Autologous Proliferative and Immunobiological Modelling of Patient-Derived Clear Cell Renal Cell Carcinoma and Kidney Organoids

Immunotherapy has emerged as the new standard of care for advanced clear cell Renal Cell Carcinoma (ccRCC), the deadliest urological malignancy. However, both efficacy and toxicity of immunotherapies is highly patient-specific and difficult to predict. This underscores the need for robust preclinical models of ccRCC-immunotherapy. Current models fail to recapitulate patient-specific tumour–immune interactions. To address this and enable testing the growing number of possible treatment combinations, I aimed to establish autologous preclinical immune modelling of ccRCC in vitro using expanded patient-derived ccRCC, kidney and peripheral blood tissues. However, patient-derived ccRCC organoids (PDO-ccRCC) proliferate poorly ex vivo and the consequences of expansion on healthy kidney organoid controls (PDO-KO) and autologous T cell expansion are poorly understood. In this dissertation I present systems for autologous preclinical modelling of ccRCC, focusing on both tumour proliferation and immunobiology. To resolve ccRCC ex vivo expansion, I used transcriptomic and proteomic identification of proliferative-niche factors and I developed a serum-free, multi-parameter, high throughput organoid formation assay to evaluate candidates for their proliferative ability. Expansive PDO-ccRCCs were characterised for their malignant transcriptional, translational and functional behaviours relative to healthy kidney organoids and primary tissue for determining their physiological relevance and value for preclinical modelling. To determine PDO-ccRCC immunogenicity I expanded and characterised cryopreserved autologous T cells in serum-free conditions for defined immune co-cultures. Additionally, healthy kidney organoids were engrafted into humanised mice to enable in vitro immune co-culture assays to be extended in vivo. Twenty seven niche-derived secreted factors were detected in ccRCC. Among these, a novel combination of PDGFb and OSM especially supported PDO-ccRCC proliferation, outperforming literature-based alternatives and contrasted the EGF/HGF dependency of non-malignant PDO-KO. Proliferating PDO-ccRCCs were counter-intuitively found to have poor in vitro characteristics of low RNA and protein content, with high shear sensitivity. This contrasted their enriched immune evasive capacity, defined by heightened expression of immune inhibitory ligands, PD-L1 and PD-L2. Immunoinhibitory PDO-ccRCCs significantly inhibited inflammatory (Interferon-γ) and cytotoxic T cell secretions (Granzyme B) of autologous and allogeneic T cells, indicating potent immune evasion in vitro. In summary, this dissertation presents the novel development of autologous preclinical models for proliferative optimisation and immune assessment of patient-derived ccRCCs in vitro, with rich potential for guiding patient-specific treatment for difficult-to-expand malignancies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Toleman, Isaac

Advisors dc:contributor.advisor

• Saeb-Parsy, Kourosh
• Stewart, Grant
• Jones, James

• Organoid
• Patient-derived
• ccRCC
• Kidney
• T cell
• Autologous
• Co-culture
• Niche-factor
• Proliferation
• Immunobiology

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ca918b3b-33c5-49aa-91d7-b536b62c400c/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.120398

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/387729