A multidimensional metabolomics workflow to image biodistribution and evaluate pharmacodynamics in adult zebrafish

An integrated evaluation of the tissue distribution and pharmacodynamic properties of a therapeutic is essential for successful translation to the clinic. To date, however, cost-effective methods to measure these parameters at the systems level in model organisms are lacking. Here, we introduce a multidimensional workflow to evaluate drug activity that combines mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing and global metabolome analysis in the adult zebrafish. As a proof of concept, we quantitatively determined the whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate (HCQ) and measured the systemic metabolic impacts of drug treatment. We found that HCQ distributed to most organs in the adult zebrafish 24 h after addition of the drug to water, with the highest accumulation of both the drug and its metabolites being in the liver, intestine and kidney. Interestingly, HCQ treatment induced organ-specific alterations in metabolism. In the brain, for example, HCQ uniquely elevated pyruvate carboxylase activity to support increased synthesis of the neuronal metabolite, N-acetylaspartate. Taken together, this work validates a multidimensional metabolomics platform for evaluating the mode of action of a drug and its potential off-target effects in the adult zebrafish. This article has an associated First Person interview with the first author of the paper

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics

Oncogenic BRAF, unrestrained by TGFβ-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells

Suppression of tumor-associated neutrophils by lorlatinib attenuates pancreatic cancer growth and improves treatment with immune checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDAC) patients have a 5-year survival rate of only 8% largely due to late diagnosis and insufficient therapeutic options. Neutrophils are among the most abundant immune cell type within the PDAC tumor microenvironment (TME), and are associated with a poor clinical prognosis. However, despite recent advances in understanding neutrophil biology in cancer, therapies targeting tumor-associated neutrophils are lacking. Here, we demonstrate, using pre-clinical mouse models of PDAC, that lorlatinib attenuates PDAC progression by suppressing neutrophil development and mobilization, and by modulating tumor-promoting neutrophil functions within the TME. When combined, lorlatinib also improves the response to anti-PD-1 blockade resulting in more activated CD8 + T cells in PDAC tumors. In summary, this study identifies an effect of lorlatinib in modulating tumor-associated neutrophils, and demonstrates the potential of lorlatinib to treat PDAC

Genomic landscape and clonal architecture of mouse oral squamous cell carcinomas dictate tumour ecology.

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression

Ex vivo treatment of patient biopsies as a novel method to assess colorectal tumour response to the MEK1/2 inhibitor, Selumetinib

Abstract Although an array of new therapeutics has emerged for the treatment of colorectal cancer, their use is significantly impacted by variability in patient response. Better pre-clinical models could substantially improve efficacy as it may allow stratification of patients into the correct treatment regime. Here we explore acute, ex vivo treatment of fresh, surgically resected human colorectal tumour biopsies as a novel pre-clinical model for identifying patient response to specific therapeutics. The MEK1/2 inhibitor, Selumetinib (AZD6244, ARRY-142886) was used as a tool compound. Firstly, we established an acute treatment protocol and demonstrated this protocol could differentiate phenotypic and pharmacodynamic responses to Selumetinib (0–3uM). We then used the protocol to evaluate Selumetinib response in tumours from 23 colon cancer patients. These studies revealed that the agent inhibited pERK1/2 phosphorylation in all tumours, caused a significant decrease in proliferation in 5/23 (22%) tumours, and that KRAS/BRAF mutant tumours were particularly sensitive to the anti-proliferative effects of the agent. These data are consistent with data from clinical trials of Selumetinib, suggesting that acute treatment of small tumour biopsies is worthy of further exploration as a pre-clinical model to evaluate colorectal cancer response to novel therapies