Bacteria-mediated tumor-targeted delivery of tumstatin (54-132) significantly suppresses tumor growth in mouse model by inhibiting angiogenesis and promoting apoptosis

doi:10.1007/s11684-022-0925-2

Front. Med.

2022, Vol. 16

Issue (6) : 873-882 https://doi.org/10.1007/s11684-022-0925-2

RESEARCH ARTICLE

Bacteria-mediated tumor-targeted delivery of tumstatin (54-132) significantly suppresses tumor growth in mouse model by inhibiting angiogenesis and promoting apoptosis

Feifei Bao¹, Mengjie Liu¹, Wenhua Gai¹, Yuwei Hua¹, Jing Li¹, Chao Han¹, Ziyu Zai¹, Jiahuang Li², Zichun Hua^1,²(

)

¹. The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, China
². Changzhou High-Tech Research Institute of Nanjing University and Jiangsu Target Pharma Laboratories Inc., Changzhou 213164, China

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Abstract

Tumor growth is an angiogenesis-dependent process and accompanied by the formation of hypoxic areas. Tumstatin is a tumor-specific angiogenesis inhibitor that suppresses the proliferation and induces the apoptosis of tumorous vascular endothelial cells. VNP20009, an attenuated Salmonella typhimurium strain, preferentially accumulates in the hypoxic areas of solid tumors. In this study, a novel Salmonella-mediated targeted expression system of tumstatin (VNP-Tum5) was developed under the control of the hypoxia-induced J23100 promoter to obtain anti-tumor efficacy in mice. Treatment with VNP-Tum5 effectively suppressed tumor growth and prolonged survival in the mouse model of B16F10 melanoma. VNP-Tum5 exhibited a higher efficacy in inhibiting the proliferation and inducing the necrosis and apoptosis of B16F10 cells in vitro and in vivo compared with VNP (control). VNP-Tum5 significantly inhibited the proliferation and migration of mouse umbilical vascular endothelial cells to impede angiogenesis. VNP-Tum5 downregulated the expression of anti-vascular endothelial growth factor A, platelet endothelial cell adhesion molecule-1, phosphorylated phosphoinositide 3 kinase, and phosphorylated protein kinase B and upregulated the expression of cleaved-caspase 3 in tumor tissues. This study is the first to use tumstatin-transformed VNP20009 as a tumor-targeted system for treatment of melanoma by combining anti-tumor and anti-angiogenic effects.

Keywords Salmonella VNP20009 tumstatin B16F10 melanoma apoptosis angiogenesis

Corresponding Author(s): Zichun Hua

Just Accepted Date: 08 April 2022 Online First Date: 22 September 2022 Issue Date: 16 January 2023

Cite this article:

Feifei Bao,Mengjie Liu,Wenhua Gai, et al. Bacteria-mediated tumor-targeted delivery of tumstatin (54-132) significantly suppresses tumor growth in mouse model by inhibiting angiogenesis and promoting apoptosis[J]. Front. Med., 2022, 16(6): 873-882.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0925-2
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I6/873

Fig.1 VNP-Tum5 and VNP delayed tumor growth and enhanced survival time in the melanoma tumor model of mice. (A) Schematic representation of the production of VNP-Tum5 and the treatment schedule. B16F10 tumor mice (n = 12 in each group) were injected i.p. with 1 × 10⁶ CFU of VNP, VNP-Tum5, or with 100 μL of PBS on day 7. (B) Detection of the expression of Tum5 in the tumor tissues of mice bearing melanoma by Western blot. α-Tubulin served as the loading control (n = 4). (C) Tumor growth curves (n = 10). (D) Representative efficacy of the recombinant VNP20009 in melanoma therapy (n = 4). (E) Kaplan–Meier survival curves of tumor-bearing mice after different treatments (n = 12). (F) Tumor doubling time of the mice. (G) Tumor growth delay of the mice (n = 8). Tumor data among different groups were compared. Data are presented as mean ± SD. *P < 0.05, ** P < 0.01, **** P < 0.0001.

Fig.2 Toxicological assessment of VNP-Tum5 and VNP in melanoma tumor mouse model. (A) Number of bacteria that colonized the tumors, spleen, and liver at day 6 post-infection (n = 4). (B) Detection of Tum5 expression in the spleen of mice bearing melanoma by Western blot analysis. α-Tubulin served as loading control (n = 3). (C) Detection of Tum5 expression in the liver of mice bearing melanoma by Western blot analysis. α-Tubulin served as loading control (n = 3). (D, E) Spleen and spleen weight after tumor incubation for 13 days (n = 4). (F, G) Liver and liver weight after tumor incubation for 13 days (n = 4). (H, I) HE staining of the spleen and liver tissues (n = 3). (J) Variations in body weight after bacterial infection. The P value was significant between the VNP group and the PBS group. Data are presented as mean ± SD. *P < 0.05, ** P < 0.01, *** P < 0.001. ns, no significance.

Fig.3 VNP-Tum5 and VNP induced the apoptosis and necrosis of melanoma in vivo and in vitro. (A) HE staining of tumor tissues (n = 3). Representative images (100×) reveal tumor necrotic areas of B16F10 tumor section treated with PBS, VNP, and VNP-Tum5 (vital tumor regions, V; necrotic tumor regions, N). (B) Quantification of necrotic tumor tissue (n = 3). (C) Ki67 and TUNEL assay of tumor tissue. Representative images showing cell proliferation and apoptosis in tumor tissues with different treatments (bright blue, nuclei; bright green, apoptotic cells; bright red, tumor aggressiveness). (D and E) Quantification of proliferation and apoptosis levels of tumor tissues (n = 3). Quantification of fluorescence by ImageJ software. (F) Representative FACS analysis of annexin V and propidium iodide (PI) staining after 2 h and 4 h of incubation with the strains (n = 3). (G, H) Quantification of apoptosis and necrosis of B16F10 melanoma cells after 2 h and 4 h of incubation with the strains (n = 3). Bar represents mean ± SD of five optical fields. *P < 0.05, ** P < 0.01. *** P < 0.001. ns, no significance.

Fig.4 VNP-Tum5 and VNP targeted at tumor blood vessels, inhibited proliferation, and inhibited angiogenesis on MUVECs. (A) Fluorescence images of tumor blood vessels. Blood vessel staining was performed and showed the co-localization of CD31 (red) and DAPI (blue) in tumor samples. (B) Quantification of CD31-positive tumor vessels. (C) VEGF-A expression in xenograft tumors from different groups at the protein level monitored by Western blot analysis. (D) Statistical analysis of VEGF-A results determined by Western blot by ImageJ. (E) Wound healing assay of MUVECs after incubation with the strain for 2 h (MOI = 1:10). (F) Statistical analysis of the wound healing experiment of MUVECs by ImageJ. (G) Inhibition of VNP and VNP-Tum5 on MUVECs at different MOI. Data are shown as mean ± SD (n = 3) with individual data points shown, analyzed by two-tailed t-test. *P < 0.05; ** P < 0.01; *** P < 0.001.

Fig.5 Mechanism of VNP-Tum5 and VNP on melanoma is associated with VEGF-A/VEGFR-2 and PI3K-AKT signaling pathways in tumor tissues. (A) Relative expression levels of VEGFR2, caspase 3, clevavead-caspase 3, p-PI3Κ/PI3Κ, and p-AΚT/AΚT were determined by Western blot analysis (n = 3). (B, C) Statistical analysis of the results of Western blot by ImageJ. (D) Possible mechanism of VNP-Tum5 in inhibiting melanoma. Data are presented as mean ± SD with individual data points shown, analyzed by two-tailed t-test. *P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001.

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