DOI: 10.5061/dryad.0m9n7
title: Data from: Ellipsoid segmentation model for analyzing light-attenuated 3D confocal image stacks of fluorescent multi-cellular spheroids

description: In oncology, two-dimensional in-vitro culture models are the standard test
 beds for the discovery and development of cancer treatments, but in the
 last decades, evidence emerged that such models have low predictive value
 for clinical efficacy. Therefore they are increasingly complemented by
 more physiologically relevant 3D models, such as spheroid micro-tumor
 cultures. If suitable fluorescent labels are applied, confocal 3D image
 stacks can characterize the structure of such volumetric cultures and, for
 example, cell proliferation. However, several issues hamper accurate
 analysis. In particular, signal attenuation within the tissue of the
 spheroids prevents the acquisition of a complete image for spheroids over
 100 micrometers in diameter. And quantitative analysis of large 3D image
 data sets is challenging, creating a need for methods which can be applied
 to large-scale experiments and account for impeding factors. We present a
 robust, computationally inexpensive 2.5D method for the segmentation of
 spheroid cultures and for counting proliferating cells within them. The
 spheroids are assumed to be approximately ellipsoid in shape. They are
 identified from information present in the Maximum Intensity Projection
 (MIP) and the corresponding height view, also known as Z-buffer. It alerts
 the user when potential bias-introducing factors cannot be compensated for
 and includes a compensation for signal attenuation.
description: S2_File: tiff image
 stack of the example 3D spheroid culture used in figures 1-4 and figure
 63D
 image stack of 3D fluorescent multi-cellular spheroid culture existing out
 of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T
 human cancer associated fibroblasts (a cell line obtained from the
 Weizmann Institute, via the PREDECT consortium).S2_File.tifS3_File: ground truth manual segmentation data on 2D MIP imagesThis is a zip-file containing manually segmented 2D ground truth labeled masks of 3D spheroid cultures. The cultures are LNCaP cancer spheroids in co-culture with CAF stromal cells (untreated, DMSO samples). For the manual segmentation 2D maximum intensity projections (MIPs) were used of the original 3D stacks. The spheroids are labeled in five distinct classes: (1) well separated, (2) overlapping with brighter spheroids in the MIP (rendering it non-separable), (3) overlapping with less bright spheroids in the MIP (rendering it well separable), (4) merely touching other spheroids, and (5) touching the border of the 2D projection of the image. Opening of the images in FIJI (ImageJ) with the ROI Manager allows visual inspection of the data. For this purpose the cases (1) to (5) are color-coded in red, green, magenta, cyan, and blue, respectively.S3_File.zipS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A06, field 13D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_001.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A06, field 23D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_002.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A06, field 33D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_003.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A06, field 43D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_004.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A08, field 13D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_001.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A08, field 23D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_002.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A08, field 33D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_003.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, control sample (DMSO), well A08, field 43D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_004.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with docetaxel, well E08, field 13D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_001.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with docetaxel, well E08, field 23D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_002.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with docetaxel, well E08, field 33D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_003.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with docetaxel, well E08, field 43D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_004.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with MDV, well F05, field 13D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_001.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with MDV, well F05, field 23D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_002.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with MDV, well F05, field 33D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_003.tifS5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with MDV, well F05, field 43D image stack of 3D fluorescent multi-cellular spheroid culture existing out of PC346c prostate cancer cells (a cell line obtained from the Erasmus Medical Center, via the PREDECT consortium) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).field_004.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A05, field 1, stack 3333D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_333.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A05, field 2, stack 3343D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_334.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A05, field 3, stack 3353D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_335.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A05, field 4, stack 3363D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_336.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A07, field 1, stack 3373D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_337.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A07, field 2, stack 3383D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_338.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A07, field 3, stack 3393D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_339.tifS4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A07, field 4, stack 3403D image stack of 3D fluorescent multi-cellular spheroid culture existing out of LNCaP human prostate cancer cells (ATCC, Rockville, USA) and CAF-PF179T human cancer associated fibroblasts (a cell line obtained from the Weizmann Institute, via the PREDECT consortium).stack_340.tifReadMeThe ReadMe file describes the 3D image data contained in this data repository.S6 File. Spot detection algorithm illustrated on the 3D image stack in S2 File.This is a multi-page tiff file which shows the EdU channel of the 3D image stack in the supplement S2 File, with the EdU positive cells annotated by small spheres. This image can serve as an example result of the spot detection algorithm (3D version). Similar to the image stack in the supplement S2 File, this image stack can be opened with an application such as ImageJ.S6_File_v2.tif