Living human lung slices for ex vivo modelling of lung cancer

Abstract

The tumor microenvironment (TME) markedly affects cancer progression, yet traditional animal models do not fully recapitulate the situation in humans. To address this, we developed tumor-derived precision lung slices (TD-PCLS), an ex vivo platform for studying the lung TME and evaluating therapies. TD-PCLS, viable for 8-10 days, preserve the heterogeneity and metabolic activity of primary tumors, as confirmed by seahorse analysis. Using multispectral FACS and phenocycler multiplex imaging, we spatially profiled TME components and cancer cell functionality. Additionally, TD-PCLS revealed patient-specific responses to chemo- and immunotherapies. To complement TD-PCLS, we established tumor-cell-seeded PCLS (TCS-PCLS) by introducing tumor and immune cells into healthy lung slices. This model highlighted macrophage-tumor interactions as critical for tumor cell proliferation, migration, and immune modulation. Together, these platforms provide a robust tool for lung cancer research, enabling precision medicine and advancing therapeutic discovery.

Keywords: Immunology; Immunotherapy; Lung cancer; Oncology.

Figure 1. Preparation and culture of TD-PCLS from fresh human lung tumor.

(A and B) Immediately after lobectomy, the removed lung was filled with 1% low-melting agar in the operating room or pathology department and waited for the agar to solidify. (C) The filled lung was evaluated and sectioned by the expert pathologist to identify the tumor area for clinical purposes and also to provide the proper piece for TD-PCLS preparation. (D) The tumor piece was transferred on ice to the molecular biology laboratory. First, the tumor was cut in the shape with sharp angle (about 90 degrees) and then stuck to the metal plate, which was then moved into the vibratome sample box filled by cold PBS. (E and F) TD-PCLS was prepared by cutting tumor pieces in various thickness, depending on tumor type and structure and subjected to further treatment, imaging, and analysis. The thickness was between 200–500 μm from lung adenocarcinoma. The criteria that defines the thickness was tumor size, thickness, solidity, and necrotic area.

Figure 2. TD-PCLS is a dynamic and vital heterogeneous ecosystem.

(A) Sytox (green) and Hoechst (blue) immunofluorescence staining as viability indicator in TD-PCLS during culture for 8 days. (n = 3 lung adenocarcinoma tumors). Scale bar: 150 μm. (B) In situ lactate dehydrogenase (LDH) activity in TD-PCLS during culture for 8 days. Scale bar: 1mm. (C) Oxygen consumption rate (OCR) in TD-PCLS cultured for 1 and 8 days measured via Seahorse analysis, (n = 2 lung adenocarcinoma patients). (D) Representative images (left panel) with quantification (right panel) of TD-PCLS cultured for 1 and 8 days, stained with EdU (red), cytokeratin 18 (CK18) and Dapi (blue). Scale bar: 50μm. n = 5 technical replicates. (E and F) Immunofluorescence staining and quantification of positive cytokeratin 18 (CK18, tumor cell marker; green) in formalin-fixed paraffin-embedded (FFPE) of lung tumor tissue immediately after surgery (day 0) and after processed to TD-PCLS and culture for 2 days. Dapi was used as nuclear dye (blue). Scale bar: 50 μm. (G and H) Immunofluorescence staining and quantification of EdU (proliferation marker; red), Tunel (apoptosis marker; red) in positive cytokeratin 18 (CK18, tumor cell and epithelial marker; orange) in TD-PCLS and healthy. Dapi was used as nuclear dye (blue) (n = 3 lung adenocarcinoma tumors). Scale bar: 50μm. (I) OCR measurement of tumor areas punched from TD-PCLS and healthy adjacent area punched from healthy PCLS (n = 3 lung adenocarcinoma tumors). Statistical significance was determined by a 2-tailed unpaired Student’s t test. Data are presented as mean ± SEM. *P < 0.05, ****P <0.0001.

Figure 3. TD-PCLS is a proxy for TME and immune constituents.

Representative plots of spectral flow cytometry of Healthy- and TD-PCLS showing the gating strategy of immune cell components including (A) macrophages, plasmacytoid dendritic cells (pDCs), classical dendritic cells (cDC), (B) natural killer (NK) cells, (C) monocytes, granulocytes, (D) gd-T cells, CD8⁺ T cells, CD4⁺ T cells and regulatory T cells (Tregs), after 2-days’ culture (n = 3 lung adenocarcinoma tumors). (E) Immunofluorescence staining of cytokeratin 18 (CK18, tumor cell marker; orange) and CD68 as macrophage pan marker (red) in healthy- TD-PCLS. Dapi was used as nuclear dye (blue) (n = 3 lung adenocarcinoma tumors). Scale bar: 50 μm.

Figure 4. Multiplex IF and multispectral imaging show distribution of structural, functional, and immune cell phenotypes in TD-PCLS.

(A) Workflow of multiplex IF staining in TD-PCLS with Akoya Bioscience technology. (B) Representative images of different cell phenotypes including tumor cells (Pan-CK), endothelial cells (CD31), smooth muscle cells (SMA), proliferating tumor cells (Pan-CK, PCNA and Ki67), apoptotic tumor cells (Pan-CK and PARP), granulocytes (CD11b), macrophages (CD68), CD4⁺ T cells (CD45, CD3e, and CD4), CD8⁺ T cells (CD45, CD3e, CD4, and CD8), Tregs (CD45, CD3e, CD4, and FoxP3), B cells (CD45 and CD20), PD1 (CD45 and PD1) cells, and PD-L1 cells (CD45 and PD-L1) in TD-PCLS. Scale bars: 2.3 mm for the whole tumor image (left) and 100 μm (right, individual cell type). (C and D) Heatmap shows a curated clustering dendrogram with cell phenotypes and pie chart showing the abundance of each cell phenotype in TD-PCLS tissue.

Figure 5. Treatment of TD-PCLS with chemo/immunotherapy regimens.

(A) Workflow of chemo/immune therapy in TD-PCLS. Created in Biorender.com. (B) EdU staining (red) as marker of proliferating tumor cells stained with CK18 (orange) as tumor cell marker in TD-PCLS treated with nivolumab (10 μg/mL) and combination of nivolumab (10 μg/mL) +carboplatin (5 μg/mL) for 48 hours (n = 2 lung adenocarcinoma tumors) followed by quantification analysis. In the quantification (right panel), each color (grey or blue) indicate one patient. Dapi was used as nuclear dye (blue). Scale bar: 100 μm. (C) Immune profile analysis including monocytes, macrophages, tumor-promoting M2 macrophages, antitumor M1 macrophages, neutrophils, T cells, CD4⁺ T cells, CD8⁺ T cells, Tregs, B cells of TD-PCLSs treated with nivolumab (10ug/mL) and combination of nivolumab (10 μg/mL) +carboplatin (5 μg/mL) for 48 hours subjected to the established tissue dissociation protocol followed by spectral flow cytometry analysis (n = 3 lung adenocarcinoma tumors).

Figure 6. Preparation and applications of TCS-PCLS.

(A) Workflow of preparation of TCS-PCLS by implementation of A549-positive GFP on nontumor/healthy PCLS. Created in Biorender.com. (B) Healthy PCLS was seeded with A549-GFP cells. After 24 hours, tissue was fixed and imaged with the Leica Thunder Imager using the lung autofluorescence (red), and the GFP signal emitted from A549 cells (green). Scale bar: 500 μm (right), 100 μm (left, magnification). (C) Healthy PCLS was cultured with A549-GFP spheroids for 24 hours. Spheroid outgrowth was imaged with the Leica SP8 confocal microscope using the Lung autofluorescence (red) and the GFP signal (green). Scale bar: 250 μm. (D) Healthy PCLS was seeded with A549-GFP cells. Time lapse imaging was done with the Keyence New All-in-One Fluorescence Microscope BZ-X800 in a stage top incubator for 24 hours. Scale bar: 500μm. (E) Healthy PCLS was seeded with A549-GFP cells and cultured for 24 hours. Prior fixation, 20 μM EdU was added overnight and visualized using the Click-iT EdU Kit. Slices were imaged using the Leica SP8 confocal microscope for the Lung autofluorescence (red), GFP signal (green), and EdU incorporation (magenta). Scale bar: 100 μm. (F) Healthy PCLS was seeded with A549-GFP and then cultured in different types of macrophage-conditioned medium (CM) for 24 hours, including naive unstimulated M0 macrophages, tumor-promoting M2 macrophages (stimulated with IL4 for 24 hours), antitumor M1 macrophages (stimulated with LPS and IFN-γ for 24 hours). Slices were imaged using the Leica SP8 confocal microscope. Scale bar: 100 μm.