Tortoise Oligopeptides Augment Cyclophosphamide's Antitumor Activity Through Dual Modulation of Therapeutic Efficacy and Hematologic Toxicity

Cyclophosphamide (CTX) is a widely used chemotherapeutic agent, but its efficacy is often limited by leukopenia, a common adverse effect for which effective preventive strategies are currently lacking. In this study, oligopeptides were prepared from a blend of three edible tortoise species (Cuora trifasciata, Mauremys mutica, and Chinemys reevesii) and evaluated for their potential to alleviate CTX-induced leukopenia. The resulting Tortoise Oligopeptides (TOPs) were characterized primarily as small molecules with molecular weights under 5 kDa and peptide lengths between 4–15 amino acids, rich in glycine, glutamic acid, and proline. In a mouse model, TOPs administration significantly ameliorated CTX-induced leukopenia in a dose-dependent manner, attenuated pathological damage in the spleen and femur, and correlated with elevated serum levels of IL-4, IL-1β, TNF-α, and IFN-γ. In CTX-treated tumor-bearing mice, TOPs not only reduced leukopenia but also enhanced the antitumor efficacy of CTX. Correlation analyses linked leukocyte recovery to increased relative abundance of gut microbiota genera such as Colidextribacter, Tyzzerella, Prevotellaceae_UCG_001, and Rikenella. KEGG pathway analysis and fecal microbiota transplantation (FMT) experiments indicated that TOPs alleviate CTX-induced granulocytopenia partly through modulation of the gut microbiota. Additionally, LC-MS/MS sequencing combined with bioinformatic prediction and molecular docking identified several peptides—including PAIPAPPVGPGPK, FSFPTLPF, and PGLPFHP—with high binding affinity to key tumor targets (BCL-2, MDM2, EGFR), suggesting intrinsic antitumor properties. These findings indicate that TOPs may serve as a specialized medical food to mitigate CTX-induced leukopenia through multimodal mechanisms involving immunonutrition, gut microbiota regulation, and direct antitumor peptide effects.