Robust multiplex cytokine profiling in second-generation humanized NOG mice using a 41-plex humanized mouse immune panel: Cross-donor validation and implications for translational oncology studies

Background: Second-generation humanized NOG mice (HIS mice) enable evaluation of immune-oncology mechanisms within a physiologically relevant human immune microenvironment. However, translational interpretation of preclinical efficacy studies requires highly specific and reproducible quantification of human cytokines. To support robust immune monitoring, we co-evaluated a 41-plex humanized mouse cytokine/chemokine Luminex panel for performance across diverse human CD34+ donor sources and multiple HIS NOG model variants. Study Aims/Hypothesis: We hypothesized that the human-specific analytes in the MILLIPLEX® 41-plex humanized mouse multiplex assay would show (i) high specificity for human cytokines with minimal cross-reactivity to host murine analytes, (ii) reproducible quantification across donor-to-donor variation, and (iii) consistent detection sensitivity across advanced HIS NOG models supporting translational oncology applications. Results: Plasma samples were collected from HIS NOG cohorts reconstituted with ≥3 unrelated CD34+ donor pools and representing multiple second-generation humanized platforms. Across all donors and models, human-restricted analytes yielded quantifiable signals above background. Murine-only control samples confirmed negligible cross-reactivity. Inter-assay and intra-assay CVs demonstrated technical reproducibility. Donor-specific cytokine patterns (e.g., Th1/Th2 balance, myeloid-associated chemokines) were preserved across models, indicating biological fidelity rather than assay drift. Baseline cytokine architecture correlated with reconstitution kinetics and human immune cell subset frequencies, supporting biological coherence. Conclusions: These validation data demonstrate that the MILLIPLEX® 41-plex Luminex assay provides reproducible, donor-consistent, and human-specific cytokine quantification in second-generation HIS NOG mice. These data support the assay's suitability for mechanistic and pharmacodynamic readouts in oncology studies requiring high-resolution human cytokine/chemokine profiling. Reliable multiplex cytokine measurement strengthens the translational bridge between HIS mouse studies and human immuno-oncology responses, enabling improved interpretation of therapeutic mechanisms, biomarkers, and treatment-induced immune modulation.