Abstract
The quantification of invasion and migration is an important aspect of cancer research, used both in the study of the molecular processes involved in this collection of diseases and the evaluation of the efficacy of new potential treatments. The transwell assay, while being one of the most widely used techniques for the evaluation of these characteristics, shows a high dependence on the operator’s ability to correctly identify the cells and a low protocol standardization. Here we present I-AbACUS, a software tool specifically designed to aid the analysis of transwell assays that automatically and specifically recognizes cells in images of stained membranes and provides the user with a suggested cell count. A complete description of this instrument, together with its validation against the standard analysis technique for this assay is presented. Furthermore, we show that I-AbACUS is versatile and able to elaborate images containing cells with different morphologies and that the obtained results are less dependent on the operator and their experience. We anticipate that this instrument, freely available (Gnu Public Licence GPL v2) at www.marilisacortesi.com as a standalone application, could significantly improve the quantification of invasion and migration of cancer cells.
Highlights
In this work the focus is on the in-vitro study of migration and invasion and on the transwell assay, one of the most common experiments used to quantify these processes[3,4,5,6], with 4198 papers in the last five years associated with the keywords “transwell assay” or “Boyden chamber” on Pubmed
In the traditional analysis the object counting functionality of ImageJ was used, while with I-AbACUS the images were elaborated following the standard workflow described in the Methods section
We have presented I-AbACUS, a computational tool designed to aid the analysis of transwell invasion/ migration assays through the automatic segmentation of the cells and the suggestion of a cell count
Summary
In this work the focus is on the in-vitro study of migration and invasion and on the transwell assay, one of the most common experiments used to quantify these processes[3,4,5,6], with 4198 papers in the last five years associated with the keywords “transwell assay” or “Boyden chamber” on Pubmed (database accessed in January 2017). As a consequence results can vary significantly with the operator’s ability to distinguish the cells from debris and pores, a task that can be quite complex especially as the number of cells in each field increases or if they exhibit a range of different phenotypes Another important aspect is the lack of standardization of the assay. This technique requires an extensive human intervention that might make it excessively time consuming for the analysis of transwell assays, where a large number of images must generally be segmented To address these limitations and improve the in-vitro quantification of migration and invasion, we have developed I- AbACUS (Invasion Assay Assisted Cell coUnting Software) a custom-made software that allows users to standardize the counting process, applying the same procedure to every image. We compare I-AbACUS and the standard approach for the analysis of transwell assays, and demonstrate the key advantage of I-AbACUS in overcoming the diversity of operator, making the results obtained with I-AbACUS reliable and robust
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