Co, I. L. | Search bioRxiv

bioRxiv2024

Single-cell analysis of an engineered organoid-based model of pancreatic cancer identifies hypoxia as a contributing factor in the determination of transcriptional subtypes

Pancreatic ductal adenocarcinoma (PDAC) is a high-mortality cancer characterized by its aggressive, treatment-resistant phenotype and a complex tumour microenvironment (TME) featuring significant hypoxia. Bulk transcriptomic analysis has identified the "classical" and "basal-like" transcriptional subtypes which have prognostic value in PDAC; however, it remains unclear how microenvironmental heterogeneity contributes to the expression of these transcriptional signatures. Here, we used single cell transcriptome analysis of the organoid TRACER platform to explore the effect of oxygen and other microenvironmental gradients on PDAC organoid cells. We found that the microenvironmental gradients present in TRACER significantly impact the distribution of organoid transcriptional phenotypes and the enrichment of gene sets linked to cancer progression and treatment resistance. More significantly, we found that microenvironmental gradients drive changes in the expression of the classical and basal-like transcriptional subtype gene signatures. This effect is likely dominated by the oxygen gradients in TRACER, as hypoxia alone induced decreases in the expression of classical marker GATA6 at both the gene and protein level in PDAC cells. This work suggests that hypoxia contributes to determining transcriptional subtypes in PDAC and broadly underscores the importance of considering microenvironmental gradients in organoid-based transcriptomic studies of PDAC.

cancer biology↗

bioRxiv2023

An Engineered 3D Co-culture Model of Primary Macrophages and Patient-Derived Tumour Cells to Explore Cellular Responses in the Graded Hypoxic Microenvironment of Pancreatic Cancer

In pancreatic ductal adenocarcinoma (PDAC), tumour associated macrophages (TAMs) are a heterogeneous immune cell population that interact with cancer cells to promote malignancy, chemo-resistance, and immunosuppression. Aside from TAMs, hypoxia is a prominent feature of PDAC that can rewire cells to survive and enhance malignancy in the tumour microenvironment (TME). Deciphering the interactions between macrophages, cancer cells and hypoxia could lead to the development of effective immune-targeted therapies for PDAC. However, there are only a few models that physiologically recapitulate the PDAC TME and allow for meaningful interrogation of cancer-immune cell interactions in hypoxia. Here, we develop a model of primary macrophages and PDAC patient organoid-derived cells by adapting TRACER, a paper-based, engineered 3D model that allows snapshot analysis of cellular response in hypoxia. In this study, we establish a direct co-culture method of primary macrophages and PDAC organoid cells in TRACER and demonstrate that TRACER co-cultures generate hypoxic gradients and show expected phenotypic responses to this hypoxic gradient. Moreover, we report for the first time in a human in vitro model that hypoxic macrophages exert a graded chemoprotective effect on gemcitabine-treated PDAC organoid cells, and that interactions between cancer cells and macrophages from the inner layers of TRACER indirectly attenuate the inflammatory response of donor-derived T-cells. Overall, the TRACER co-culture system is a novel, fully human 3D in vitro cancer-immune model for evaluating the response of macrophages and cancer cells in a hypoxic gradient.

bioengineering↗