Spatial mapping of pancreatic cancer subtypes and their microenvironmental niches

Introduction: Transcriptomic studies have identified two major subtypes of pancreatic ductal adenocarcinoma
(PDAC), i.e. a ‘classical’ and a ‘basal-like’ subtype. These subtypes have differential expression of GATA6, with
prognostic and potentially predictive value. The basal-like subtype has worse prognosis and is characterized with
increased gene dosage of mutant KRAS. In this project, I perform high resolution spatial subtyping in human PDAC
and (multicellular) organoids to gain novel insights of tissue heterogeneity and subtype plasticity.
Preliminary results: Part 1 of the project was in collaboration with a biotechnological company. For their purpose
to start up a biobank of tumor derived PDAC models, I successfully set up a clinical flow to receive PDAC
resections in the lab. Patient tissue was used for DNA/RNA omics analysis and I generated tumor-derived organoid
cultures. I collected 45 PDAC samples in 3 years of which half of them were successful to derive organoids from.
During part 2, I exploited tumor paraffin sections to deeply investigate tumor heterogeneity on the level of individual
patients. I optimized a Basescope™ assay for specific in situ detection of KRASG12D, the most prevalent point
mutation in human PDAC. It needs to be highlighted that this was never done before in the field of pancreatic
cancer. BaseScope™ and multiplex RNAScope™ are combined with immunostainings to perform in-depth spatial
analysis on single-cell level, using HALO (Indica™) software. Apart from inter-patient and intra-patient inter-ductal
heterogeneity, I observed and quantified phenotypic diversity of tumor cells within individual pancreatic tumor
ducts. Except for the negative correlation between GATA6 and KRASG12D, with my colleagues, we introduced
novel subtype-specific mRNA panels for multiplex in situ hybridization that better capture the basal-like cell state
compared to widely used surrogate markers. I further demonstrated that the intra-ductal heterogeneity is related
to functional diversity with proliferative classical regions and epithelial to mesenchymal transition in basal-like
regions. PDAC organoids recapitulated the intra-ductal phenotypic and functional diversification that can be shifted
experimentally by co-culturing with cancer-associated fibroblasts (CAFs).
Preliminary conclusion: I successfully established quantitative in situ profiling of single point mutations and
transcriptomic subtypes, revealing extensive intra-tumor phenotypic and functional heterogeneity down to the level
of single pancreatic tumor ducts (E. Michiels et al. submitted for publication to J Pathol). I now aim to complement
the findings by performing additional in-depth spatial analysis to investigate tumor microenvironmental (TME)
niches (detailed below in 1b) in relation to the subtype-associated intra-ductal heterogeneity. I will address this
research question using hyperplex spatial biology platforms with focus on different subpopulations of CAFs and
immune cells.

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