High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

Abstract

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no readyto-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application -SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary "Manual Initialize" and "Hand Draw" tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

TASI: A software tool for spatial-temporal quantification of tumor spheroid dynamics

“…SpheroidSizer [26] Both No Open source, Matlab Celigo [5,22,25] ; Imaris & Velocity [24] ; VTT_Acca [4] Both No [5,22,24,25] ; No mathematical models, and statistical tests are performed to compare parameters across conditions, and visualizations are generated. These steps are described in greater detail below.…”

“…Software for spheroid image analysis has largely focused on static images generated by high throughput screening [4][5][6][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Existing software programs for analyzing spheroid imaging are described in Table 1.…”

“…Software for measuring spheroid dynamics has received relatively less attention [4,13,[23][24][25][26][27][28][29]]. An interactive system for segmenting and measuring spheroid volume and dimensions was developed in [26]. Of the software packages available for spheroid analysis, none provides end-to-end statistical analysis and visualization of imaging measurements.…”

ImageJ macros for the user-friendly analysis of soft-agar and wound-healing assays

“…For instance, microscopy is an important tool for analyzing changes in cell morphology in response to specific agents, but the amount of data generated by automated techniques greatly exceeds manual processing capacity (2). …”

“…These techniques are necessarily subjective since the researcher has to manually trace the regions of interest in each image (i.e., areas lacking cells or the areas of the colonies). This is particularly problematic for large-scale drug screening experiments with hundreds of images, when the task becomes laborious and time-consuming (2). The algorithms presented here stand out because they overcome these two problems, allowing precise, rapid, and unbiased analyses of both assays.…”