, USA) [18].…”

Comparison of the Inhibitory Effect of PEG-Liposomal and Conventional Doxorubicin on Migration and Extravasation Efficacy on Canine Osteosarcoma Cell Line- in Vitro and Ex Ovo Studies.

“…Wound healing assays serve as useful tools in quantifying alterations in cell migratory capabilities following experimental manipulation with chemotherapeutic agents. (Grada et al 2017) , (Varankar and Bapat 2018) To the best of our knowledge, wound healing assays have not been performed to assess the inhibitory effect of liposomal doxorubicin on the migration of any canine OSA cell line. In this study, PEG-liposomal doxorubicin inhibited the migration of canine osteosarcoma.…”