Biology subjects

Lodrini, M.

Publications and source records attributed to Lodrini, M..

3 recordsLinked to original sources

bioRxiv2026

Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

cancer biology↗

bioRxiv2025

Cryo-mtscATAC-seq for single-cell mitochondrial DNA genotyping and clonal tracing in archived human tissues

High-throughput clonal tracing of primary human samples relies on naturally occurring barcodes, such as somatic mitochondrial DNA (mtDNA) mutations detected via single-cell ATAC-seq (mtscATAC-seq). Fresh-frozen clinical specimens preserve tissue architecture but compromise cell integrity, thereby precluding their use in multi- omic approaches such as mitochondrial genotyping at single-cell resolution. Here, we introduce Cryo-mtscATAC-seq, a broadly applicable method for diverse pathophysiological contexts to isolate nuclei with their associated mitochondria ("CryoCells") from frozen samples for high-throughput clonal analysis. We applied Cryo-mtscATAC-seq to the neurodegenerated human brain, glioblastoma (GBM), pediatric neuroblastoma, and human aorta, and implemented mitobender, a computational tool to reduce ambient mtDNA in single-cell assays. Our approach revealed regional clonal gliogenesis and microglial expansions in amyotrophic lateral sclerosis (ALS), persistence of oligodendrocyte progenitor cell (OPC)-like clones in GBM recurrence, mtDNA depth heterogeneity after neuroblastoma chemotherapy, and oligoclonal proliferation of smooth muscle cells in human aorta. In conclusion, Cryo-mtscATAC-seq broadly extends mtDNA genotyping to archival frozen specimens across tissue types, opening new avenues for investigation of cell state- informed clonality in human health and disease.

systems biology↗

bioRxiv2024

Targeting MYCN upregulates L1CAM tumor antigen in MYCN-dysregulated neuroblastoma to increase CAR T cell efficacy

BackgroundCurrent treatment protocols have only limited success in pediatric patients with neuroblastomas harboring amplifications of the central oncogene, MYCN. Adoptive T cell therapy presents an innovative strategy to improve cure rates. However, L1CAM-targeting CAR T cells achieved only limited response against refractory/relapsed neuroblastoma in an ongoing phase I trial to date. Here, we investigate how oncogenic MYCN levels influence tumor cell response to CAR T cells, as one possible factor limiting success in trials. MethodsHigh MYCN levels were induced in SK-N-AS cells harboring the normal diploid MYCN complement using a tetracycline-inducible system. The inducible MYCN cell model or MYCN-amplified neuroblastoma cell lines were cocultured with L1CAM-CAR T cells. CAR T cell effector function was assessed via activation marker expression (flow cytometry), cytokine release and tumor cytotoxicity (biophotonic signal assessment). The cell model was characterized using RNA sequencing, and our data compared to publicly available RNA and proteomic data sets from neuroblastomas. ChIP-sequencing data was used to determine transcriptional L1CAM regulation by MYCN using public data sets. Synergism between CAR T cells and the MLN8237 AURKA inhibitor, which indirectly inhibits MYCN activity, was assessed in vitro using the Bliss model and in vivo in an immunocompromised mouse model. ResultsInducing high MYCN levels in the neuroblastoma cell model reduced L1CAM expression and, consequently, L1CAM-CAR T cell effector function (activation, cytokine release and cytotoxicity) in vitro. Primary neuroblastomas possessing high MYCN levels expressed lower levels of both the L1CAM transcript and L1CAM tumor antigen. Indirectly inhibiting MYCN via AURKA using MLN8237 treatment restored L1CAM expression on tumor cells in vitro and restored L1CAM-CAR T cell effector function. Combining MLN8237 and L1CAM-CAR T cell treatment synergistically increased neuroblastoma-directed killing in MYCN-overexpressing cells in vitro and in vivo concomitant with severe in vivo toxicity. ConclusionWe shed new light on a primary resistance mechanism in MYCN-driven neuroblastoma against L1CAM-CAR T cells via target antigen downregulation. These data suggest that combining L1CAM-CAR T cell therapy with pharmacological MYCN inhibition may benefit patients with high-risk neuroblastomas harboring MYCN amplifications.

immunology↗