OBJECTIVE To explore the synergistic anti-tumor effect and mechanism of oncolytic adenovirus(rAd.Nulli) combined with atezolizumab on thyroid cancer cells.

METHODS The CCK-8 assay and flow cytometry were used to detect the proliferation inhibition and apoptosis induction of the combined treatment on TPC-1 cells(thyroid cancer cells) and Nthy-ori 3-1 cells(normal thyroid cells). The key role of AMPK was verified by intervention with the AMPK inhibitor Compound C. Western blotting was used to detect the phosphorylation levels of AMPK, p-AMPK, and downstream mTOR and ULK1. Co-IP was used to verify the interaction between rAd.Nulli and AMPK. A nude mouse thyroid cancer xenograft model was established by subcutaneous injection of TPC-1 cells or TT cells, and six intervention groups were set up(control group, rAd.Nulli group, atezolizumab group, combination group, cisplatin group, and combination+Compound C group). The tumor volume and weight were evaluated, and the histopathological changes of the tumor tissue were observed by HE staining.

RESULTS The combination group significantly inhibited the proliferationsurvival rate (18.68±1.10)% and promoted apoptosis(30.73±2.34)% of TPC-1 cells compared with the single drug groups(P<0.01), and had no significant toxicity to Nthy-ori 3-1 cells survival rate(98.15±3.58)%. After AMPK inhibition, the anti-tumor effect of the combination treatment was significantly reversed(cell survival rate increased to 88.96%, apoptosis rate decreased to 3.92%, P<0.01). Western blotting showed that the expression of p-AMPK and p-ULK1 was upregulated, and the expression of p-mTOR was downregulated in the combination group, and Compound C could block this regulatory effect(P<0.01). Co-IP confirmed the specific binding of rAd.Nulli and AMPK. In the in vivo experiments of TPC-1 cells and TT cells, the tumor volume and weight in the combination group were significantly lower than those in the single drug groups(P<0.01), and the proportion of necrotic areas in the tumor tissue was significantly increased; while the tumor suppression effect of the combination+Compound C group was significantly weakened.

CONCLUSION rAd.Nulli directly binds to and activates the AMPK signaling pathway, downregulating mTOR and upregulating ULK1 phosphorylation, thereby synergizing with atezolizumab to inhibit proliferation and promote apoptosis in thyroid cancer cells. The combination therapy demonstrates good safety in normal thyroid cells, and AMPK serves as the core target of this strategy, providing a novel molecular mechanism and experimental basis for immune-combination therapy in thyroid cancer.