Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.
Background Despite major interest in tyrosine kinase inhibitors (TKIs) as a treatment option for metastatic colorectal cancer (mCRC), almost all TKIs tested for mCRC fail in early-phase clinical trials. Although showing specific target inhibition at low concentrations, TKIs have a much broader kinase inhibitory potency at higher concentrations. In an attempt to leverage these many additional, low-affinity targets, high-dose regimens that may trigger efficacy are explored. Here, we studied unprecedented drug exposure–response relationships in vitro using mCRC patient-derived tumour organoids (PDTOs). Methods We established patient-derived tumor organoids (PDTOs) from mCRC biopsies and, based on favorable physicochemical and pharmacokinetic properties, selected 3 TKIs (sunitinib, cediranib and osimertinib). Following standard IC50 assessment using continuous dosing with a concentration range, we investigated the cytotoxic antitumor effect of high-dose, short-term (HDST) treatment. Five PDTOs were exposed to 20 µM TKI for 1–24h, washed and given normal medium, and PDTO-outgrowth was determined 1 week later. At exposures of 1, 3 and 6 h, we measured intra-tumoroid TKI concentrations using a clinically validated LC/MS-MS method. PDTO cell death was observed using live-cell microscopy, and quantified by both caspase 3/7 enzyme activity assay and cleaved caspase-3 immunofluorescent staining. Results We show that most PDTOs tested are sensitive to multikinase TKIs sunitinib and cediranib, and all to osimertinib. Furthermore, we demonstrate that high-dose, short-term(HDST) TKI treatment effectively blocks organoid growth. In line with recent clinical data of high-dose sunitinib tumour accumulation, HDST treatment led to markedly elevated intra-tumoroid TKI concentrations, which correlated with PDTO sensitivity. This suggests exposure-dependent cytotoxicity and supports the concept that efficacy is induced by a broad kinase inhibitory spectrum. Mechanistically, HDST osimertinib treatment for just 3 hours induced regulated cell death in treated organoids. Conclusion Our work provides a better understanding of TKI exposure vs response and can be used to determine patient-specific sensitivity. In addition, these results may guide both mechanistic elucidation in organotypic translational models and the translation of target drug exposure to clinical dosing strategies. Moreover, HDST osimertinib treatment warrants clinical exploration for mCRC.
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