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    Biomedical Imaging and Instrumentation

    Longitudinal Biomechanical Imaging of Tumor Spheroids using Brillouin Microscopy

    (G-258) Longitudinal Biomechanical Imaging of Tumor Spheroids using Brillouin Microscopy

    Friday, October 13, 2023
    3:30 PM – 4:30 PM PDT
    Location: Exhibit Hall - Row G - Poster # 258
    Introduction::

    Breast cancer is the most common cancer diagnosis in women in the United States as of 20231. Mammograms are currently used for early breast cancer detection and while effective, are limited by the inability to detect small cancerous tumors and the generation of false-negative results due to increased breast tissue density. Typical biomarkers such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), have been extensively used as prognostic and predictive biomarkers to optimize patient therapy treatments, and for the prediction of patient outcomes. More recently, there has been increased interest in understanding the underlying cellular mechanisms facilitating the metastatic potential of breast cancer, with studies observing cell mechanical properties as a potential biomarker2. Changes in elastic and viscoelastic properties of living cells induced by the progression of disease have been strongly correlated to the way cells respond to structural and molecular modifications3. The mechanical regulation of cancer progression has been extensively studied in vitro, however, most of these studies have been restricted to 2D environments lacking the important features of 3D physiological tissues. This study examines the mechanical properties  of 2D cells and 3D tumor spheroids relative to cancer progression using an innovative all-optical method known as Brillouin Microscopy (BM). Spheroids will also undergo a cytotoxicity/viability assay to assess the presence of photodamage on day 9. 



    Materials and Methods:: Healthy non-tumorigenic breast epithelial cell line MCF10A (M1), and metastatic MCF10CA1h (M3) cells were used for spheroid colony formation, following established protocols4. All cells used did not exceed 10 passages. To obtain spheroids, the 3D on top method was employed, in which 200 μl of ice-cold Matrigel (Corning, 354230) was added to a chilled 35 mm gridded dish (Ibidi, 81166) and then placed in the incubator for 30 minutes allowing the layer of Matrigel to solidify. Approximately 5,000 cells were suspended in 500 μl of assay medium and placed on top of the Matrigel layer and incubated for 30 minutes. An additional 500 μl of assay medium containing 10% Matrigel was added on top of the cell seeded Matrigel layer to obtain a final concentration of 5% Matrigel. Cells form 3D tumor-like spheroid colonies over the course of several days. 2D Brillouin images of select spheroids were acquired by a confocal Brillouin microscope on days 0, 2, 5, and 8 as seen in Figures 1 and 2, and the average Brillouin shift was used to represent the mechanical properties of the spheroids.

    Results, Conclusions, and Discussions::

    The average Brillouin Shift of M1 spheroids is higher than that of M3 on day 0, 2, and day 8, however, no difference is observed between healthy and metastatic cells on day 5, as seen in Table 1. Statistical significance was observed when comparing M1 to M3 on day 0, and day 8 (p< 0.05). The Brillouin shift of M1 and M3 spheroids tends to increase from day 0 to day 5, however, M3 exhibits a decrease in Brillouin shift on day 8 whereas M1 continues to increase, as seen in Table 1. It has been established that cellular mechanical properties play a significant role in pathogenesis and pathophysiology, and it is evident that cancer cells display distinct mechanical properties when compared to healthy cells for many types of cancer. Biomarkers have played a significant role in demonstrating the molecular mechanisms involved in breast cancer progression but have limited sensitivity and specification for cancer diagnosis. In recent years, biomechanics has been recognized as a new biomarker for early detection of cancer. Therefore, there is a significant need for a method that allows the measurements of the mechanical properties of the cells in physiologically relevant environments. Here, we demonstrated a non-contact and label-free optical method for measuring the mechanical properties of cancer cells in 3D conditions. We envision this method can pave the way for understanding the underlying biomechanical mechanism that facilitate cancer cell progression. Future work will focus on the confirmation of the observed trends in both M1 and M3 cell lines and validate the Brillouin data with Atomic Force Microscopy. We investigated the mechanical evolution of breast cancer cells in 3D conditions using non-contact optical Brillouin microscopy. The longitudinal mechanical measurement of spheroids for 8 days revealed that cancer cells and healthy cells have different mechanical features during growth. This new data could help us understand the role of biomechanics in cancer progression.



    Acknowledgements (Optional): :

    References (Optional): :

    1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA: A Cancer Journal for Clinicians 2023; 73(1):17-48.
    2. Baker EL, Lu J, Yu D, Bonnecaze RT, Zaman MH. Cancer cell stiffness: integrated roles of three-dimensional matrix stiffness and transforming potential. Biophys J. 2010 Oct 6;99(7):2048-57. doi: 10.1016/j.bpj.2010.07.051. PMID: 20923638; PMCID: PMC3042573.


    3. Suresh S, Spatz J, Mills JP, Micoulet A, Dao M, Lim CT, Beil M, Seufferlein T. Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria. Acta Biomater. 2005 Jan;1(1):15-30. doi: 10.1016/j.actbio.2004.09.001. PMID: 16701777.


    4. Lee GY, Kenny PA, Lee EH, Bissell MJ. Three-dimensional culture models of normal and malignant breast epithelial cells.     Nat Methods. 2007 Apr;4(4):359-65. doi: 10.1038/nmeth1015. PMID: 17396127; PMCID: PMC2933182.