Noninvasive in vivo imaging of NF-κB activation predicts immunotherapy response in solid tumors

Cancer immunotherapy improves survival, yet many patients exhibit primary or acquired resistance. We established a combinatorial immunotherapy (COMBO) consisting of tumor antigen-specific Th1 cells and dual immune checkpoint blockade and aimed to noninvasively identify sites of immune activation associated with therapeutic response. Using ^NF-κBLuc-reporter mice, we longitudinally monitored NF-κB activation by using in vivo bioluminescence imaging in the tumor microenvironment (TME) and bone marrow (BM) of mice bearing OVA-expressing MC38 adenocarcinoma (responder) or B16 melanoma (non-responder). COMBO treatment induced tumor regression in OVA-MC38 but not OVA-B16 tumors. Responsive tumors showed significantly increased NF-κB activation in the TME, whereas resistant melanomas displayed no therapy-induced NF-κB activation. In contrast, BM NF-κB activity was reduced upon COMBO treatment in both models. Immunofluorescence and flow cytometry analyses revealed NF-κB activation in tumor-infiltrating MPO⁺ neutrophils and a concomitant reduction of CD11b⁺Gr-1^high neutrophils in the BM in OVA-MC38 bearing mice, suggesting therapy-driven myeloid cell egress. Thus, therapeutic efficacy strongly correlated with NF-κB activation within the TME, while systemic BM changes reflected immune mobilization. Longitudinal imaging of NF-κB activity may enable early discrimination between therapy-sensitive and -resistant tumors in preclinical models.