Biology subjects

Trusolino, L.

Publications and source records attributed to Trusolino, L..

9 recordsLinked to original sources

bioRxiv2026

Pdk1-dependent metabolic heterogeneity promotes aggressive colorectal cancer through cell-intrinsic interferon signaling

Tumor heterogeneity is a major determinant of tumor progression and therapeutic resistance, yet the contribution of metabolism to intratumoral heterogeneity still remains poorly explored. Here, we find that pyruvate dehydrogenase kinase-1 (Pdk1) expression defines metabolically distinct cancer cell populations in colorectal tumors. Image-based spatial single-cell phenotyping shows that Pdk1-high cells are associated with tumor progression in orthotopic models of colorectal cancer (CRC). Genetic ablation experiments demonstrate the role of Pdk1-high cells in tumor growth, 5-fluorouracil chemoresistance and tumor relapse. Mechanistically, we identify a link between Pdk1-driven metabolic states and specific cell phenotypes, where Pdk1-high cancer cells exhibit an EMT, stem cell-like phenotype characterized by increased interferon (IFN) signaling. Mitochondrial remodeling in Pdk1-high cells leads to the release of mitochondrial DNA to the cytosol and activation of IFN pathways, promoting Pdk1-driven phenotypes. Inhibition of IFN signaling impairs the tumorigenic and aggressive features of Pdk1-high cancer cells, thus establishing a functional connection between metabolic heterogeneity, cell-intrinsic IFN signaling and aggressive CRC.

cancer biology↗

bioRxiv2026

TGF-β1-induced differentiation enhances chemotherapy response in metastatic colorectal cancer organoids

BackgroundIn metastatic colorectal cancer, systemic therapies frequently fail, partly due to underlying phenotypic plasticity rooted in pre-existing multi-type cell populations. Intratumoral lineage hierarchies within colorectal tumors require renovated efforts to decode growth principles, design rational therapeutic approaches, and accurately interpret drug response. Understanding the cell-state dynamics of untreated tumors and the degree of cell responsiveness to exogenous stimuli is therefore crucial to improving currently underwhelming therapeutic outcomes. MethodsHere, we leveraged patient-derived organoids established from hepatic metastases of colorectal cancer patients to deconstruct population hierarchies by combining single-cell transcriptomics with single-molecule RNA fluorescent in situ hybridization. Computational frameworks were used to identify independent gene modules. We then employed flow cytometry analysis to track cytokine-induced population shifts using a cell surface marker, validating our findings through bulk RNA analysis, functional assays, and viability assays in response to oxaliplatin. ResultsOur data substantiate the existence of a dual population configuration within untreated metastatic colorectal cancer organoids with diverse genetic backgrounds. Gene modules detected via single-cell transcriptomics delineate a stem-like (LGR5+) and a differentiated-like (KRT20+) population that fluctuate dynamically over time. Spatially and temporally resolved, single-cell level analysis through single molecule FISH captures the inherent stochasticity in cell fate decisions revealing surprising phenotypic variability even across different organoids derived from the same patient. By using GABRA2 as a surface marker we track the emergence of differentiated cells over the course of time and investigate the respective roles of TGF-{beta}1 and IL-6 in the differentiation of organoids. Our findings indicate that IL-6 exerts no major effect within our cell autonomous setting. In stark contrast, TGF-{beta}1 triggered cell cycle arrest and differentiation, while simultaneously reducing clonogenic capacity and significantly amplifying the cytotoxic potency of oxaliplatin. ConclusionsOur findings provide evidence of the dual Stem and Differentiated population hierarchy in metastatic colorectal cancer organoids, and demonstrate how this axis can be effectively hijacked by TGF-{beta}1 to suppress tumor growth. Our results suggest that further mechanistic exploitation of this cell-autonomous, tumor-suppressive arm of TGF-{beta}1 signalling could open unappreciated therapeutic windows in advanced colorectal cancer.

cancer biology↗

bioRxiv2025

On-target mutations confer resistance to WRN helicase inhibitors in Microsatellite Unstable Cancer Cells.

Werner helicase inhibitors (WRNi) are in clinical development for microsatellite-unstable (MSI) tumors with defective DNA mismatch repair. Here, we investigate how cancer cell evolution shapes response to WRN inhibition and informs potential resistance mechanisms. Genome-wide CRISPR screens combined with WRN knockout did not identify bypass mechanisms, underscoring WRNs essential, non-redundant function in MSI cells. Pharmacogenomic screens identified modulators of WRNi sensitivity, including SMARCAL1, which links it to WRN-MSI synthetic lethality. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving acquired resistance to multiple WRNi in vitro and in vivo, which was mitigated by combination with standard chemotherapies. Some resistance mutations conferred broad cross-resistance, whereas others preserved sensitivity to alternative clinical-grade WRNi with distinct mechanism of action. Our findings could inform clinical trial design by suggesting the feasibility of real-time tracking of emerging resistance and enabling early therapeutic adaptations. SignificanceWe present the first exploration of how MSI cancer cells evolve under the selective pressure of WRN helicase inhibition, providing a framework for understanding adaptive responses to this newly identified synthetic-lethal dependency. This study identifies on-target WRN mutations as key drivers of resistance in MSI cancers, supporting the use of combination strategies with other standard-of-care treatments to prevent resistance. It highlights how mutation tracking can guide therapeutic switching to clinically available WRN inhibitors with distinct mechanisms of action, thereby refining clinical development and potentially improving biomarker-informed patient outcomes.

cancer biology↗

bioRxiv2024

Heterogeneity and evolution of DNA mutation rates in microsatellite stable colorectal cancer

DNA sequence mutability in tumors with chromosomal instability is conventionally believed to remain uniform, constant, and low, based on the assumption that further mutational accrual in a context of marked aneuploidy is evolutionarily disadvantageous. However, this concept lacks robust experimental verification. We adapted the principles of mutation accumulation experiments, traditionally performed in lower organisms, to clonal populations of patient-derived tumoroids and empirically measured the spontaneous rates of accumulation of new DNA sequence variations in seven chromosomally unstable, microsatellite stable colorectal cancers (CRCs) and one microsatellite unstable CRC. Our findings revealed heterogeneous mutation rates (MRs) across different tumors, with variations in magnitude within microsatellite stable tumors as prominent as those distinguishing them from microsatellite unstable tumors. Moreover, comparative assessment of microsatellite stable primary tumors and matched synchronous metastases consistently documented a pattern of MR intensification during tumor progression. Therefore, wide-range diversity and progression-associated evolvability of DNA sequence mutational instability emerge as unforeseen hallmarks of microsatellite stable CRC, complementing karyotype alterations as selectable traits to increase genetic variation. One sentence summaryTumors with chromosomal instability accrue DNA sequence mutations at highly variable rates, which increase during metastatic progression.

cancer biology↗

bioRxiv2023

XENTURION, a multidimensional resource of xenografts and tumoroids from metastatic colorectal cancer patients for population-level translational oncology

The breadth and depth at which cancer models are interrogated contribute to successful translation of drug discovery efforts to the clinic. In colorectal cancer (CRC), model availability is limited by a dearth of large-scale collections of patient-derived xenografts (PDXs) and paired tumoroids from metastatic disease, the setting where experimental therapies are typically tested. XENTURION is a unique open-science resource that combines a platform of 129 PDX models and a sister platform of 129 matched PDX-derived tumoroids (PDXTs) from patients with metastatic CRC, with accompanying multidimensional molecular and therapeutic characterization. A PDXT-based population trial with the anti-EGFR antibody cetuximab revealed variable sensitivities that were consistent with clinical response biomarkers, mirrored tumor growth changes in matched PDXs, and recapitulated the outcome of EGFR genetic deletion. Adaptive signals upregulated by EGFR blockade were computationally and functionally prioritized, and inhibition of top candidates increased the magnitude of response to cetuximab. These findings illustrate the probative value and accuracy of large ex vivo and in vivo living biobanks, highlight the importance of cross-platform and cross-methodology systematic validation, and offer avenues for molecularly informed preclinical research.

cancer biology↗

bioRxiv2023

Integrative ensemble modelling of cetuximab sensitivity in colorectal cancer PDXs

AO_SCPLOWBSTRACTC_SCPLOWPatient-derived xenografts (PDXs) are tumour fragments engrafted into mice for preclinical studies. PDXs offer clear advantages over simpler in vitro cancer models - such as cancer cell lines (CCLs) and organoids - in terms of structural complexity, heterogeneity, and stromal interactions. We characterised 231 colorectal cancer PDXs at the genomic, transcriptomic, and epigenetic level and measured their response to cetuximab, an EGFR inhibitor in clinical use for metastatic colorectal cancer. After assessing PDXs quality, stability, and molecular concordance with publicly available patient cohorts, we trained, interpreted, and validated an integrated ensemble classifier (CeSta) which takes in input the PDXs multi-omic characterisation and predicts their sensitivity to cetuximab treatment (AUROC > 0.9). Our study shows that large PDX collections can be used to train accurate, interpretable models of drug sensitivity, which 1) better recapitulate patient-derived therapeutic biomarkers than other models trained on CCL data, 2) can be robustly validated across independent PDX cohorts, and 3) can be used for the development of novel therapeutic biomarkers.

bioinformatics↗

bioRxiv2022

CONNECTOR, fitting and clustering of longitudinal data to reveal a new risk stratification system.

The transition from the evaluation of a single time point to the examination of the entire dynamic evolution of a system is possible only in the presence of the proper framework. The strong variability of dynamic evolution makes the definition of an explanatory procedure for data fitting and data clustering challenging. Here we present CONNECTOR, a data-driven framework able to analyze and inspect longitudinal data in a straightforward and revealing way. When used to analyze tumor growth kinetics over time in 1599 patient-derived xenograft (PDX) growth curves from ovarian and colorectal cancers, CONNECTOR allowed the aggregation of time-series data through an unsupervised approach in informative clusters. Through the lens of a new perspective of mechanism interpretation, CONNECTOR shed light onto novel model aggregations and identified unanticipated molecular associations with response to clinically approved therapies.

bioinformatics↗

bioRxiv2022

Molecular phenotyping of colorectal neoplasia shows dynamic and adaptive cancer stem cell population admixture

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterised by fetal transcriptional profiles. Neoplasia hijacks regenerative signalling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signalling disruption, and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis, and is influenced by cell-intrinsic, extrinsic and therapeutic selective pressures.

cancer biology↗

bioRxiv2019

Evolving neoantigen profiles in colorectal cancers with DNA repair defects

BackgroundNeoantigens that arise as a consequence of tumour-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumour types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.\n\nMethodsWe performed Whole Exome Sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures and predicted neoantigens.\n\nResultsThe majority of CRC models showed remarkably stable mutational and neoantigen profiles, however those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures, and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly-evolving CRC.\n\nConclusionsThese results indicate that CRC carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

cancer biology↗