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

Enhancing Targeted Cancer Diagnosis by Quantifying Single-Cell Mechanics(단일 세포 역학을 정량화하여 표적 암 진단 강화)

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.

저자 스포트라이트: 단일 세포 재료 특성 측정을 위한 전단 분석 프로토콜  

단세포 물질 특성 측정을 위한 전단 분석 프로토콜

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  

단일 세포 전단 분석 이미징 데이터 처리

DIC 소프트웨어를 사용하여 디지털 이미지 상관 관계 또는 DIC를 수행하려면 순전히 분석 기록에서 파생된 이미지를 DIC 소프트웨어(가급적이면 TIF 파일 형식)로 가져옵니다. 최적화된 상관관계를 위해 마지막 영상에 대한 새 영상의 변형을 추적하는 차분의 합 옵션을 선택합니다. 31 x 31 픽셀의 부분 집합 크기와 20픽셀의 스텝 크기를 선택합니다.선택한 단일 셀에 대한 관심 영역을 매핑합니다. 셀과 같은 불규칙한 모양의 경우 다각형 마스크를 사용하여 셀의 기하 도형을 매핑합니다. 매핑된 셀 내에서 임의의 점을 선택하여 변형을 추적합니다.매핑 후 Add a Strain Gauge(스트레인 게이지 추가)를 클릭하고 정의된 세포 경계 내의 지점에서 개별 스트레인 게이지를 그려 분석할 핵 또는 세포질과 같은 세포의 특정 지점을 선택합니다. 시작을 클릭하여 변형률 처리를 시작하고 변형률 대 시간 데이터를 얻습니다. 이 상관 관계의 결과로 생성된 변형률 시간 플롯은 MATLAB에서 추가 분석을 위해 CSV 파일로 내보낼 수 있습니다.생성된 변형률 시간 플롯을 두 번 클릭하거나 마우스 오른쪽 버튼으로 클릭하고 데이터 내보내기(Export Data)를 선택합니다. 순전히 분석 기록에서 추출한 이미지 프레임의 DIC는 크리프 응력 반응과 호환되는 변형 시간 데이터를 성공적으로 생성했습니다. 순전히 분석 기술을 사용한 연구 결과에 따르면 암세포는 일반적으로 정상 세포보다 기계적으로 더 적합하고 점성이 적습니다.세포의 강성 및 점도는 정상 세포 상태에서 약간 전이 상태로, 그리고 최종적으로 고도로 전이성 상태로 암 진행이 증가함에 따라 감소하는 것으로 관찰되었다. 그러나, 이들 세포 유형에 대한 이완 시간은 유의한 변화를 나타내지 않았다.