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Patient derived organoid established under hypoxic is preferrentially enriched for Moffitt, basal like subtype that corresponse to the dedifferentiate portion of the tumour section.

ABSTRACT: It is widely accepted that the desmoplastic feature of pancreatic ductal adenocarcinoma (PDAC) and the associated hypoxic microenvironment strongly correlate with a more malignant phenotype that is more resistance to conventional-, targeted- and immuno-therapies. Whether chronic hypoxia in the PDAC stromal gives rise to distinct populations driven by genetics diversity or adoption of distinct phenotypic states in response to low oxygen-tension is poorly understood. To overcome potential selection bias during the establishment of primary 3D organoids lines, single cell dissociated from each surgically resected tumour were split and established directly in 20% O2 (normoxia) and 1% O2 (hypoxia), yielding cystic and solid organoid morphology respectively. Organoid established under hypoxia (HYPO-PCO) displayed higher expression of EMT-related proteins, a Moffitt basil-like subtype transcriptomes and higher resistance to 5-FU than organoid established under normoxia (NORMO-PCO). Strikingly, while NORMO-PCO subjected to hypoxia displayed the expected gene signatures enriched including inflammatory response, mTORC1 signalling and glycolysis, these signatures were also enriched when compared to HYPO-PCO under hypoxia. This indicates that the two organoid lines captured distinct population from the same tumour which they were derived from. Given recent appreciation for the better understanding of and the ability to target hypoxia to improve PDAC therapeutic responses, this study highlights the importance of our approach to study hypoxic population over the more conventional model which study hypoxic response in organoids established under normoxia.

ORGANISM(S): Homo sapiens

PROVIDER: GSE240649 | GEO | 2024/01/25

REPOSITORIES: GEO

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Project description:Oxygen fluctuation during tissue remodeling imposes a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastomas are believed to possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. To identify adaptive response mechanism, we compared the effects of acute and chronic hypoxia versus oxygenation on stem-like glioblastoma (GS) cells. GS cell lines were established and propagated either under normoxia (21% O2) or hypoxia (1% O2) and subsequently exposed to acute normoxia or hypoxia, respectively. Gene expression profiling revealed that acute hypoxia predominantly induced metabolic pathways and cell cycle arrest, whereas chronic hypoxia activated neurodevelopmental processes. In particular, we found increased expression of glycolytic enzymes, especially of the preparatory phase of glycolysis, under acute hypoxia, whereas pentose phosphate pathway (PPP) enzymes were downregulated. The opposite was found for acute oxygenation of hypoxic GS cells. Findings were confirmed by qPCR and immunoblot analyses. Despite downregulation by hypoxia, expression of PPP enzymes is increased in GBMs compared to normal brain, whereas expression of hypoxia-inducible enzymes of the parallel preparatory phase of glycolysis is decreased. Immunohistochemistry revealed strong staining for PPP enzymes in the bulk of GBM tissue, especially in highly proliferative areas, but not in pseudopalisading (hypoxic) cells. Glycolytic enzymes displayed an inverse pattern. Mass spectrometric analysis using [1,2-13C2]-D-glucose showed reduced glucose flux through the PPP under hypoxia in favor of flux through glycolysis. Acute and chronic hypoxia increased cell migration but reduced proliferation, whereas normoxia had opposite effects. Our findings extend Warburg’s observations by showing that in most tumor cells the PPP, which supplies metabolites for biomass production, is favored over the parallel preparatory phase of glycolysis, but is suppressed under acute severe hypoxia, causing a switch to direct glycolysis to protect against hypoxic stress.

Project description:Purpose: Due to its high metastatic proclivity, pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer. Therefore, it is imperative to better understand how the disease spreads as it progresses. Using a novel genetically engineered mouse model that allows us to isolate a subpopulation of cancer cells with superior metastatic capacity, we show that this aggressive phenotype correlates exclusively with a strong hypoxia signature. We subsequently identified the novel hypoxia-inducible gene Blimp1, which appears to play a critical role in regulating the hypoxic response upon its induction. Furthermore, genetic ablation of Blimp1 greatly reduces the level of metastasis in a PDAC mouse model. The nature of this Blimp1-regulated hypoxia signature is very unstable, since the seeded metastatic lesions mostly re-adopt similar transcriptomic profiles as the primary tumors. In conclusion, our results offer a potential mechanistic insight into how hypoxia drives metastasis in PDAC. Methods: The liver metastasis cell line 688M was subjected to control or knockdown of Blimp1 before assay. Cells ere validated for knockdown efficiency and then cultured under normoxia and hypoxia (0.5% O2) for 24 hours before preparation for RNA-Seq (total of 4 groups with duplicates, overall 8 samples). Results: For the control knockdown group, 0.5% O2 culture(hypoxia) for 24 hours induced dramatic changes in global gene expression, and Blimp1 knockdown potently mitigated changes of significant amount of genes induced under hypoxia. In brief, cell cycle-related genes that are normally suppressed by hypoxia (which contributes to hypoxia-induced cell cycle arrest) are not suppressed under hypoxia in the Blimp1 knockdown cells. In contrast, ~35% of genes that are induced under hypoxia in the control knockdown cells are instead induced less than 50% (under hypoxia) upon Blimp1 knockdown. Conclusions: Blimp1 is a critical regulator of hypoxic response in pancreatic cancer.

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Patient derived organoid established under hypoxic is preferrentially enriched for Moffitt, basal like subtype that corresponse to the dedifferentiate portion of the tumour section.

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