A recombinant adeno-associated virus expressing secretory TGF‑β type II receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice

doi:10.12122/j.issn.1673-4254.2024.05.03

Abstract

Abstract:

Objective To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-β (TGF-β) type II receptor (sTβRII) extracellular domain-IgG2a Fc fusion protein (sTβRII-Fc) on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice. Methods The pAAV-sTβRII-Fc vector expressing sTβRII-Fc fusion protein constructed by molecular cloning, the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTβRII virus, which was purified by density gradient centrifugation with iodixanol. Western blotting was used to examine the effects of AAV-sTβRII virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin, vimentin and p-Smad2/3 in 4T1 cell xenografts in mice. BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTβRII virus, AAV-GFP virus or PBS (n=6) through the tail vein, and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging. Ki67 expression in the tumor tissues and sTβRII protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining, and tumor metastases in the vital organs were examined with HE staining. Results The recombinant pAAV-sTβRII-Fc vector successfully expressed sTβRII in HEK 293T cells. Infection with AAV2-sTβRII virus significantly reduced TGF-β1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice (P<0.05). In the tumor-bearing mice, intravenous injection of AAV-sTβRII virus significantly increased E-cadherin expression, reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues (P<0.05 or 0.01), and induced liver-specific sTβRII expression without causing body weight loss or heart, liver, spleen or kidney pathologies. Conclusion The recombinant AVV2 vector encoding sTβRII extracellular domain is capable of blocking the TGF-β signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.

Key words: triple negative breast cancer, TGF?βII, migration, transforming growth factor?β signaling pathway, adeno-associated virus

Zhi CUI, Cuijiao MA, Qianru WANG, Jinhao CHEN, Ziyang YAN, Jianlin YANG, Yafeng LÜ, Chunyu CAO. A recombinant adeno-associated virus expressing secretory TGF‑β type II receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice[J]. Journal of Southern Medical University, 2024, 44(5): 818-826.

Figures/Tables 5

Fig.1 Preparation and functional identification of AAV2-sTβRII.

Fig.2 AAV2-sTβRII inhibits proliferation of transplanted 4T1 xenograft tumors in mice. A: Gene therapy model in mice. B: Representative images of bioluminescence in mice on days 5 and 21 after Intravenous Injections. C: Statistical diagram of tumors fluorescence signal value. D: Images of tumors from the experiment group (AAV2-sTβRII) and the control groups (PBS and AAV2-GFP). E: Growth curve of orthotopic transplantation tumors. F: Tumor weight of each group. G: Representative images of the immunohistochemical staining for Ki67 proteins in tumor sections. *P<0.05, **P<0.01.

Fig.3 AAV2-sTβRII inhibits Smad2/3 phosphorylation in transplanted 4T1 xenograft tumors in mice. A: Expression of p-Smad2/3 in tumor tissues. B: Immunofluorescence staining for sTβRII-Fc proteins in mice liver. *P<0.05, ***P<0.001.

Fig.4 Effects of AAV2-sTβRII virus on lung metastasis of 4T1 cell xenograft in mice. A: Representative images of the lungs from the mice in different groups (black arrows indicate visible metastatic nodules). B: HE staining of the lung sections. C: E-cadherin and vimentin protein expressions in 4T1 cell xenograft. *P<0.05, **P<0.01.

Fig.5 Toxic effects of intravenously injected AAV2-sTβR II virus in mice. A: Body weight changes of the mice. B: HE staining of the heart, liver, spleen and kidney of the mice.

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