Combining vincristine with interferon-gamma suppresses neuroblastoma tumor growth in an orthotopic mouse model

Background: Applying immunotherapy to neuroblastoma has been challenging, especially within an immune‐suppressive tumor microenvironment. Tumor-associated macrophages (TAM) within neuroblastoma can be cancer-inhibiting (M1) or cancer-promoting (M2). Optimal integration of immunotherapy with chemotherapy is needed to harness both the immune response and cytotoxic effect. We hypothesize that 1) IFN-gamma induces M1 TAM in neuroblastoma 2) combining vincristine with IFN-gamma suppresses tumor growth while vincristine administration after IFN-gamma diminishes the suppression.

Methods: IFN-gamma (2µg) and vincristine (50µg) were loaded into separate silk films. Orthotopic, syngeneic neuroblastoma xenografts were generated by injecting 9464D cells into left adrenal gland of immunocompetent C57BL/6 mice. Drug-loaded silk film implantation was initiated once tumor size was greater than 100mm3. Treatments included control silk film, vincristine, IFN-gamma, vincristine followed by IFN-gamma five days later, IFN-gamma followed by vincristine five days later, or combination vincristine and IFN-gamma. Tumor volume was measured with ultrasound. Tumors were homogenized for quantitative-PCR (q-PCR). Formalin preserved paraffin-embedded tumor sections were stained with hematoxylin and eosin (H&E).

Results: Time to reach tumor size 800 mm3 was significantly longer with the combination of vincristine and IFN-gamma compared to control (mean±SD days: 12.2±2.7 vs. 5.7±1.2, p=0.012). IFN-gamma alone also significantly delayed tumor growth compared to control (10.9±1.5 vs. 5.7±1.2, p=0.001). Delayed administration of vincristine after IFN-gamma advanced tumor growth compared to combination treatment (7.5±1.1 vs. 12.2±2.7, p=0.017). Q-PCR demonstrated increased expression of M1 markers with IFN-gamma. H&E staining of tumor sections demonstrated tumor necrosis adjacent to the IFN-gamma silk film.

Conclusions: Local delivery of sustained-release IFN-gamma increases M1 TAM and suppresses neuroblastoma tumor growth. Concurrent therapy of vincristine and IFN-gamma significantly delays tumor growth but not IFN-gamma followed by vincristine, suggesting an interplay between immune response and chemotherapy-induced cytotoxicity.