S3_File: ground truth manual segmentation data on 2D MIP images

This 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

ReadMe

The 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

S5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with docetaxel, well E08, field 3

3D 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)

S5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with docetaxel, well E08, field 1

3D 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)

S2_File: tiff image stack of the example 3D spheroid culture used in figures 1-4 and figure 6

3D 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)

S5_File: 3D image stack of PC346c cancer spheroids in co-culture with CAFs, treated with MDV, well F05, field 4

3D 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)

Comparison of the proposed analysis method with a baseline 2D MIP analysis method.

<p>Spheroid cell culture proliferation, quantified by EdU positive cells, from a 3D homogeneous spheroid culture treated with a cytotoxic compound (Docetaxel, 1e-8 M) and a cytostatic compound (MDV-3100, 1e-7 M). In (a) the spheroid volume is shown, and in (b) the number of EdU positive cells per spheroid. In (c) the total number of EdU positive cells per image volume is shown, where the size of the dots represent the total foreground / background ratio of the MIP of the image stack. In (d), the EdU positive cell count, based both on the proposed and a 2D MIP analysis method, is compared. The normalized difference in EdU positive cells, weighted with the spheroid volume, is shown.</p

Light attenuation in multi-cellular spheroids.

<p>(a) Top view from the xy-plane of a spheroid with an ellipsoid fitted, where the RFP (561 nm), Hoechst (405 nm), and EdU (640 nm) channels are shown. (b) The same spheroid, in a side view (xz-plane). No signal is detected from the lower part of the spheroid (assuming that the spheroid is of an ellipsoidal shape). (c) Parameters of the spheroid derived from the vertical profile curve of the RFP signal through the ellipsoid center: top z-coordinate, maximum intensity, analyzable depth (corresponding to the user-defined minimum intensity percentage).</p

S4_File: 3D image stack, LNCaP cancer spheroids in co-culture with CAFs, control sample (DMSO), well A07, field 3, stack 339

3D 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)