Metastatic microtissues were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) for 40 min, after either 3 or 24 hours of coculture in the various conditions. SDC microscopy (X-1 Yokogawa with Borealis modification), fitted with a 10× Plan Apo objective and a red channel filter set (excitation 561 nm), was used to collect z-stacks of the coculture microtissues. Maximal intensity projections were made from z-stacks using 1 μm as the step size and 70 μm as the thickness (>2 microtissues per condition analyzed with >4 fields of view, for 365 cells analyzed per condition on average). Cancer cell volume and sphericity were obtained from Imaris imaging analysis software (version 9.1.0, Bitplane AG, Zurich, Switzerland) according to the algorithm of (16 (link)).
Imaris imaging analysis software
Manufactured by Oxford Instruments
Sourced in United Kingdom, Switzerland
Imaris imaging analysis software is a powerful tool for visualizing and analyzing 3D and 4D microscopy data. It provides a comprehensive suite of image processing and analysis functions to help researchers extract meaningful insights from their data.
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5 protocols using imaris imaging analysis software
1
Imaging and Analysis of Metastatic Microtissues
2
3D Co-culture Microtissue Imaging
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Co-culture microtissues were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) for 40 min, after 24 h of co-culture in PCa-Norm or PCa-AD media, and stained for DAPI and phalloidin. SDC was used to image the cancer cells (mKO2, red), the nuclei (DAPI, blue), and the F-actin filaments (phalloidin, green). The 10 × Plan Apo objective was used, with the red (ex 561 nm), green (ex 488 nm), and blue (ex 405 nm) filter sets. Maximal intensity projections were made from z-stacks using 1 µm as step size and 70 µm thickness (>2 microtissues/condition analyzed with > 3 fields of view, for ~335 cells analyzed/condition). Cancer cell volume and shape factor were obtained from Imaris imaging analysis software (version 9.1.0, Bitplane AG, Zurich, Switzerland) and cancer cell orientation on hOBMT was obtained from ImageJ software (1.51j8. NIH, USA,55 (link)). Algorithm details are found in the Methods Supplement.
3
Measuring Cell Volume Dynamics
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To measure the volumes of COLO205, HT29, HCT116 and LoVo cells ± selumetinib and their selumetinib-resistant derivatives ± selumetinib, cells were plated in their normal growth medium in chamber slides (Thistle Scientific, Glasgow, UK) and 24 h later washed with media only and treated with or without selumetinib for a further 72 h. Cells were then incubated for 5 min with CellMask Orange Plasma Membrane Stain (Thermo Fisher Scientific, Loughborough, UK), which was added directly to the media at a dilution of 1:1000. Cells were then imaged using an Andor Revolution confocal spinning disk system (Andor, Oxford Instruments, Oxfordshire, UK) taking 100 images per cell, over a depth of 50 µm and a step interval of 0.5 µm. At least 300 cells were imaged per condition and cell volumes were determined using Imaris imaging analysis software (Bitplane, Oxford Instruments, Oxfordshire, UK).
4
Quantifying Neuronal Mitochondrial Abundance
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All imaging analysis was done using coded cell lines, so the observer was blinded to the genotypes. Only after data collection and analysis were cell lines decoded. For each cell line (≥ 5 individuals per group), ≥15 neurons were imaged for analysis of mitochondrial abundance within the neuron. To quantify mitochondrial area, cells were labeled with anti-Beta Tubulin (total neuronal area) and anti-TOMM20 (mitochondrial area), images were loaded into the Imaris imaging analysis software (Oxford Instruments, Abingdon, United Kingdom) and surfaces for each marker were created (see Figure 6 ). The mitochondrial area (anti-TOMM20) was then divided by the total neuronal area (anti-Beta Tubulin) to give the percentage of the neuron that contains mitochondria. After collecting all data, significance testing was performed by both ordinary one-way ANOVA and Turkey’s multiple comparisons test with a single pooled variance.
5
Quantifying Neuronal Mitochondrial Density
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All imaging analysis was done using coded cell lines, so the observer was blinded to the genotypes. Only after data collection and analysis were cell lines decoded. For each cell line (>5 individuals per group), >15 neurons were imaged for analysis of mitochondrial abundance within the neuron. To quantify mitochondrial area, cells were labeled with α-Beta Tubulin (total neuronal area) and α-TOMM20 (mitochondrial area), images were loaded into the Imaris imaging analysis software (Oxford Instruments, Abingdon, UK) and surfaces for each marker were created (see Fig. 6). The mitochondrial area (α-TOMM20) was then divided by the total neuronal area (α-Beta Tubulin) to give the percentage of the neuron that contains mitochondria. After collecting all data, significance testing was performed by both ordinary one-way ANOVA and Turkey's multiple comparisons test with a single pooled variance.
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