1. Mission

The field of biomedical sciences in general, and oncology in particular, has reached a level of complexity that can no longer progress through one-dimensional approaches. The development of new preventive strategies, potential therapeutic compounds, diagnostic protocols, and follow-up programs cannot rely exclusively on traditional in vitro or in silico (computational) systems. Instead, an integrated approach is required, encompassing the full range of available methodologies: from precision medicine applied to patient care, to animal models—an essential test bed—without neglecting in vitro and in silico experimentation.

The value of animal models is further demonstrated by the use of genetically modified mice, which can reproduce, in a controlled manner, the onset and progression of numerous neoplastic pathologies. These models enable both an in-depth study of molecular mechanisms and the preclinical evaluation of new pharmacological compounds. At the same time, the engraftment of human tumors derived from patients into immunodeficient mice has led to the development of patient-derived xenograft (PDX) models, which allow researchers to study tumor biology and therapeutic responses in vivo. These models make it possible to conduct clinical studies in patients while simultaneously monitoring disease evolution in animals, thus promoting a real-time transfer of knowledge from experimental models to humans. Within this framework, specific clinical studies are currently being conducted simultaneously in humans and animals, with the aim of accelerating the development of precision medicine and personalized treatments.

The scientific community remains strongly committed to minimizing the use of animals by enforcing stringent regulations designed to limit the number of specimens employed, while promoting the development of alternative experimental approaches.

The concept of precision medicine is based on the ability to identify individual susceptibility to certain genetic diseases through advanced molecular technologies such as next-generation sequencing (NGS), followed by targeted therapeutic interventions aimed at the specific molecular alterations identified. This strategy brings numerous advantages: improved preventive capacity, clinical decision-making supported by detailed molecular information, more effective therapeutic outcomes with fewer side effects, the possibility of earlier and more targeted treatments, and significant costreductions for healthcare systems. Moreover, innovative approaches such as liquid biopsy now allow for non-invasive monitoring of disease progression.

The mission of UOSD SAFU is centered on the study of human tumors through innovative experimental models, including both the transplantation of primary human tumors into immunocompromised mice at orthotopic sites (PDX models) and the use of genetically engineered mouse models (GEMMs). The use of such models is indispensable for obtaining reliable data on both the efficacy and safety of new drugs and therapeutic combinations, since only whole-organism experiments can provide information that cannot be derived from in vitro systems alone.

Currently, UOSD SAFU has at its disposal a wide range of animal models that allow researchers to follow disease progression from onset through to therapeutic response. Research activities are particularly focused on models for breast, colon, pancreatic, intracranial tumors, and multiple myeloma. In addition, athymic (nude) mice are available for studies on UV-induced carcinogenesis (melanoma and non-melanoma skin cancers).

Alongside its research activities, UOSD SAFU is also responsible for the daily management of animal experimentation within the Institute. In this context, the Unit coordinates the activities of the Animal Welfare Body (in compliance with Legislative Decree No. 26/2014), which oversees the ethical and scientific evaluation of all projects involving animal experimentation.

The staff of the Unit operate as a facility supporting research activities across the entire Institute. In particular, two researchers are permanently dedicated to animal welfare and to assisting investigators within the animal facility located at the Castel Romano branch, while a veterinary oncologist regularly monitors the health status of all housed animals.

In parallel with its core activities, the SAFU Unit is deeply engaged in the development and validation of next-generation sequencing (NGS) technologies in the context of exploratory research. This includes not only laboratory-based applications but also the design and implementation of dedicated software for the analysis and interpretation of complex datasets.

Current scientific evidence indicates that genomic analysis alone is not sufficient to improve diagnosis and therapy. The response of a neoplasm to treatment is influenced not only by its genomic features but also by its transcriptome and epigenome.

RNA analysis provides crucial insights into transcriptional variations associated with tumor phenotypes, enabling the identification of gene expression dysregulation and pathogenic gene fusions. Importantly, when integrated with exome sequencing, these approaches make it possible to identify tumor-specific neoantigens as well as fusion proteins generated within the tumor. Furthermore, RNA-seq analyses are used to profile the immune infiltrate in tumors, while exome sequencing is often applied to assess the mutational burden of individual cancers.

More recently, NGS methodologies have been extended to the study of non-coding RNAs, including miRNAs and long non-coding RNAs (lncRNAs), across various tumor types, with the aim of identifying novel, tumor-specific biomarkers.

Unlike the genome, the epigenome is not static: epigenetic alterations may precede or follow disease onset, reflect environmental exposures, or be shaped by specific lifestyle factors. This makes the epigenome an attractive field for biomarker discovery and for translational applications in oncology. Through these studies, SAFU researchers are investigating how alterations in chromatin conformation drive neoplastic transformation by modifying DNA accessibility to transcription factors and RNA polymerases.

Within this framework, three researchers are exclusively dedicated to nucleic acid sequencing activities, supported by five bioinformaticians who oversee computational analyses and data integration.

2. Research activities

Animal Facility

The conventional animal facility has been located since December 2015 at the Plaisant Company site in Castel Romano. The facility currently houses only mice, all genetically controlled, which are used as experimental models.

Colonies include immunosuppressed animals transplanted with human tumors (orthotopic and xenograft models), such as breast, prostate, mesothelioma, lung, kidney, colon, and melanoma. These models are employed for preclinical pharmacokinetic studies with newly synthesized molecules (drugs, peptides, oligonucleotides), as well as with combinations of different compounds.

Studies may also be performed using tumors derived from in vitro cell lines previously engineered with a bioluminescent reporter gene, allowing non-invasive imaging to monitor neoplastic development at different growth stages and to track metastasis formation.

In addition, the facility maintains several patient-derived xenograft (PDX) models from colon cancers, along with approximately 20 genetically engineered mouse models (GEMMs).

All procedures involving animals and their care comply with institutional guidelines and are approved by the Italian Ministry of Health.

At present, the animal facility hosts around 700 mice. Animals are monitored daily, with routine litter changes and ad libitum access to water. Housing is provided in eight racks of individually ventilated cages (IVCs). The animal rooms occupy two spaces of 25 m² each.

The facility also contains two laboratories of approximately 20 m² each:

• Laboratory 1 is equipped with two horizontal laminar flow hoods, a centrifuge, two microscopes, and two refrigerated cabinets (+4 °C and –20 °C).
• Laboratory 2 houses a bio/fluorescence imaging system, a gas anesthesia system, a CO₂incubator, a refrigerated centrifuge, and a hot-air oven.

A ^137Cs irradiator is currently allocated at the Mostacciano site.

Next-Generation Sequencing Facility

The NGS Facility is a core research infrastructure designed to provide access to state-of-the-art next-generation DNA and RNA sequencing technologies. Its mission is to deliver high-quality data and analytical support to collaborators while ensuring comprehensive assistance throughout the entire project workflow—from experimental design to sequencing and bioinformatic analysis.

Instrumentation and Capabilities

The facility is equipped with a wide range of sequencing and molecular profiling platforms, enabling flexibility across project scales and applications:

• llumina MiSeq (1–25 million read-pairs) for small-scale sequencing.
• Illumina NextSeq 500 (130–400 million read-pairs) for medium-throughput projects.
• Illumina NovaSeq 6000 (0.8–20 billion read-pairs per run; acquired in 2021) for large-scale sequencing.
• Oxford Nanopore GridION and PromethION (acquired in 2023) for long-read sequencing and real-time analysis.
• NanoString nCounter system for multiplex analysis of up to 800 RNA, DNA, or protein targets.
• NanoString GeoMX Digital Spatial Profiler and 10x Genomics Visium for spatial transcriptomics and proteomics.
• 10x Genomics Chromium controllers (acquired in 2021) for single-cell and single-nucleus library preparation.
• DEPArray system for rare cell recovery and single-cell analysis.
• Agilent TapeStation and Bioanalyzer for nucleic acid integrity assessment.

During these years, the NGS Facility supported a broad range of research projects, generating the following outputs:

• RNA sequencing: 2,526 samples
• Whole-exome sequencing: 1,502 samples
• Epigenetic assays: 664 samples
• Targeted DNA-seq library preparation: 96 samples
• SARS-CoV-2 sequencing: 2,942 samples
• Single-cell sequencing: 81 samples
• GeoMX spatial profiling: 504 samples
• 10x Visium spatial assays: 20 samples
• Nanopore sequencing: 5 samples
• Liquid biopsy genomic samples: 106
• Methylome panel analysis: 13

The progressive expansion of the facility’s platform portfolio, particularly with the recent introduction of single-cell, spatial, and long-read sequencing technologies, has significantly enhanced its capacity to support integrated multi-omic analyses. These developments strengthen the facility’s role in enabling comprehensive molecular characterization across genomic, transcriptomic, and epigenomic dimensions, with increasing relevance for translational research and precision medicine.

In addition to its core activities, the SAFU Unit devoted substantial efforts to the study of COVID-19 and its impact on patient care. In collaboration with the Microbiology Unit of the San Gallicano Institute, as well as the Oncogenomic, Epigenetic, and Bioinformatics Units, the group conducted genomic sequencing of more than 3,000 SARS-CoV-2–positive patients. This large-scale effort enabled the systematic monitoring of viral variants circulating in the Lazio region and provided critical epidemiological insights into the evolution of the pandemic.

Parallel to these activities, Dr. Piaggio investigated the broader effects of the COVID-19 pandemic on healthcare access for cancer and immunocompromised patients. Her work compared the impact of the pandemic on therapeutic accessibility across three Italian regions, analyzing how institutional reorganizational measures influenced key performance metrics.

Furthermore, Dr. Piaggio established a COVID-19 Biobank, collecting biological samples from SARS-CoV-2–infected patients. This resource was designed to support ongoing and future studies on immunological alterations in patients with immune frailty, offering a valuable platform for translational research and potential therapeutic innovations.

Multiple Myeloma (MM) Research and Related Activities

Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal expansion of plasma cells in the bone marrow and excessive production of immunoglobulins. Over the past years, the SAFU Unit has established an optimized protocol for the collection and storage of viable CD138⁺ plasma cells from patients.

Through collaborations with several hematology units, bone marrow samples have been obtained from approximately 350 MM patients under clinical monitoring, as well as from individuals with MGUS, smoldering myeloma, relapsed disease, and post-transplantation states, alongside healthy donors. Importantly, this dataset includes over 50 longitudinally matched samples from patients at first onset and following therapy.

Chromatin accessibility profiling by ATAC-seq has been carried out on 55 MM, 11 MGUS, and 4 healthy samples. These analyses identified approximately 90,000 accessible regions in MM, about 20% of which were also detected in MGUS, highlighting both heterogeneity and shared chromatin features (Sharing Index). Motif enrichment and footprinting analyses revealed that canonical MM transcription factors (e.g., IRF4, MEF2C) were frequently engaged at private loci, while NRF1 binding motifs were significantly enriched at highly shared loci.

Follow-up ChIP-seq analyses in MM cell lines confirmed NRF1 binding at over 7,000 accessible regions. Functional experiments demonstrated that NRF1 expression is upregulated in MM through the activation of a disease-specific enhancer. A transcriptional program comprising 103 NRF1- bound genes—enriched for pathways related to oxidative phosphorylation, ubiquitination, and mitochondrial dysfunction—was identified and shown to correlate with disease aggressiveness and patient mortality.

NRF1 depletion in MM cells led to impaired ubiquitination, increased ER stress, and heightened sensitivity to proteasome inhibition. Both in vitro and in vivo studies supported a model in which NRF1 sustains survival under proteasome stress, contributing to resistance to proteasome inhibitors.

In parallel, the Unit investigated epigenetic regulation in MM progression and bone disease. For example, miR-590-3p was characterized as a regulator of plasma cell proliferation and bone resorption. Ongoing collaborative projects are examining chromatin structure dynamics across disease progression and treatment.

Through the analysis of ATAC-seq datasets from MGUS (n=7), primary and post-treatment MM (n=26), and relapse (n=11), a temporal co-accessibility network was developed to identify communities of non-coding regulatory elements with coordinated activity. Integration with publicdatasets and machine learning approaches highlighted a subset of enhancer-like elements associated with treatment resistance. These elements stratified patients into disease-like and healthy-like groups, even among post-treatment samples, suggesting persistence of epigenetic reprogramming despite clinical response.

Target gene analysis linked these enhancers to transcriptional programs defining bortezomib response. In scRNA-seq data from 48,032 plasma cells (18 MM patients), these genes were predictive of suboptimal versus optimal responses. Resistant cells were marked by sustained activation of the unfolded protein response.

Collectively, these studies define NRF1 as a key transcriptional regulator of proteasome stress adaptation and reveal non-coding enhancer elements that drive epigenetic reprogramming and bortezomib resistance in MM. These findings establish a framework for integrating chromatin accessibility, transcriptional profiling, and functional genomics to uncover therapeutic vulnerabilities and biomarkers of treatment resistance.

The Unit’s research objectives are further advanced through the integrated activities of several specialized groups: