DOI: 10.5281/zenodo.1414528
title: Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation: Confocal and atomic force microscopy

description: <strong>Summary</strong>

Dataset of imaging data related to the publication  Raudenska, M., Kratochvilova, M., Vicar, T., Gumulec, J., Balvan, J., Polanska, H. Pribyl, J. & Masarik, M.:Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation. <em>Scientific Reports </em><strong>2019, </strong>9, 1660

This dataset includes image data of <em>atomic force microcopy</em> (Young modulus) and <em>confocal microscopy</em>(staining of F-actin and β-tubulin) of prostate cell lines PNT1A, 22Rv1, and PC-3. 

<strong>Materials and Methods</strong>

<em>Cells, cell culture conditions</em>

Cells confluent up to 50–60% were washed with a FBS-free medium and treated with a fresh medium with FBS and required antineoplastic drug concentration (IC50 concentration for the particular cell line). The cells were treated with 93 µM (PC-3), 38 µM (PNT1A), and 24 µM (22Rv1) of cisplatin (Sigma-Aldrich, St. Louis, Missouri), respectively. IC50 concentrations used for treatment with docetaxel (Sigma-Aldrich, St. Louis, Missouri) were 200nM for PC-3, 70nM for PNT1A, and 150nM for 22Rv1. 

<em>Long-term zinc (II) treatment of cell cultures</em>

Cells were cultivated in the constant presence of zinc(II) ions. Concentrations of zinc(II) sulphate in the medium were increased gradually by small changes of 25 or 50 µM. The cells were cultivated at each concentration no less than one week before harvesting and their viability was checked before adding more zinc. This process was used to select zinc resistant cells naturally and to ensure better accumulation of zinc within the cells (accumulation of zinc is usually poor during the short-term treatment of prostate cancer cells). Total time of  the cultivation of cell lines in the zinc(II)-containing media exceeded one year. Resulting concentrations of zinc(II) in the media (IC50 for the particular cell line) were 50 µM for the PC-3 cell line, 150 µM for the PNT1A cell line, and 400 µM for the 22Rv1 cell line. The concentrations of zinc(II) in the media and FBS were taken into account. 

<em>Actin and tubulin staining</em>

β-tubulin was labeled with anti- β tubulin antibody [EPR1330] (ab108342) at a working dilution of 1/300. The secondary antibody used was Alexa Fluor® 555 donkey anti-rabbit (ab150074) at a dilution of 1/1000. Actin was labeled with Alexa Fluor™ 488 Phalloidin (A12379, Invitrogen); 1 unit per slide. For mounting Duolink® In Situ Mounting Medium with DAPI (DUO82040) was used. The cells were fixed in 3.7% paraformaldehyde and permeabilized using 0.1% Triton X-100. 

<em>Confocal microscopy</em>

The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm.

<em>Atomic force microscopy</em>

We used the bioAFM microscope JPK NanoWizard 3 (JPK, Berlin, Germany) placed on the inverted optical microscope Olympus IX‑81 (Olympus, Tokyo, Japan) equipped with the fluorescence and confocal module, thus allowing a combined experiment (AFM‑optical combined images). The maximal scanning range of the AFM microscope in X‑Y‑Z range was 100‑100‑15 µm. The typical approach/retract settings were identical with a 15 μm extend/retract length, Setpoint value of 1 nN, a pixel rate of 2048 Hz and a speed of 30 µm/s. The system operated under closed-loop control. After reaching the selected contact force, the cantilever was retracted. The retraction length of 15 μm was sufficient to overcome any adhesion between the tip and the sample and to make sure that the cantilever had been completely retracted from the sample surface. Force‑distance (FD) curve was recorded at each point of the cantilever approach/retract movement. AFM measurements were obtained at 37°C (Petri dish heater, JPK) with force measurements recorded at a pulling speed of 30 µm/s (extension time 0.5 sec).

The Young's modulus (E) was calculated by fitting the Hertzian‑Sneddon model on the FD curves measured as force maps (64x64 points) of the region containing either a single cell or multiple cells. JPK data evaluation software was used for the batch processing of measured data. The adjustment of the cantilever position above the sample was carried out under the microscope by controlling the position of the AFM‑head by motorized stage equipped with Petri dish heater (JPK) allowing precise positioning of the sample together with a constant elevated temperature of the sample for the whole period of the experiment. Soft uncoated AFM probes HYDRA-2R-100N (Applied NanoStructures, Mountain View, CA, USA), i.e. silicon nitride cantilevers with silicon tips are used for stiffness studies because they are maximally gentle to living cells (not causing mechanical stimulation). Moreover, as compared with coated cantilevers, these probes are very stable under elevated temperatures in liquids – thus allowing long-time measurements without nonspecific changes in the measured signal.

<em>Image analysis</em>

Fluorescence microscopy data were analyzed in ImageJ 1.52h and Python 3.7.1 as follows: cells were manually segmented using actin fluorescence channel, two regions were created for analysis: whole cell and cell periphery, lining a 4 μm thick region around cell border and including most of periphery actin cytoskeleton. In these two regions following parameters were measured for both actin and tubulin fluorescence: Integrated intensity, median intensity, and following regions were measured to describe cell morphology: Cell area, Maximum caliper (max feret diameter), roundness, and aspect ratio. Moreover, stress fibers were manually segmented in every cell and following parameters were measured: number of fibers per cell, feret angle of fiber, integrated intensity, fiber length, mean intensity. Next, a standard deviation of feret angles of individual fibers was calculated relatively to mean of feret angle using a circstd function from scipy package for Python.

<strong>Identification of files</strong>

<em>Microscopy data</em>

Files are separated into individual zip files. The dataset of <em>confocal microscopy </em>is separated based on treatments: untreated control, docetaxel-treated cells, cisplatin-treated cells, zinc-treated cells. Filenames actin_tubulin_Zstack_cisplatin.zip, actin_tubulin_Zstack_untreated_control.zip, actin_tubulin_Zstack_zinc.zip, actin_tubulin_Zstack_docetaxel.zip. Files included in these ZIP archives are named as follows: "cellline_treatment_FOV". Files are 3-layer 16bit tiff files with layer sequence as follows: F-Actin (Phalloidin)/b-tubulin/Hoechst 33342. The dataset contains 242 FOVs of three cell line types/three treatments + one control, files are Z-stacks made of 50 slices.

The dataset of <em>atomic force microscopy </em>(AFM) is included in one ZIP archive "AFM_YoungModulus_SetpointHeight.zip", which includes data on Young modulus and Setpoint Height of cell lines 22Rv1, PNT1A and PC-3 and treatments zinc, docetaxel, cisplatin (+control), i.e. identical like for confocal microscopy. The file naming is as follows: "AFM_cellline_treatment_FOV_Youngmodulus.tif"  for Young modulus and "AFM_cellline_treatment_FOV_setpointheight.tif" for setpoint height. The data are filtered 32-bit tiff images, where the pixel value correspond to cell stiffness (young modulus) in Pa or setpoint height in m.

<em>Confocal microscopy analysis files</em>

Following files are csv tables including image analysis of actin/tubulin staining captured by confocal microscope:

Cytoskeleton_fluo_analysis_Cell_Cell_periphery_morphology.csv: table includes analyzed data for actin and tubulin staining in following cellular regions: cell, cell periphery. Standard ImageJ parameters regarding intensity and morphology included.

Cytoskeleton_fluo_analysis_Fibers.csv: table includes results of manual segmentation and consequent analysis of actin stres fibers in the cells. Apart from standard ImageJ parameters, also number of stress fibers per cell and standard deviation of fiber angle relative to the cell mean angle (for details see methods) are included.
description: This work was supported by funds from the Faculty of Medicine, Masaryk University to Junior researcher (Martina Raudenska), by Grant Agency of the Czech Republic (18-24089S) and by CIISB research infrastructure project LM2015043 funded by MEYS CR (support to AFM measurements). We thank Dr. Martin Falk from the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic for the performance of confocal microscopy and Marek Feith and Tomas Slaby from Masaryk University, Brno for analysis and processing of fluorescence microscopy data.
description: {"references": ["Raudenska M, Kratochvilova M, Vicar T, Gumulec J, Balvan J, Polanska H, Pribyl J, Masarik M: Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation. Scientific Reports 2019, 9:1660"]}