Biology subjects

Puliafito, A.

Publications and source records attributed to Puliafito, A..

6 recordsLinked to original sources

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

Inference of lineage hierarchies, growth and drug response mechanisms in cancer cell populations without tracking

Lineage hierarchies and plasticity regulate development and tissue homeostasis, while diverted lineage dynamics and aberrant phenotypic plasticity are among the causes of incomplete drug response and secondary resistance in cancer. Knowing the dynamics of phenotypically plastic populations is therefore central to understand growth regulation principles and to rationally design therapeutic approaches that might anticipate drug-tolerant states. Lineage inference however largely relies on single-cell tracking techniques, which are notoriously difficult in complex biological models. To overcome these limitations, we developed a method to infer active phenotypic transitions in a multi-lineage tumor or clone and to quantify them, solely relying on counting lineage abundances with no pedigree. We demonstrate the effectiveness of our approach to cancer cell plasticity and drug treatment in silico. We then perform experiments on cancer cell populations and show that our method correctly predicts growth mechanisms and transition probabilities.

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

Two-fluid dynamics and micron-thin boundary layers shape cytoplasmic flows in early Drosophila embryos

Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. Here, we combine hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, our model explains observations that remained elusive, and makes a series of new predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially whilst the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, we use our model to analyze the effects of flows on the transport of the morphogen Bicoid, and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, our two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.

biophysics↗

bioRxiv2022

Collective directional migration drives the formation of heteroclonal cancer cell clusters

Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multi-clonal cell aggregates are not fully elucidated. Here we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors consistently with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, with evidence that conditioned culture media act as chemoattractant and corroborated by a wide screening on secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.

cancer biology↗

bioRxiv2022

Cullin-5 regulates nuclear positioning and reveals insights on the sensing of the nuclear-to-cytoplasmic ratio in Drosophila embryogenesis

In most metazoans, early embryonic development is characterized by rapid division cycles which pause before gastrulation at the mid-blastula transition (MBT).1 These early cleavage divisions are accompanied by cytoskeletal rearrangements which ensure proper nuclear positioning. Yet, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium, and nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows.2,3 Previously, shackleton (shkl) mutants were identified in which this axial spreading is impaired.4 Here, we show that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity genetically, suggesting that a major target of Cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis at the MBT which we exploit to study cell cycle control as a function of the N/C ratio. We found that the N/C ratio is sensed collectively in neighborhoods of about 100m and such collective sensing is required for a precise MBT in which all the nuclei in the embryo pause their division cycle. Moreover, we found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to the spatiotemporal regulation of Cdk1 activity. Collectively, our results reveal a new pathway controlling nuclear spreading and provide a quantitative dissection of how nuclear cycles respond to the N/C ratio.

developmental biology↗