The administration of passive and active immunotherapy against Syntenin-1 decreased the tumoral growth and pulmonary metastasis in a murine model of triple-negative breast cancer

Abstract

Breast cancer remains the leading cause of cancer-related deaths worldwide, with the triple-negative breast cancer (TNBC) subtype exhibiting a particularly high mortality rate. Conventional immunotherapy treatments have proven ineffective for this subtype, highlighting the need for the identification of novel tumor antigens, such as Syntenin-1. This 32 kDa protein is linked to cellular proliferation, angiogenesis, and metastasis. Recent research has proposed both active (vaccines) and passive (antibodies) immunotherapy as potential complementary treatments for breast cancer. The primary objective of this study was to assess the efficacy of targeting Syntenin-1 through active and passive immunity as a strategy for developing new immunotherapies for TNBC. We conducted an in silico analysis to select a peptide derived from the amino acid sequence of Syntenin-1, which was synthesized chemically as MAP8. This peptide was administered to Balb/c mice to induce a humoral immune response. Immunized mice were then used to obtain polyclonal antibodies for evaluating active immunity. A total of twenty-eight Balb/c mice were divided into seven experimental groups. Tumor induction was achieved by administering the 4T1 cell line (5 × 10⁴ cells) for 30 days in groups 3-7. Passive treatment was given at low (1 mg/kg) and high (1 mg/kg) doses on days 8, 15, and 22 following tumor induction. Mice were sacrificed to collect blood and organs for analyzing tumor growth, metastasis, and the humoral immune response. The KA-11-MAP8 peptide, derived from the PDZ-2 domain of Syntenin-1, successfully induced antibody production in Balb/c mice after administration. Purified antibodies were able to recognize the native protein in both the 4T1 cell line and the brain. Both passive and active treatments targeting Syntenin-1 resulted in reduced tumor size and fewer metastatic nodules in the lungs. This study provides evidence for the efficacy of the KA-11-MAP8 peptide derived from Syntenin-1 in eliciting a humoral immune response, which in turn impacts tumor development and metastasis in a murine model of TNBC.

Keywords: 4T1 cell line; Active immunotherapy; Breast cancer; MAP8; Passive immunotherapy; Peptides; Sintenin-1.

Fig. 1

In silico analysis and identification of the KA-11-MAP8 peptide derived from Syntenin-1. (A) Ab initio modeling of the 3D structure of Syntenin-1. This structure illustrates the four functional domains of Syntenin-1: the N-terminal (amino acids 1–109), PDZ1 (amino acids 110–193), PDZ2 (amino acids 194–274), and the C-terminal (amino acids 275–298). Ramachandran plots were utilized to assess the quality of the model. (B,C) In silico analysis for the identification of immunogenic and antigenic sites. B) The number of peptides with the capacity to bind to MHC I/II for T cell activation. (C) The number of peptides exhibiting physical and antigenic properties for B cell activation. Bepipred was used for linear epitope prediction, Emini for surface accessibility prediction, and K&T (Kolaskar & Tongaonkar) for antigenicity. (D) Localization of the preselected peptides. This section provides a schematic representation of the preselected peptides. The image was created in Protter (https://wlab.ethz.ch/protter/start/).

Fig. 2

Immunogenicity of the KA-11-MAP8 and purification of polyclonal antibodies. (A) Antibody levels in mice inoculated with the KA-11-MAP8 peptide were evaluated using an indirect ELISA, with serum samples serving as the primary antibody. (B) Antibody sub-isotype evaluation was conducted using serum samples collected on day 120. The levels of IgM (anti-Mu, 1:6,000; Jackson ImmunoResearch Cat#AB2340064) and IgG (anti-Gamma, 1:8,000; Thermo Fisher Scientific Cat#A16084) were assessed via indirect ELISA. (C) Antibody purity was tested using SDS-PAGE (12%) stained with Coomassie blue. The SDS-PAGE results displayed two lanes: one for the molecular weight marker (BioRad Cat#1610373EDU) and another showing the denatured purified polyclonal antibody with both heavy chain (HC) and light chain (LC) bands. (D) Antibody isotype analysis. (E) IgG sub-isotypes in the polyclonal antibody were evaluated through an indirect ELISA, using the purified polyclonal antibody as the primary antibody. For the evaluation of antibody subtypes, anti-Mu (IgM, 1:6,000; Jackson ImmunoResearch Cat#AB2340064) and anti-Gamma (IgG, 1:8,000; Thermo Fisher Scientific Cat#A16084) were utilized as secondary antibodies. Additionally, for IgG sub-isotypes, anti-IgG1 (Abcam Cat#AB98693), anti-IgG2a (Abcam Cat#AB98698), anti-IgG2b (Abcam Cat#AB98703), and anti-IgG3 (Abcam Cat#AB98708) coupled to HRP were diluted 1:4,000.

Fig. 3

Recognition of the KA-11-MAP8 peptide and the native form of Syntenin-1 by the polyclonal antibody. (A) Dot blot and Western blot analysis were performed. The positive control consisted of total protein extracts derived from the brain, while the conjugate control (CC) was used to assess the biological activity of the secondary antibody. A commercial antibody, anti-Syntenin-1 C-3 (Santa Cruz Biotechnology, Cat#sc-515538), served as an additional positive control. The Western blot included the 4T1 cell line and total protein extracts from the brain. (B) Immunocytochemistry was conducted using the 4T1 cell line, which is derived from a spontaneous breast tumor in mice. Staining was performed using the streptavidin–biotin peroxidase-based immunocytochemical method. The primary antibodies used were the polyclonal antibody and the anti-Syntenin-1 C3 commercial antibody. In the negative control, the primary antibody was omitted. (C) Schematic representation of the cell-ELISA method used to assess antibody isotypes and sub-isotypes. This image was created using Biorender (https://www.biorender.com/). (D) Antibody subtypes and sub-isotypes present in the polyclonal antibody. A cell-ELISA was performed using 4T1 cell cultures grown in 96-well plates. The primary antibody employed was the polyclonal antibody, while the secondary antibodies included monoclonal antibodies: anti-IgG (H + L whole molecule 1:8,000), anti-Mu (IgM 1:6,000; Jackson ImmunoResearch, Cat#AB2340064), anti-Gamma (IgG 1:8,000; Thermo Fisher Scientific, Cat#A16084), as well as sub-isotypes anti-IgG1 (Abcam, Cat#AB98693), anti-IgG2a (Abcam, Cat#AB98698), anti-IgG2b (Abcam, Cat#AB98703), and anti-IgG3 (Abcam, Cat#AB98708) conjugated to HRP, and diluted to 1:4,000.

Fig. 4

The effect of active and passive treatment with the KA-11-MAP8 peptide and polyclonal antibody on tumor growth and lung metastasis in Balb/c mice. (A) Representative images of breast tissue and lungs from the control and experimental groups. (B) Volume of the primary tumor, with comparisons made between Group 3 and the other groups. The statistical significance is noted as follows: *p = 0.0104 (Group 4), **p = 0.0082 (Group 5), and ***p = 0.0077 (Group 6). (C) Tumor weight measurements. (D) Weight of the lungs, expressed in grams. Groups 1 to 7 (G1-G7) are indicated. The plus sign (+) in the passive treatment denotes the dose of polyclonal antibody administered: + : 1 mg/kg; ++: 3 mg/kg; +++: 5 mg/kg.

Fig. 5

Morphological Evaluation and Syntenin-1 Expression in Control and Experimental Groups. (A) This panel shows representative images of H&E staining and immunohistochemistry (IHC) for the primary tumor and lungs in both control and experimental groups. For the IHC, we used the commercial antibody anti-Syntenine-1 C-3 (Santa Cruz Biotechnology, Cat#sc-515538), which was stained using a streptavidin–biotin peroxidase-based immunohistochemical method. (B) The panel highlights the anatomopathological characteristics of the analyzed tissues. The left panel displays the percentage of necrosis, the middle panel shows the percentage of mitosis per area, and the right panel illustrates the number of metastatic nodules. Statistical analysis was performed using one-way ANOVA, revealing significant results: *p = 0.0245 (between Groups 5 and 6) and *p = 0.0456 (between Groups 6 and 7). Groups 1 to 7 are denoted as G1-G7. The symbol "+" in the passive treatment indicates the dose of the administered polyclonal antibody: +denotes 1 mg/kg; ++ denotes 3 mg/kg; and +++ denotes 5 mg/kg.

Fig. 6

Detection of antibody isotypes and sub-isotypes in serum collected from both control and experimental groups. The measured antibody levels include total IgG (heavy + light chains), IgM, and specific IgG sub-isotypes: IgG1, IgG2a, IgG2b, and IgG3. Groups 1 to 7 are labeled as G1 through G7. In the passive treatment section, the '+' symbol indicates the dose of the administered polyclonal antibody: + indicates 1 mg/kg, ++ indicates 3 mg/kg, and +++ indicates 5 mg/kg.