Investig Clin Urol.  2020 Mar;61(2):216-223. 10.4111/icu.2020.61.2.216.

Establishment of patient-derived three-dimensional organoid culture in renal cell carcinoma

• Na JC
• Kim JH
• Kim SY
• Gu YR
• Jun DY
• Lee HH
• Yoon YE
• Choi KH
• Hong SJ
• Han WK

• KMID: 2471078
• DOI: http://doi.org/10.4111/icu.2020.61.2.216

Abstract

PURPOSE
Renal cell carcinoma is a heterogeneous kidney cancer, and over 403,000 cases were reported worldwide in 2018. Current methods for studying renal cell carcinoma are limited to two-dimensional (2D) culture of primary cell lines and patient-derived xenograft models. Numerous studies have suggested that 2D culture poorly represents the diversity, heterogeneity, and drug-resistance of primary tumors. The time and cost associated with patient-derived xenograft models poses a realistic barrier to their clinical utility. As a biomimetic model, patient-derived three-dimensional (3D) organoid culture can overcome these disadvantages and bridge the gap between in vitro cell culture and in vivo patient-derived xenograft models. Here, we establish a patient-derived 3D organoid culture system for clear cell renal cell carcinoma and demonstrate the biomimetic characteristics of our model with respect to both primary kidney cancer and conventional 2D culture.
MATERIALS AND METHODS
Normal renal tissues and tumor tissues were collected from patients with clear cell renal cell carcinoma. The dissociated cells were cultured as conventional 2D culture and 3D organoid culture. The biomimetic characteristic of the two cultures were compared.
RESULTS
Compared with 2D culture, the 3D organoid cultures retained the characteristic lipid-rich, clear cell morphology of clear cell renal cell carcinoma. Carbonic anhydrase 9 and vimentin were validated as biomarkers of renal cell carcinoma. Expression of the two validated biomarkers was more enhanced in 3D organoid culture.
CONCLUSIONS
Patient-derived 3D organoid culture retains the characteristics of renal cell carcinoma with respect to morphology and biomarker expression.

Keyword

Carcinoma, renal cell; Organoids; Precision medicine

MeSH Terms

Biomarkers
Biomimetics
Carbonic Anhydrases
Carcinoma, Renal Cell*
Cell Culture Techniques
Cell Line
Heterografts
Humans
In Vitro Techniques
Kidney Neoplasms
Organoids*
Population Characteristics
Precision Medicine
Vimentin
Biomarkers
Carbonic Anhydrases
Vimentin

Figure

• 

  Fig. 1 Morphologic characteristics in patient-derived three-dimensional (3D) organoid culture of renal cell carcinoma. (A) Clear cell renal cell carcinoma morphology in primary tissue by hematoxylin and eosin (H&E) stain. (B) 3D organoid culture under light microscopy shows diverse colonies. (C) Paraffin blocked samples of 3D organoid culture show morphology similar to that of clear cell renal cell carcinoma in primary tissue. All scale bars, 20 µm.

• 

  Fig. 2 Biomarker comparison in two-dimensional (2D) culture, patient-derived three-dimensional (3D) organoid culture, and primary tissue. (A) Immunofluorescence analysis of carbonic anhydrase 9 (CA9) (green), keratin (red), vimentin (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) expression in 2D cultured cancer and normal cells. Scale bars, 10 µm. (B) CA9 and keratin immunofluorescence in 3D organoid culture of cancer and normal cells. Scale bars, 10 µm. (C) Immunohistochemistry (IHC) staining of CA9 and vimentin in primary tissue from normal and renal cancer patients. (D) IHC scores of renal cancer tissue in 5 patients. (E) Real-time polymerase chain reaction fold induction of CA9 and vimentin in primary tissue of renal cancer patients relative to normal tissue.

• 

  Fig. 3 Patient-derived three-dimensional (3D) organoid culture of renal cancer cells exhibits increased expression of carbonic anhydrase 9 (CA9) and vimentin. (A) Expression of CA9 and vimentin in 3D organoid culture (passage 4) and two-dimensional (2D) culture by an immunofluorescence assay. Scale bars, 20 µm. (B) Real-time polymerase chain reaction (RT-PCR) relative mean intensity scores of CA9 and vimentin in 3D organoid culture compared to 2D culture (p<0.05). Error bar represents standard error of mean. (C) RT-PCR fold induction of CA9 and vimentin in 3D culture relative to 2D culture. (D) Western blot of CA9 in 3D organoid culture and 2D culture. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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