Raman-guided analysis of drug response combined with chemometrics helps monitor the effect of ruxolitinib on acute lymphoblastic leukemia.

Ruxolitinib (RUX), a selective JAK1/JAK2 inhibitor, is considered a therapeutic option for childhood B-cell precursor acute lymphoblastic leukemia (B-ALL) with JAK2 gain-of-function mutations. This study aimed to evaluate whether Raman spectroscopy combined with chemometric analysis can monitor the biochemical effects of RUX treatment in B-ALL cell lines. We employed single-cell confocal Raman imaging, flow cytometry, and Western blotting to assess the response of JAK2-mutated (MUTZ-5 and MHH-CALL-4) and wild-type (SEM) B-ALL cells to 10 μM RUX treatment over 48 h. Dimensionality reduction methods (PCA, t-SNE) and classification approach (o-PLS-DA) were applied to the spectral data to identify treatment-induced changes. RUX selectively reduced STAT5 phosphorylation and induced distinct Raman spectral shifts in JAK2-mutant cells, particularly in DNA- and protein-related bands. No significant changes were observed in JAK2 wild-type cells. The results demonstrate that Raman spectroscopy, when integrated with multivariate analysis, enables the non-destructive tracking of leukemia cell responses to targeted therapy and may support the development of phenotyping tools for drug monitoring in precision oncology.