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.

Keywords: 3D cell culture; co-culture; fibroblasts; macrophages; spheroids; tumor microenvironment.

Figure 1

Homotypic and heterotypic spheroid generation. (A) Homotypic and heterotypic spheroids on different days in cell culture. Scale bar 400 µm. (B) Measurement of spheroid diameters. (C) Number of cells/spheroid on different days in culture. Data are shown as mean relative to control in three independent experiments (SD). Statistical analysis was performed using Dunnett’s test with an ANOVA test for multiple comparisons vs. control. * p value < 0.05 and ** p value < 0.001.

Figure 2

Variation in the diameter of spheroids by density and the size of well. (A) homotypic spheroids of the A549 lineage formed with a density of 1000 cells per well. Scale bar 400 µm. (B) Measurement of the diameter of A549 and LC-HK2 homotypic spheroids. Scale bar 400 µm. (C) Measurement of the diameter of the A549 and LC-HK2 homotypic spheroids on different days in the culture. Data are shown as mean relative to day 5 in three independent experiments (SD). Statistical analysis was performed using Dunnett’s test with an ANOVA test for multiple comparisons vs. day 5. * p value < 0.05 and ** p value < 0.001.

Figure 3

Confocal laser scanning microscope (LSM) images of actin filaments and tubulin microfilaments: image of the spheroids showing separate channels (actin and microtubules), and merging. Immunofluorescence of microtubules (green) of spheroid cells was performed with primary antibody anti-mouse and secondary antibody Alexa 488, the nucleus (blue) stained with DAPI, and the actin cytoskeleton (red). Cellular organization is evidenced by the actin cytoskeleton staining and mitotic cells, by microtubules in the mitotic spindle. Scale Bar: 100 µm.

Figure 4

Morphological characterization of homotypic and heterotypic spheroids. (A,B) To visualize the cytokeratin 18 filaments within the heterotypic spheroids containing carcinoma cells, immunofluorescence was performed using an antibody anti-cytokeratin 18 mouse and anti-mouse alexa 488. Additionally, nuclei were stained with DAPI and with red actin cytoskeleton. Scale bar 100 µm. (C) Semi-thin sections (from TEM) of both homotypic and heterotypic spheroids. Scale bar: left, 50 µm and right, 100 µm.

Figure 5

Transmission electron microscopy representative images of homotypic spheroids: cells from spheroids A549 and LC-HK2 have organelles such as nucleus (Nu); asterisks show nuclear pores (*); mitochondria (Mt), endoplasmic reticulum (ER), lipid droplets (dl), apoptosis (Ap), lamellar bodies (Lb).

Figure 6

Transmission electron microscopy representative images of heterotypic spheroids with fibroblast: the cells of the spheroids A549/FDH and LC-HK2/FDH present organelles such as the nucleus (Nu), mitochondria (Mt), endoplasmic reticulum (ER), vesicles (V), lipid droplets (dl), lysosomes (L), autophagy (Au), Golgi apparatus (AG), lamellar bodies (Lb), filaments (F). Blue dotted lines show contact between two neighboring cells (----).

Figure 7

Transmission electron microscopy representative images of heterotypic spheroids with macrophages: LC-HK2/Mcf spheroid cells have organelles such as the nucleus (Nu), mitochondria (Mt), endoplasmic reticulum (ER), vesicles (V), double membranes (dm), lipid droplets (dl), lysosomes (L).

Figure 8

Viability and cell cycle of homotypic and heterotypic spheroids: (A) cell viability of spheroids with 7 days in cell culture, stained with Hoechst and propidium iodide; (B) cell cycle of spheroids with 7 days in cell culture. Data are shown as mean relative to control in three independent experiments (SD).

Figure 9

Characterization of spheroid cell migration. (A) Images after 72 h of spheroids placed on an adherent surface. Scale bar: 400 µm. (B) Measurement of the diameter occupied by the cells that migrated from the spheroids after 72 h; three spheroids per treatment were measured in triplicate. Statistical analysis was performed using Dunnett’s test with an ANOVA test for multiple comparisons vs. control. (C) Spheroids after 7 days of generation shown on their surface cells with much larger cytoplasm, in bubble format. Scale bar: 400 µm. (D) Immunofluorescence of cells that migrated from LC-HK2 and LC-HK2/FDH spheroids and nuclei were stained with IP (red), with microtubules in green and cytoplasm in blue. Scale bar LC-HK2 (image of laser scanning confocal microscopy): Right—50 µm, Left—100 µm; LC-HK2/FDH (image obtained by LionHeart microscopy): Right 30 µm Left—100 µm. ** p value < 0.001.

Figure 10

Differences in response to anti-cancer drugs of LC-HK2 heterotypic spheroids treated for 48 h. (A) Cell viability assay with HO and IP of spheroids treated for 48 h (merge). Blue cells represent spheroid-forming cells, and red areas represent non-viable cells. Bar Scale: 200 µm. (B) Measurement of spheroid diameter after 48 h of treatment. Three spheroids per treatment were measured in triplicate. (C) Measurement of ATP production of spheroids treated with Dox, CDDP, CA5 and Sts (used as a control for cell death). The measurements were normalized with respect to the control. The experiments were performed in triplicate. Statistical analysis was performed using Dunnett’s test with an ANOVA test for multiple comparisons vs control. * p value < 0.05 and ** p value < 0.001.