Abstract
Bioengineered three-dimensional (3D) tumor models that incorporate heterotypic cellular communication are gaining interest as they can recapitulate key features regarding the intrinsic heterogeneity of cancer tissues. However, the architectural complexity and heterogeneous contents associated with these models pose a challenge for toxicological assays to accurately report treatment outcomes. To address this issue, we describe a comprehensive image analysis procedure for structurally complex organotypic cultures (CALYPSO) applied to fluorescence-based assays to extract multiparametric readouts of treatment effects for heterotypic tumor cultures that enables advanced analyses. The capacity of this approach is exemplified on various 3D models including adherent/suspension, mono-/heterocellular cultures and several disease types. The subsequent analysis revealed specific morphological effects of oxaliplatin chemotherapy, radiotherapy, and photodynamic therapy. The procedure can be readily implemented in most laboratories to facilitate high-throughput toxicological screening of pharmaceutical agents and treatment regimens on organotypic cultures of human disease to expedite drug and therapy development.
Highlights
Background subtractionTo limit the role of the cell debris or detached cells in the quantification of the background intensity, images from the no treatment group were used and the mask was slightly dilated, using a disk shaped structuring element with a radius of 2px
We demonstrate the capacity of CALYPSO to extract multiparametric readouts from various 3D culture methods and disease types as exemplified on (1) adherent and (2) suspended 3D cultures of pancreatic cancer, as well as (3) an adherent 3D culture model of micrometastatic ovarian carcinoma
In order to provide a robust treatment response assay for architecturally complex 3D tumor models, we established an image analysis workflow that enables the extraction of functional area- and fluorescence intensity-based parameters
Summary
Background subtractionTo limit the role of the cell debris or detached cells in the quantification of the background intensity, images from the no treatment group were used and the mask was slightly dilated, using a disk shaped structuring element with a radius of 2px. To extract the residual live area of each nodule, the calcein AM fluorescence intensity was binarized for quantification of the absolute live area and fractional live area of the tumor organoids. For this step, a fixed threshold value was applied to all images. The threshold value was obtained by applying Otsu’s method to the no treatment group, which was multiplied by a factor of 0.4 to facilitate optimal analysis of our images, i.e., to set up the dynamic range for the fractional live area between 0 (based on TK controls) and 1 (based on no treatment controls). The amount of pixels exceeding the threshold was summed to provide the area of the live residual disease per tumor organoid
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