3D Tumor Models in Urology

Abstract

Three-dimensional tumor models have become established in both basic and clinical research. As multicellular systems consisting of tumor and tumor-associated cells, they can better represent tumor characteristics than monocellular 2D cultures. In this review, we highlight the potential applications of tumor spheroids and organoids in the field of urology. Further, we illustrate the generation and characteristics of standardized organoids as well as membrane-based 3D in vitro models in bladder cancer research. We discuss the technical aspects and review the initial successes of molecular analyses in the three major urologic tumor entities: urinary bladder carcinoma (BCa), prostate carcinoma (PCa), and renal cell carcinoma (RCC).

Keywords: 3D cell culture technique; bladder cancer; prostate cancer; renal cell carcinoma.

Figure 1

Proliferation of HT-1376 in strata membranes. Cells were grown in Alvetex Strata, fixed with PFA after 3 days (a), 13 days (b), and 19 days (c,d), embedded in paraffin, cut into 7 μm thick sections and labeled with fluorescent dyes; cell nuclei blue, beta-actin green, Ki-67 red; (a) HT-1376 cells already migrate from the apical side (asterisk) into the membrane (arrows), the proliferation is moderate (red nuclei); (b,c) many more cells are found within the membrane and the proliferation rate is especially high in those migrating cells; (d) higher magnification shows the formation of a superficial multilayered tissue-like structure and indicates the migration of single cells through the membranous pores (arrows); images taken with an CKX53 microscope (Olympus, Hamburg, Germany); calculation of the proliferation rate using Image J. [23]; (d) scale bar indicates 20 µm.

Figure 2

Urinary bladder organoid composed of three different human urinary bladder cell types. (a) The organoid shows a clear stratification into outer non-malignant urothelial cells (HBLAK, green) and a core of urinary bladder fibroblasts and smooth muscle cells (HBF, HBSMC); urothelial cells are positive for cytokeratin (green, CKPAN), whereas HBF and HBSMC are negative (asterisk), the tight junction protein zonula occludens-1 (ZO-1) is depicted in orange, nuclei are shown in blue color (stained with TO-PRO-3); (b) detail of (a) demonstrate that the expression of ZO-1 (orange) is mostly restricted to the top cell layer of HBLAK cells (arrows in (a,b)), while vinculin (VCL, orange) expression (c) is very strong in both, HBLAK and stromal cells (asterisk); antibodies used (Table S1).

Figure 3

Organoid composed of HBF, HBSMC, HUVEC, and HBLAK, cultured for 14 days with 10 days of differentiation. (a) Immunohistochemical staining of cytokeratin 5 & 6 (CK5+6, green), von Willebrand factor (VWF, orange), nuclei are stained with TO-PRO-3 (blue), note the tubular structures formed by HUVEC (arrows); (b) detail of (a) showing the decoration of HUVEC with VWF; (c) zonula occludens protein-1 (ZO-1, orange) indicating barrier formation (arrowheads) by HBLAK stained with an antibody against CKPAN (green), note the labeling of tubular structures within the core of the organoid, probably HUVEC (arrows); antibodies used (Table S1).

Figure 4

Urinary bladder tumor organoids from RT-112 (initial tumor grade 2) and T-24 (grade 3), HBF and HBSMC. (a–c) Hematoxylin-eosin staining (HE) of human BCa organoids; (a,b) RT-112 organoids show a clear urothelial stratification, T-24 organoids show a loose structure (c); (d–f) immunohistochemical staining of cytokeratin (green, CKPAN), Ki-67 (orange), nuclei are stained with DAPI (blue); (e,f) detail of (d) showing T-24 cells by their cytokeratin expression in the organoid (green, CKPAN); T-24 BCa cells show a high proliferation index (red, Ki-67); purple by overlay with nuclei); scale bar: 100 µm; antibodies used (Table S1).