Characterization of 3D NSCLC Cell Cultures with Fibroblasts or Macrophages for Tumor Microenvironment Studies and Chemotherapy Screening

Abstract

The study of 3D cell culture has increased in recent years as a model that mimics the tumor microenvironment (TME), which is characterized by exhibiting cellular heterogeneity, allowing the modulation of different signaling pathways that enrich this microenvironment. The TME exhibits two main cell populations: cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAM). The aim of this study was to investigate 3D cell cultures of non-small cell lung cancer (NSCLC) alone and in combination with short-term cultured dermal fibroblasts (FDH) and with differentiated macrophages of the THP-1 cell line. Homotypic and heterotypic spheroids were morphologically characterized using light microscopy, immunofluorescence and transmission electron microscopy. Cell viability, cycle profiling and migration assay were performed, followed by the evaluation of the effects of some chemotherapeutic and potential compounds on homotypic and heterotypic spheroids. Both homotypic and heterotypic spheroids of NSCLC were generated with fibroblasts or macrophages. Heterotypic spheroids with fibroblast formed faster, while homotypic ones reached larger sizes. Different cell populations were identified based on spheroid zoning, and drug effects varied between spheroid types. Interestingly, heterotypic spheroids with fibroblasts showed similar responses to the treatment with different compounds, despite being smaller. Cellular viability analysis required multiple methods, since the responses varied depending on the spheroid type. Because of this, the complexity of the spheroid should be considered when analyzing compound effects. Overall, this study contributes to our understanding of the behavior and response of NSCLC cells in 3D microenvironments, providing valuable insights for future research and therapeutic development.

A Novel ceRNET Relying on the lncRNA JPX, miR-378a-3p, and Its mRNA Targets in Lung Cancer

“…In particular, JPX promoted cell proliferation, migration, and invasion, confirming data already reported; conversely, miR-378a gave the opposite effect, indicating an oncosuppressive activity; this activity was completely counteracted by JPX overexpression, showing results similar to control experiments (Figure 3). We also decided to explore the functional relevance of JPX/miR-378a interaction by using spheroid models, which are commonly used in cancer research for their ability to better recapitulate the biological and molecular features of the complex tumor architecture, and which thus limit the use of animals for in vivo studies [38,39]. Results that were consistent with the above-described assays were obtained using the 3D-spheroid formation assay.…”

“…Different cancer cell lines are able to form anchorage-independent aggregations, called spheroids, indicative of their ability to proliferate as cancer stem-like cells and to successfully establish clones with tumor-initiating potential at distant niches for metastasizing [38,39]. The ability to form 3D-spheroids was tested with A549 cells transfected with the control molecules, miR-378a mimic, JPX overexpressing vector, or a combination of both.…”