Periodic Reporting for period 1 - artFibro (Artifying fibroblasts: Perturbation modelling in the lung tumor phase space to rewire fibroblasts for immunotherapy.)
Periodo di rendicontazione: 2023-04-01 al 2025-09-30
Lung cancer remains the leading cause of cancer-related mortality. While immunotherapy has revolutionized treatment, its efficacy is restricted to a subset of patients, highlighting an urgent need for novel strategies to overcome resistance. Our recent groundbreaking discovery of universal antigen-presenting fibroblasts (apFibros) within human and murine lung tumors presents a paradigm shift. These apFibros directly stimulate cancer-specific CD4 T cells, fostering immune-rich environments that promote tumor rejection and suggesting a novel mechanism for T cell activation independent of lymph nodes. Preliminary data further indicate their potential to mitigate resistance to checkpoint inhibitors. The central challenge for therapeutic exploitation of apFibros lies in their low numbers and the incomplete understanding of the molecular cues that govern their immunogenic configurations. This project, "Artifying Fibroblasts" (artFibro), addresses these critical bottlenecks by integrating cutting-edge computational and laboratory approaches. We will generate comprehensive perturbation datasets across single-cell/cell systems, transcriptomics/epigenomics, and spatial/temporal dimensions in both human and mouse models. Our overarching objective is to dissect the molecular landscape regulating fibroblast states and, crucially, to identify perturbations that can reprogram cancer-associated fibroblasts into antigen-presenting states. The expected impact of artFibro is profound. By unravelling the mechanisms that drive apFibro formation and function, we aim to unlock new avenues for immunotherapy. Successfully "artifying" fibroblasts to enhance their antigen-presenting capabilities could transform the tumor microenvironment, rendering unresponsive tumors susceptible to immunotherapy and expanding its benefits to a broader patient population. This project has the potential to significantly improve survival rates for lung cancer patients by providing a novel, fibroblast-centric immunotherapeutic strategy.
During the first period, the artFibro project already made significant strides in dissecting the origins, heterogeneity, and immunomodulatory functions of apFibros in lung cancer. Using lineage tracing, single-cell transcriptomics, and spectral cytometry, we identified diverse apFibro subsets each with distinct gene regulatory networks (GRNs). These was validated in vivo through comprehensive Cre-loxP tracing and bone marrow chimera models. Spatial profiling via Xenium and CODEX in human NSCLC samples revealed proximity of apFibros to CD4⁺ T cells, implicating potential roles in local immune modulation. Ligand–receptor modeling and spatial neighborhood analysis are being applied to infer cell–cell interactions. To identify apFibro regulators, we are employing single cell multi-omics, CRISPR-based perturbations and floxed mouse lines. Functional implications are being tested using newly generated Col1a2-LSL-DTR-EGFP mice for targeted fibroblast ablation. In vivo and ex vivo systems have been established to reprogram fibroblasts toward antigen-presenting states. These include AAV-mediated in vivo gene editing and primary cell CRISPR workflows, coupled with T cell co-culture assays. Together, these efforts yield new mechanistic insights and foundational models for immunotherapeutic development.
The discovery of apFibros in tumors significantly advances stromal biology, redefining immune–stromal boundaries. apFibros possess antigen-presenting capacities, and influence T cell dynamics, opening new paradigms in cancer immunology. This chimeric identity challenges established tumor microenvironment models and reveals a novel regulatory axis for immunotherapeutic intervention. These findings lay the groundwork for next-generation therapies targeting stromal–immune interactions. Early data from CRISPR perturbation and co-culture assays suggest actionable pathways that could be leveraged to enhance anti-tumor immunity. Two immunotherapeutic concepts have already emerged with potential patenting. Further uptake requires completion of in vivo functional studies, validation in additional tumor types, and development of delivery strategies for stroma-targeted interventions. Commercialization will depend on establishing preclinical efficacy and safety, ideally via partnerships with biotech/pharma. Access to scalable gene editing technologies, supportive IP strategies, and regulatory frameworks that recognize stroma-targeted immunotherapies will be essential. The generation of novel mouse models, spatial atlases, and open-source software tools positions artFibro as a driver of interdisciplinary innovation. These outputs, once fully validated and disseminated, will enable the broader community to explore apFibros as a therapeutic target, potentially transforming current approaches to cancer treatment.