Despite the significant advancements in the therapeutic management of renal cell carcinoma (RCC), there is still a pressing need for patient specific platforms that can predict personalised treatment response. Tumouroids are tissue-engineered 3D in vitro models that mimic the physiological tumour microenvironment, can reproduce cell-cell and cell-matrix interactions and incorporate patient derived tumour material.
Our aim was to manufacture compartmentalised biomimetic renal cancer tumouroids using collagen type I, that incorporate primary cells isolated from RCC surgical specimens in the inner compartment, surrounded by a stromal compartment that recapitulates the extracellular matrix (ECM) density, composition and cell populations of the in situ tumour microenvironment. We hypothesized that tumouroids will recapitulate the original tissue in terms of phenotype, genotype and response to treatment.
RCC samples were collected post-surgery (n=16), different tissue disaggregation methods were used and the isolated cells were grown in 2D and tumouroids. Tumouroids were manufactured either as simple (one compartment, populated with primary RCC cells) or complex (two compartments: a simple tumouroid as the inner compartment surrounded by a stroma compartment populated with fibroblasts and endothelial cells). RAFT absorbers were used to remove excess liquid to increase the matrix density reproducibly, to near human cancer ranges. Immunofluorescence and confocal microscopy were used to determine whether the patient derived tumouroids retain the expression of RCC markers and whole exome sequencing (WES) was performed to investigate resemblance to the parental tumour. Cell proliferation was compared among different culture conditions by measuring ATP production. Tumouroids were also challenged with a clinically used tyrosine kinase inhibitor, pazopanib.
We showed that the patient derived RCC tumouroids, made by cells isolated via different methods, retain the expression of renal cancer associated gene markers, including mutant VHL and characteristic protein markers, including cytokeratins CK8/18 and alpha smooth muscle actin (αSMA). Non linear increase in proliferation was observed in the tumouroids which may be indicative of 3D organization, differentiation and other cellular functions. Tumouroids’ response to pazopanib ranged from none to strong (40%). Additionally, when treatment response was compared among tumouroids of different levels of complexity, tumouroids that incorporated a stromal compartment responded more than simpler tumouroid models (n=3).
Our results indicate that RCC cells maintain their phenotype and genotype when grown as tumouroids. Future work will compare the response from tumouroids to the responses from both xenografts and the actual patients to determine the suitability of tumouroids as personalised cancer treatment platforms.