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. 2021 May 31;19(1):165.
doi: 10.1186/s12951-021-00904-6.

The targetable nanoparticle BAF312@cRGD-CaP-NP represses tumor growth and angiogenesis by downregulating the S1PR1/P-STAT3/VEGFA axis in triple-negative breast cancer

Ke Gong #  1 , Juyang Jiao #  2 , Chaoqun Xu  3 , Yang Dong  1 , Dongxiao Li  3 , Di He  1 , De Zhao  1 , Jian Yu  1 , Ying Sun  1 , Wei Zhang  4 , Min Bai  5 , Yourong Duan  6
Affiliations

The targetable nanoparticle BAF312@cRGD-CaP-NP represses tumor growth and angiogenesis by downregulating the S1PR1/P-STAT3/VEGFA axis in triple-negative breast cancer

Ke Gong et al. J Nanobiotechnology. .

Abstract

Background: Overexpressed vascular endothelial growth factor A (VEGFA) and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) cause unrestricted tumor growth and angiogenesis of breast cancer (BRCA), especially triple-negative breast cancer (TNBC). Hence, novel treatment strategy is urgently needed.

Results: We found sphingosine 1 phosphate receptor 1 (S1PR1) can regulate P-STAT3/VEGFA. Database showed S1PR1 is highly expressed in BRCA and causes the poor prognosis of patients. Interrupting the expression of S1PR1 could inhibit the growth of human breast cancer cells (MCF-7 and MDA-MB-231) and suppress the angiogenesis of human umbilical vein endothelial cells (HUVECs) via affecting S1PR1/P-STAT3/VEGFA axis. Siponimod (BAF312) is a selective antagonist of S1PR1, which inhibits tumor growth and angiogenesis in vitro by downregulating the S1PR1/P-STAT3/VEGFA axis. We prepared pH-sensitive and tumor-targeted shell-core structure nanoparticles, in which hydrophilic PEG2000 modified with the cyclic Arg-Gly-Asp (cRGD) formed the shell, hydrophobic DSPE formed the core, and CaP (calcium and phosphate ions) was adsorbed onto the shell; the nanoparticles were used to deliver BAF312 (BAF312@cRGD-CaP-NPs). The size and potential of the nanoparticles were 109.9 ± 1.002 nm and - 10.6 ± 0.056 mV. The incorporation efficacy for BAF312 was 81.4%. Results confirmed BAF312@cRGD-CaP-NP could dramatically inhibit tumor growth and angiogenesis in vitro and in MDA-MB-231 tumor-bearing mice via downregulating the S1PR1/P-STAT3/VEGFA axis.

Conclusions: Our data suggest a potent role for BAF312@cRGD-CaP-NPs in treating BRCA, especially TNBC by downregulating the S1PR1/P-STAT3/VEGFA axis.

Keywords: BAF312; P-STAT3; S1PR1; Targeted nanoparticle; Triple-negative breast cancer; VEGFA.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Scheme 1
Scheme 1
The schematic illustration of BAF312@cRGD-CaP-NPs for tumor treatment through downregulating the S1PR1/P-STAT3/VEGFA axis. The BAF312@cRGD-CaP-NPs are gradually accumulated at the tumor sites in vivo via the EPR effect and active targeting mediated by cRGD and then uptaken by tumor cells and tumor blood vessel cells. Next, the nanoparticles release BAF312 in the acidic tumor microenvironment. Finally, BAF312@cRGD-CaP-NPs induce tumor cell apoptosis and destroy tumor blood vessel to maximize their antitumor efficacy through S1PR1/P-STAT3/VEGFA pathway
Fig. 1
Fig. 1
S1PR1 overexpression in BRCA causes poor survival and is positively correlated with STAT3 and VEGFA. a–e TCGA database suggest the correlation between patient survival and S1PR1 expression in every stage of breast cancer. f Oncomine database analysis of S1PR1 expression in breast cancer and adjacent normal tissue. Mean ± SEM, *P < 0.05. g–i Oncomine database analysis of VEGFA expression in breast cancer and adjacent normal tissue. ICBA is intraductal cribriform breast adenocarcinoma, DBC is ductal breast carcinoma, IDBC is invasive ductal breast carcinoma, and LBC is lobular breast carcinoma. Mean ± SEM, **P < 0.01, ***P < 0.001. j, k TIMER database analysis of the relationship between S1PR1 and STAT3 or STAT3 and VEGFA. “Cor” represents the correlation value, and p is the p value, with values less than 0.05 being considered meaningful. BRCA is breast invasive carcinoma. l String database suggest the connection of S1PR1 and STAT3 and VEGFA in breast cancer, p value less than 0.05 is considered meaningful
Fig. 2
Fig. 2
Downregulation of S1PR1 improves the chemosensitivity of BRCA and inhibits vascular formation by decreasing P-STAT3/VEGFA. a–c qPCR analysis of S1PR1 expression in MCF-7 and MDA-MB-231 and HUVECs after treated with S1PR1-siRNA for 48 h. Mean ± SEM, ***P < 0.001. d–f Western blotting assays show the S1PR1, P-STAT3 and VEGFA protein expression in MCF-7 and MDA-MB-231 cells and HUVECs after treatment with S1PR1-siRNA for 48 h. g–j MTT analysis of the viabilities of MCF-7 and MDA-MB-231 cells following treatment with the cisplatin or doxorubicin for 2 days. k Vascular formation assay analysis of the tube formation of HUVECs pretreated with S1PR1-siRNA for 20 h. l The statistical results show the tube formation number of HUVECs. Mean ± SEM, **P < 0.01
Fig. 3
Fig. 3
BAF312 promotes apoptosis of BRCA and inhibits vascular formation of HUVECs via downregulating S1PR1/P-STAT3/VEGFA. a The structure of BAF312. b, c MTT assays suggest the cells viability of MCF-7 and MDA-MB-231 and HUVEC following treatment with the BAF312 for 72 h. d–f qPCR results show the S1PR1 expression of MCF-7 and MDA-MB-231 and HUVECs after treatment with the BAF312 for 48 h. Mean ± SEM, ***P < 0.001. g Apoptosis assays indicate the apoptotic rates of MCF-7 and MDA-MB-231 after treatment with the BAF312 for 48 h. h The statistical results show the apoptotic ratio of MCF-7 and MDA-MB-231. Mean ± SEM, ***P < 0.001. i Calcein-AM/PI staining analysis of apoptosis rates of MCF-7 and MDA-MB-231 following treatment with 10 µM BAF312 for 72 h. The red fluorescence means PI positivity, and the green fluorescence means calcein positivity. j Vascular formation assay analysis of the tube formation of HUVECs incubated with 10 µM BAF312 for 20 h. k The statistical results of the tube formation number for HUVECs. Mean ± SEM, **P < 0.01. l Western blotting assays show S1PR1 and P-STAT3 and VEGFA expression levels in HUVECs after treatment with 10 µM BAF312 for 48 h
Fig. 4
Fig. 4
Formation process and characterization of pH-sensitive shell–core BAF312@cRGD-CaP-NPs. a Schematic diagram of the BAF312@cRGD-CaP-NP formation process. b Nanosize indicated that the BAF312@cRGD-CaP-NP size was 109.9 ± 1.002 nm, with a PDI of 0.253 ± 0.006. TEM results indicated BAF312@cRGD-CaP-NPs were spherical and homogeneous. c, d The stability of BAF312@cRGD-CaP-NPs were tested for 7 days. e, g The average potentials of BAF312@cRGD-CaP-NPs in aqueous solutions of pH 7.4 and pH 6.0 were − 10.6 ± 0.056 mV and + 6.80 ± 0.013 mV, respectively. f, h HPLC assay showed the BAF312 release profiles from NPs or NPs modified with cRGD at pH 7.4 and 6.0
Fig. 5
Fig. 5
NPs modified with cRGD enhanced BRCA and HUVECs uptake of NPs and lysosomal escape occurred. a–c The cellular uptake of free RB and RB@CaP-NPs and RB@cRGD-CaP-NPs in MCF-7 and MDA-MB-231 and HUVECs were detected by the fluorescence microscope. d–f Flow cytometry assays show the fluorescence intensities of RB and RB@CaP-NPs and RB@cRGD-CaP-NPs in cells for 4 h. g Blue fluorescence indicates the nucleus; Red fluorescence indicates the RB or RB@cRGD-CaP-NPs; Green fluorescence indicates the lysosome (LysoTracker); The overlap of green fluorescence and red fluorescence indicate that most free RBs were entrapped in the lysosome, while the green and red fluorescence separately existed in the NPs group indicated that most NPs were successfully escaped from lysosomes
Fig. 6
Fig. 6
BAF312@cRGD-CaP-NPs boost the apoptosis of BRCA and inhibit the vascular formation via downregulating S1PR1/P-STAT3/VEGFA. a, c The apoptotic ratio of MCF-7 and MDA-MB-231 cells were detected by the apoptosis assay after treatment with 10 µM BAF312, 10 µM BAF312@CaP-NPs, and 10 µM BAF312@cRGD-CaP-NPs for 2 days. b, d The statistical results revealed the apoptotic ratio for cells. Mean ± SEM, *P < 0.05, *** P < 0.001. e, f Calcein-AM/PI staining indicated the apoptosis of MCF-7 and MDA-MB-231 cells after treatment with 10 µM BAF312, 10 µM BAF312@CaP-NPs, and 10 µM BAF312@cRGD-CaP-NPs for 3 days. The red fluorescence means PI positivity, and the green fluorescence means calcium positivity. g Vascular formation assay analysis of the tube formation of HUVECs after treatment with 10 µM BAF312, 10 µM BAF312@CaP-NPs, and 10 µM BAF312@cRGD-CaP-NPs for 20 h. h The statistical results showed tube formation rates for HUVECs. Mean ± SEM, * P < 0.05, ***P < 0.001. i–k Western blot analyzed proteins expression of S1PR1, P-STAT3, VEGFA, and Bcl-2 in breast cancer cells and HUVECs after treatment with 10 µM BAF312, 10 µM BAF312@CaP-NPs, and 10 µM BAF312@cRGD-CaP-NPs for 2 days
Fig. 7
Fig. 7
The tumor targeting ability of cRGD-NPs and the antitumor efficiency of BAF312@cRGD-CaP-NPs in vivo. a Upper: After intravenous injection for 2, 12, 24 and 96 h, the distribution of Dir@CaP-NPs and Dir@cRGD-CaP-NPs in MDA-MB-231 tumor-bearing nude mice (Dir dose is 1 μg/mL) (n = 3). Below: Fluorescence images of anatomic tumors and other major organs (spleen, heart, liver, lung, and kidney) at 96 h after the intravenous injection of Dir@CaP-NPs and Dir@cRGD-CaP-NPs. b Quantitative analysis of the mean Dir fluorescence intensity of tumor sites of intravenous injection of Dir@CaP-NPs and Dir@cRGD-CaP-NPs for 2, 12, 24, and 96 h. Mean ± SEM, n = 3, **P < 0.01, ***P < 0.001. c Quantitative analysis of the mean Dir fluorescence intensity of the anatomic tumors and major organs (spleen, heart, liver, lung, and kidney) at 96 h after the intravenous injection of Dir@CaP-NPs and Dir@cRGD-CaP-NPs. d Schematic illustration of the MDA-MB-231 tumor implantation and the treatments in nude mice. Twenty days after tumor implantation, these mice were treated with the indicated treatments on the indicated days. e Fig. of tumors collected from the different treatment groups. f The tumor weights collected from different groups after anatomy (n = 6). g The tumor growth curves. h The changes of body weights
Fig. 8
Fig. 8
BAF312@cRGD-CaP-NPs inhibit tumor growth and angiogenesis in vivo via downregulating the expression of the S1PR1/P-STAT3/VEGFA. a Immunofluorescence assay analysis of TUNEL in the tumors. Scale bar: 50 µm. b IHC analysis of Ki67 expression in the tumors. Scale bar: 50 µm. c Immunofluorescence assay analysis of CD31 in the tumors. Scale bar: 50 µm. d The statistical results of TUNEL positivity in MDA-MB-231 tumors. Mean ± SEM, n = 3, **P < 0.01, ***P < 0.001. e The statistical results of Ki67 positivity in MDA-MB-231 tumors. Mean ± SEM, n = 3, **P < 0.01, ***P < 0.001. f The statistical results of CD31 for MDA-MB-231 tumors. Mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. g The proteins expression of S1PR1, P-STAT3, VEGFA, and Bax were determined by IHC in tumors of which collected at the end point. Scale bar: 50 µm
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