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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I5 - Quantifying Breast Cancer Cellular Morphology and its Link to Metastasis

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Detection of metastatic breast cancer will not happen until after the cancer has spread throughout the body, resulting in delayed treatment and often, more advanced cancer cases. Thus, early identification of metastatic cancer could significantly alter therapeutic approaches worldwide. Morphology, or cell shape, is a quantifiable readout of the unique molecular and biochemical reactions occurring within a cell and can be used as an indicator of molecular functioning. As such, we propose that cell morphology could be used as a diagnostic tool for determining cancer aggressiveness, though, there is still debate on what cell structures aid in migration and invasion out of the primary tumor. We hypothesize that cell morphology can be used as a metric for predicting cell migratory potential. Elongated mesenchymal-like cells are associated with cancer aggressiveness since the epithelial-to-mesenchymal transition is a hallmark of breast cancer metastasis. Additionally, increased ploidy, which appears as larger cell/nucleus size is associated with more aggressive tumor phenotype. We would expect cell lines with a more elongated morphology (i.e. mesenchymal-like) or with a higher presence of larger cells to achieve higher rates of migration. The present work has quantitatively evaluated cell morphology characteristics using high-resolution microscopy and high-throughput data analysis software, providing a complex study of cancer cell morphology parameters. The implication of this study is that cell morphology can be used as an additional parameter in determining metastatic potential. This analysis establishes that there are unique and differentiable morphological characteristics within low metastatic and high metastatic breast cancer cells.

Materials and

Methods:  MCF7 (non-invasive adenocarcinoma, ER+, PR+) and MDA-MB-231 (highly-invasive adenocarcinoma ER-/PR-/HER2-) cells were cultured and seeded at density of 10,000 cells/cm2 so the individual cells and any potential cytoskeletal projections could be easily identified. After a 14-hour incubation period, cells were fixed with a 4% paraformaldehyde solution, and the cells and their nuclei were dyed with Hoechst and Fluorescin-5-Malemide, respectively. Cells were subsequently imaged on the Cytation 1 Cell Imager (Agilent BioTek), with 16 images taken per well for a total of 1536 images per cell line.

CellProfiler, a free open-source software provided by the Broad Institute, was used to quantitatively measure cell morphology from the images obtained. A pipeline was developed to detect the edge of both cytoplasm and nuclei, quantifying several morphological parameters. The algorithm enhanced the pixel intensity for both nuclear and cytoplasm signals. Pixels above a specified intensity on the DAPI (nuclear, blue) channel were grouped to identify nuclei, and pixels above a specific intensity on the GFP (cytoplasm, green) channel were grouped to identify cytoplasm. Overlays of nuclear and cytoplasm annotations identified individual cells, as shown in Figure 1A. The pipeline quantified cell and nucleic area, cell eccentricity, cell form factor, cell solidity, and nucleus-to-cytoplasm ratio for each individual cell. MATLAB was used to convert cellular data into a matrix, then 50,000 cells per cell line were randomly selected to be analyzed. The MCF7 and MDA-MB-231 data for each morphological characteristic was compared using unpaired t-tests, displayed in Figure 1B.

Results, Conclusions, and Discussions: A distinct difference in cell morphology was observed between the MCF7 and MDA-MB-231 cell lines, which can be linked to cell function and migration potential. The triple-negative and highly aggressive MDA-MB-231 cell line shows significantly larger cell and nucleus areas (Figure 1B) as compared to the lowly metastatic MCF7 cell line, potentially indicating increased ploidy. Ploidy increase is most often observed in high-grade tumors in comparison to low-grade tumors and is associated with tumor cell proliferation and overall worse prognosis. While MDA-MB-231 cells and their nuclei were on average larger than their MCF7 counterparts, CellProfiler measured a significantly lower nucleus-to-cytoplasm ratio. This is linked to cell morphology as cells with more cell-surface projections (e.g. lamellipodia and filopodia) and increased focal adhesions have a much larger 2D cell area as compared to cells more epithelial-like in morphology that have higher circularity and lower eccentricity. Future work will confirm this and the hypothesis that more elongated (mesenchymal-like) cells are more aggressive. Overall, this analysis pipeline quantified breast cancer cells with a low metastasis (MCF7) rate as smaller and with a higher nucleus to cytoplasm ratio, than breast cancer cells with a high metastasis rate (MDA-MB-231).

In conclusion, this study establishes quantifiable morphological characteristics that vary between highly metastatic MDA-MB-231 cells and lowly metastatic MCF7 breast cancer cells. By quantifying distinct differences in cell morphology across breast lines, we are able to gain a more wholistic view of the individual cells within a sample. Future work includes evaluating additional cell morphology parameters (e.g. eccentricity, form factor, perimeter, etc.), quantifying these morphological characteristics within other breast cell lines (cancerous and noncancerous), and comparing this data to previously recorded 2D cell migration data. This work will strengthen our knowledge on the relationship between quantifiable morphology parameters and breast cancer aggressiveness.

Acknowledgements (Optional):  This work is supported by the National Science Foundation CAREER Award #2145521.