Screening and identification of vascular endothelial cell targeting peptide in gastric cancer through novel integrated in vitro and in vivo strategy
Authors: Zhang Yan-ting , Wang Sheng-hui , Chen Bei 
Published: July 2024

Purpose Antiangiogenesis therapy has become a hot field in cancer research. Since tumor blood vessels carry specific markers that are usually related to angiogenesis, study of these heterogeneous molecules in different tumor vessels may be beneficial for promoting antiangiogenic therapy. Previously using phage display technology, we identified a targeted peptide named GX1 homing to gastric cancer vessels for the first time. However, GX1 also showed some non-specific binding with normal gastric vessels, which can lead to toxic side effects on normal endothelial cells. Therefore, we urgently need to adopt new screening strategies to avoid non-specific binding to normal vessels and obtain gastric cancer vascular targeting peptides with higher specificity.Methods In this study, we designed a new strategy which combined “negative screening” in vitro and “positive screening” in vivo for the first time. An in vivo positively screening was conducted using tumor bearing nude mice to identify peptides that were specifically enriched within the vasculature of gastric cancer. Concurrently, an in vitro negative screening process was conducted on normal vasculature endothelial cells, including human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs), to eliminate peptides binding to normal vasculature. After four rounds of iterative screening, a target peptide specifically targeting gastric cancer vasculature was obtained. In addition, we established an in vitro co-culture model of tumor vascular endothelial cells and studied the affinity of these peptides. We labeled the targeting peptides with fluorescein isothiocyanate (FITC) for the competitive and inhibitory assay.Results Blood vessel density analysis confirmed redundant capillary vessels in the xenografts, indicating that the mouse model was suitable for positive screening. After four rounds of panning, there was an obvious enrichment for the phages specifically binding to vasculature of gastric cancer while minimally binding to normal vascular endothelial cells. The peptide CNTGSPYEC exhibited the highest reproducibility. In vitro immunofluorescence staining confirmed that the peptide CNTGSPYEC could specifically enrich in Co-HUVECs while showing no binding to normal vascular endothelial cells. In vivo immunofluorescence staining revealed that the peptide CNTGSPYEC could only bind to vascular endothelial cells specifically in gastric cancer but show no non-specific binding with normal tissue. Competitive and inhibitory assay also verified the targeting characteristics of the peptide with the fluorescence intensity of 17.13. As the concentration increases, the competitive inhibition rate can be incrementally raised to 93% (p < 0.05). Endothelial tube formation assay indicated that the peptide could suppress neovascularization, with the microvessel count reducing by 40% (p < 0.05). Furthermore, Cell Counting Kit-8 assay (CCK8) showed that the targeted peptide could partly inhibit cell proliferation of Co-HUVEC (44%).Conclusion Our novel strategy of the combined in vitro and in vivo screening outperforms previous methods that relied solely on negative/positive screening. In vivo and in vitro test confirmed the high targeting characteristic of the new peptide. Therefore, the peptide CNTGSPYEC may be a potential candidate in diagnosis and anti-angiogenesis therapy of gastric cancer. Our further exploration employs it as a vehicle for mediating drug accumulation in gastric cancer tissue.