Name: Processing the Single-Cell Shear Assay Imaging Data
Uploaded: 2023-05-19T13:30:00
Description: Watch this Scientific Journal Video about Processing the Single-Cell Shear Assay Imaging Data at JoVE.com

processing the single-cell shear assay imaging data

Verbesserung der gezielten Krebsdiagnose durch Quantifizierung der Einzelzellmechanik

Studying the biophysical differences between cancerous and non-cancerous cells allows for novel diagnostic and therapeutic opportunities1. Understanding how differences in biomechanics/mechanobiology contribute to tumor progression and treatment resistance will reveal new avenues for targeted therapy and early diagnosis2.
While it is known that cancer cell mechanical properties differ from normal cells (e.g., viscoelasticity of the plasma membrane and nuclear envelope)3,4,5, robust and reproducible methods for measuring these properties in live cells are lacking6. The shear assay method is used to quantify the mechanical properties of cells by subjecting single cells to fluid shear stress and analyzing their individual responses and resistance to the applied stress3,4,5,7,8,9. Although several methods and techniques have been used to characterize the mechanical properties of single cells, these tend to affect cell material properties by i) perforating/damaging the cell membrane due to the indentation depth, complex tip geometries, or substrate stiffening associated with atomic force microscopy (AFM)10,11, ii) inducing cellular photodamage during optical trapping12,13, or iii) inducing complex stress states associated with micropipette aspiration14,15. These external effects are associated with significant uncertainties in the accuracy of cell viscoelasticity measurements6,16,17.
To address these limitations, the shear assay method described here provides a highly controllable and simple approach to simulate physiological flow in the body without affecting cellular material properties in the process. Fluid shear stresses in this assay represent mechanical stresses experienced by cells in the body either by fluids within the tumor interstitium or in the blood during circulation18,19,20. Further, these fluid stresses promote various malignant behaviors in cancer cells, including progression, migration, metastasis, and cell death19,21,22,23&#160;which vary between tumorigenic and non-tumorigenic cells. Moreover, the altered mechanical features of cancer cells (i.e., they are often &#34;softer&#34; than normal cells found within the same organ) allow them to persist in hostile tumor microenvironments, invade surrounding normal tissues, and metastasize to distant sites24,25,26. By creating a pseudo-biological environment where cells experience physiological levels of fluid shear stress, a process that is physiologically relevant and not destructive to the cell is achieved. The cellular responses to these applied fluid shear stresses allow us to characterize cell mechanical properties.
This paper provides a shear assay protocol for the extensive study of the mechanical properties and behavior of cancerous and non-cancerous cells under applied shear stress. Cells respond to external forces in an elastic and viscous manner and can therefore be idealized as a viscoelastic material3. This technique is categorized into: (i) cell culture of dispersed single cells, (ii) controlled application of fluid shear stress, (iii) in situ imaging and observation of cellular behavior (including resistance to stress and deformation), (iv) strain analysis of cells to determine the extent of deformation, and (v) characterization of the viscoelastic properties of single cells. By interrogating these mechanical properties and behaviors, complex cellular mechanobiology can be distilled to quantifiable data. A protocol outlining this method allows for the cataloging of and comparison between various malignant and non-malignant cell types. Quantifying these differences has the potential to establish diagnostic and therapeutic biomarkers.

Autor im Rampenlicht: Scher-Assay-Protokoll zur Bestimmung von Einzelzell-Materialeigenschaften  

Scherprobenprotokoll zur Bestimmung von Einzelzell-Materialeigenschaften

The physical hallmarks of cancer are relatively new and exciting topics in cancer research. Our research seeks to specifically characterize the mechanical properties of both cancers and non-cancer cells to better understand the physical mechanisms by which cancer cells survive shear forces in the body and invade the immune system. Recent development in my period of research is the use of microfluidic devices and high-throughput techniques to understudy in a larger scale, cellular deformability and the viscoelastic properties of cells, and also in the identification of novel biomarkers such as the regulation of cytoskeletal proteins and extracellular matrix proteins for early cancer diagnosis.Current experimental challenges involved in this field are the complexity of the tumor microenvironment, scaling throughput, clinical translation, standardization and reproducibility, and integration of multimodal data. So, some of the significant findings I've established in this field from my past research and from my current research is that cancer cells can be distinguished from normal cells based on their responses to mechanical stressors. And the extent to which resist mechanical stressors is dependent on structural integrity, which is dictated to a reasonable extent by the presence of cytoskeletal proteins like the filamentous actin and other structural proteins within the cell.Our protocol offers a simple, repeatable, and non-destructive approach to characterize cultured cells mechanically. This is in contrast to other techniques like AFM and optical tweezers, which require a certain level of technical expertise in order to perform an experiment in a relatively low throughput. Within the use of the shear acid technique, will greatly advance the single cell mechanical characterization, and will also aid in many different cross-sectional areas such as rare disease diagnosis, personalized diagnosis and care, and monitoring single-cell drug interactions.

Watch this Scientific Journal Video about Processing the Single-Cell Shear Assay Imaging Data at JoVE.com

Processing the Single-Cell Shear Assay Imaging Data  

Verarbeitung der Bildgebungsdaten des Einzelzell-Schertests

Um eine digitale Bildkorrelation (DIC) mit der DIC-Software durchzuführen, importieren Sie die Bilder, die aus der reinen Assay-Aufzeichnung stammen, in die DIC-Software, vorzugsweise im TIF-Dateiformat. Für eine optimierte Korrelation wählen Sie die Option Summe der Differenziale, die die Verformung eines neuen Bildes in Bezug auf das letzte Bild verfolgt. Wählen Sie eine Teilmengengröße von 31 x 31 Pixel mit einer Schrittweite von 20 Pixeln aus.Ordnen Sie den Interessenbereich für eine ausgewählte einzelne Zelle zu. Verwenden Sie für eine unregelmäßige Form, z. B. eine Zelle, eine Polygonmaske, um die Geometrie der Zelle darzustellen. Wählen Sie beliebige Punkte innerhalb der zugeordneten Zelle aus, um die Verformung zu verfolgen.Wählen Sie nach dem Mapping bestimmte Punkte auf der Zelle aus, z. B. den Zellkern oder das Zytoplasma, die analysiert werden sollen, indem Sie auf Dehnungsmessstreifen hinzufügen klicken und einzelne Dehnungsmessstreifen an Punkten innerhalb der definierten Zellgrenze zeichnen. Klicken Sie auf Start, um die Dehnungsverarbeitung zu starten und Dehnungs-Zeit-Daten zu erhalten. Das aus dieser Korrelation generierte Dehnungs-Zeit-Diagramm kann als CSV-Datei zur weiteren Analyse in MATLAB exportiert werden.Doppelklicken Sie oder klicken Sie mit der rechten Maustaste auf das generierte Dehnungszeitdiagramm und wählen Sie Daten exportieren aus. DIC von Bildbildern, die aus reinen Assay-Aufnahmen extrahiert wurden, lieferten erfolgreich Dehnungszeitdaten, die mit der Kriechspannungsantwort kompatibel sind. Die Ergebnisse von Studien, bei denen die Sheer-Assay-Technik verwendet wurde, zeigten, dass Krebszellen im Allgemeinen mechanisch nachgiebiger und weniger viskos waren als normale Zellen.Es wurde beobachtet, dass die Steifigkeit und Viskosität der Zellen mit zunehmender Krebsprogression von einem normalen Zellzustand zu einem leicht metastasierten Zustand und schließlich zu einem stark metastasierten Zustand abnimmt. Die Relaxationszeit für diese Zelltypen zeigte jedoch keine signifikanten Variationen.

Research

JoVE Journal

Cancer Research

Processing the Single-Cell Shear Assay Imaging Data

Shear Assay Protocol for the Determination of Single-Cell Material Properties

Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a ...

Forschung

Krebsforschung

Microfluidic Shear Apparatus Setup for Single-Cell Fluid Shear Assay

Aufbau eines mikrofluidischen Scherapparats für den Einzelzell-Fluid-Shear-Assay

Performing Single-Cell Shear Assay and Imaging

Durchführung von Einzelzell-Schertests und Bildgebung