Introduction: Heterogeneity in patient response to cancer treatment can be influenced by many factors, including the time-of-day treatments are administered. Emerging research suggests chronotherapy, the alignment of treatment with molecular circadian rhythm oscillations, can enhance response to immunotherapy (Fey, RM. Cancers. 2025. 21;17 (5):732). Chronotherapy in murine melanoma models treated with immune checkpoint inhibitor (ICI) significantly impacts tumor control when given during peaks in CD8 T cell activity (Wang, Chen. Cell. 2024. 187;11), highlighting the importance of understanding circadian effects on immune responses. 

The 24-hour circadian cycle is regulated by the suprachiasmatic nucleus (SCN) and is entrained via light stimuli. However, synchronized molecular clock genes throughout the body influence biological systems, including our immune system. Core clock genes like Per2 and Bmal1 are regulated by vasoactive intestinal peptide (VIP). Additionally, VIP is known to be immunosuppressive and is overexpressed in various cancers. Targeting this pathway with a VIP-receptor antagonist shows significant tumor control in murine acute myeloid leukemia and mastocytoma models (Peterson, CT. Oncoimmunology. 2017. 16;6 (5):e1304336). Given VIP's dual-role in circadian regulation and immunosuppression, we hypothesized that the anti-cancer immune responses induced by ANT308 would vary according to the diurnal cycle.

Methods: DBA2/J mice were inoculated with 1 x 105 murine P815 mastocytoma cell line and treated by subcutaneous injection with 20 ug ANT308 daily for 10 days based on the Zeitgeber time (ZT) system. The mice are entrained in a 12-hour light-dark cycle, with ZT0 marking the light cycle at 7AM and ZT12 the dark cycle at 7PM, when mice are most active. Mice were treated with ANT308 at either timepoint and blood was collected throughout a 24-hour clock period in tumor-bearing and non-tumor-bearing mice. Corticosterone and VIP levels were measured in plasma by ELISA and peripheral blood samples were analyzed by flow cytometry. Mice were monitored for tumor volume and survival. 

Results: Plasma corticosterone levels reflect a strong circadian oscillation with concentrations of 3,528 pg/ml at 7PM and 227 pg/ml at 7AM. VIP plasma levels did not demonstrate a significant change throughout a 24-hour period, likely due to the short half-life of VIP in blood. However, flow cytometry analysis of blood from tumor-bearing mice showed peak frequencies of VIP+ CD8 and CD4 T cells at 3AM with a trough at 7PM. The frequency of Ki67+ CD8 T cells were lowest at 7AM and gradually increased to peak at 3AM. In the P815 tumor model, mice receiving ANT308 treatment at 7PM (ZT12) had delayed tumor growth compared to the 7AM (ZT0) treatment group. The 7PM group had significantly improved tumor control and survival (p=0.029) compared to the control, whereas the 7AM group did not. Interestingly, mice treated with ANT308 at 7AM, before rest phase, displayed hyperactive behavior compared to the other groups.  

These data indicate the role of VIP-receptor antagonist chronotherapy to treat hematologic malignancies and suggest the importance of aligning cancer patient therapy regimens with innate molecular clocks. Future studies will explore the effects of ANT308 chronotherapy in alternate tumor models and will further characterize immune cell phenotypes when treated with ANT308 at various times of day. This study highlights environmental and innate biological impacts on heterogeneity in patient response to therapy and introduces alternate avenues for optimizing care in cancer patients. 

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2025
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