Fasting-mimicking diet enhances EGFR-TKI efficacy in oral cancer through dual mechanisms: direct cancer cell sensitization and tumorassociated macrophage crosstalk

Lei Wang¹Yujie Wang¹Rong Wang¹Fu-Lian Gong²Ji-Yuan Chen¹Ya-Ting Yu¹Ze-Rong Qiu¹Yongfang Yuan^1*

• ¹Department of Pharmacy, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
• ²Shanghai Children's Medical Center Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China

Background: Emerging evidence suggests the fasting-mimicking diet (FMD) offers a promising alternative to traditional calorie restriction and intermittent fasting, mitigating associated adverse effects including cachexia. Clinical trials have demonstrated the safety and efficacy of FMD, highlighting its considerable potential for translational applications. Future research should focus on assessing with molecularly targeted therapies to enhance therapeutic outcomes. The present study investigates the efficacy of FMD combined with EGFR-TKI therapy in oral cancer.Methods: M2-polarized macrophages derived from THP-1 cells were used to model TAMs. 2D and 3D oral cancer cell cultures (Cal-27 and OECM-1) were treated with gefitinib under standard or FMDconditioned media. TAMs recruitment and interaction with tumor spheroids were assessed via coculture and Transwell assays. Cal-27 xenograft mouse model was used to evaluate in vivo effects of FMD and gefitinib. Gene expression and signaling pathways were analyzed through bioinformatics, ELISA, RT-PCR, Western blot, and immunohistochemistry.Results: FMD enhanced the anti-proliferative effect of gefitinib in vitro in both 2D and 3D oral cancer models directly. Bioinformatics and 3D models identified CCL2 as a gefitinib-induced chemokine reversed by FMD, which suppressed CCL2-mediated TAMs recruitment and tumor spheroid growth. In vivo, combined FMD and gefitinib treatment significantly reduced tumor volume, Ki-67+ proliferating cells, and M2-like TAMs density, accompanied by decreased serum CCL2 levels. Mechanistically, FMD inhibited gefitinib-induced STAT3 phosphorylation, leading to reduced CCL2 expression. Pharmacological modulation of STAT3 confirmed its role in regulating CCL2 secretion.