Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 µm intervals, covering a depth of 90 µm in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Comparison of monocytic cell lines U937 and THP-1 as macrophage models for in vitro studies.

Understanding macrophage biology can improve comprehension of diverse biological processes and provide insights into novel therapeutic immunomodulatory strategies. Due to limited yield and technical difficulty in isolating primary macrophages, in vitro studies commonly use monocytes as precursor cells. Monocytic cell lines are a virtually unlimited source of macrophage precursors and two of the most frequently used cell lines are THP-1 and U937. Besides a great variability in macrophage differentiation protocols there is scarce information on possible differences in the biological responses of these cell lines. In this study, we used a standardized differentiation protocol using PMA and compared the response of macrophages derived from THP-1 and U937 cells to M1-and M2-polarizing conditions. THP-1-derived macrophages are more responsive to M1 stimuli and skewed towards M1 phenotype, whereas U937-derived macrophages were more responsive to M2 stimuli and skewed towards M2 phenotype. THP-1-derived macrophages also had greater production of ROS and phagocytic activity. Under M1-polarizing conditions, macrophages derived from both THP-1 and U937 reduced phagocytosis activity and the increased production of ROS. This information should be considered to make an informed choice on the cell line used as in vitro macrophage model, according to the experimental goals and biological context.