Identification of the immunotherapy susceptibility of solid cancer using noninvasive in vivo bioluminescence optical imaging of NF-κB activation for precision medicine

Introduction : Immunotherapy has substantially improved the overall survival of cancer patients. Unfortunately, many patients do not benefit from cancer immunotherapy because of primary or acquired therapy resistance. We have established a highly efficient tumor antigen-specific (TA) Th1 cell- and immune checkpoint inhibitor (ICI)-based combinatory immunotherapy (COMBO). Here, we aimed to identify the sites of immune cell activation under COMBO therapy by performing longitudinal, noninvasive in vivo optical imaging (OI) of NF-κB activation in the tumor stroma, endogenous infiltrating immune cells within the tumor microenvironment (TME), and bone marrow (BM) and uncovered an enhanced NF-κB activation exclusively in responsive tumors. Methods : We used ^NF−κB Luc-reporter mice, which express luciferase (luc) upon NF-κB activation, thereby allowing us to study NF-κB activation patterns in the TME and BM. We subcutaneously ( s.c. ) inoculated these mice with ovalbumin-expressing (OVA)-MC38 adenocarcinoma (ICI responder) or OVA-B16 melanoma cells (ICI nonresponder). Four days after tumor cell inoculation, the mice underwent baseline in vivo bioluminescence optical imaging (BLI) followed by COMBO treatment composed of 2 Gy whole-body radiation, OVA-specific IFN-γ-producing CD4 ⁺ T cells (OVA-Th1), programmed cell death ligand-1 (αPD-L1) and lymphocyte-activation gene 3 (αLAG-3)-specific monoclonal antibodies (mAbs). Sham-treated tumor-bearing mice received PBS and isotype mAbs instead of OVA-Th1 cells or ICI. Tumor growth or regression was monitored by determining the tumor volume with a caliper. BLI of the tumors and the BM of the hind limbs was performed daily until the end of the experiment. For ex vivo cross correlation, we performed histopathology (H&E), immunohistochemistry (IHC; CD3 ⁺ T cells and B220 ⁺ B cells) and immunofluorescence microscopy (IFM; p50, luciferase) of the tumors. Additionally, we performed multicolor flow cytometry analysis of tumors and lymphatic organs, with a focus on immune cell composition and activation state. Results : COMBO treatment of ^NF−κB Luc-reporter mice efficiently suppressed the growth of OVA-MC38 adenocarcinomas but not of OVA-B16 melanomas. We detected significantly enhanced NF-κB activity in the TME of responsive OVA-MC38 tumors in ^NF−κB Luc-reporter mice upon COMBO treatment compared to that observed with sham treatment. In sharp contrast, we could not determine whether COMBO treatment induced NF-κB activation in the resistant OVA-B16 melanoma TME of ^NF−κB Luc-reporter mice compared with that in the sham-treated group. Next, we measured NF-kB activity in the BM of the hind limbs, which showed a significantly reduced signal intensity (SI) in COMBO-treated OVA-MC38 and OVA-B16 tumor-bearing ^NF−κB Luc-reporter mice compared with the BM of their sham-treated tumor-bearing littermates. Furthermore, IFM analysis of OVA-MC38 tumors from COMBO-treated mice revealed the treatment-induced activation of NF-κB in MPO⁺Luc⁺ neutrophils. Consistently, flow cytometry analysis of BM cells from the OVA-MC38 tumor model demonstrated a reduced number of CD11b⁺Gr-1 ^high neutrophils upon COMBO treatment, suggesting that systemic immune activation promoted their egress from the BM. Conclusion : The treatment efficacy of COMBO-sensitive adenocarcinomas was strongly associated with the therapy-induced activation of NF-κB in the TME. In contrast, in therapy-resistant melanomas, COMBO treatment did not alter NF-κB activity in the TME. Furthermore, COMBO treatment was associated with a reduction in NF-κB activity in the BM of tumor-bearing mice, most likely indicating therapy-associated egress of myeloid cells. In summary, our study revealed that longitudinal monitoring of NF-κB activation in the tumor stroma and infiltrating endogenous immune cells could be a powerful tool for early identification of therapy sensitivity and therapeutic resistance in the preclinical setting.