Biology subjects

Franciosa, G.

Publications and source records attributed to Franciosa, G..

6 recordsLinked to original sources

bioRxiv2025

Acute IFN-γ elicits long-term proteomic IFN-γ signature in melanoma cells

Interferon-{gamma} (IFN-{gamma}) is a central mediator of antitumor immunity, and its downstream effects are widely assumed to require continuous cytokine exposure. Here, we applied state-of-the-art mass spectrometry (MS)-based proteomics to patient-derived melanoma cells exposed either continuously to IFN-{gamma} or to a 3-hour pulse followed by cytokine removal. A transient IFN-{gamma} exposure was sufficient to establish a stable proteomic state indistinguishable from continuous stimulation, demonstrating that IFN-{gamma}-induced remodeling is rapidly triggered and durably maintained independently of ligand persistence. Proximal JAK-STAT signaling was rapid, saturable at low cytokine concentrations, and fully reversible upon washout, yet downstream proteomic programs remained durably imprinted. In contrast, functional consequences such as cytostasis remained dose-dependent, revealing a disconnect between signaling activation and phenotypic sensitivity. Together, these findings challenge long-standing assumptions about IFN-{gamma} signaling, redefine the temporal requirements for cytokine-driven tumor cell programming, and provide a mechanistic framework for improving immunotherapies primarily working via IFN-{gamma}-signaling and physiologically relevant in vitro models of tumor-immune interactions.

biochemistry↗

bioRxiv2025

The proteomics and phosphoproteomics landscape of melanoma under T cell attack

Understanding how tumor cells interact with tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) is crucial for identifying targetable immune checkpoints and predictive biomarkers for immunotherapy. While transcriptional responses have been characterized, protein-level changes remain largely unexplored. Here, we used a system reproducing the interaction of TILs with cancer cells occurring in the TME, by co-culturing patient-derived cancer cells with matched autologous TILs at sub-lethal ratios. Using this system, we profiled the early response that cancer (melanoma) cells and TILs activate following autologous T cell attack. To distinguish melanoma from TIL proteomes, we applied stable isotope labeling by amino acids in cell culture (SILAC) combined with Orbitrap Astral-based data-independent acquisition (DIA) mass spectrometry, enabling cell type-specific profiling of protein and phosphorylation dynamics without FACS sorting. This approach also captured the global newly synthesized proteome of the mixed cultures. Our analyses resolved interferon-{gamma}-dependent proteome changes occurring in melanoma cells, identified the cytotoxic and regulatory T-cell molecule (CRTAM) as a selective marker of reactive cytotoxic T lymphocytes, and revealed tumor-intrinsic kinase activation signatures. Among these, multiple DNA damage response-associated kinases were activated during immune attack, suggesting potential therapeutic vulnerabilities. Overall, this framework enables proteomic dissection of tumor-immune interactions and provides a resource for guiding biomarker discovery and therapeutic strategies to improve immunotherapy outcomes.

cancer biology↗

bioRxiv2023

Trafficking Machinery is Rapidly Primed to Facilitate Polarised IL-6 Secretion in Dendritic Cells

The mounting of an adaptive immune response is critical for removing pathogens from the body and generating immunological memory. Central to this process are myeloid cells, which sense pathogens through a variety of cell surface receptors, engulf and destroy pathogens and become activated. Activation is essential for the release of cytokines as well as the cell-surface presentation of pathogen-derived-antigens. Activation-induced cytokine release by myeloid cells requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is becoming increasingly clear that trafficking machinery has to be re-programmed through post-translational modifications to dynamically regulate cytokine secretory events. We demonstrate through quantitative total internal-resonance fluorescence (TIRF) microscopy that short-term stimulation with the pathogenic stimulus lipopolysaccharide (LPS) is sufficient to up-regulate IL-6 secretion rates in human blood monocyte-derived dendritic cells and that this secretion is asymmetric and thus polarised. Using bioinformatics analysis of our phosphoproteomic data, we demonstrate that LPS stimulation of monocyte-derived dendritic cells rapidly reprograms SNARE-associated membrane trafficking machinery, through phosphorylation/dephosphorylation events. Finally, we link this enhanced rate of secretion to the phosphorylation of the SNARE protein VAMP3 at serine 44 (48 in mice), by showing that this phosphorylation drives the release of VAMP3 by its chaperone WDFY2 and the complexing of VAMP3 with STX4 at the plasma membrane.

cell biology↗

bioRxiv2023

Cofilin-Driven Nuclear Deformation Drives Dendritic Cell Migration through the Extracellular Matrix

To mount an adaptive immune response, dendritic cells must process antigens, migrate to lymph nodes and form synapses with T cells. Critical to 3D migration and mechano-sensing is the nucleus, which is the size-limiting barrier for navigation through gaps in the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed, adopting a raison-like shape and enables dendritic cells to overcome the typical 2 - 3-micron pore limit for 3D-migration. We show that the nuclear shape-change is partially attained through reduced cell adhesion, whereas improved migration through extracellular matrix is achieved through reprogramming of the actin cytoskeleton. Specifically we show that cofilin-1 is phosphorylated at serine 41 drives the assembly of a Cofilin-ActoMyosin (CAM)-ring proximal to the nucleus and enhancing migration through 3D collagen gels. In summary, these data describe novel signaling events through which dendritic cells simultaneously deform their nucleus and enhance their migratory capacity; molecular events that may be re-capitulated in other contexts such as wound healing and cancer.

cell biology↗

bioRxiv2022

Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging when analyzing tiny sample amounts. We present a novel hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem MS scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmarked hybrid-DIA against state-of-the-art targeted MS methods (i.e. SureQuant) using EGF-stimulated HeLa cells and found the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiled the global phosphoproteome. To demonstrate the robustness, sensitivity and potential of hybrid-DIA, we profiled chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluated the difference of cancer cells in 2D vs 3D culture. Altogether, we showed that hybrid-DIA is the way-to-go method in highly sensitive phospho-proteomics experiments.

systems biology↗

bioRxiv2022

Matrix Gla Protein acts as a driver of stemness and tumor initiation in ovarian cancer

Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression. To this aim, a better understanding of the molecular and functional makeup of OCSC in clinically relevant model systems is essential. We have profiled the transcriptome of OCSC vs. their bulk cell counterpart from a panel of patient-derived OC cell cultures. This revealed that Matrix Gla Protein (MGP), classically known as a calcification-preventing factor in cartilage and blood vessels, is markedly enriched in OCSC. Functional assays showed that MGP confers several stemness-associated traits to OC cells, including a transcriptional reprogramming. Patient-derived organotypic cultures pointed to the peritoneal microenvironment as a major inducer of MGP expression in OC cells. Furthermore, MGP was found to be necessary and sufficient for tumor initiation in OC mouse models, by shortening tumor latency and increasing dramatically the frequency of tumor-initiating cells. Mechanistically, MGP-driven OC stemness was mediated by the stimulation of Hedgehog signaling, in particular through the induction of the Hedgehog effector GLI1, thus highlighting a novel MGP/Hedgehog pathway axis in OCSC. Finally, MGP expression was found to correlate with poor prognosis in OC patients, and was increased in tumor tissue after chemotherapy, supporting the clinical relevance of our findings. Thus, MGP is a novel driver in OCSC pathophysiology, with a major role in stemness and in tumor initiation.

cancer biology↗