Organoids as a new model for improving regenerative medicine and cancer personalized therapy in renal diseases

Abstract

The pressure towards innovation and creation of new model systems in regenerative medicine and cancer research has fostered the development of novel potential therapeutic applications. Kidney injuries provoke a high request of organ transplants making it the most demanding system in the field of regenerative medicine. Furthermore, renal cancer frequently threaten patients' life and aggressive forms still remain difficult to treat. Ethical issues related to the use of embryonic stem cells, has fueled research on adult, patient-specific pluripotent stem cells as a model for discovery and therapeutic development, but to date, normal and cancerous renal experimental models are lacking. Several research groups are focusing on the development of organoid cultures. Since organoids mimic the original tissue architecture in vitro, they represent an excellent model for tissue engineering studies and cancer therapy testing. We established normal and tumor renal cell carcinoma organoids previously maintained in a heterogeneous multi-clone stem cell-like enriching medium. Starting from adult normal kidney specimens, we were able to isolate and propagate organoid 3D-structures composed of both differentiated and undifferentiated cells while expressing nephron specific markers. Furthermore, we were capable to establish organoids derived from cancer tissues although with a success rate inferior to that of their normal counterpart. Cancer cultures displayed epithelial and mesenchymal phenotype while retaining tumor specific markers. Of note, tumor organoids recapitulated neoplastic masses when orthotopically injected into immunocompromised mice. Our data suggest an innovative approach of long-term establishment of normal- and cancer-derived renal organoids obtained from cultures of fleshly dissociated adult tissues. Our results pave the way to organ replacement pioneering strategies as well as to new models for studying drug-induced nephrotoxicity and renal diseases. Along similar lines, deriving organoids from renal cancer patients opens unprecedented opportunities for generation of preclinical models aimed at improving therapeutic treatments.

Fig. 1. Establishment of normal kidney organoid cultures.

a Schematic drawing of organoid culture isolation. Fresh tissues are dissociated, cultured for 72 h in the stem-cell-enriching medium and finally plated in Matrigel into organoid medium. b Representative images of normal kidney organoid cultures at passage (P) 3, 6, and 9. Two different populations can be distinguished: well structured (top panels) and round-shaped aggregates (lower panels). Microscope magnification ×4. c Scheme of the cultures trends over passages. d Representative confocal images of immunofluorescence stainings for SOX2, Ki67, Ck8–18, E-cadherin (green) and actin (red). Nuclei are stained with DAPI (blue). Microscope magnification ×20. Scale bars 100 μm

Fig. 2. Characterization of normal kidney organoid cultures.

a Cartoon of renal markers specific for each region of the nephron. b Representative confocal images stained for Aquaporin 1 (green) and Actin (red). Nuclei are stained with DAPI (blue). Microscope magnification ×20. Scale bars 100 μm. c mRNA expression assessed by qPCR of Aquaporine 1 (Aqp1) in the same normal kidney organoid culture. “786” commercial RCC cell line is used as negative control. Results are shown as a mean ± SD of at least three independent experiments. d Representative confocal images stained for Aquaporin 2 (green) and Actin (red). Nuclei are stained with DAPI (blue). Microscope magnification ×20. Scale bars 100 μm. e mRNA expression assessed by qPCR of Aquaporine 2 in the same normal kidney organoid culture. “786” RCC line is used as negative control. Results are shown as a mean ± SD of at least three independent experiments. Immunofluorescence images are representative of at least five independent experiments. f Organoids were included in cryomatrix (OCT), cutted in 8 μM slides and stained for WT1, LTL, Actin and DAPI markers. Representative confocal images were reported. g–h Fresh fixed and permeabilized organoids evaluated for LTL, WT1, Actin and DAPI markers by immunofluorescence assay. Representative confocal images were reported. i Fresh fixed and permeabilized organoids evaluated for CD-31, Actin and DAPI markers by immunofluorescence assay. Representative confocal images were reported

Fig. 3. Normal kidney organoids as cisplatin cytotoxicity assay.

a Representative phase contrast images of organoid cultures after 72 h of 100 μM Cisplatin exposition. Organoid untreated cultures (NT) were used as control. b Representative images of 100 μM cisplatin treated and untreated (NT) organoid cultures evaluated after 72 h for LTL, Cleaved-Caspase 3, Actin and DAPI markers by immunofluorescence stainings. c Cleaved-Caspase 3 expression evaluated in 100 μM cisplatin treated and untreated (NT) organoid cultures after 72 h by Western blotting. Representative images of two organoids cultures were reported. GAPDH protein was used as internal control

Fig. 4. Establishment of RCC organoid cultures.

a Representative images of RCC kidney organoid cultures at passage (P) 3, 6 and 9. Microscope magnification ×4. b Percentage of organoid culture formation efficiency starting from normal and RCC fresh tissues. c Kaplan-Meier plot of culture persistence over passages of normal (black) and RCC (red) organoids. d Illustration of the number of cells counted in normal (black) and RCC (red) organoids from passages 6 to 9. Results are expressed as mean ± SD of three independent cultures. e Representative confocal microscope images of normal (upper panel) and tumor (lower panel) organoids stained for Ki67 (green) and actin (red). Nuclei are stained with DAPI (blue). Microscope magnification ×60. Scale bars 50 μm. Immunofluorescence images are representative of at least six independent experiments

Fig. 5. Characterization of RCC organoid cultures.

a Representative confocal microscope images of normal (left panel) and tumor organoid (right panel) stained for SOX2, Ck8-18, E-cadherin and HIF1α (green), respectively. Nuclei are stained with DAPI (blue) and Actin staining is pseudo-colored in red. Microscope magnification ×60. Scale bars 50 μm. b–h mRNA expression, assessed by qPCR, of Aquaporine 1, Aquaporine 2, ClC-K1, PGK1, NDRG1, VEGFR2, CAIX in normal kidney and RCC organoid cultures. Results are shown as a mean ± SD of at least three independent experiments. Immunofluorescence images are representative of at least six independent experiments

Fig. 6. Organoids in vivo propagation.

a Representative stereomicroscopic image of the tumor mass (Tumor, red arrow) excised 120 days after injection of a RCC organoid culture, under the renal capsule of immunocompromised mice. The cancer mass is highlighted by a yellow line. Scale bars 1 mm. b Representative H&E images of a xenograft derived from a G4 ccRCC organoid culture. Blue lines and red arrows indicate human tumor invading the mouse parenchyma. Microscope magnification ×10 (left and central panels) and 20 (right panel) (c) H&E staining of the original matrigel organoid culture included in OCT and cut in 5 μm slides for staining. Microscope magnification x20 Scale bars 50 μm. d H&E staining of the original matrigel organoid cultures and xenograft tumor masses included in OCT, cut in 5 μm slides for staining. Microscope magnification x20 Scale bars 50 μm