Biology subjects

Salvucci, M.

Publications and source records attributed to Salvucci, M..

5 recordsLinked to original sources

bioRxiv2022

Identification and characterization of four bacteriome- and mycobiome-derived subtypes in tumour and adjacent mucosa tissue of colorectal cancer patients

ObjectiveHere, we systematically investigated alterations in the bacteriome and mycobiome of CRC patients in tumours and matched adjacent mucosa resulting in the identification of microbiome-based subtypes associated with host clinico-pathological and molecular characteristics. DesignDiversity and composition of bacteriome and mycobiome of tumour and adjacent mucosa, resulting subtypes were computationally deconvoluted from RNA sequencing, using >10000 samples from in-house and publically available patient cohorts. ResultsThe bacteriome of tumours had higher dominance and lower -diversity compared to matched adjacent local and distant mucosa. Tumours were enriched with Proteobacteria (Gammaproteobacteria class), Fusobacteria (including Fusobacterium Nucleatum species) and Basidiomycota fungi (Malasseziaceae family). Tumours were depleted of Bacteroidetes (Bacteroidia class), Firmicutes (Clostridia class) and Ascomycota (Sordariomycetes and Saccharomycotina). Tumours and adjacent mucosa samples were classified into 4 microbial subtypes, termed C1 to C4, based on the bacteriome and mycobiome composition. The bacterial Propionibacteriaceae, Enterobacteriaceae, Fusobacteriaceae, Bacteroidaceae and Ruminococcaceae and the fungal Malasseziaceae, Saccharomycetaceae and Aspergillaceae were among the key families driving the microbial subtyping. Microbial subtypes were associated with distinct tumour histology and patient phenotypes and served as an independent prognostic marker for disease-free survival. Key associations between microbial subtypes and alterations in host immune response and signalling pathways were validated in the TCGA pan-cancer cohort. The microbial subtyping demonstrated stratification value in the pan-cancer settings beyond merely representing differences in survival by cancer type. ConclusionsThis study demonstrates the translational potential of microbial subtyping in CRC patient stratification, and provides avenues to design tailored microbiota modulation therapy to further precision oncology. Statement of significanceO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIThe microbiome has been implicated in the pathogenesis, progression and therapeutic response in patients diagnosed with CRC and other cancers. C_LIO_LIThe vast majority of studies to date has focussed on investigating the bacteriome while the critical role played by the mycobiome in shaping cancer has begun to be explored more recently. C_LIO_LIThe bacterial and fungal composition and diversity in on-tumour tissue compared to matched local and distal off-tumour mucosa is largely unexplored. C_LIO_LITumorigenesis may be promoted via alterations of the microbiome ecosystem that may be better recapitulated by a multi-kingdom microbial signature rather than by the abundance of individual bacterial or fungal microorganisms. C_LI What are the new findings?O_LIOn-tumour tissue was enriched with Fusobacteria, Proteobacteria, Basidyomicota and depleted of Bacteroidetes, Firmicutes, Ascomycota compared with adjacent off-tumour mucosa. C_LIO_LIWe stratified >600 CRC patients into four distinct microbial-based subtypes (C1-C4) according to their bacteriome and mycobiome composition. The microbial subtypes were associated with distinct prognosis, clinical phenotypes such as staging, tumour location, history, lymphovascular invasion, TP53 status and clinical outcome. C_LIO_LIFurthermore, the majority of matched adjacent mucosa samples were classified as C1 and paired tumour-matched normal samples demonstrated a robust shift towards the C1 subtype in off-tumour tissue, suggesting that the C1 subtype may recapitulate a healthier-like microbiome. This hypothesis was supported by the microbial subtyping of colon samples from healthy subjects that were categorised as C1 almost exclusively. C_LIO_LIThe identified microbial subtyping demonstrated stratification value in the pan-cancer settings (n=28 additional solid cancer indications spanning >9000 samples) beyond merely representing differences in survival by cancer type, providing the strongest stratification in liver cancer. C_LI How might it impact on clinical practise in the foreseeable future?O_LIThis study laids the foundation to develop a microbial signature as biomarker to clinically manage CRC and other solid cancers and potentially lead to microbiome-based companion diagnostics to design microbiota modulation therapy tailored to specific patients subgroups with distinct bacteriomes and mycobiomes. C_LI

cancer biology↗

bioRxiv2021

Patients with mesenchymal tumours and high Fusobacteriales prevalence have worse prognosis in colorectal cancer (CRC)

ObjectiveTranscriptomic-based subtyping, Consensus Molecular Subtyping (CMS) and CRC Intrinsic Subtyping (CRIS), identify a patient subpopulation with mesenchymal traits (CMS4/CRIS-B) and poorer outcome. Here, we investigated the relationship between prevalence of Fusobacterium nucleatum (Fn) and Fusobacteriales, CMS/CRIS subtyping, cell type composition, immune infiltrates and host contexture to refine patients stratification and identify druggable context-specific vulnerabilities. DesignWe coupled cell culture experiments with characterization of Fn/Fusobacteriales prevalence and host biology/microenviroment in tumours from 2 independent CRC patient cohorts (Taxonomy: n=140; TCGA-COAD-READ: n=605). ResultsIn vitro, Fn infection induced inflammation via NF{kappa}B/TNF in HCT116 and HT29 cancer cell lines. In patients, high Fn/Fusobacteriales were found in CMS1, MSI tumours, with infiltration of macrophages M1, reduced macrophages M2, and high IL6/IL8/IL1{beta} signaling. Analysis of the Taxonomy cohort suggested that Fn was prognostic for CMS4/CRIS-B patients, despite having lower Fn load than CMS1 patients. In the TCGA-COAD-READ cohort, we likewise identified a differential association between Fusobacteriales relative abundance and outcome when stratifying patients in mesenchymal (either CMS4 and/or CRIS-B) vs. non-mesenchymal (neither CMS4 nor CRIS-B). Patients with mesenchymal tumours and high Fusobacteriales had approximately 2-fold higher risk of worse outcome. These associations were null in non-mesenchymal patients. Modelling the 3-way association between Fusobacteriales prevalence, molecular subtyping, and host contexture with logistic models with an interaction term disentangled the pathogen/host-signaling relationship and identified aberrations (including EMT/WNT/NOTCH) as candidate targets. ConclusionThis study identifies CMS4/CRIS-B patients with high Fn/Fusobacteriales prevalence as a high-risk subpopulation that may benefit from therapeutics targeting mesenchymal biology. Significance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIFusobacterium nucleatum (Fn), a commensal Gram-negative anaerobe from the Fusobacteriales order, is an onco-bacterium in CRC as a causal relationship between Fn prevalence and CRC pathogenesis, progression and treatment response has been reported in vivo. C_LIO_LIBroad spectrum antibiotics has proven moderately successful in reducing tumour growth in preclinical models. However, the use of antibiotics to treat bacterium-positive cases in the clinic is not a viable option as it may further alter the already dysbiotic gut microbiome of CRC patients and may also have limited efficacy against Fn which penetrates and embeds deeply within the tumour. C_LIO_LIThe highly heterogenous CRC patient population can be classified into distinct molecular subtypes (CMS and CRIS) based on gene expression profiles mirroring the underlying transcriptional programs. Patients classified as CMS4 and CRIS-B exhibit a mesenchymal phenotype and have poorer outcome. C_LI What are the new findings?O_LIFn/Fusobacteriales prevalence is associated with immune involvement (decrease in macrophages M1 and increase in macrophages M2) and activation of specific signalling programs (inflammation, DNA damage, WNT, metastasis, proliferation, cell cycle) in the host tumours. C_LIO_LIThe prevalence of bacteria from the Fusobacteriales order, largely driven by Fn species, play an active or opportunistic role depending on the underlying host tumour biology and microenvironment. C_LIO_LIFn and other species of the Fusobacteriales order are enriched in CMS1 (immuno, microsatellite unstable) patients compared to CMS2-4 cases. C_LIO_LIFn/Fusobacteriales prevalence is associated with worse clinical outcome in patients with mesenchymal-rich CMS4/CRIS-B tumours, but not in patients with other molecular subtypes. C_LI How might it impact on clinical practice in the foreseeable future?O_LIFn/Fusobacteriales screening and transcriptomic-based molecular subtyping should be considered to identify patients with mesenchymal-rich tumours and high bacterium prevalence and to inform disease management. C_LIO_LIFn/Fusobacteriales prevalence may need to be addressed exclusively in patients with mesenchymal-rich high-stromal infiltrating tumours rather than a blanket-approach to treat all pathogen-positive patients. C_LIO_LIClinical management of the disease for this subpopulation of high-risk patients with unfavourable clinical outcome could be attained by administering compounds currently in clinical trials that target aberrations in the host signaling pathways (NOTCH, WNT, EMT) and tumour microenviroment (inflammasome, activated T cells, complement system, and macrophage chemotactism and activation). C_LI

cancer biology↗

bioRxiv2021

An atlas of inter- and intra-tumor heterogeneityof apoptosis competency in colorectal cancertissue at single cell resolution

Cancer cells ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells and quantification of 20 cell lineage and apoptosis proteins. Ordinary differential equation-based modelling of apoptosis sensitivity at single cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. We identified an enrichment for BCL2 in immune, and BAK, SMAC and XIAP in cancer cells. ODE-based modelling at single cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, with significant inter- and intra-tumor heterogeneity. However, we did not find increased spatial heterogeneity of apoptosis signaling in cancer cells, suggesting that such heterogeneity is an intrinsic, non-genomic property not increased by the process of malignant transformation.

cancer biology↗

bioRxiv2021

Stratification Of Chemotherapy-Treated Stage III Colorectal Cancer Patients Using Multiplexed Imaging And Single Cell Analysis Of T Cell Populations

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Predictive biomarkers for identification of patients with increased risk for disease recurrence are currently lacking, with progress in biomarker discovery hindered by the diseases inherent heterogeneity. The immune profile of colorectal tumors has previously been found to have prognostic value. The aims of this study were to evaluate immune signatures in the tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single cell analysis technology (Cell DIVE). Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin chemotherapy. Single sections underwent multilplexed immunofluorescence with Cy3- and Cy5-conjugated antibodies for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase and S6). We applied a probabilistic multi-class, multi-label classification algorithm based on multi-parametric models to build statistical models of protein expression to classify immune cells. Expert annotations of immune cell markers were made on a range of images, and Support Vector Machines (SVM) were used to derive a statistical model for cell classification. Images were also manually scored independently by a Pathologist as high, moderate or low, for stromal and total immune cell content. Excellent agreement was found between manual and total automated scores (p<0.0001). Higher levels of multi-marker classified regulatory T cells (CD3+CD4+FOXP3+PD1-) were significantly associated with disease-free survival (DFS) and overall-survival (OS) (p=0.049 and 0.032), compared to FOXP3 alone. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cells with greater potential for predicting patient outcomes.

cancer biology↗

bioRxiv2020

TRAIL signalling promotes entosis in colorectal cancer

Entosis is a form of non-phagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumours show evidence of entosis, however factors controlling entosis remain to be elucidated. Here we find that the death receptor ligand TRAIL is a potent activator of entosis in colon cancer cells. CLEM/3D confocal microscopy analysis revealed ultrastructural features of entosis and subsequent entotic cell death of inner cells upon TRAIL treatment. Induction of entosis and apoptosis by TRAIL were mutually exclusive events but both required the presence of caspase-8. Bax/Bak double knock-out or caspase inhibition altered the fate of inner cells from entotic cell death to survival and escape. Analysis of colorectal cancer tumours showed a significant association between expression levels of TRAIL and CICs. Notably, the presence of CICs in the invasive front regions of colorectal tumours was significantly correlated with adverse patient prognosis.

cancer biology↗