Microenvironment of a tumor-organoid system enhances hepatocellular carcinoma malignancy-related hallmarks

Abstract

Organ-like microenviroment and 3-dimensional (3D) cell culture conformations have been suggested as promising approaches to mimic in a micro-scale a whole organ cellular functions and interactions present in vivo. We have used this approach to examine biologic features of hepatocellular carcinoma (HCC) cells. In this study, we demonstrate that hepatocellular carcinoma (HCC) cells, fibroblasts, endothelial cells and extracellular matrix can generate organoid-like spheroids that enhanced numerous features of human HCC observed in vivo. We show that the addition of non-parenchymal cells such as fibroblast and endothelial cells is required for spheroid formation as well as the maintenance of the tissue-like structure. Furthermore, HCC cells cultured as spheroids with non-parenchymal cells express more neo-angiogenesis-related markers (VEGFR2, VEGF, HIF-α), tumor-related inflammatory factors (CXCR4, CXCL12, TNF-α) and molecules-related to induced epithelial-mesenchymal transition (TGFβ, Vimentin, MMP9) compared with organoids containing only HCC cells. These results demonstrate the importance of non-parenchymal cells in the cellular composition of HCC organoids. The novelty of the multicellular-based organotypic culture system strongly supports the integration of this approach in a high throughput approach to identified patient-specific HCC malignancy and accurate anti-tumor therapy screening after surgery.

Keywords: endothelial cell; fibroblast; hepatocellular carcinoma; organoid; tumor microenvironment.

Figure 1.

Self-assembly of tumor-organoids and proliferation assay. (A) Phase contrast microscopy of tumor-organoid in a 24-well culture plate. Time-lapse of HCC cells and human non-parenchymal cells (fibroblast and endothelial cells) or only HCC cells cultured on matrigel at 24h. Immunofluorescence analysis of the resulting tumor-organoids, Vimentin; red, nuclei; blue. Scale bar 100 µm. (B) Immunofluorescence stainings of endothelial cells in 2 mixed cells tumor-organoids and human HCC tissue, vWF; red, nuclei; blue. Scale bar 100 µm. (C) Proliferation analysis by Ki67 immunefluorescence staining, GPC-3; red, nuclei; blue, Ki67; green. Scale bar 200 µm. (n = 3) (D) Ki67-GPC3 positive cell quantification (n = 3).

Figure 2.

Characterization of tumor-organoids for epithelial-mesenchymal transition associated markers. (A) Immunofluorescence analysis of the resulting tumor-organoids and human HCC tissue, white arrow; strong double positive cells in human HCC tissue, GPC-3; green, nuclei; blue, vimentin; red. Scale bar 200 µm. (n = 3). (B) Quantitative PCR analysis of Vimentin, MMP9 and TGFβ in human tumor-organoids and controls at 24 h after self-assembly. Results represent mean  ±  s.d., (n = 3) * p < 0.05.

Figure 3.

Characterization of tumor-organoids for neo-angiogenesis-related markers. (A) Immunofluorescence analysis of the resulting tumor-organoids and human HCC tissue, white arrows; strong double positive cells in human HCC tissue, GPC-3; red, nuclei; blue, VEGFR2; green. Scale bar 200 µm. (n = 3). (B) Quantitative PCR analysis of VEGFR2, VEGF, HIF-α in human tumor-organoids and controls at 24 h after self-assembly. Results represent mean  ±  s.d., (n = 3) * p < 0.05.

Figure 4.

Characterization of tumor-organoids for tumor-related inflammatory factors. (A) Immunofluorescence analysis of the resulting tumor-organoids and human HCC tissue, white arrows; CXCR4 positive fibroblasts in human HCC tissue, GPC-3; green, nuclei; blue, CXCR4; red. Scale bar 200 µm. (n = 3). (B) Quantitative PCR analysis of CXCR4, CXCL12, TNF-α in human tumor-organoids and controls at 24 h after self-assembly. Results represent mean  ±  s.d., (n = 3) * p<0.05.

Figure 5.

Expression of long-term cultured tumor-organoids. (A) Quantitative PCR analysis of Vimentin, MMP9, TGFβ, VEGFR2, VEGF, HIF-α, CXCR4, CXCL12 and TNF-α in human tumor-organoids and controls at 7 d after self-assembly. Results represent mean  ±  s.d., (n = 3) * p<0.05.