A standardized imaging and analysis workflow for quantitative evaluation of cutaneous neurofibromas in Nf1-KO mice

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder in which cutaneous neurofibromas (cNFs) represent one of the most common and burdensome manifestations. No approved pharmacological treatment exists. Preclinical studies are essential to evaluate candidate therapies, but reliable outcome and endpoint measures for cNFs in animal models remain limited. We developed and validated a standardized methodology to assess drug efficacy in the Prss56Cre Nf1-KO mouse model which recapitulates key features of cNFs. In this model, Nf1 inactivation and tdTomato (Tom) reporter expression were specifically targeted to Schwann cells (SCs) responsible for cNF development. This approach enables real-time monitoring, isolation, and manipulation of tumor SCs at any time. We defined macroscopic (tumor count, total Tom+ fluorescent surface area, fluorescence intensity) and microscopic (cell-type composition defined by immunolabeling with a panel of specific markers, area quantification) endpoints, developed dedicated ImageJ scripts for automated image analysis, and compared the results with those obtained using the conventional manual method. Both automated measurements showed excellent reproducibility (ICC = 1) and strong correlation with manual analysis (Spearman's coefficient > 0.90), while significantly reducing analysis time (up to 100-fold faster). Bland-Altman analyses confirmed the absence of systematic bias compared with manual scoring. The standardized image naming and metadata integration further facilitated data consolidation and statistical analysis. This validated approach provides a reliable, reproducible, and time-efficient framework for evaluating drug effects on cNFs in preclinical studies. It establishes a foundation for robust efficacy testing of candidate therapies, facilitates cross-study comparability, and accelerates therapeutic development and clinical translation.