I en studie publicerad för 2 dagar sen, 11/11, visar forskare från University of Michigan-Dearborn
att HoloMonitor kan särskilja mellan vanliga och ofarliga hudceller (melanocyter) och aggressiva hudcancerceller (SK-MEL-28 Melanoma Cells). Upptäckten har betydelse för framtida bättre och snabbare diagnosställning samt behandling av en aggressiv hudcancer.
Morphological and Optical Profiling of Melanocytes and SK-MEL-28 Melanoma Cells Via Digital Holographic Microscopy and Quantitative Phase Imaging
Abstract
Melanoma, which originates from pigment-producing melanocytes, is an aggressive and deadly skin cancer. Despite extensive research, its mechanisms of progression and metastasis remain unclear. This study uses quantitative phase imaging via digital holographic microscopy, Principal Component Analysis (PCA), and t-distributed Stochastic Neighbor Embedding (t-SNE) to identify the morphological, optical, and behavioral differences between normal melanocytes and SK-MEL-28 melanoma cells. Our findings reveal significant differences in cell shape, size, and internal organization, with SK-MEL-28 cells displaying greater size variability, more polygonal shapes, and higher optical thickness. Phase shift parameters and surface roughness analyses underscore melanoma cells' uniform and predictable textures. Violin plots highlight the dynamic and varied migration of SK-MEL-28 cells, contrasting with the localized movement of melanocytes. Hierarchical clustering of correlation matrices provides further insights into complex morphological and optical relationships. Integrating label-free imaging with robust analytical methods enhances understanding of melanoma's aggressive behavior, supporting targeted therapies and highlighting potential biomarkers for precise melanoma diagnostics and treatment.
1 Introduction
Melanoma is a highly aggressive form of skin cancer originating from melanocytes, the pigment-producing cells in the epidermis.Despite extensive research, the biological mechanisms underlying melanoma progression and metastasis remain incompletely understood, necessitating more detailed studies on cellular morphology, behavior, and molecular characteristics.
These differences are pivotal for understanding the disease and developing targeted therapeutic strategies.Recent advances in holographic microscopy have enabled detailed visualization of cellular structures, revealing significant differences in cell shape, size, and internal organization.These imaging techniques allow for the examination of parameters such as cell area, optical volume, and thickness, offering a comprehensive view of cellular morphology.
HoloMonitor visar här skillnaderna mellan dessa 2 celltyper.
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| Holographic Microscopic Images A) and Single Cell Thickness Measurements Across Length and Width B) of Melanocytes and SK-MEL-28 cells. |
Cell Culture and Digital Holographic Microscopy
The melanoma cell line SK-MEL-28 (ATCC HTB-72) was cultured in RPMI 1640 (ATCC 30–2001) supplemented with 10% Fetal Bovine Serum (ATCC) and 1% Penicillin-Streptomycin (Thermo Fisher) at 37 °C with 5% CO2. Primary epidermal melanocytes (HEMa, ATCC PCS-200-013) were cultured in Dermal Cell Basal Medium (ATCC PCS-200-030) combined with the Adult Melanocyte Growth Kit (ATCC PCS-200-042) at 37 °C with 5% CO2, with the complete growth medium containing 5 µg mL−1 rh Insulin, 50 µg mL−1 Ascorbic Acid, 6 mM L-Glutamine, 1.0 µM Epinephrine, 1.5 mM Calcium chloride, 0.2% Peptide Growth Factor (proprietary formulation), and 1% M8 Supplement (proprietary formulation). Both cell lines were seeded in 24-well plates and analyzed over a 58-h period using a HoloMonitor M4 holographic imaging microscope (Phase Holographic Imaging, Lund, Sweden). The HoloMonitor M4 captured images at 15-min intervals in each well, with each interval consisting of 30 frames. The App Suite Cell Imaging software analyzed these frames, conducting Guided End-Point Assays (including Cell Quality Control), Guided Kinetic Assays (including Kinetic Proliferation Assay and Kinetic Motility Assay), and In-Depth Assays (including Cell Morphology), providing detailed insights into cell behavior and morphology over the observation period.
2D, pseudo 3D Cell Holographic Microscopy
Microscopic 2D images of the Melanocytes and SK-MEL-28 cells were captured via the HoloMonitor M4, which automatically snapped photos of the cells at 15-min intervals over a 58-h time period. Under the “Experiment Overview” feature of the App Suite Cell Imaging software, the image color palette was modified via the Coloring panel from a monochromatic scale to a blue-yellow coloring, with the blue indicative of the cells and the yellow indicative of the growth medium. The colors applied were relative to the thickness of the cells, with the scale ranging from -0.5 µm to 20.1 µm.
Individual pseudo-3D images of the Melanocytes and SK-MEL-28 cells were analyzed via the App Suite Cell Imaging software of the HoloMonitor M4.
Cell Movement Plot
In order to identify patterns of cell movement over time between Melanocytes and SK-MEL-28 cells, the movement of the cells in their respective wells on a Cell Movement Plot was visualized. The analysis was completed using the App Suite Cell Imaging software of the HoloMonitor M4. Using the “In-Depth Analysis: Single Cell Tracking” package of the software, 235 frames of the wells containing viable cells, which were taken at 15-min intervals, via the “Identify Cells” function were analyzed. These frames were then applied to the “Single Cell Tracking” function and integrated into an analysis to track cell movements across a 58-h period. Every cell in the well under the “Cell Movement Plot” feature, thus ensuring that each cell was incorporated into the plot was opted to include.
3 Discussion
Quantitative phase imaging (QPI) has become a formidable technique for characterizing cells without the need for labels, utilizing the principles of digital holographic microscopy (DHM) to offer comprehensive insights into cellular morphology and dynamics. In the last twenty years, there have been notable advancements in QPI methods, which have broadened their use in cell biology. Various QPI methodologies have been developed highlighting their dependability and quantitative mapping capabilities.These advancements have streamlined the use of interference microscopy, making it more accessible for biological applications. Quantitative Phase-Digital Holographic Microscopy (QP-DHM) has the potential to non-invasively visualize cellular structures and monitor dynamic processes, such as neuronal activity and spine dynamics, which are crucial for understanding cellular functions in neuroscience and psychiatry.Additionally, advancements in QPI have shown how specific biophysical parameters can be extracted from quantitative phase signals, offering critical biological insights for cell characterization.Despite these advances, challenges remain in interpreting quantitative phase signals in relation to biological processes. Accurately determining parameters such as absolute cell volume and membrane fluctuations is essential for furthering our understanding of cellular behavior.This body of work addresses these challenges by employing advanced numerical algorithms to enhance the classification and characterization of cells based on their morphological and physical properties. The novelty of this approach is the integration of a commercial DHM QPI system with advanced numerical techniques, allowing for more accurate cell classification. Deriving specific biophysical parameters and correlating them with cellular functions contributes to the development of quantitative microscopy and opens new avenues for research in cell biology and related fields.
Jag kommer osökt att tänka på ett av mina besök hos min hudcancerläkare där ett misstänkt melanom skars bort och skickades till patologen för att få svar på om/hur aggressivt det var. Det tog labbet ca 2 veckor att få fram det svaret. Forskarna visar här i denna studie att de med HoloMonitor kan få fram ett första preliminärt svar på 58 h. Femtioåtta timmar!!!
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