Patient-derived spheroids and patient-derived organoids simulate evolutions of lung cancer

Abstract

Cancer cells harbor many genetic mutations and gene expression profiles different from normal cells. Patient-derived cancer cells (PDCC) are preferred materials in cancer study. We established patient-derived spheroids (PDSs) and patient-derived organoids (PDOs) from PDCCs isolated from the malignant pleural effusion in 8 patients. The morphologies suggested that PDSs may be a model of local cancer extensions, while PDOs may be a model of distant cancer metastases. The gene expression profiles differed between PDSs and PDOs: Gene sets related to inflammatory responses and EMT were antithetically regulated in PDSs or in PDOs. PDSs demonstrated an attenuation of the pathways that contribute to the enhancement of transforming growth factor beta (TGF-β) induced epithelial mesenchymal transition (EMT), while PDOs demonstrated an attenuation of it. Taken together, PDSs and PDOs have differences in both the interaction to the immune systems and to the stroma. PDSs and PDOs will provide a model system that enable intimate investigation of the behavior of cancer cells in the body.

Keywords: Extracellular matrix; Immune system; Lung cancer; Organoid; Spheroid.

Graphical abstract

Fig. 1

Formation of PDS and its morphology. (A) Observation using a phase contrast microscope. A representative result was shown (patient IDEN 202002). (i) Cells transferred to a flask without a feeder layer (passage 1, day 0). (ii) Cells at passage 1, day 10, (iii) Cells at passage 15, day 10, (iv) Cells at passage 30, day 10. A larger congregation was formed as the passage number increases, suggesting an increased proliferation rate and an advancement to a more malignant phenotype. A scale bar indicates 200 μm. (B) Cell morphology. A representative result was shown (patient IDEN 202002). The appearance of large, multinucleated cells (indicated by an arrow in Giemsa staining, and cytokeratin 7 + Hoechst 33258 double immunofluorescence staining) and a fainter TFF-1 staining (indicated by an arrow) suggests an advancement to a more malignant phenotype. A scale bar indicates 50 μm.

Fig. 2

Formation of PDO and its morphology. (A) Observation using a phase contrast microscope. A representative result was shown (patient IDEN 202006). (i) PDCCs were embedded in Matrigel matrix (passage 0, day 0). (ii) Cells at passage 0, day 10, (iii) Cells at passage 0, day 20, (iv) Cells after a subculture, at passage 5, day 20. A spherical structure was evident, (v) Cells at passage 10, day 20, (vi) Cells at passage 15, day 20. A scale bar indicates 200 μm. (B) Cell morphology. A representative result was shown (patient IDEN 202006). The cell congregate has a structure and has a diameter consistent of an organoid (i.e., >200 μM). A scale bar indicates 50 μm.

Fig. 3

(A) (B) Principle component analysis (PCA) for PDCCs, PDSs and PDOs. The figure displays a three-dimensional scatter plot of the first three principal components (PCs) of the data based on the log transformed TPM. Each point represents an RNA-Seq sample. Each pair of PDCC and PDS or PDO are clustered together.

Fig. 4

(A) Heatmaps demonstrate the difference in the gene expression profiles. PDS and PDOs were compared to their parental PDCCs. Each column represents a single sample. Upregulated genes were colored by shades of red, while downregulated genes were colored by shades of blue according to the Z-score. (B) (C) Volcano plot demonstrate the difference in the expression of individual genes. PDSs and PDOs were compared to their parental PDCCs. Red plots represented upregulated genes (log2 fold change >1, p value < 0.05). Blue plots represented downregulated genes (log2 fold change >1, p value < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Gene set enrichment analysis (GSEA). Comparisons were based on the transcripts per million (TPM). (A) PDSs and their originating PDCCs. (B) PDOs and their originating PDCCs. (C) PDSs and PDOs. The green curve shows the enrichment score (ES) for the gene set. The bar at the bottom represents the genes. The far left (red) correlated with the most up-regulated genes and the far right (blue) correlated with the most down-regulated genes, the vertical black lines indicated the position of the gene sets. The heatmap indicated the core enriched gene sets. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Heatmaps demonstrating the difference in curated gene sets proofed by experts (C2 in MSigDB collections: http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). (A) PDS and its parental PDCC (B) PDO and its parental PDCC (C) PDS and PDO. Each column represented a single sample. Upregulated pathways were colored by shades of red, while downregulated pathways were colored by shades of blue according to the Z-score. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)