Shear Stress-Induced Pre-Cytokinetic Block: A New Cellular Response Revealed by an Innovative Shear Stress Generator

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To overcome the current methodological limitations of such research, here we present a new device that replicates similar conditions by applying shear stress on cultured cells. The device provides a less complex, easily accessible alternative to traditional microfluidics while generating fluid shear stress values comparable to those in human veins and capillaries. The device allows analyses of large cell numbers in standard cell culture flasks and incubators. Using this device to explore the shear stress-induced responses of various human cell lines, we discovered a previously unknown, reversible pre-cytokinetic block occurring in cells that lose anchorage during mitosis and are kept under constant shear stress. Notably, some cancer cell lines appear to bypass this unorthodox cell-cycle block, suggesting its role as a safety checkpoint to restrict the proliferation of cancer cells in the bloodstream and their overall spreading potential. These findings provide new insights into the diverse responses of normal and cancer cells to shear stress and highlight the potential of our technology for research on circulating tumor cells and metastatic spread. Biological sciences/Biological techniques Biological sciences/Biological techniques/Biological models Biological sciences/Biological techniques/Biological models/Cancer models Health sciences/Medical research Health sciences/Medical research/Pre clinical studies fluid shear stress circulating tumor cells microfluidic systems metastasis mitosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cancer metastasis, an often incurable, terminal disease stage, is responsible for approximately 90% of cancer-related deaths 1 . Cancer cells are subjected to numerous factors and stressors throughout their journey from primary tumors to distal tissues, including specific physical forces. Fluid shear stress, experienced within the blood and lymphoid vessels and interstitial fluid, significantly impacts circulating cancer cells 2 , 3 . Despite its acknowledged importance, the effect of shear stress on cancer cells, and more broadly on human cells, is not well understood 4 . This gap in our knowledge is primarily due to currently limited technological possibilities. Aside from sophisticated single-cell analysis methods applied to cells isolated from a patient's peripheral blood circulation 5 , 6 , most studies addressing shear stress rely on microfluidic systems that closely simulate fluid circulation in vessels. However, these systems typically aren't compatible with suspension cells, limiting their application to adherent cells only 2 , 7 . A further constraint of microfluidics is the limited number of cells that can be examined at any given time. This results in a limited understanding of the biology of circulating cells, including circulating tumor cells (CTCs). Our present study aims to fill this crucial knowledge gap by designing, constructing, validating, and applying a novel device - the shear stress generator (SSG), an experimental tool capable of mimicking the physiological fluid shear stress conditions the cells experience in blood vessels. We document here that this pioneering technology promises to provide new insights into how shear stress impacts the behavior of cancer cells, thereby paving the way for a better understanding of cancer metastasis and contributing to development of more effective anticancer treatment strategies. Leveraging this versatile approach, here we have examined the impact of shear stress on significantly larger numbers of cells and discovered a previously unknown biological phenomenon critical for the fate of the mitotic cells that are transiting into a suspension mode. Results Construction and characterization of the shear stress generator (SSG) The device is composed of a clip-like platform that holds a standard 150 cm 2 cell culture flask connected to the rotor of a stepper motor (Fig. 1 a,b). A custom-made program drives the stepper motor to oscillate the flask in a horizontal plane. The final optimized settings for the flask movement—used in all subsequent experiments—involve rotor oscillations (i.e., flask movement) of ± 35° for 2.47s. This movement acceleration triggers a recurring wave of media within the flask, reaching a surface flow speed of approximately 0.8 m/s, as determined by image analysis of floating air bubbles (Fig. 2 a). The flow speed's primary component aligns with the flask's direction. However, near the flask's neck, a perpendicular component to this motion deviates the overall trend from perfect linearity. Upon hitting the flask's sidewalls, the wave also generates slight turbulence accompanied by bubble formation. Overall, this setup offers a relatively straightforward analogy to various mechanical forces expected within the venous system. We used slow-motion camera footage of dispersed air bubbles spontaneously forming in the culture medium during the operation to provide a detailed analysis of the forces induced by the media movement. The measured bubble accelerations were recalculated into shear stress values, peaking at 10 dyne/cm 2 with a mean value of 1.8 dyne/cm 2 (Fig. 2 b). Importantly, such shear stress aligns with the levels reported for the human venous system 8 , 9 , 10 . An essential feature of our experimental setup is its long-term stability, capable of continuous operation for several days without any failures or undesirable changes in the movement and thus shear stress values. The system also requires a relatively low amount of energy—approximately 7W for two 150cm 2 flasks—making it suitable for use in cell incubators, which are not commonly equipped with active cooling and thus limit the placement of high-power devices. Biological Relevance and Impact of Generated Shear Stress Following the mechanical characterization of the device, we next investigated the occurrence of some typical shear stress-induced phenotypes on cultured mammalian cells. Initially, we evaluated the elongation of endothelial cells (ECs) according to the flow vector 11 . This physiological response naturally occurs in the vasculature and can be simulated in microfluidic systems using a specialized cultivation chamber, pumps, and valves to ensure a defined media flow 8 . Indeed, the media flow under our standardized SSG experimental conditions was sufficient to trigger this typical cellular elongation process (Fig. 3 a,b). In order to see if cells are perceiving shear stress at the molecular level, we examined selected ECs' mechanosensory and signaling pathways. Several pathways have been established in flow sensing by EC 12 . These mechanosensitive pathways become activated within seconds to hours and may return to baseline state within 24 hours of exposure to flow 13 – 18 . First, we determined the expression of general fluid flow markers in EC: ICAM-1 and VCAM-1 in two typical time points reflecting expression changes (48 hours) and adaptation of the cells to the flow (72 hours). Whereas the ICAM-1 transcript showed a transient increase at 48 hours, returning to normal levels at 72 hours, the VCAM-1 transcript stayed permanently elevated (Fig. 4 a). Such data can be interpreted to mean cells indeed perceived the flow to which they adapted (ICAM-1) 15 ; however, the flow has rather a turbulent character, at least to some extent, as demonstrated by persistently elevated VCAM 19 over 72 hours. Further, the immediate shear stress perception onset was assayed through the phosphorylation status of major protein kinases involved in response to flow 16 – 18 . The Erk1/2 kinase showed transient phosphorylation within five minutes from flow onset. The maximum phosphorylation of Akt kinase was delayed to 30 minutes – 2 hours. Instead, the phosphorylation of JNK and P38 kinases showed a steady increase up to 6 hours of the experiment duration (Fig. 4 b). Interestingly, the early transient phosphorylation of ERK1/2 indicates the perception of steady flow 16 , whereas the phosphorylation pattern of Akt, JNK and P38 kinases was consistent with the general perception of flow 17 , 18 . Taken together, the cells were able to sense the shear stress generated by the device. Such shear stress was rather perceived as a blend of laminar and turbulent characteristics. In summary, these findings suggest that the presented device is applicable in various studies assessing cellular responses to shear stress and thus provides a possible alternative or an addition to experimental strategies currently using complicated, less versatile, and costly microfluidic systems. Fluid Shear Stress Induces a Pre-Cytokinetic Block If exposed to the mechanical forces generated by our device, several adherently growing cell lines release cells into suspension during mitosis. This effect is due to the relatively weaker adhesion of mitotic cells to the cultivation surface, commonly referred to as mitotic shake-off 20 . Importantly, this mechanism might be highly relevant for potential tumor cell dissemination. At least some circulating tumor cells (CTCs) may originate from mitotic cells dislodged from the primary tumor just by the mechanical forces caused by blood or plasma flow. Taking advantage of our device's compatibility with cells in suspension and long-term applications, we followed the fate of the released mitotic cells under continuous fluid shear stress conditions. In human primary fibroblast strain BJ, as well as some cancer cell lines such as U-2-OS osteosarcoma, MDA-MB-231, MCF7 and BT549 breast cancers, and A549 lung cancer, the released mitotic cells were unable to complete the final phase of cytokinesis which under normal circumstances leads to daughter cells separation. The resulting binucleate-like cells were subjected to microscopic analysis, revealing incomplete cleavage with a persisting actin-myosin contractile ring (Fig. 5 a,b). Next, we tested the ability of these bi-nucleated cells to survive under conditions mimicking those that cells likely experience in the bloodstream, i.e. under continuous shear stress cultivation over prolonged periods. For this experiment, we collected the bi-nucleated cells after 18 hours of cultivation under shear stress conditions. Cells were counted, placed into a new cultivation flask, and cultivated for another 24 and 48 hours. The number of cells was then compared to that at the starting point of this long-term experiment. In all models tested, the prolonged cultivation of bi-nucleated cells under continuous shear stress turned to be highly cytotoxic (Fig. 6 a), suggesting an important role of this newly identified cell cycle pre-cytokinetic block in regulating cell survival under shear stress and thereby possibly limiting the potential of cancerous cells for metastatic spread. Interestingly, and in contrast to the cell models mentioned above, some of the other human cancer cell lines that we tested, including SiHA and HeLa S3 (established from a cervical carcinoma), PC3 (prostate cancer), and Cal51 (breast cancer), showed a different behavioral pattern. Thus, while the latter cancer models also accumulated cells in suspension under continuous shear stress conditions, the occurrence of binucleated cells with incomplete cytokinesis within the suspension fraction, as described above, was sporadic, reflecting a relatively normal mitotic process resulting into timely and complete daughter cell separation. After exposing such cellular suspensions to continuous shear stress over prolonged periods, we found a cytostatic rather than cytotoxic effects for SiHA, PC3, and Cal51 cell lines and surprisingly normal ongoing proliferation for HeLa S3 cells (Fig. 6 b). In conclusion, our findings presented in this section show that most human cell lines of our panel subjected to continuous shear stress release mitotic cells, and furthermore, the shear stress triggers mechanisms that interrupt the cell cycle just before cytokinesis. At the same time, the released cells are incapable of long-term survival in such a state. On the other hand, some cancer cells can bypass this safety mechanism, which might have significant consequences for tumor dissemination potential. Reversibility of the Pre-Cytokinesis Cell Cycle Block As nothing is known about this pre-cytokinesis cell cycle block induced by shear stress, we next examined its reversibility. We used U-2-OS cells as a model cell line and cultivated them on the shear stress device for 18h. The suspension fraction of cellular doublets was collected and immediately seeded. The re-seeded cells reattached and completed the cell separation process (cytokinesis). Subsequently, such divided cells entered the S phase and resumed relatively synchronized cell growth, as evidenced by the flow cytometry analysis of the DNA content at different time points (Fig. 7 a). We also performed a colony formation assay (CFA) on these cells, including cells exposed to the shear stress for 18, 24, and 48 hours. For the CFA, the exact amounts of live cells were seeded and compared with the control (U-2-OS cells grown under standard conditions and trypsinized just before seeding for CFA analysis). Compared to the control, the amount of colonies originating from cellular doublets was slightly reduced in the case of 18 hours, and the ability to give rise to colonies decreased for the more extended cultivation periods (Fig. 7 b,c). In conclusion, we found that under continuous shear stress, the released mitotic cells were blocked just before cytokinesis, and this block is reversible in early time points. Discussion We believe our present study provides a significant advancement in understanding the effects of shear stress on various cell types, both normal and cancerous. Our newly designed and applied shear stress generator provides a unique cell culture tool that conveniently replicates some physiological conditions cells experience in the bloodstream. The technique's simplicity, cost-effectiveness, and compatibility with standard laboratory equipment and long-term operation enable new studies in cancer research, particularly in elucidating the often life-threatening process of tumor cell dissemination via the bloodstream. For example, little is known about the fate of circulating tumor cells (CTCs) in the venous system, particularly in the context of their survival mechanisms and seeding potential under the mechanical stress they inevitably experience. In-vitro studies on CTCs have traditionally employed highly sophisticated microfluidic systems to simulate fluid shear. However, such approaches are often limited by sample size, as the cultivation area of commonly available microfluidic chambers is relatively small, and the complexity of these systems can lead to loss or damage of suspension cellular fraction. Our shear stress generator addresses most of these issues while effectively imitating the shear stress environment inside human veins. Notably, our SSG can be applied to large cell quantities, providing a cultivation surface that is two orders of magnitude larger than conventional microfluidic chambers. This innovation facilitates applications in laboratory techniques that require large numbers of cells, such as western blot analysis. Additionally, our device is designed so that cells released into the suspension during the shear stress treatment are not lost or damaged within the tubing and valves, a typical issue prevalent in microfluidic systems. The presented solution's simplicity also has its limitations, primarily the inability to simulate complex conditions, such as defined blood flow and pressure changes. Despite these limitations, our device can produce a fluid shear stress of 0–10.5 dyne/cm 2 , corresponding to values observed within the venous and possibly arterial system 8 , 9 , 10 . As such, our method effectively generates some of the expected shear stress-induced cellular responses, proving its potential to replace or complement some of the microfluidics-based experiments. The shear stress generated by the device was rather a blend of laminar and turbulent characteristics, as indicated by observed bubble trajectories and endothelial cell response. A pivotal finding of our study is the discovery of a cellular phenomenon related to the final stages of the mitotic process: a reversible pre-cytokinetic block observed in cells that lose anchorage during mitosis and continue to experience fluid shear stress. We propose that this block serves as a safety checkpoint, preventing cells from their potentially hazardous proliferation within the blood or lymphatic system. Our discovery of this reversible pre-cytokinetic block adds a new dimension to the current understanding of cellular response to mechanical stress. Our data suggest existence of cell survival mechanisms activated under harsh, shear-stress conditions, highlighting the potential relevance for cancer metastasis. Another noteworthy finding that we present was the apparent bypass of this safety cell-cycle block in some cancer cell lines. These observations raise a plethora of new questions regarding the regulation of this process and the potential genetic and epigenetic factors that allow the latter subset of cancer cells to bypass this block and its consequences for the survival and overall fate of circulating cancer cells. Of relevance, it was observed that binucleated tetraploid cells induced chemically displayed higher degrees of genomic instability and tumorigenic potential 21 . In conclusion, our newly constructed SSG device and its application in mimicking some of the physical forces inside the venous system might pave the way for more comprehensive studies on the impact of shear stress on cells. This could lead to significant progress in cancer biology, particularly in understanding cancer cell survival under specific stress conditions and during metastasis. Moreover, while our study sheds new light on the effect of shear stress on the cell cycle and cellular function, the mechanism behind the observed pre-cytokinetic block and how some tumor cells can bypass this blockade remains to be elucidated. Future research in this area is needed to explore these exciting directions. Methods Cell cultures All used cell lines were obtained from ATCC, and mycoplasma was regularly tested. The list of cell lines included mice endothelial cells MS1, human breast cancer-derived MDA-MB-231, Cal51, MCF7, and BT549 cell lines, human osteosarcoma-derived U-2-OS cell line, human cervical cancer-derived SiHa and HeLa S3 cell lines, human prostate cancer-derived PC3 cell line, human lung cancer-derived A549 cell line, and human primary foreskin fibroblasts BJ. All cell lines were cultivated in DMEM (Lonza) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich). Fetal serum for MS1 cell line cultivation was thermally inactivated (56°C, 30 min.) before addition to media. Determination of cellular elongation index The determination of cell elongation was carried out using image analysis 22 . Cells in microphotographs in the transmitted light mode of observation were localized by the "Analyze Particles" function in ImageJ 23 . An ellipse was circumscribed to each cell. The elongation index was calculated as the ratio of the major to the minor axis of such an ellipse. Experimental workflow for the shear stress generator usage All tested cell lines were seeded on a 150 cm2 cultivation flask for 24 hours before applying continuous fluid shear stress. Seeding numbers were optimized to reach approximately 80% confluency at the beginning of the experiment. The cultivation flask was located on the clip-like platform horizontally, and the device was placed in a standard cell culture CO2 incubator. Cultivation parameters were 37°C, 5% CO2, and 100% humidity. The device controller was set to reach rotor oscillations (i.e., flask movement) of ± 35° within 2.47s, which corresponds to fluid shear stress mean value 1.8 dyne/cm2, median 1.4 dyne/cm2, and range 0 – 10.5 dyne/cm2. Depending on the experiment, the cells were exposed to fluid shear stress conditions for 18, 24, 48, or 72 hours. Adherent cellular fractions for analysis of MS1 cells were harvested by trypsinization. Quantitative polymerase chain reaction (qPCR) Total mRNA was extracted using the Qiagen extraction kit according to the manufacturer's instructions, and the extraction yield was quantified by Nanodrop (Implen). Reverse transcription to obtain cDNA was performed according to the protocol previously described 24 . qPCR was performed on LightCycler 480 Instrument II (Roche). Master mix for qPCR (total volume 12,5 µl) was prepared according to the recommended protocol for Platinum Taq DNA Polymerase used for PCR reaction (Invitrogen, cat.no 15966005). Eva Green (Biotium) intercalation chemistry was used to detect fluorescence signals. Primers used for qPCR were custom synthesized (Eurofins): VCAM-1 fw: 5´-TCTTACCTGTGCGCTGTGAC-3'; rw: 5´-GACCTCCACCTGGGTTCTCT-3', ICAM-1 fw: 5´-CTTCCAGCTACCATCCCAAA-3', rw: 5´-CTTCCAGGGAGCAAAACAAC-3', ACTB fw: 5´-GCGCAAGTACTCTGTGTGGA-3'; rw: 5´-CCGGACTCATCGTACTCCTG -3'. Each reaction was performed twice using 1 µl of cDNA. qPCR cycling parameters were denaturation at 95°C for 10 min., 45 cycles of amplification at 95°C for 15 s, 60 °C for 20 s, and 72°C for 25 s. Relative quantification of gene expression was calculated using the 2-ΔΔCp method. The graphical processing and statistical significance calculation were performed in GRAPHPAD Prism 8.0.1. Western blot analysis Cells exposed to fluid shear stress were lysed directly into 1x Laemmli sample buffer. The proteins in the samples were quantified with Bradford assay (ThermoFisher Scientific) according to the manufacturer's protocol. An equal amount of proteins were separated on commercial gradient 4-15% Mini Protean TGX precast gel (BIO-RAD) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine milk in Tris-buffered saline containing 0.1% Tween 20 for 30 minutes at room temperature. Membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 h at room temperature. Secondary antibodies were visualized by ELC detection reagent (ThermoFisher Scientific). Primary antibodies used in this study: SMC1 (Abcam ab9262; 1:2000), p-Akt(ser403)(Cell Signaling 9271S; 1:1000), Akt (Cell Signalling 9272S; 1:1000), p-JNK (Santa Cruz sc-6254;1:250), JNK (Santa Cruz sc-7345;1:250), p44/42-MAPK (ERK1/2)(thr202/tyr204) (Cell Signaling 4370S; 1:500), p44/42-MAPK (ERK1/2)(Cell Signaling 4696S; 1:1000). Secondary antibodies used in this study: goat-anti mouse IgG-HRP (GE Healthcare; 1:1000) and goat-anti rabbit (GE Healthcare; 1:1000). Colony formation assay (CFA Cells released by continuous shear stress were collected and centrifuged. Only live cells were counted based on trypan blue staining (Sigma Aldrich). CFA was performed by seeding 300 cells per well of 6-well plates and cultivated under standard cultivating conditions. The cells were grown for eight days until discreet colonies were visible in a microscope. Fixation of colonies was done with 70 % ice-cold ethanol, and colonies were further stained with crystal violet (Sigma Aldrich). After washed with H2O and air-drying, the colonies were covered with edible white powdered sugar to increase the contrast. Wells were scanned using a table scanner, and colonies were counted with custom-made software analysis. The graphical processing of obtained data and statistical significance calculation were performed in GRAPHPAD Prism 8.0.1. Flow cytometry analysis Cells (0,5 .10 6 ) were fixed with cold 4% formaldehyde for 15 minutes. After fixation, the cellular suspension was resuspended in PBS+ 5 % FBS +2mM EDTA with 0.5 µg/ml DAPI. Samples were analyzed using a BD FACSverse flow cytometer. Declarations Author Contribution LB, MM, and JB conceived the study; LB and MM designed most of the cellular experiments, which were performed mainly by LB and IP; MM designed and built the shear stress generator; MD built and programmed the controller for the shear stress generator; JV performed the physical and contributed to the biological qPCR-based characterization of the shear stress generator; KC performed most of the western blot analyses; TB and ZS contributed the microscopy, flow-cytometry and cellular toxicity experiments; MM, LB, ZS, and JB interpreted the results and wrote the manuscript, which was approved by all authors. Acknowledgment The study was supported by Large RI Project LM2023050 funded by MEYS CR, the National Institute for Cancer Research project (Program EXCELES, ID Project No. LX22NPO5102), and the Grant agency of the Czech Republic: GACR 17-25976S. 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Supplementary Files SupplementFulllenghtgelsblots.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 05 Apr, 2024 Reviews received at journal 29 Mar, 2024 Reviewers agreed at journal 25 Mar, 2024 Reviewers invited by journal 25 Mar, 2024 Editor assigned by journal 25 Mar, 2024 Editor invited by journal 25 Mar, 2024 Submission checks completed at journal 21 Mar, 2024 First submitted to journal 09 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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09:03:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4053852/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4053852/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-83058-3","type":"published","date":"2025-02-22T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53414973,"identity":"e6fddc74-c987-4493-9339-96d7f5812445","added_by":"auto","created_at":"2024-03-25 17:29:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3757677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDesign of the shear stress generator\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Schematic representation of the shear stress generator involving the stepper motor, two clips holding the standard 150 cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cell cultivation flasks, and stepper motor controller. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Photograph of the actual device used for the study \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Placement of the device inside a standard CO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e cell culture incubator\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/bcf7611ca52cbb58271c4b76.jpg"},{"id":53414445,"identity":"c2245e6b-c47c-4fcd-b37b-ff957412f17f","added_by":"auto","created_at":"2024-03-25 17:21:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":874183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eQuantification of generated fluid shear stress. a\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Image from a high-speed camera used for tracking air bubbles in the cultivation media in time during the operation of the shear stress generator. Tracks of individual bubbles are marked by color lines \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Calculation of shear stress values generated by the device. The fluid shear stress time course was estimated based on liquid surface velocity and an average medium depth per one cycle of flask dangling (2.47 s) inside the flask employing image analysis of each of the analyzed air bubble. The calculated fluid shear stress showed the following characteristics: mean value 1.8 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, median 1.4 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2,\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and range 0 – 10.5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/e68fb49316ec78995d8cee54.jpg"},{"id":53414446,"identity":"57b273bd-b9b3-4638-ad33-2abc5acf3c7c","added_by":"auto","created_at":"2024-03-25 17:21:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe medium flow effect on elongation of the MS1 murine endothelium cells:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Cells were subjected to standard cultivation conditions or continuous fluid shear stress using the shear stress generator. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) images depicting the alignment and elongation of cells subjected to the generator-induced fluid flow compared to control. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) The comparison of the elongation indexes (ratio of the major to the minor axis of an ellipse circumscribed to cells). The elongation index was determined by image analysis. Data are given as mean±SD. Each data point represents descriptors of at least 9000 cells in two biological replicates, each having at least three technical replicates. An unpaired two-tailed t-test was used for p-value calculation, with statistical significance ****p\u0026lt;0.0001. Microscopic images show the typical shape of cells after 48 hours of treatment. The bar indicates 50 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/2a9eb303a44477dbe54eb801.jpg"},{"id":53414450,"identity":"0cd0c08c-1009-4a61-8802-ebd63c26ab84","added_by":"auto","created_at":"2024-03-25 17:21:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2503907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMechanosensitive response of MS1 murine endothelium cells to generated fluid shear stress.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Transcriptional response of mechanosensing markers. Control cells and cells cultivated on the shear stress generator (0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) for 48h and 72 hours were analyzed using qPCR. Data are given as relative values compared to the control and displayed as mean±SD. The experiment was performed in three independent biological replicates (n=3). An ordinary one-way ANOVA test with Sidak's multiple comparison test was used for p-value calculation. Relevant comparisons are shown (ns, not significant; * P \u0026lt; 0.05; *** P \u0026lt; 0.001) **** p\u0026lt;0.0001). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Western blot analysis of posttranslationally activated (phosphorylated) mechanosensing markers after exposure of MS1 cells to the fluid shears stress (0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) at different time points. SMC1 protein was used as a loading control.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/cdf087dbeb009cfa415e32c2.jpg"},{"id":53414449,"identity":"3527ffae-f4e4-4c52-9df5-284f3cd77bec","added_by":"auto","created_at":"2024-03-25 17:21:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":972062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInterrupted cytokinesis in mitotic cells under fluid shear stress conditions. a\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Fluorescence images depicting suspension of binuclear-like cells accumulating under continuous fluid shear stress conditions. Cells were cultivated on the shear stress generator generating fluid shear stress 0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e for 18 hours; the suspension fraction was stained by HOECHST DNA stain and evaluated under a fluorescent microscope (upper image U-2-OS cells, lower image BJ primary fibroblasts). The population also contains mononuclear cells representing freshly released mitotic cells (recognizable by condensed chromosomes). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Detail of the binuclear-like cells (U-2-OS) using phase contrasted transmission light microscopy with visible actin-myosin contractile rings (marked by arrows).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/4ffd00b09cf7ced27acada67.jpg"},{"id":53414453,"identity":"ef6e5ebc-76ac-4b3f-a22f-97f202405b31","added_by":"auto","created_at":"2024-03-25 17:21:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":440232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSurvival of released suspension cells in different cell lines under continuous shear stress conditions. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eSelected cell lines were cultivated on the shear stress generator generating fluid shear stress 0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e for 18h. Cell suspension fraction was collected and placed in a new flask cultivated on the shear stress generator (0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) for another 24 or 48 hours. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Cell lines that respond to the shear stress by accumulating cells with unfinished cytokinesis in suspension. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Cell lines that respond to the shear stress by accumulating cells with finished mitosis (binuclear-like cells are absent).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/c239aef85a9d3dd9512c8a9c.jpg"},{"id":53414448,"identity":"471800da-ee9a-49d4-a808-45f3f4feaa5f","added_by":"auto","created_at":"2024-03-25 17:21:55","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":986524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eReversibility of the cytokinesis block analyzed via colony formation assay. a) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eU-2-OS cells were cultivated on the shear stress device generating fluid shear stress (FSS) 0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e for 18h. Cell suspension fraction was collected and immediately seeded for fluorescence cytometry-based cell cycle analysis. Cells were collected, fixed, and analyzed for DNA content by DAPI stain at indicated time points. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb,c) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eU-2-OS cells were cultivated on the shear stress device generating fluid shear stress (FSS) 0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e for 18h. The suspension fraction of cells was collected and either immediately seeded for colony formation assay or placed in a new flask, which was further cultivated on the shear stress generator (0-10,5 dyne/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) for another 24 or 48 hours, after which it was seeded for colony formation assay. Three hundred live cells were seeded into each well and compared with the control - trypsinized U-2-OS cells grown under standard conditions. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Example of scored colonies for different time points. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) colony formation assay quantification.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"figurePage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/b3478456757301c90e5692b0.jpg"},{"id":77052501,"identity":"6835e5bd-c092-45fd-bc56-1b37a07af06f","added_by":"auto","created_at":"2025-02-24 16:12:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10599006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/6e2ffda8-f0da-487f-a3c8-80ec2cfc8d2f.pdf"},{"id":53414452,"identity":"9099456b-7e02-42b5-8a78-cf5d152d3619","added_by":"auto","created_at":"2024-03-25 17:21:55","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2185195,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementFulllenghtgelsblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4053852/v1/70cbcdeea692f2508867ffae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Shear Stress-Induced Pre-Cytokinetic Block: A New Cellular Response Revealed by an Innovative Shear Stress Generator","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer metastasis, an often incurable, terminal disease stage, is responsible for approximately 90% of cancer-related deaths \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Cancer cells are subjected to numerous factors and stressors throughout their journey from primary tumors to distal tissues, including specific physical forces. Fluid shear stress, experienced within the blood and lymphoid vessels and interstitial fluid, significantly impacts circulating cancer cells \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Despite its acknowledged importance, the effect of shear stress on cancer cells, and more broadly on human cells, is not well understood \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This gap in our knowledge is primarily due to currently limited technological possibilities. Aside from sophisticated single-cell analysis methods applied to cells isolated from a patient's peripheral blood circulation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, most studies addressing shear stress rely on microfluidic systems that closely simulate fluid circulation in vessels. However, these systems typically aren't compatible with suspension cells, limiting their application to adherent cells only \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A further constraint of microfluidics is the limited number of cells that can be examined at any given time. This results in a limited understanding of the biology of circulating cells, including circulating tumor cells (CTCs).\u003c/p\u003e \u003cp\u003eOur present study aims to fill this crucial knowledge gap by designing, constructing, validating, and applying a novel device - the shear stress generator (SSG), an experimental tool capable of mimicking the physiological fluid shear stress conditions the cells experience in blood vessels. We document here that this pioneering technology promises to provide new insights into how shear stress impacts the behavior of cancer cells, thereby paving the way for a better understanding of cancer metastasis and contributing to development of more effective anticancer treatment strategies. Leveraging this versatile approach, here we have examined the impact of shear stress on significantly larger numbers of cells and discovered a previously unknown biological phenomenon critical for the fate of the mitotic cells that are transiting into a suspension mode.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction and characterization of the shear stress generator (SSG)\u003c/h2\u003e \u003cp\u003eThe device is composed of a clip-like platform that holds a standard 150 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e cell culture flask connected to the rotor of a stepper motor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). A custom-made program drives the stepper motor to oscillate the flask in a horizontal plane. The final optimized settings for the flask movement\u0026mdash;used in all subsequent experiments\u0026mdash;involve rotor oscillations (i.e., flask movement) of \u0026plusmn;\u0026thinsp;35\u0026deg; for 2.47s. This movement acceleration triggers a recurring wave of media within the flask, reaching a surface flow speed of approximately 0.8 m/s, as determined by image analysis of floating air bubbles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe flow speed's primary component aligns with the flask's direction. However, near the flask's neck, a perpendicular component to this motion deviates the overall trend from perfect linearity. Upon hitting the flask's sidewalls, the wave also generates slight turbulence accompanied by bubble formation. Overall, this setup offers a relatively straightforward analogy to various mechanical forces expected within the venous system.\u003c/p\u003e \u003cp\u003eWe used slow-motion camera footage of dispersed air bubbles spontaneously forming in the culture medium during the operation to provide a detailed analysis of the forces induced by the media movement. The measured bubble accelerations were recalculated into shear stress values, peaking at 10 dyne/cm\u003csup\u003e2\u003c/sup\u003e with a mean value of 1.8 dyne/cm\u003csup\u003e2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Importantly, such shear stress aligns with the levels reported for the human venous system\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn essential feature of our experimental setup is its long-term stability, capable of continuous operation for several days without any failures or undesirable changes in the movement and thus shear stress values. The system also requires a relatively low amount of energy\u0026mdash;approximately 7W for two 150cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e flasks\u0026mdash;making it suitable for use in cell incubators, which are not commonly equipped with active cooling and thus limit the placement of high-power devices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBiological Relevance and Impact of Generated Shear Stress\u003c/h2\u003e \u003cp\u003eFollowing the mechanical characterization of the device, we next investigated the occurrence of some typical shear stress-induced phenotypes on cultured mammalian cells. Initially, we evaluated the elongation of endothelial cells (ECs) according to the flow vector\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This physiological response naturally occurs in the vasculature and can be simulated in microfluidic systems using a specialized cultivation chamber, pumps, and valves to ensure a defined media flow \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Indeed, the media flow under our standardized SSG experimental conditions was sufficient to trigger this typical cellular elongation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to see if cells are perceiving shear stress at the molecular level, we examined selected ECs' mechanosensory and signaling pathways. Several pathways have been established in flow sensing by EC \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These mechanosensitive pathways become activated within seconds to hours and may return to baseline state within 24 hours of exposure to flow\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. First, we determined the expression of general fluid flow markers in EC: ICAM-1 and VCAM-1 in two typical time points reflecting expression changes (48 hours) and adaptation of the cells to the flow (72 hours). Whereas the ICAM-1 transcript showed a transient increase at 48 hours, returning to normal levels at 72 hours, the VCAM-1 transcript stayed permanently elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Such data can be interpreted to mean cells indeed perceived the flow to which they adapted (ICAM-1)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e; however, the flow has rather a turbulent character, at least to some extent, as demonstrated by persistently elevated VCAM\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e over 72 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther, the immediate shear stress perception onset was assayed through the phosphorylation status of major protein kinases involved in response to flow\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The Erk1/2 kinase showed transient phosphorylation within five minutes from flow onset. The maximum phosphorylation of Akt kinase was delayed to 30 minutes \u0026ndash; 2 hours. Instead, the phosphorylation of JNK and P38 kinases showed a steady increase up to 6 hours of the experiment duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Interestingly, the early transient phosphorylation of ERK1/2 indicates the perception of steady flow\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, whereas the phosphorylation pattern of Akt, JNK and P38 kinases was consistent with the general perception of flow \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Taken together, the cells were able to sense the shear stress generated by the device. Such shear stress was rather perceived as a blend of laminar and turbulent characteristics.\u003c/p\u003e \u003cp\u003eIn summary, these findings suggest that the presented device is applicable in various studies assessing cellular responses to shear stress and thus provides a possible alternative or an addition to experimental strategies currently using complicated, less versatile, and costly microfluidic systems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFluid Shear Stress Induces a Pre-Cytokinetic Block\u003c/h2\u003e \u003cp\u003eIf exposed to the mechanical forces generated by our device, several adherently growing cell lines release cells into suspension during mitosis. This effect is due to the relatively weaker adhesion of mitotic cells to the cultivation surface, commonly referred to as mitotic shake-off \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Importantly, this mechanism might be highly relevant for potential tumor cell dissemination. At least some circulating tumor cells (CTCs) may originate from mitotic cells dislodged from the primary tumor just by the mechanical forces caused by blood or plasma flow.\u003c/p\u003e \u003cp\u003eTaking advantage of our device's compatibility with cells in suspension and long-term applications, we followed the fate of the released mitotic cells under continuous fluid shear stress conditions. In human primary fibroblast strain BJ, as well as some cancer cell lines such as U-2-OS osteosarcoma, MDA-MB-231, MCF7 and BT549 breast cancers, and A549 lung cancer, the released mitotic cells were unable to complete the final phase of cytokinesis which under normal circumstances leads to daughter cells separation. The resulting binucleate-like cells were subjected to microscopic analysis, revealing incomplete cleavage with a persisting actin-myosin contractile ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we tested the ability of these bi-nucleated cells to survive under conditions mimicking those that cells likely experience in the bloodstream, i.e. under continuous shear stress cultivation over prolonged periods. For this experiment, we collected the bi-nucleated cells after 18 hours of cultivation under shear stress conditions. Cells were counted, placed into a new cultivation flask, and cultivated for another 24 and 48 hours. The number of cells was then compared to that at the starting point of this long-term experiment. In all models tested, the prolonged cultivation of bi-nucleated cells under continuous shear stress turned to be highly cytotoxic (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), suggesting an important role of this newly identified cell cycle pre-cytokinetic block in regulating cell survival under shear stress and thereby possibly limiting the potential of cancerous cells for metastatic spread.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, and in contrast to the cell models mentioned above, some of the other human cancer cell lines that we tested, including SiHA and HeLa S3 (established from a cervical carcinoma), PC3 (prostate cancer), and Cal51 (breast cancer), showed a different behavioral pattern. Thus, while the latter cancer models also accumulated cells in suspension under continuous shear stress conditions, the occurrence of binucleated cells with incomplete cytokinesis within the suspension fraction, as described above, was sporadic, reflecting a relatively normal mitotic process resulting into timely and complete daughter cell separation. After exposing such cellular suspensions to continuous shear stress over prolonged periods, we found a cytostatic rather than cytotoxic effects for SiHA, PC3, and Cal51 cell lines and surprisingly normal ongoing proliferation for HeLa S3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn conclusion, our findings presented in this section show that most human cell lines of our panel subjected to continuous shear stress release mitotic cells, and furthermore, the shear stress triggers mechanisms that interrupt the cell cycle just before cytokinesis. At the same time, the released cells are incapable of long-term survival in such a state. On the other hand, some cancer cells can bypass this safety mechanism, which might have significant consequences for tumor dissemination potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReversibility of the Pre-Cytokinesis Cell Cycle Block\u003c/h2\u003e \u003cp\u003eAs nothing is known about this pre-cytokinesis cell cycle block induced by shear stress, we next examined its reversibility. We used U-2-OS cells as a model cell line and cultivated them on the shear stress device for 18h. The suspension fraction of cellular doublets was collected and immediately seeded. The re-seeded cells reattached and completed the cell separation process (cytokinesis). Subsequently, such divided cells entered the S phase and resumed relatively synchronized cell growth, as evidenced by the flow cytometry analysis of the DNA content at different time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). We also performed a colony formation assay (CFA) on these cells, including cells exposed to the shear stress for 18, 24, and 48 hours. For the CFA, the exact amounts of live cells were seeded and compared with the control (U-2-OS cells grown under standard conditions and trypsinized just before seeding for CFA analysis). Compared to the control, the amount of colonies originating from cellular doublets was slightly reduced in the case of 18 hours, and the ability to give rise to colonies decreased for the more extended cultivation periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, we found that under continuous shear stress, the released mitotic cells were blocked just before cytokinesis, and this block is reversible in early time points.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe believe our present study provides a significant advancement in understanding the effects of shear stress on various cell types, both normal and cancerous. Our newly designed and applied shear stress generator provides a unique cell culture tool that conveniently replicates some physiological conditions cells experience in the bloodstream. The technique's simplicity, cost-effectiveness, and compatibility with standard laboratory equipment and long-term operation enable new studies in cancer research, particularly in elucidating the often life-threatening process of tumor cell dissemination via the bloodstream.\u003c/p\u003e \u003cp\u003eFor example, little is known about the fate of circulating tumor cells (CTCs) in the venous system, particularly in the context of their survival mechanisms and seeding potential under the mechanical stress they inevitably experience. In-vitro studies on CTCs have traditionally employed highly sophisticated microfluidic systems to simulate fluid shear. However, such approaches are often limited by sample size, as the cultivation area of commonly available microfluidic chambers is relatively small, and the complexity of these systems can lead to loss or damage of suspension cellular fraction. Our shear stress generator addresses most of these issues while effectively imitating the shear stress environment inside human veins. Notably, our SSG can be applied to large cell quantities, providing a cultivation surface that is two orders of magnitude larger than conventional microfluidic chambers. This innovation facilitates applications in laboratory techniques that require large numbers of cells, such as western blot analysis. Additionally, our device is designed so that cells released into the suspension during the shear stress treatment are not lost or damaged within the tubing and valves, a typical issue prevalent in microfluidic systems.\u003c/p\u003e \u003cp\u003eThe presented solution's simplicity also has its limitations, primarily the inability to simulate complex conditions, such as defined blood flow and pressure changes. Despite these limitations, our device can produce a fluid shear stress of 0\u0026ndash;10.5 dyne/cm\u003csup\u003e2\u003c/sup\u003e, corresponding to values observed within the venous and possibly arterial system\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As such, our method effectively generates some of the expected shear stress-induced cellular responses, proving its potential to replace or complement some of the microfluidics-based experiments. The shear stress generated by the device was rather a blend of laminar and turbulent characteristics, as indicated by observed bubble trajectories and endothelial cell response.\u003c/p\u003e \u003cp\u003eA pivotal finding of our study is the discovery of a cellular phenomenon related to the final stages of the mitotic process: a reversible pre-cytokinetic block observed in cells that lose anchorage during mitosis and continue to experience fluid shear stress. We propose that this block serves as a safety checkpoint, preventing cells from their potentially hazardous proliferation within the blood or lymphatic system.\u003c/p\u003e \u003cp\u003eOur discovery of this reversible pre-cytokinetic block adds a new dimension to the current understanding of cellular response to mechanical stress. Our data suggest existence of cell survival mechanisms activated under harsh, shear-stress conditions, highlighting the potential relevance for cancer metastasis. Another noteworthy finding that we present was the apparent bypass of this safety cell-cycle block in some cancer cell lines. These observations raise a plethora of new questions regarding the regulation of this process and the potential genetic and epigenetic factors that allow the latter subset of cancer cells to bypass this block and its consequences for the survival and overall fate of circulating cancer cells. Of relevance, it was observed that binucleated tetraploid cells induced chemically displayed higher degrees of genomic instability and tumorigenic potential\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, our newly constructed SSG device and its application in mimicking some of the physical forces inside the venous system might pave the way for more comprehensive studies on the impact of shear stress on cells. This could lead to significant progress in cancer biology, particularly in understanding cancer cell survival under specific stress conditions and during metastasis. Moreover, while our study sheds new light on the effect of shear stress on the cell cycle and cellular function, the mechanism behind the observed pre-cytokinetic block and how some tumor cells can bypass this blockade remains to be elucidated. Future research in this area is needed to explore these exciting directions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll used cell lines were obtained from ATCC, and mycoplasma was regularly tested. The list of cell lines included mice endothelial cells MS1, human breast cancer-derived MDA-MB-231, Cal51, MCF7, and BT549 cell lines, human osteosarcoma-derived U-2-OS cell line, human cervical cancer-derived SiHa and HeLa S3 cell lines, human prostate cancer-derived PC3 cell line, human lung cancer-derived A549 cell line, and human primary foreskin fibroblasts BJ. All cell lines were cultivated in DMEM (Lonza) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich). Fetal serum for MS1 cell line cultivation was thermally inactivated (56°C, 30 min.) before addition to media.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of cellular elongation index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe determination of cell elongation was carried out using image analysis\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e. Cells in microphotographs in the transmitted light mode of observation were localized by the \"Analyze Particles\" function in ImageJ\u0026nbsp;\u003csup\u003e23\u003c/sup\u003e. An ellipse was circumscribed to each cell. The elongation index was calculated as the ratio of the\u0026nbsp;major to the minor axis of such an ellipse.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental workflow for the shear stress generator usage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tested cell lines were seeded on a 150 cm2 cultivation flask for 24 hours before applying continuous fluid shear stress. Seeding numbers were optimized to reach approximately 80% confluency at the beginning of the experiment. The cultivation flask was located on the clip-like platform horizontally, and the device was placed in a standard cell culture CO2 incubator. Cultivation parameters were 37°C, 5% CO2, and 100% humidity. The device controller was set to reach rotor oscillations (i.e., flask movement) of ± 35° within 2.47s, which corresponds to fluid shear stress mean value 1.8 dyne/cm2, median 1.4 dyne/cm2, and range 0 – 10.5 dyne/cm2. Depending on the experiment, the cells were exposed to fluid shear stress conditions for 18, 24, 48, or 72 hours. Adherent cellular fractions for analysis of MS1 cells were harvested by trypsinization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative polymerase chain reaction (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal mRNA was extracted using the Qiagen extraction kit according to the manufacturer's instructions, and the extraction yield was quantified by Nanodrop (Implen). Reverse transcription to obtain cDNA was performed according to the protocol previously described\u0026nbsp;\u003csup\u003e24\u003c/sup\u003e. qPCR was performed on LightCycler 480 Instrument II (Roche). Master mix for qPCR (total volume 12,5 µl) was prepared according to the recommended protocol for \u0026nbsp;Platinum Taq DNA Polymerase used for PCR reaction (Invitrogen, cat.no 15966005). Eva Green (Biotium) intercalation chemistry was used to detect fluorescence signals. Primers used for qPCR were custom synthesized (Eurofins): VCAM-1 fw: 5´-TCTTACCTGTGCGCTGTGAC-3'; rw: 5´-GACCTCCACCTGGGTTCTCT-3', ICAM-1 fw: 5´-CTTCCAGCTACCATCCCAAA-3', rw: 5´-CTTCCAGGGAGCAAAACAAC-3', ACTB fw: 5´-GCGCAAGTACTCTGTGTGGA-3'; rw: 5´-CCGGACTCATCGTACTCCTG -3'. Each reaction was performed twice using 1 µl of cDNA. qPCR cycling parameters were denaturation at 95°C for 10 min., 45 cycles of amplification at 95°C for 15 s, 60 °C for 20 s, and 72°C for 25 s. Relative quantification of gene expression was calculated using the 2-ΔΔCp method. The graphical processing and statistical significance calculation were performed in GRAPHPAD Prism 8.0.1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells exposed to fluid shear stress were lysed directly into 1x Laemmli sample buffer. The proteins in the samples were quantified with Bradford assay (ThermoFisher Scientific) according to the manufacturer's protocol. An equal amount of proteins were separated on commercial gradient 4-15% Mini Protean TGX precast gel (BIO-RAD) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine milk in Tris-buffered saline containing 0.1% Tween 20 for 30 minutes at room temperature. Membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 h at room temperature. Secondary antibodies were visualized by ELC detection reagent (ThermoFisher Scientific). Primary antibodies used in this study: SMC1 (Abcam ab9262; 1:2000), p-Akt(ser403)(Cell Signaling 9271S; 1:1000), Akt (Cell Signalling 9272S; 1:1000), p-JNK (Santa Cruz sc-6254;1:250), JNK (Santa Cruz sc-7345;1:250), p44/42-MAPK (ERK1/2)(thr202/tyr204) (Cell Signaling 4370S; 1:500), p44/42-MAPK (ERK1/2)(Cell Signaling 4696S; 1:1000). Secondary antibodies used in this study: goat-anti mouse IgG-HRP (GE Healthcare; 1:1000) and goat-anti rabbit (GE Healthcare; 1:1000).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay (CFA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells released by continuous shear stress were collected and centrifuged. Only live cells were counted based on trypan blue staining (Sigma Aldrich). CFA was performed by seeding 300 cells per well of 6-well plates and cultivated under standard cultivating conditions. The cells were grown for eight days until discreet colonies were visible in a microscope. Fixation of colonies was done with 70 % ice-cold ethanol, and colonies were further stained with crystal violet (Sigma Aldrich). After washed with H2O and air-drying, the colonies were covered with edible white powdered sugar to increase the contrast. Wells were scanned using a table scanner, and colonies were counted with custom-made software analysis. The graphical processing of obtained data and statistical significance calculation were performed in GRAPHPAD Prism 8.0.1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (0,5 .10\u003csup\u003e6\u003c/sup\u003e) were fixed with cold 4% formaldehyde for 15 minutes. After fixation, the cellular suspension was resuspended in PBS+ 5 % FBS +2mM EDTA with 0.5 µg/ml DAPI. Samples were analyzed using a BD FACSverse flow cytometer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLB, MM, and JB conceived the study; LB and MM designed most of the cellular experiments, which were performed mainly by LB and IP; MM designed and built the shear stress generator; MD built and programmed the controller for the shear stress generator; JV performed the physical and contributed to the biological qPCR-based characterization of the shear stress generator; KC performed most of the western blot analyses; TB and ZS contributed the microscopy, flow-cytometry and cellular toxicity experiments; MM, LB, ZS, and JB interpreted the results and wrote the manuscript, which was approved by all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eThe study was supported by Large RI Project LM2023050 funded by MEYS CR, the National Institute for Cancer Research project (Program EXCELES, ID Project No. LX22NPO5102), and the Grant agency of the Czech Republic: GACR 17-25976S.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChaffer, C. 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Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 671\u0026ndash;675 (2012).\u003c/li\u003e\n\u003cli\u003eKudlova, N. \u003cem\u003eet al.\u003c/em\u003e An efficient, non-invasive approach for in-vivo sampling of hair follicles: design and applications in monitoring DNA damage and aging. \u003cem\u003eAging\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 25004\u0026ndash;25024 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"fluid shear stress, circulating tumor cells, microfluidic systems, metastasis, mitosis","lastPublishedDoi":"10.21203/rs.3.rs-4053852/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4053852/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring metastasis, cancer cells navigate through harsh conditions, including various mechanical forces in the bloodstream, highlighting the need to understand the impact of mechanical and shear stresses on cancer cells. To overcome the current methodological limitations of such research, here we present a new device that replicates similar conditions by applying shear stress on cultured cells. The device provides a less complex, easily accessible alternative to traditional microfluidics while generating fluid shear stress values comparable to those in human veins and capillaries. The device allows analyses of large cell numbers in standard cell culture flasks and incubators. Using this device to explore the shear stress-induced responses of various human cell lines, we discovered a previously unknown, reversible pre-cytokinetic block occurring in cells that lose anchorage during mitosis and are kept under constant shear stress. Notably, some cancer cell lines appear to bypass this unorthodox cell-cycle block, suggesting its role as a safety checkpoint to restrict the proliferation of cancer cells in the bloodstream and their overall spreading potential. These findings provide new insights into the diverse responses of normal and cancer cells to shear stress and highlight the potential of our technology for research on circulating tumor cells and metastatic spread.\u003c/p\u003e","manuscriptTitle":"Shear Stress-Induced Pre-Cytokinetic Block: A New Cellular Response Revealed by an Innovative Shear Stress Generator","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 17:21:50","doi":"10.21203/rs.3.rs-4053852/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-05T11:52:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-29T12:21:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5bca6e3c-c9ea-4106-983f-d6424eddcadd","date":"2024-03-25T18:36:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-25T14:02:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-25T13:57:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-25T07:59:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-21T05:57:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-09T08:24:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9819d4d-d190-497e-9c33-e5aebf7f8604","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29702882,"name":"Biological sciences/Biological techniques"},{"id":29702883,"name":"Biological sciences/Biological techniques/Biological models"},{"id":29702884,"name":"Biological sciences/Biological techniques/Biological models/Cancer models"},{"id":29702885,"name":"Health sciences/Medical research"},{"id":29702886,"name":"Health sciences/Medical research/Pre clinical studies"}],"tags":[],"updatedAt":"2025-02-24T15:59:46+00:00","versionOfRecord":{"articleIdentity":"rs-4053852","link":"https://doi.org/10.1038/s41598-024-83058-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-02-22 15:56:54","publishedOnDateReadable":"February 22nd, 2025"},"versionCreatedAt":"2024-03-25 17:21:50","video":"","vorDoi":"10.1038/s41598-024-83058-3","vorDoiUrl":"https://doi.org/10.1038/s41598-024-83058-3","workflowStages":[]},"version":"v1","identity":"rs-4053852","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4053852","identity":"rs-4053852","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}