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Biosensors are aimed at detecting tiny physical and chemical stimuli in biological systems. Physical forces are ubiquitous, being implied in all cellular processes, including cell adhesion, migration, and differentiation. Given the strong interplay between cells and their microenvironment, the extracellular matrix (ECM) and the structural and mechanical properties of the ECM play an important role in the transmission of external stimuli to single cells within the tissue. Vice versa, cells themselves also use self-generated forces to probe the biophysical properties of the ECM. ECM mechanics influence cell fate, regulate tissue development, and show peculiar features in health and disease conditions of living organisms. Force sensing in biological systems is therefore crucial to dissecting and understanding complex biological processes, such as mechanotransduction. Atomic Force Microscopy (AFM), which can both sense and apply forces at the nanoscale, with sub-nanonewton sensitivity, represents an enabling technology and a crucial experimental tool in biophysics and mechanobiology. In this work, we report on the application of AFM to the study of biomechanical fingerprints of different components of biological systems, such as the ECM, the whole cell, and cellular components, such as the nucleus, lamellipodia and the glycocalyx. We show that physical observables such as the (spatially resolved) Young’s Modulus (YM) of elasticity of ECMs or cells, and the effective thickness and stiffness of the glycocalyx, can be quantitatively characterized by AFM. Their modification can be correlated to changes in the microenvironment, physio-pathological conditions, or gene regulation.

Metformin is a widely used and well-tolerated anti-diabetic drug that can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear. We studied the anti-cancer activity of metformin on colorectal cancer (CRC) by using the drug to treat HT29, HCT116 and HCT116 p53−/− CRC cells. Metformin reduced cell proliferation and migration by inducing cell cycle arrest in the G0/G1 phase. This was accompanied by a sharp decrease in the expression of c-Myc and down-regulation of IGF1R. The anti-proliferative action of metformin was mediated by two different mechanisms: AMPK activation and increase in the production of reactive oxygen species, which suppressed the mTOR pathway and its downstream targets S6 and 4EBP1. A reduction in CD44 and LGR5 expression suggested that the drug had an effect on tumour cells with stem characteristics. However, a colony formation assay showed that metformin slowed the cells’ ability to form colonies without arresting cell growth, as confirmed by absence of apoptosis, autophagy or senescence. Our finding that metformin only transiently arrests CRC cell growth suggests that efforts should be made to identify compounds that combined with the biguanide can act synergistically to induce cell death.

Peritoneal metastases (PM) are common routes of dissemination for colorectal cancer (CRC) and remain a lethal disease with a poor prognosis. The properties of the extracellular matrix (ECM) are important in cancer development; studying their changes is crucial to understand CRC-PM development. We studied the elastic properties of ECMs derived from human samples of normal and neoplastic PM by atomic force microscopy (AFM); results were correlated with patient clinical data and expression of ECM components related to metastatic spread. We show that PM progression is accompanied by stiffening of the ECM, increased cancer associated fibroblasts (CAF) activity and increased deposition and crosslinking in neoplastic matrices; on the other hand, softer regions are also found in neoplastic ECMs on the same scales. Our results support the hypothesis that local changes in the normal ECM can create the ground for growth and spread from the tumour of invading metastatic cells. We have found correlations between the mechanical properties (relative stiffening between normal and neoplastic ECM) of the ECM and patients’ clinical data, like age, sex, presence of protein activating mutations in BRAF and KRAS genes and tumour grade. Our findings suggest that the mechanical phenotyping of PM-ECM has the potential to predict tumour development.

Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are commonly treated with radio-ligand therapy (RLT) but reliable biomarkers for predicting early disease progression are lacking. In this exploratory study we investigated whether microRNA (miRNA) expression in archival samples is associated with early response to RLT and with tumour origin and grading. Forty-eight formalin-fixed, paraffin-embedded samples from G1–G2 GEP-NETs patients treated with RLT ( 177 Lu-Oxodotreotide (Lutathera ® ) were analyzed. Two CT or MRI scans per patient, performed within three months before and after RLT, were used to assess early progression (PD) according to RECIST v1.1. Thirteen miRNAs previously implicated in NET biology were quantified by qRT-PCR. Multiple logistic regression evaluated associations between miRNA expression and early progression, as well as relationships with tumour origin and grade. We observed trends suggesting that lower expression of miR-21-5p (OR = 0.51, 90% CI: 0.26–1.00) and miR-196a (OR = 0.78, 90% CI: 0.59–1.02), and higher expression of miR‑30a-5p (OR = 2.62, 90% CI: 0.1.14–6.05) may be associated with a reduced likelihood of early progression. Lower miR-196a and higher miR‑30a-5p expression was also more frequent in pancreatic tumours and lower miR-196a expression is associated to G1 lesions. These findings indicate that miRNA profiling in GEP-NETs archival samples is feasible and support the potential role of miR-21-5p, miR-196a, and miR‑30a-5p as exploratory biomarkers for early progression afterRLT. Further validation in independent cohorts is required to confirm these preliminary observations.

The risk of colorectal cancer (CRC) depends on environmental and genetic factors. Among environmental factors, an imbalance in the gut microbiota can increase CRC risk. Also, microbiota is influenced by host genetics. However, it is not known if germline variants influence CRC development by modulating microbiota composition. We investigated germline variants associated with the abundance of bacterial populations in the normal (non-involved) colorectal mucosa of 93 CRC patients and evaluated their possible role in disease. Using a multivariable linear regression, we assessed the association between germline variants identified by genome wide genotyping and bacteria abundances determined by 16S rRNA gene sequencing. We identified 37 germline variants associated with the abundance of the genera Bacteroides, Ruminococcus, Akkermansia, Faecalibacterium and Gemmiger and with alpha diversity. These variants are correlated with the expression of 58 genes involved in inflammatory responses, cell adhesion, apoptosis and barrier integrity. Genes and bacteria appear to be involved in the same processes. In fact, expression of the pro-inflammatory genes GAL , GSDMD and LY6H was correlated with the abundance of Bacteroides , which has pro-inflammatory properties; abundance of the anti-inflammatory genus Faecalibacterium correlated with expression of KAZN, with barrier-enhancing functions. Both the microbiota composition and local inflammation are regulated, at least partially, by the same germline variants. These variants may regulate the microenvironment in which bacteria grow and predispose to the development of cancer. Identification of these variants is the first step to identifying higher-risk individuals and proposing tailored preventive treatments that increase beneficial bacterial populations.

Background Peritoneal metastases from colorectal cancer (CRCPM) are related to poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have been reported to improve survival, but peritoneal recurrence rates are still high and there is no consensus on the drug of choice for HIPEC. The aim of this study was to use patient derived organoids (PDO) to build a relevant CRCPM model to improve HIPEC efficacy in a comprehensive bench-to-bedside strategy. Methods Oxaliplatin (L-OHP), cisplatin (CDDP), mitomycin-c (MMC) and doxorubicin (DOX) were used to mimic HIPEC on twelve PDO lines derived from twelve CRCPM patients, using clinically relevant concentrations. After chemotherapeutic interventions, cell viability was assessed with a luminescent assay, and the obtained dose–response curves were used to determine the half-maximal inhibitory concentrations. Also, induction of apoptosis by different HIPEC interventions on PDOs was studied by evaluating CASPASE3 cleavage. Results Response to drug treatments varied considerably among PDOs. The two schemes with better response at clinically relevant concentrations included MMC alone or combined with CDDP. L-OHP showed relative efficacy only when administered at low concentrations over a long perfusion period. PDOs showed that the short course/high dose L-OHP scheme did not appear to be an effective choice for HIPEC in CRCPM. HIPEC administered under hyperthermia conditions enhanced the effect of chemotherapy drugs against cancer cells, affecting PDO viability and apoptosis. Finally, PDO co-cultured with cancer-associated fibroblast impacted HIPEC treatments by increasing PDO viability and reducing CASPASES activity. Conclusions Our study suggests that PDOs could be a reliable in vitro model to evaluate HIPEC schemes at individual-patient level and to develop more effective treatment strategies for CRCPM.

BackgroundImmune checkpoint inhibitors (ICI) improved survival of patients with non-small cell lung cancer (NSCLC), yet many patients do not respond to treatment. The identification of markers for ICI response remains an unmet clinical need. This study hypothesizes that host genetics influences the response to ICI, contributing to the variability in efficacy among individuals.MethodsWe conducted a genome-wide association study (GWAS) in patients with NSCLC on ICI monotherapy with nivolumab, pembrolizumab, or atezolizumab, to identify germline variants associated with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) at 24 months after the start of ICI therapy. Genomic DNA was genotyped using Axiom Precision Medicine Research Arrays. Raw data were processed with Axiom Analysis Suite, and quality checked with PLINK software. Imputation to the whole genome was done on the Michigan Imputation Server. Association analyses were performed for ORR (logistic regression with PLINK2 software) and survival (Cox proportional hazards model, with GenAbel package in R environment), with appropriate covariates. Variants were annotated for functional significance using SNPnexus and FUMA. Post-GWAS analyses, including colocalization, were performed to explore the function of the identified variants. Their possible role as expression quantitative trait loci was investigated in different databases (GTEx, eQTLGen, TCGA).ResultsNo genome-wide significant associations were found for ORR or PFS, while a locus on chromosome 2 (lead variant: rs111648355) showed near genome-wide significance (p value=6.3×10⁻⁸) for OS. Patients with minor alleles of these variants exhibited significantly worse OS (HR=5.1, 95% CI: 2.9 to 9.2). Functional annotation linked these variants to regulatory effects on genes including MSH2, MSH6, PPP1R21, FBXO11, and STON1. These genes play a role in mismatch repair, endosomal trafficking, or major histocompatibility complex class II regulation, and might influence the response to immunotherapy.ConclusionsThis study identifies an association between a genomic locus on chromosome 2 and OS in patients with NSCLC treated with ICI. Although these results need validation in larger cohorts and functional studies to elucidate the underlying mechanisms, they highlight the potential of germline variants as predictive biomarkers of response to ICI.

Patient-derived organoids (PDOs) are tridimensional cultures derived from the stem component of a tissue. They preserve the genetic and phenotypic characteristics of the tissue of origin, and represent valuable in vitro models for drug screening, biomarker discovery, cell therapy and genetic modification. Importantly, PDOs reproduce the tumor behavior and can predict therapeutic responses, making them relevant for clinical applications for personalized therapies. PDOs may also be used for studying the interactions between cancer cells and the tumor microenvironment (TME). These interactions are driven by biochemical factors released by the cells, and biomechanical events such as the remodeling of the extracellular matrix (ECM). In recent years, it has become evident that the interactions between cancer cells and the TME have an impact on tumor development and on the efficacy of cancer therapy Therefore, targeting both tumor cells and the TME may improve patient response to treatment. Most PDO culture protocols are limited to epithelial cells. However, recent advances such as use of decellularized ECM (dECM) scaffolds have allowed for the development of in vivo -like environments that host diverse cell types, both normal and pathological, in a tridimensional (3D) manner that closely mimics the complexity of the TME. dECM-based models effectively replicate the interactions between tumor cells, ECM and the microenvironment, are easy to analyze and adaptable for drug testing. By incorporating TME components and therapeutic agents, these models offer an advanced platform for preclinical testing.

Introduction Disease recurrence after surgery is a crucial predictor of poor prognosis in colorectal cancer, where disseminated disease at the time of intervention can also be observed in localized early-stage cases. We evaluated the ability to predict disease recurrence of miRNAs from two signatures that we have found linked to the presence of colorectal cancer (CL signature) or adenoma (HgA signature) in higher-risk subjects. Methods miRNAs from the signatures were studied longitudinally by quantitative real-time polymerase chain reaction in plasma from 24 patients with resectable colorectal cancer collected at the time of surgery and during scheduled follow-up across 36 months. Patients either showed relapse within 36 months (alive with disease (AWD)), or remained disease-free (no evidence of disease (NED)) for the same period. Results Although the signatures did not predict recurrence, expression of the miRNAs from the CL signature decreased 1 year after surgery, and one miRNA of the signature, miR-378a-3p, almost reached significance in the NED subgroup (Wilcoxon signed-rank test: p-value = 0.078). Also, miR-335-5p from the HgA signature was higher in AWD patients before surgery (Kruskal–Wallis test: p-value = 0.019). Conclusions These data, although from a small cohort of patients, support the possible use of miRNAs as non-invasive biomarkers in liquid biopsy-based tests to identify patients at risk of relapse and to monitor them during follow-up.

A miRNAs profiling on a group of familial and sporadic breast cancers showed that miRNA-342 was significantly associated with estrogen receptor (ER) levels. To investigate at functional level the role of miR-342 in the pathogenesis of breast cancer, we focused our attention on its \"in silico\" predicted putative target gene ID4, a transcription factor of the helix-loop-helix protein family whose expression is inversely correlated with that of ER. ID4 is expressed in breast cancer and can negatively regulate BRCA1 expression. Our results showed an inverse correlation between ID4 and miR-342 as well as between ID4 and BRCA1 expression. We functionally validated the interaction between ID4 and miR-342 in a reporter Luciferase system. Based on these findings, we hypothesized that regulation of ID4 mediated by miR-342 could be involved in the pathogenesis of breast cancer by downregulating BRCA1 expression. We functionally demonstrated the interactions between miR-342, ID4 and BRCA1 in a model provided by ER-negative MDA-MB-231 breast cancer cell line that presented high levels of ID4. Overexpression of miR-342 in these cells reduced ID4 and increased BRCA1 expression, supporting a possible role of this mechanism in breast cancer. In the ER-positive MCF7 and in the BRCA1-mutant HCC1937 cell lines miR-342 over-expression only reduced ID4. In the cohort of patients we studied, a correlation between miR-342 and BRCA1 expression was found in the ER-negative cases. As ER-negative cases were mainly BRCA1-mutant, we speculate that the mechanism we demonstrated could be involved in the decreased expression of BRCA1 frequently observed in non BRCA1-mutant breast cancers and could be implicated as a causal factor in part of the familial cases grouped in the heterogeneous class of non BRCA1 or BRCA2-mutant cases (BRCAx). To validate this hypothesis, the study should be extended to a larger cohort of ER-negative cases, including those belonging to the BRCAx class.