Abstract
BackgroundStrategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis (CRC-pc).MethodsWe established organoid models from peritoneal metastases of two naïve CRC patients. A standard paraffin inclusion was conducted to compare their 3D structure and immunohistochemical profile with that of the corresponding surgical samples. RNA expression levels of the CRC stem cell marker LGR5 was measured by in situ hybridization. The secretome of organoids was profiled by mass spectrometry. Energy homeostasis of organoids was interfered with 4-IPP and metformin. Biochemical and metabolic changes after drug treatments were investigated by western blot and mass spectrometry. Mitochondria impairment was evaluated by electron microscopy and mitotraker staining.ResultsThe two organoids recapitulated their corresponding clinical samples in terms of 3D structure and immmunoistochemical profile and were positive for the cancer stem cells marker LGR5. Proteomic analyses of organoids highlighted their strong dependence on energy producing pathways, which suggest that their targeting could be an effective therapeutic approach.To test this hypothesis, we treated organoids with two drugs that target metabolism acting on AMP-activated protein kinase (AMPK), the main regulator of cellular energy homeostasis, which may act as metabolic tumour suppressor in CRC. Organoids were treated with 4-IPP, an inhibitor of MIF/CD74 signalling axis which activates AMPK function, or metformin that inhibits mitochondrial respiratory chain complex I.As a new finding we observed that treatment with 4-IPP downregulated AMPK signalling activity, reduced AKT phosphorylation and activated a JNK-mediated stress-signalling response, thus generating mitochondrial impairment and cell death. Metformin treatment enhanced AMPK activation, decreasing the activity of the anabolic factors ribosomal protein S6 and p4EBP-1 and inducing mitochondrial depolarization.ConclusionWe provide evidence that the modulation of AMPK activity may be a strategy for targeting metabolism of CRC-pc organoids.
Highlights
Strategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis (CRC-pc)
We found the prominent expression of metabolic pathways mainly related to oxidative homeostasis and glucose metabolism and focused on the macrophage migration inhibitory factor (MIF)/CD74/mitochondria axis, and on the relative 4-iodo-6-phenylpyrimidine (4-IPP) inhibitor, as playing an important role in JNK modulation of the cellular response to reactive oxygen species [7]
Mitochondria impairment after 4-IPP and metformin treatments in Colorectal cancer peritoneal carcinomatosis organoid 2 (C2) organoids Because mitochondria are known to produce significant amounts of ROS that may generate oxidative stress contributing to mitochondrial damage, affecting signal transduction pathways, cellular functions and viability, we studied the effects of the two compounds on ROS production (Fig. 6a)
Summary
Strategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis (CRC-pc). Intraperitoneal dissemination is a common progression feature for colorectal cancer (CRC). For the past two decades, aggressive treatments of CRC peritoneal carcinomatosis (CRC-pc), such as cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy, has improved long-term survival [1]. Tumour-initiating, or cancer stem cells (CSCs), have a key role in metastatic CRC (hepatic and/or lung) and chemotherapy resistance [2]; limited information is known about the role of CSCs in CRC-pc development. A CRC organoid is obtained by allowing cells (derived from a primary tumour specimen) to self-organize into a 3D structure that recapitulates the original glandular organization commonly observed in human CRC surgical samples. We have established (in serum-free medium) two CRC-pc organoid cultures of peritoneal metastatic lesions from two CRC patients that showed enrichment in the expression of the CRC CSC marker leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) a member of the canonical WNT pathway and a well-recognized marker of the cell progenitor population located at the crypt-base [4, 5]
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