Protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments

Abstract

3D culture models better replicate tumor structure and interactions than 2D methods, enhancing cancer therapy research. Here, we present a protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments. We describe steps for optimizing tumor spheroids, isolating CD4+ T cells and monocytes, and differentiating T regulatory (Treg) cells and M2-like macrophages. We then detail procedures for seeding tumor-immune spheroids, performing fluorescence-activated cell sorting (FACS) analysis, and characterizing spheroids, including their response to radiotherapy.

Keywords: Microscopy; cancer; cell biology; cell culture; cell isolation; cell-based assays; flow cytometry; immunology; model Organisms.

Graphical abstract

Figure 1

Casting process of agarose molds and functional characterization of murine lung TSp (A) Heat agarose in the microwave and prepare MicroTissues 3D Petri Dish. Add 600 μL of melted agarose and wait until complete solidification. (B) Introduce each agarose molds in individual wells of 12-well culture plate. (C) Fill the well up to 2 mL of complete medium. Place the culture plate in a humidified incubator until seeding TSp or TImS. Hydration of the molds needs to be done at least 2h before seeding. (D) Diagram summarizing the functional characterization assays of cells within murine lung TSp. Plate #1 is used to measure cell viability while plate #2 is used to measure mitochondrial activity.

Figure 2

Morphological and physical characterization of TSp (A) Representative images of TSp formation using different cell number seeding (1,000, 2,500, and 5,000 cells). Images were taken on days 1, 3, 6, and 8, for Lacun3 and CMT167 lung cancer cells. (B) Pipeline followed in AnaSP software to analyze diameter, compactness and circularity of different spheroids. We first create a mask of the spheroid. Then the software automatically calculates different parameters of each TSp. (C) Diameter, compactness, and circularity parameters were used to characterize the physical properties of TSp. AnaSP software was employed for the analysis. Data are expressed as mean ± SD and were analyzed with a two-way ANOVA followed by a Bonferroni test. ∗: p <0.05; ∗∗: p <0.01; ∗∗∗: p <0.001.

Figure 3

Functional characterization of TSp Graphs showing cell viability (A) proliferation rate (B) and mitochondrial activity (expressed in MFI) (C) of Lacun3 or CMT167 TSp. Data are expressed as mean ± SD and were analyzed with student’s t-test. ∗: p <0.05; ∗∗: p <0.01.

Figure 4

Diagram representing the isolation of murine CD4+ T cells and monocytes and the establishment of co-culture conditions (A) The scheme illustrates the isolation of murine bone marrow cells and splenocytes, followed by differentiation into M2-like macrophages and T regulatory cells, respectively. (B) Gating strategy used to show the yield of the conversion into T regulatory cells. (C) Gating strategy and yield obtained in the macrophage differentiation step.

Figure 5

Irradiation of TImS and analysis of cell death (A) Example of the gating strategy showing the different populations included in the TImS: Tumor cells (CD45-); Non-converted CD4 T cells (CD45+CD4+CD25-FoxP3-), T regulatory cells (CD45+CD4+CD25+FoxP3+) and M2-like macrophages (CD45+CD11b+F4/80+CD206+). Viakrome 808 is used to stain non-viable cells. (B) Graphical representation of the total number of immune cells (non-converted CD4+ T cells, T regulatory cells, and M2-like macrophages) present in each mold, with or without radiation, in both models studied. Data are expressed as mean ± SD and were analyzed with a student’s t-test. ∗∗∗: p <0.001.

Figure 6

Irradiation of TSp and TImS and analysis of cell death (A and B) Representative brightfield images of Lacun3- and CMT167-derived TSp and TImS with or without irradiation. (C and D) Representative fluorescence images of Lacun3 TImS (C) and CMT167 TImS (D) at day 1 (untreated) and day 3 (after irradiation). Tumor cells are seen in blue (CellTrace Violet Cell Proliferation), M2-like macrophages in red (CellTrace Far Red Cell Proliferation) and T regulatory cells in green (CellTrace CFSE Cell Proliferation). Scale bar: 100 μm.