Autophagy-Targeting Stapled Peptide Utilizes Macropinocytosis for Cell Entry to Potentiate Anti-Proliferative Autosis in Small-Cell Lung Cancer

<b>Background</b>: Small-cell lung cancer (SCLC) is an aggressive malignancy marked by rapid progression, early metastasis, and frequent relapse despite chemotherapy. Due to its genetic complexity, targeted therapies have limited success. Autophagy, a lysosome-dependent cellular degradation process, plays a key role in SCLC, yet effective autophagy-targeting strategies are lacking. This study evaluates Tat-SP4, an autophagy-targeting stapled peptide, for its anti-proliferative effects in SCLC. <b>Method</b>: We assessed Tat-SP4's impact on autophagy in SCLC cells by measuring p62 and LC3 levels. Mitochondrial function was evaluated via mitochondrial membrane potential (Δψm) and oxygen consumption rate (OCR). Anti-proliferative effects were determined using cell viability assays in vitro and xenograft models in vivo. Cellular uptake mechanisms were investigated using Ca²⁺ imaging and pharmacological inhibitors. <b>Result</b>: Tat-SP4 induced a strong autophagic response and triggered autosis, a form of autophagy-dependent necrotic cell death, impairing SCLC cell proliferation. It also caused mitochondrial dysfunction with impaired oxidative phosphorylation (OXPHOS). Tat-SP4 entered cells predominantly via macropinocytosis, triggering extracellular Ca²⁺ influx measurable by live-cell imaging. Digoxin, an Na⁺, K⁺-ATPase inhibitor, partially reversed the effect of Tat-SP4 on Ca²⁺ influx, cell death, and OXPHOS activity. Lastly, Tat-SP4 inhibited tumor growth in a xenograft-based animal model for SCLC. <b>Conclusions</b>: The autophagy-targeting stapled peptide Tat-SP4 inhibited the proliferation of SCLC cells in vitro and inhibited the growth of the SCLC tumor in vivo. Macropinocytosis facilitates cell entry for Tat-SP4, which can be monitored by influx of extracellular Ca²⁺. By exploiting macropinocytosis for cell entry and converting the pro-survival autophagy process into a death pathway, Tat-SP4 represents a novel therapeutic strategy against SCLC.