Cancer resistance to immune checkpoint inhibitors motivated investigations into leveraging the immunostimulatory properties of radiotherapy to overcome immune evasion and to improve treatment response. However, clinical benefits of radiotherapy-immunotherapy combinations have been modest. Routine concomitant tumor-draining lymph node irradiation (DLN IR) might be the culprit. As crucial sites for generating anti-tumor immunity, DLNs are indispensable for the in situ vaccination effect of radiotherapy. Simultaneously, DLN sparing is often not feasible due to metastatic spread. Using murine models of metastatic disease in female mice, here we demonstrate that delayed (adjuvant), but not neoadjuvant, DLN IR overcomes the detrimental effect of concomitant DLN IR on the efficacy of radio-immunotherapy. Moreover, we identify IR-induced disruption of the CCR7-CCL19/CCL21 homing axis as a key mechanism for the detrimental effect of DLN IR. Our study proposes delayed DLN IR as a strategy to maximize the efficacy of radio-immunotherapy across different tumor types and disease stages.

FLASH RT, which employs ultra-high dose rates (UHDR), has shown potential in reducing irradiation-induced damage to normal tissue while maintaining effective tumor targeting. For successful clinical translation, mechanistic explanation behind the so-called FLASH effect has yet to be deciphered and new in vivo model systems for mechanistic studies are therefore of high demand. We investigated the differential effects of UHDR (3000–7000 Gy/s) and conventional (CONV) (0.5 Gy/s) irradiation in D. melanogaster by irradiating adult female flies with 16 MeV and 9 MeV electron beams using an adapted clinical linear accelerator. Substantial lifespan prolongations were observed in single high-dose UHDR-irradiated groups compared to CONV irradiation groups with increasing doses (1000–1500 Gy). Split-dose UHDR irradiation further increased the lifespan compared to single high-dose UHDR irradiation. In addition, climbing deficits were induced by 300 Gy of CONV irradiation, but not by single high-dose UHDR irradiation. Additionally, we identified increased levels of lipid peroxidation in D. melanogaster brains indicating ferroptosis following CONV irradiation, which was not observed after single high-dose UHDR irradiation. Using relevant biological endpoints, we here demonstrate D. melanogaster with its advantageous characteristics to be a highly practical preclinical model organism to mechanistically investigate differential responses to UHDR and CONV irradiation.

Colorectal carcinoma is the third most common cancer globally and a major cause of cancer-related deaths. Many patients develop radiotherapy resistance, underscoring the urge for new therapies. The second messenger cGMP is crucial to maintain intestinal homeostasis, and its lack correlates with epithelial dysfunction and increased tumorigenesis. We investigated the role of TOP-V122, a dual-mode NO-releasing PDE-5 inhibitor, as an anti-tumoral drug alone and in combination with ionizing radiation. The therapeutic effect of TOP-V122 alone and combined with radiotherapy was assessed using patient-derived colorectal cancer organoids in vitro and in an orthotopic murine tumor model. Preformed CRC organoids treated with TOP-V122 and radiotherapy showed smaller organoid size, lighter cystic structures, appeared less compact and covered a smaller area in the wells, with increased spacing between individual structures. ATP-based viability and luciferase assays confirmed enhanced anti-tumoral efficacy of the combined treatment. TOP-V122 alone did not induce DNA double-strand breaks or ROS, and while it did not prevent radiotherapy-induced double-strand breaks formation in the combined treatment, it significantly abrogated γH2AX foci formation, suggesting interference with early DNA damage signaling. KAP-1 phosphorylation was unaffected, indicating specificity for γH2AX pathways. Combined treatment further reduced the proliferative status (Ki67) and upregulated p57^Kip2 expression, whereas p21 and Wnt/β-catenin signaling (CCDN1, MYC, β-catenin) remained unchanged. In the orthotopic CRC model, oral TOP-V122 alone reduced tumor growth, and the combined treatment with radiotherapy further enhanced anti-tumoral efficacy. IVIS imaging showed a 55 % early tumor shrinkage rate with the combination, compared with 33 % and 23 % for TOP-V122 and radiotherapy alone, respectively. Terminal tumor weights confirmed these findings, with a 40 % reduction for the combined treatment versus controls. All treatments were well tolerated with stable body weight. The PDE-5 inhibitor TOP-V122 represents a novel therapeutic option for colorectal cancer alone and in combination with radiotherapy.