The establishment of kidney cancer organoid line in drug testing

Abstract

Introduction: Kidney cancer is a common urological malignancy worldwide with an increasing incidence in recent years. Among all subtypes, renal cell carcinoma (RCC) represents the most predominant malignancy in kidney. Clinicians faced a major challenge to select the most effective and suitable treatment regime for patients from a wide range of modalities, despite improved understanding and diagnosis of RCC.

Objective: Recently, organoid culture gained more interest as the 3D model is shown to be highly patient specific which is hypothetically beneficial to the investigation of precision medicine. Nonetheless, the development and application of organotypic culture in RCC is still immature, therefore, the primary objective of this study was to establish an organoid model for RCC.

Materials and methods: Patients diagnosed with renal tumor and underwent surgical intervention were recruited. RCC specimen was collected and derived into organoids. Derived organoids were validated by histological examminations, sequencing and xenograft. Drug response of organoids were compared with resistance cell line and patients' clinical outcomes.

Results: Our results demonstrated that organoids could be successfully derived from renal tumor and they exhibited high concordance in terms of immunoexpressional patterns. Sequencing results also depicted concordant mutations of driver genes in both organoids and parental tumor tissues. Critical and novel growth factors were discovered during the establishment of organoid model. Besides, organoids derived from renal tumor exhibited tumorigenic properties in vivo. In addition, organoids recapitulated patient's in vivo drug resistance and served as a platform to predict responsiveness of other therapeutic agents.

Conclusion: Our RCC organoid model recaptiluated histological and genetic features observed in primary tumors. It also served as a potential platform in drug screening for RCC patients, though future studies are necessary before translating the outcomes into clinical practices.

Keywords: 3D culture; disease modeling; drug screening; organoid model; renal cell carcinoma.

FIGURE 1

Schematic of establishment of organoid, validation methods, and potential applications. (Created in BioRender.com).

FIGURE 2

Immunofluorescent images of normal and tumor organoids with different markers. Normal organoids could be morphologically differentiated from tumor organoids as the former exhibited a regular structure while latter demonstrated an irregular architecture. Both normal and tumor organoids expressed urothelial markers while only tumor organoids expressed cancer markers. The levels of Ki67 was higher in tumor than normal organoids.

FIGURE 3

Representative IHC images of parental tumor tissue and derived organoid lines. IHC staining indicated that high degree of association of immunoexpressions were generally observed between tumor and organoids. Tumor IHC images were at 20×, scale bar indicated 50 μm. Organoid IHC images were at 40×, scale bar indicated 50 μm.

FIGURE 4

Brightfield and HE staining images of normal organoid and tumor organoid cultures. Normal organoid cultures exhibited a clear lumen while tumor organoid cultures exhibited cell aggregates or mixed morphologies. Brightfield images were at 10×, scale bar indicated 50 μm. HE staining images were at 40×, scale bar indicated 50 μm.

FIGURE 5

Concordance of mutations detected in RCC42, RCC52, RCC76 and RCC1TT1 parental tumor and organoid in terms of SNV and INDEL.

FIGURE 6

Mutation landscape of RCC organoids. Somatic mutations and copy number alterations identified in organoids and parental tumors with whole exome sequencing. Representative genes were selected from TCGA database, and they were known to be mutated in kidney cancer.

FIGURE 7

In vivo validation of tumorigenic ability of organoids in ectopic xenografts. (A) HE and IHC images of RCC42 parental tumor, organoid and ODX. IHC results were comparable between organoid lines and tumors from xenograft, indicating that the tumor was possibly derived from organoids. CA‐IX and PAX8 expressions were specific to organoid and ODX only. TFE3 should exhibit as nuclear stain, therefore, that of organoid was regarded as negative. (B) HE and IHC images of RCC52 parental tumor, organoid and ODX. IHC results were comparable between organoid lines and tumors from xenograft, indicating that the tumor was possibly derived from organoids. CK7 and TFE3 expressions were specific to organoid and ODX only. HE and IHC images were at 40×, scale bar indicated 50 μm.

FIGURE 8

Dose response curve of RCC42 organoid lines and ccRCC cell lines upon indicated drug administration. 786‐O denotes sunitinib‐sensitive condition while 786‐O‐R denotes sunitinib‐resistant condition.