Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation

We focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository (https://zenodo.org/, Digital Object Identifiers:10.5281/zenodo.1494935)

Cell segmentation methods for label-free contrast microscopy: review and comprehensive comparison

Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities

Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGF ss 1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-a, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators

Quantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light

Increased exposition to blue light may induce many changes in cell behavior and significantly affect the critical characteristics of cells. Here we show that multimodal holographic microscopy (MHM) within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light. We focused on the effect of blue light with a wavelength of 485 nm on morphological and dynamical parameters of four cell lines, malignant PC-3, A2780, G361 cell lines, and the benign PNT1A cell line. We used MHM with blue light doses 24 mJ/cm2, 208 mJ/cm2 and two kinds of expositions (500 and 1000 ms) to acquire real-time quantitative phase information about cellular parameters. It has been shown that specific doses of the blue light significantly influence cell motility, cell dry mass and cell density. These changes were often specific for the malignant status of tested cells. Blue light dose 208 mJ/cm2 × 1000 ms affected malignant cell motility but did not change the motility of benign cell line PNT1A. This light dose also significantly decreased proliferation activity in all tested cell lines but was not so deleterious for benign cell line PNT1A as for malignant cells. Light dose 208 mJ/cm2 × 1000 ms oppositely affected cell mass in A2780 and PC-3 cells and induced different types of cell death in A2780 and G361 cell lines. Cells obtained the least damage on lower doses of light with shorter time of exposition

Magnetically Driven Self-Degrading Zinc-Containing Cystine Microrobots for Treatment of Prostate Cancer

Prostate cancer is the most commonly diagnosed tumor disease in men, and its treatment is still a big challenge in standard oncology therapy. Magnetically actuated microrobots represent the most promising technology in modern nanomedicine, offering the advantage of wireless guidance, effective cell penetration, and non-invasive actuation. Here, new biodegradable magnetically actuated zinc/cystine-based microrobots for in situ treatment of prostate cancer cells are reported. The microrobots are fabricated via metal-ion-mediated self-assembly of the amino acid cystine encapsulating superparamagnetic Fe3O4 nanoparticles (NPs) during the synthesis, which allows their precise manipulation by a rotating magnetic field. Inside the cells, the typical enzymatic reducing environment favors the disassembly of the aminoacidic chemical structure due to the cleavage of cystine disulfide bonds and disruption of non-covalent interactions with the metal ions, as demonstrated by in vitro experiments with reduced nicotinamide adenine dinucleotide (NADH). In this way, the cystine microrobots served for site-specific delivery of Zn2+ ions responsible for tumor cell killing via a "Trojan horse effect". This work presents a new concept of cell internalization exploiting robotic systems' self-degradation, proposing a step forward in non-invasive cancer therapy

Self-propelled magnetic dendrite-shaped microrobots for photodynamic prostate cancer therapy

Photocatalytic micromotors that exhibit wireless and controllable motion by light have been extensively explored for cancer treatment by photodynamic therapy (PDT). However, overexpressed glutathione (GSH) in the tumor microenvironment can down-regulate the reactive oxygen species (ROS) level for cancer therapy. Herein, we present dendrite-shaped light-powered hematite microrobots as an effective GSH depletion agent for PDT of prostate cancer cells. These hematite microrobots can display negative phototactic motion under light irradiation and flexible actuation in a defined path controlled by an external magnetic field. Non-contact transportation of micro-sized cells can be achieved by manipulating the microrobot's motion. In addition, the biocompatible microrobots induce GSH depletion and greatly enhance PDT performance. The proposed dendrite-shaped hematite microrobots contribute to developing dual light/magnetic field-powered micromachines for the biomedical field.Web of Science614

Quantitative phase microscopy timelapse dataset of PNT1A, DU-145 and LNCaP cells with annotated caspase 3,7-dependent and independent cell death

Time-lapse dataset of prostatic cell lines (DU-145, PNT1A, LNCaP) exposed to cell death-inducing compounds (staurosporine, doxorubicin) and black phosphorus. The time-lapse dataset is annotated as follows: (1) cell masks and cell numbers, (2) by cell death type and timepoint of death in the attached xlsx file. This dataset is supplementary to the article: Tomas Vicar, Martina Raudenska, Jaromir Gumulec, Michal Masarik, Jan Balvan. Detection and characterization of apoptotic and necrotic cell death by time-lapse quantitative phase image analysis. bioRxiv, 589697; DOI: https://doi.org/10.1101/589697 Code is available at https://github.com/tomasvicar/CellDeathDetect Methods Cell culture and cultured cell conditions LNCaP cell line was established from a lymph node metastase of the hormone-refractory patient and contains a mutation in the AR gene. This mutation creates a promiscuous AR that can bind to different types of steroids. LNCaP cells are AR-positive, PSA-positive, PTEN-negative and harbor wild-type p53 {Skjoth, 2006 #150; Mitchell, 2000 #149}. PNT1A is immortalized non-tumorigenic epithelial cell line. PNT1A cells harbour wild-type p53. However, SV40 induced T-antigen expression inhibits the activity of p53. This cell line had lost the expression of androgen receptor (AR) and prostate-specific antigen (PSA) (Raudenska, 2019). DU-145 cell line is derived from the metastatic site in the brain and contains P223L and V274F mutations in p53. This cell line is PSA and AR-negative and androgen independent (Chappell, 2012). All cell lines used in this study were purchased from HPA Culture Collections (Salisbury, UK). and were cultured in RPMI-1640 medium with 10 % FBS. The medium was supplemented with antibiotics (penicillin 100 U/ml and streptomycin 0.1 mg/ml). Cells were maintained at 37°C in a humidified (60%) incubator with 5% CO2 (Sanyo, Japan). Correlative time-lapse quantitative phase-fluorescence imaging QPI and fluorescence imaging were performed by using multimodal holographic microscope Q-PHASE (TESCAN, Brno, Czech Republic). To determine the amount of caspase-3/7 product accumulation, cells were loaded with 2 µM CellEventTM Caspase-3/7 Green Detection Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol and visualized using FITC 488 nm filter. To detect the cells with a loss of plasma membrane integrity, cells were stained with 1 ug/ml propidium iodide (Sigma Aldrich Co., St. Louis, MO, USA) and visualized using TRITC 542 nm filter. Nuclear morphology and chromatin condensation were analyzed using Hoechst 33342 nuclear staining (ENZO, Lausen, Switzerland) and visualized using DAPI 461 nm filter. Cells were cultivated in Flow chambers μ-Slide I Lauer Family (Ibidi, Martinsried, Germany). To maintain standard cultivation conditions (37°C, humidified air (60%) with 5% CO2) during time-lapse experiments, cells were placed in the gas chamber H201 - for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). To image enough cells in one field of view, lens Nikon Plan 10/0.30 were chosen. For each cell line and each treatment, seven fields of view were observed with the frame rate 3 mins/frame for 24 or 48 h respectively. Holograms were captured by CCD camera (XIMEA MR4021 MC-VELETA), fluorescence images were captured using ANDOR Zyla 5.5 sCMOS camera. Complete quantitative phase image reconstruction and image processing were performed in Q-PHASE control software. Cell dry mass values were derived according to {Prescher, 2005 #177} and {Park, 2018 #178} from the phase (eq. (1)), where m is cell dry mass density (in pg/μm2), φ is detected phase (in rad), λ is wavelength in μm (0.65 μm in Q-PHASE), and α is specific refraction increment (≈0.18 μm3/pg). All values in the formula except the Phi are constant. Phi (Phase) is the value measured directly by the microscope. Integrated phase shift through a cell is proportional to its dry mass, which enables studying changes in cell mass distribution (Park et al., 2018). File description There are three archives included for particular cell lines: QPI_annotated_timelapse_DU145.zip for DU-145 cells QPI_annotated_timelapse_PNT1A.zip for PNT1A cells QPI_annotated_timelapse_LNCaP.zip for LNCaP cells The archive includes of following files: Tiff with time-lapse quantitative phase image (32-bit files 600x600px with values in pg/um2 with framerate 1 frame/3minutes with 1.59 px/um), named QPI_cellline_treatment_FOV.tiff Tiff file with segmentation mask for particular cells named mask_cellline_treatment_FOV.tiff xlsx table with cell death type (1 for apoptosis, 2 for necrosis, 3 for ambiguous/surviving) and time of death for representative cell number from mask, named labels_cellline_treatment_FOV.xlsx file naming has following conventions: cell names: DU145, PNT1A, LNCaP for particular cell line treatments: st, bp, do for staurosporine, black phosphorus and doxorubicin fields of view: 1 to 7 e.g. QPI_DU145_st_4.tif, mask_DU145_st_4.tif, labels_DU145_st_4.xls

Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation: dataset of confocal and atomic force microscopy

Summary Dataset of imaging data of the experiment "Cisplatin enhances cell stiffness: Biomechanical profiling of prostate cancer cells". This dataset includes image data of atomic force microcopy (Young modulus) and confocal microscopy(staining of F-actin and β-tubulin) of prostate cell lines PNT1A, 22Rv1, and PC-3. Materials and Methods Cells, cell culture conditions 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. Long-term zinc (II) treatment of cell cultures 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. Actin and tubulin staining 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. Confocal microscopy 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. Atomic force microscopy 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. Identification of files Files are separated into individual zip files. The dataset of confocal microscopy 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 atomic force microscopy (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