Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear. Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry. Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an “in situ tumor vaccine” and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells. Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

Purpose: Radioresistance is an important factor for unsatisfactory prognosis in Nasopharyngeal carcinoma (NPC) patients. Ubiquitous mitochondrial creatine kinase (CKMT1) is always associated with malignancy in a variety of cancers. However, its significance in NPC progression and radiosensitivity remains unclear.The present study focused on investigating the effects of CKMT1 on NPC cell radiosensitivity. Materials and methods: CKMT1 was overexpressed in NPC cell line CNE-1 or knocked out in CNE-2. Biological changes were detected after cells exposing to different doses of X-ray to determine the role of CKMT1 on NPC cell radiosensitivity. Results: CKMT1 promotes proliferation and migration in NPC cell lines CNE-1 and CNE-2. Overexpression of CKMT1 in CNE-1 cells enhanced colony formation rates, reduced G2/M phase cell cycle arrest, lowered apoptosis rate and c-PARP level and elevated STAT3 phosphorylation level after radiation treatment. While knocking out CKMT1 using the CRISPR/Cas9 system in CNE-2 cells lowered colony formation rates, increased G2/M phase cell cycle arrest, apoptosis rates and c-PARP levels, and decreased STAT3 phosphorylation in response to radiation treatment. Conclusions: NPC cells with higher CKMT1 exhibited lower radiosensitivity through promoting phosphorylation of STAT3. Our findings suggest that CKMT1 may be an alternative radiotherapeutic target in NPC therapy.

Liver cancer is one of the most common malignant tumors with no available satisfactory treatment. The aim of the present study was to investigate the anti‑tumor effect of an irradiated hepatocellular carcinoma (HCC) whole‑cell vaccine and its underlying mechanisms. Hepa1‑6 and H22 HCC cell lines were irradiated in preparation for whole‑cell vaccine production. Subsequently, two HCC tumor‑bearing mouse models were created by injecting these Hepa1‑6 and H22 cells into the abdominal skin of C57BL/6 and ICR mice, respectively. The mice were immunized with the corresponding whole‑cell vaccine the next day, and then once a week until the end of the experimental period. Tumor growth, blood T helper (Th)9 cells and plasma interleukin (IL)‑9 levels were monitored during the immunization period. Th9 cells were also induced by in vitro co‑culture of the whole‑cell vaccine with lymphocytes from the spleen and lymph nodes of the corresponding mice. Alterations of gene expression in transcription factor (TF) were determined by reverse transcription‑quantitative PCR, and Th9 cells were detected using flow cytometry. The whole‑cell vaccine effectively suppressed HCC tumor growth, as indicated by slower tumor growth and a smaller tumor size in the immunized group compared with the control. The percentage of blood Th9 cells and the concentration of plasma IL‑9 were significantly increased in the immunized group. The whole‑cell vaccine also induced Th9 cell differentiation and upregulated the expression of TFs PU.1, interferon regulatory factor 4 and basic leucine zipper transcriptional factor ATF‑like. These results suggest that the irradiated HCC whole‑cell vaccine inhibited tumor growth by increasing Th9 cell numbers in HCC mice.

Conventional fractionated radiotherapy (CFRT) for hepatocellular carcinoma (HCC) is limited by intrinsic radioresistance. In this study, we investigated the radiosensitizing potential of maraviroc, a chemokine receptor 5 (CCR5) antagonist, and its mechanistic basis in HCC. A murine HCC model was established by subcutaneous implantation of H22 cells into the hind limbs of mice. Tumor-bearing mice received CFRT with or without maraviroc, and tumor growth kinetics were evaluated. Systemic levels of myeloid-derived suppressor cells (MDSCs) in peripheral blood and plasma chemokine ligand 5 (CCL5) were longitudinally monitored post-irradiation. In vitro mechanistic studies utilized maraviroc combined with conditioned media from 2 Gy-irradiated H22 cells to dissect its radiosensitizing effects. H22 cell viability, proliferation and migration were assessed following irradiation with or without maraviroc. Flow cytometry was employed to quantify polymorphonuclear MDSC (PMN-MDSC) proliferation, differentiation and immunosuppressive capacity via T-cell proliferation assays. Compared to monotherapy with either CFRT or maraviroc alone, maraviroc combined with CFRT significantly inhibited HCC growth in the mouse model. In vitro, maraviroc did not directly enhance irradiation-induced H22 cell death or suppress proliferation but reversed PMN-MDSC-mediated immunosuppression by attenuating PMN-MDSC migration and abrogating PMN-MDSC suppression of T-cell proliferation. Maraviroc combined with CFRT significantly inhibited the differentiation of bone marrow cells into PMN-MDSCs. In conclusion, the synergistic application of CCR5 antagonist with CFRT significantly enhanced radiosensitivity in HCC, primarily through suppression of PMN-MDSCs differentiation and migration, coupled with blockade of their T-cell proliferation inhibitory functions.

Background: Radiation-induced pulmonary fibrosis (RIPF) is a serious complication in patients treated with transthoracic irradiation. To date, there are no effective drugs for RIPF treatment. In this study, we attempted to explore the function of miR-761 in RIPF, further investigate its potential mechanism and evaluate its effectiveness in the treatment of RIPF. Methods: qRT-PCR analysis was used to detect miR-761 and peroxisome proliferator-activated receptor gamma (PPARg) coactivator-1 (PGC-1α) expression. Western Blot (WB) assay was applied to verify the regulation of PGC-1α by miR-761 and the expression of fibrosis-related proteins. Gel contraction assay was performed to demonstrate the level of fibroblast activation in vitro. A mouse RIPF model was used to validate the anti-fibrotic effect of Antagomir761. Bioinformatics analysis and dual-luciferase reporter assays were utilized to confirm the regulation relationship between miR-761 and PGC-1α. Results: The results showed that miR-761 was significantly elevated in irradiated mice lungs and fibroblasts. Overexpression of miR-761 in vitro promoted fibroblast activation. Whereas inhibition of miR-761 attenuated the degree of RIPF and inhibited fibroblast activation. Mechanistically, PGC-1α was a direct and functional target of miR-761, overexpression of PGC-1α inhibited irradiation-induced fibroblast activation, and knockdown of PGC-1α caused miR-761 inhibitor loses its anti-activation ability in irradiated cells. Conclusion: Our findings demonstrated that miR-761 regulated RIPF by targeting PGC-1α. Inhibition of miR-761 restored PGC-1α expression and attenuated RIPF damage, and miR-761 was a potential target for preventing the development of RIPF.

Objectives: The aim of this study is to determine whether thrombin time (TT) could be used as diagnostic biomarkers and predict the prognosis for sudden sensorineural hearing loss (SSNHL). Methods: Sixty-one patients diagnosed with SSNHL and 65 people who underwent physical examination were recruited. Data on the patient’s background, clinical course, and laboratory findings were collected. SSNHL patients were divided into the effective and ineffective groups according to the hearing recovery from the treatment and were assessed by binary logistic regression. Receiver-operating characteristic (ROC) analysis was carried out for the best discriminating cutoff value of the biomarker with the corresponding sensitivity and specificity was calculated. Results: The SSNHL group exhibited prolonged TT (19.11 ± 1.12 seconds) compared to the control group (17.58 ± 2.18 seconds, P < .001). Binary logistic regression analysis found a significant positive association between TT and SSNHL and was observed with an odds ratio (OR) 1.769 [95% confidence interval (CI) 1.344-2.330, P < .001] in the unadjusted model. Even after adjustment using the variables included in the multivariate models, TT was significantly predictive of SSNHL. A TT cutoff value of 17.65 seconds provides optimal separation between patients with SSNHL and controls in the ROC analysis [Area Under the Curve (AUC) 0.773, 95% CI 0.689-0.856; sensitivity, 0.918; and specificity, 0.569]. TT in the effective group of SSNHL patients was shorter (18.76 ± 1.06 seconds) than that in the ineffective group (19.43 ± 1.09 seconds, P = .018). The cutoff value of TT as progress predictors was 19.85 seconds. The TT < 19.85 seconds showed an effective rate 59.09% (26/44) higher than 17.65% (3/17) of TT ≥ 19.85 seconds. Conclusions: TT is a potential biomarker of SSNHL and is independently associated with the prognosis of patients with SSNHL.