Irradiated Cell-derived Exosomes Enhance Cell Proliferation and Radioresistance via the MAPK/Erk Pathway.

BACKGROUND/AIM: Radiation therapy is pivotal in cancer treatment; however, its efficacy is limited by challenges such as tumor recurrence. This study delves into the role of exosomes, which are molecular cargo-bearing vesicles, in influencing cell proliferation, radioresistance, and consequent post-irradiation tumor recurrence. Given the significance of exosomes from irradiated malignancies in diagnostics and therapy, it is vital to delineate their functional dynamics, especially in breast and cervical cancer cell lines, where the impact of irradiation on exosome behavior remains enigmatic. MATERIALS AND METHODS: Using MDA-MB-231 and HeLa cell lines, exosomes were isolated from the culture supernatant via ultracentrifugation. The bicinchoninic acid assay was used to measure exosome quantities in irradiated and non-irradiated cells. Radiosensitivity was assessed using colony formation assays, while the role of the MAPK/Erk signaling pathway in recipient cell proliferation and radioresistance was probed using western blotting. RESULTS: Irradiated cells, in both MDA-MB-231 and HeLa lines, produced significantly more exosomes than their non-irradiated counterparts. Co-culturing irradiated cells with exosomes led to increased cell survival post-irradiation and enhanced cell proliferation in both cell lines. Western blotting indicated elevated p-Erk expression in such cells, underscoring the influence of the MAPK/Erk pathway in radioresistance and proliferation. CONCLUSION: The study establishes a potential nexus between exosome secretion and tumor resurgence following radiotherapy. The spotlight falls on the MAPK/ERK signaling conduit as a key influencer. This new knowledge provides an innovative strategy for counteracting cancer recurrence after radiotherapy, emphasizing the importance of understanding the multifaceted roles of exosomes in this context.

Biological effects of prostaglandin E2-EP4 antagonist (AAT-008) in murine colon cancer in vivo: enhancement of immune response to radiotherapy and potential as a radiosensitizer.

Background: Prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four receptors (EP1-4). We investigated the radiosensitizing effects of a newly developed antagonist of PGE2-EP4 (AAT-008) in mouse colon cancer cells in vivo and explored the mechanism using flow cytometry (FCM). Methods: CT26WT cells grown in Balb/c mice were used. AAT-008 at doses of 0, 3, 10, and 30 mg/kg/day was orally administered once or twice daily for up to 19 days. On day 3, the tumors were irradiated at 9 Gy in the radiotherapy (RT) group. Tumor sizes were measured every other day. For the first FCM series, AAT-008 (10 mg/kg/day) was administered from day 0 to 18 and RT (9 Gy) was given on day 3. The population of effector T cells (Teff), defined as CD45+CD8+CD69+, in the tumors was investigated on day 19. For the second FCM series, AAT-008 (30 mg/kg/day) was administered from day 0 to 12. The populations of Teff and regulatory T cells (Treg), and the ratio of Teff/Treg were investigated on day 13. Results: The growth delay effect of AAT-008 administered alone (3-30 mg/kg/day) appeared minimal. In the first growth delay experiment where AAT-008 was administered once daily, the combined effect of AAT-008 (30 mg/kg/day) and RT appeared additive. In the second growth delay experiment where AAT-008 was administered twice daily, the combined effect appeared additive at 3 and 10 mg/kg/day and supra-additive at 30 mg/kg/day. In the first FCM series, the mean Teff proportions in the tumors were 43% and 31% in the 10 mg + RT and 0 mg + RT groups, respectively. Notably, 67% Teff was observed in responsive mice in the 10 mg + RT group. In the second FCM series, the mean Treg proportion and Teff/Treg ratio in the 0 mg + RT and 30 mg + RT groups were 4.0% and 1.5%, respectively (P=0.04) and 10 and 22, respectively (P=0.04). Conclusions: AAT-008 potentially enhances the radiosensitivity of colon cancer cells, apparently by stimulating the immune system against the cancer cells.

Radiation therapy enhances systemic antitumor efficacy in PD-L1 therapy regardless of sequence of radiation in murine osteosarcoma.

Recent studies demonstrate that immune checkpoint blockade (ICB) increases the chances of the abscopal effect, an anti-tumor effect outside the radiation field in radiation therapy. However, the optimal sequence between radiation and ICB remains unclear. To investigate the impact of sequence of radiation in anti-PD-L1 antibody (P1) therapy on immune microenvironments and antitumor efficacies in local and abscopal tumors, metastatic LM8 osteosarcoma cells were inoculated into both legs of C3H mice. For irradiation, only one side leg was irradiated at 10 Gy. Then mice were divided into four groups: administrated anti-PD-L1 antibody three times (P1 monotherapy), receiving radiation 3 days prior to P1 therapy (P1+pre-Rad), and receiving concurrent radiation with P1 therapy (P1+conc-Rad). Thereafter, tumor immune microenvironment and tumor volume changes were analyzed in irradiated and unirradiated tumors. The P1+pre-Rad regimen increased the proportion of CD8+ programmed cell death 1 (PD-1)+ granzyme B (GzmB)+ reinvigorated T cells and decreased the proportion of CD8+ PD-1+ GzmB- exhausted T cells than P1+conc-Rad regimen in unirradiated tumors. Combination regimens suppressed tumor growth in irradiated tumors compared with that in P1 monotherapy. In both irradiated and unirradiated tumors, significant tumor growth suppression and prolonged overall survival were observed under both combination treatment regimens compared with P1 monotherapy. However, no distinct differences in unirradiated tumor volume and survival were observed between P1+pre-Rad and P1+conc-Rad groups. These results suggest that local irradiation is necessary to improve systemic treatment efficacy in P1 therapy regardless of sequence of local irradiation.

High Dose Local Photon Irradiation Is Crucial in Anti-CTLA-4 Antibody Therapy to Enhance the Abscopal Response in a Murine Pancreatic Carcinoma Model.

Pancreatic cancer is an extremely treatment-resistant neoplasm to chemotherapy and immunotherapy. The combination of photon beam irradiation and anti-CTLA-4 antibody (C4) for the anti-tumor effect enhancement at local and distant tumors (abscopal tumors) was investigated using the pancreatic ductal adenocarcinoma (PDAC) mouse model. Pan02 cells were bilaterally inoculated to both legs of C57BL/6 mice. High dose photon beams in a hypofractionation or a single fraction were delivered to the tumors on one leg. Monotherapy with C4 via i.p. was not effective for PDAC. The high dose irradiation to the local tumors produced significant shrinkage of irradiated tumors but did not induce the abscopal responses. In contrast, the combination therapy of high dose photon beam irradiation in both hypofractionation and a single fraction with C4 enhanced the anti-tumor effect for abscopal tumors with significantly prolonged overall survival. The flow cytometric analysis revealed that the combination therapy dramatically decreased the regulatory T cell (Treg) proportion while increasing the cytotoxic T lymphocytes in both local and abscopal tumors. These results suggest that high dose photon beam irradiation plays an important role in C4 therapy to enhance the abscopal response with immune microenvironment changes in PDAC, regardless of the fractionation in radiation therapy.

Radiation Dose Escalation is Crucial in Anti-CTLA-4 Antibody Therapy to Enhance Local and Distant Antitumor Effect in Murine Osteosarcoma.

We previously reported that a combination of 10 Gy of X-ray irradiation and dual immune checkpoint blockade with anti-CTLA-4 (C4) and anti-PD-L1 antibodies produced a significant shrinkage of irradiated and unirradiated tumors (abscopal effect) and prolonged overall survival. However, the optimal radiation delivery regimen combined with single immune checkpoint blockade of C4 for inducing a maximum systemic antitumor response still remains unclear, particularly for patients with osteosarcoma. We used syngeneic C3H mice that were subcutaneously injected with LM8 osteosarcoma cells into both legs. C4 was administered three times, and one side of the tumor was irradiated by X-ray beams. The optimal radiation dose required to induce the abscopal effect was explored with a focus on the induction of the type-I interferon pathway. Radiation delivered in a single fraction of 10 Gy, 4.5 Gy × 3 fractions (fx), and 2 Gy × 8 fx with C4 failed to produce significant inhibition of unirradiated tumor growth compared with monotherapy with C4. Dose escalation to 16 Gy in a single fraction, or the equivalent hypofractionated dose of 8 Gy × 3 fx, which significantly increased secretion of IFN-ß in vitro, produced a dramatic regression of both irradiated and unirradiated tumors and prolonged overall survival in combination with C4. Furthermore, irradiation at 16 Gy in both a single fraction and 8 Gy × 3 fx diminished regulatory T cells in the unirradiated tumor microenvironment. These results suggest that total dose escalation of radiation is crucial in C4 therapy to enhance the antitumor response in both local and distant tumors and prolonged overall survival regardless of fractionation for osteosarcoma.