Background: Anti-angiogenic therapy has been widely applied to the clinical treatment of malignant tumors. However, the efficacy of such treatments has been called into question, especially in triple-negative breast cancer (TNBC). Bevacizumab, the first anti-angiogenic agent approved by FDA, actually increases invasive and metastatic properties of TNBC cells, resulting from the activation of Wnt/β-catenin signaling in response to hypoxia. As a critical receptor of Wnt/β-catenin signaling, Frizzled-7 (Fzd7) is aberrantly expressed in TNBC, indicating Fzd7 a potential target for developing drugs to be combined with anti-angiogenic agents. Methods: Hybridoma technique and antibody humanization technique were utilized to generate a Fzd7-targeting antibody (SHH002-hu1). Biolayer interferometry (BLI) assay and near infrared (NIR) imaging were conducted to detect the affinity and targeting ability of SHH002-hu1. Next, whether SHH002-hu1 could suppress the invasion and migration of TNBC cells induced by Bevacizumab were validated, and the underlying molecular mechanisms were elucidated by luciferase reporter and western blot assays. The nude-mice transplanted TNBC models were established to assess the anti-TNBC activities of SHH002-hu1 when combined with Bevacizumab. Then, the effects on putative TNBC stem-like cells and Wnt/β-catenin signaling were evaluated by immunofluorescence (IF). Further, the tumor-initiating and self-renew capacity of TNBC cells were studied by secondary nude mouse xenograft model and sphere formation assay. In addition, the effects of SHH002-hu1 on the adaptation of TNBC cells to hypoxia were evaluated by the detection of vasculogenic mimicry (VM) and hypoxia-inducible factor-1α (HIF-1α) transcriptional activity. Results: The novel humanized antibody targeting Fzd7 (SHH002-hu1) exhibited extremely high affinity with Fzd7, and specifically targeted to Fzd7⁺ cells and tumor tissues. SHH002-hu1 repressed invasion, migration and epithelial-mesenchymal cell transformation (EMT) of TNBC cells induced by Bevacizumab through abating Wnt/β-catenin signaling. SHH002-hu1 significantly enhanced the capacity of Bevacizumab to inhibit the growth of TNBC via reducing the subpopulation of putative TNBC stem-like cells, further attenuating Bevacizumab-enhanced tumor-initiating and self-renew capacity of TNBC cells. Moreover, SHH002-hu1 effectively restrained the adaptation of TNBC cells to hypoxia via disrupting Wnt/β-catenin signaling. Conclusion: SHH002-hu1 significantly enhances the anti-TNBC capacity of Bevacizumab, and shows the potential of preventing TNBC recurrence, suggesting SHH002-hu1 a good candidate for the synergistic therapy together with Bevacizumab.

Non–small‐cell lung cancer (NSCLC) is one of the deadliest cancers worldwide, and metastasis is considered one of the leading causes of treatment failure in NSCLC. Wnt/β‐catenin signaling is crucially involved in epithelial–mesenchymal transition (EMT), a crucial factor in promoting metastasis, and also contributes to resistance developed by NSCLC to targeted agents. Frizzled‐7 (Fzd7), a critical receptor of Wnt/β‐catenin signaling, is aberrantly expressed in NSCLC and has been confirmed to be positively correlated with poor clinical outcomes. SHH002‐hu1, a humanized antibody targeting Fzd7, was previously successfully generated by our group. Here, we studied the anti‐tumor effects of SHH002‐hu1 against NSCLC and revealed the underlying mechanism. First, immunofluorescence (IF) and near‐infrared (NIR) imaging assays showed that SHH002‐hu1 specifically binds Fzd7⁺ NSCLC cells and targets NSCLC tissues. Wound healing and transwell invasion assays indicated that SHH002‐hu1 significantly inhibits the migration and invasion of NSCLC cells. Subsequently, TOP‐FLASH/FOP‐FLASH luciferase reporter, IF, and western blot assays validated that SHH002‐hu1 effectively suppresses the activation of Wnt/β‐catenin signaling, and further attenuates the EMT of NSCLC cells. Finally, the subcutaneous xenotransplanted tumor model of A549/H1975, as well as the popliteal lymph node (LN) metastasis model, was established, and SHH002‐hu1 was demonstrated to inhibit the growth of NSCLC xenografts and suppress LN metastasis of NSCLC. Above all, SHH002‐hu1 with selectivity toward Fzd7⁺ NSCLC and the potential of inhibiting invasion and metastasis of NSCLC via disrupting Wnt/β‐catenin signaling, is indicated as a good candidate for the targeted therapy of NSCLC.