Introduction: Current primary liver cancer models fail to truly encompass the human tumour immune microenvironment, exacerbating a recognised discord between the preclinical and clinical successes of emerging (immuno)therapeutics. The organotypic 3D culture of human precision-cut tumour slice (PCTS) is a cancer explant model which retains tumour specific histoarchitecture, aetiological background, disease phenotype, resident immune landscape, and checkpoint expression for up to 7 days ex-vivo. Our study aims to advance culture conditions of PCTS using a proprietary Multi-well Plate (MuPL) perfusion bioreactor to extend culture lifetime and allow perfusion of immune cells through PCTS, in order to validate PCTS as a tool to assess patient-specific therapeutic responses.
Methodology: PCTS generated from primary liver cancer (hepatocellular carcinoma and cholangiocarcinoma) were treated with approved single agent or combinatory checkpoint inhibitor monoclonal antibody (CPI-mAb) or kinase inhibitor (KI) therapy for up to 7 days. PCTS/immune cells co-cultures in the MuPL bioreactor were longitudinally assessed for viability, histology, and tissue integrity. Therapeutic response was determined by evaluating histology (H&E and Sirius red), apoptosis/cell death (TUNEL, lactate-dehydrogenase release, cytokeratin 18), and proliferative capacity (PC; Ki67). Gene expression was assessed using QuantiGene RNA Assay. Resident immune cells were assessed by immunofluorescence and FACS.
Results: PCTS and immune cells co-cultured in the MuPL bioreactor maintained viability, structural integrity and histoarchitecture for >7 days. Doxorubicin was used as a positive cell death control in all treated patients, decreasing PCTS viability and PC by day 5. Compared to monotherapy, nivolumab (CPI-mAb) + regorafenib (KI) therapy decreased the tumour-to-stroma ratio and PC in all patients by day 7. Also, significantly increased apoptosis was detected in one patient, who comparatively showed higher checkpoint expression including PD-1, PD-L1 and CTLA-4. Other combinatorial immunotherapies, including atezolizumab + bevacizumab and nivolumab + ipilimumab (CPI-mAbs), reduced PC without affecting histology or viability. The overall immunotherapeutic response was patient-specific.
Conclusion: PCTS can be used as a powerful tool to study personalised responses to (immune)therapeutics. In addition, PCTS can be successfully cultured in our proprietary perfusion system, recapitulating tumour-immune cell interactions, allowing assessment of response to cell and vaccine therapy ex-vivo.