Exosomes Derived from Mouse Breast Carcinoma Cells Facilitate Diabetic Wound Healing

Abstract

Background: Exosomes derived from breast cancer have been reported to play a role in promoting cell proliferation, migration, and angiogenesis, which has the potential to accelerate the healing process of diabetic wounds. The aim of this investigation was to examine the function of exosomes originating from 4T1 mouse breast carcinoma cells (TEXs) in the process of diabetic wound healing.

Methods: The assessment of primary mouse skin fibroblasts cell proliferation and migration was conducted through the utilization of CCK-8 and wound healing assays, while the tube formation of HUVECs was evaluated by tube formation assay. High-throughput sequencing, RT-qPCR and cell experiments were used to detect the roles of miR-126a-3p in HUVECs functions in vitro. The in vivo study employed a model of full-thickness excisional wounds in diabetic subjects to explore the potential therapeutic benefits of TEXs. Immunohistochemical and immunofluorescent techniques were utilized to evaluate histological changes in skin tissues.

Results: The findings suggested that TEXs facilitate diabetic wound healing through the activation of cell migration, proliferation, and angiogenesis. An upregulation of miR-126a-3p has been observed in TEXs, and it has demonstrated efficient transferability from 4T1 cells to HUVEC cells. The activation of the PI3K/Akt pathway has been attributed to miR-126a-3p derived from TEXs.

Conclusions: The promotion of chronic wound healing can be facilitated by TEXs through the activation of cellular migration, proliferation, and angiogenesis. The activation of the PI3K/Akt pathway by miR-126a-3p originating from TEXs has been discovered, indicating a potential avenue for enhancing the regenerative capabilities of wounds treated with TEXs.

Keywords: Angiogenesis; Cell proliferation and migration; Exosomes; Wound healing; miR-126a-3p.

Fig. 1

Identification and characterization of TEXs. A TEM analysis of structures of exosomes (× 40.0 k). Scale bar: 100 nm. B Exosome concentration and size distribution determined using NTA. C The protein levels of VEGF-A and a series of exosome markers in TEXs were verified by western blot

Fig. 2

TEXs enhance cell proliferation and migration. A Cell proliferation was examined by CCK-8 assay after different treatments (n = 4). B Representative graphs of experiments for mouse skin fibroblasts migration (n = 4). Scale bar: 100 μm. C Quantitative analysis of the migration area in (B) (n = 4). ***P < 0.001; ns indicates no significant difference

Fig. 3

Effect of Matrigel on tube formation. A Representative images of tube formation assay in HUVECs (n = 3). Scale bar: 100 μm. B Quantitative analyses of the branch points, total loops, and the total tube length in A. C Representative images and CD31 staining, H&E staining photographs, scale bar: 50 μm of the Matrigel plugs, scale bar: 2 mm of the HE staining photographs. D Histogram representing the red fluorescence intensity, (n = 3). ***P < 0.001; ns represents no significant difference

Fig. 4

Exosomes-associated miRNA expression profiles. A Venn diagram showing the distribution of the miRNAs in HEXs and TEXs. B Heat map showing the expression profile of miRNA of two exosomes. C miR-126a-3p in TEXs and HEXs as detected by qRT-PCR (n = 3). D Protein expressions in different treatments. E Histograms of protein expression. **P < 0.01; ***P < 0.001; ns indicates no significant difference

Fig. 5

Exosomes-derived miR-126a-3p. A 4T1 transfected with a Cy3-labelled miR-126a-3p mimics (red) and co-cultured with HUVECs in a transwell (0.4 μm) plate. B Representative image of HUVECs incubated with 4T1 transfected with a Cy3-labelled miR-126a-3p mimics with or without GW4869 treatment (20 μM) in a transwell (0.4 μm) plate. Scale bar: 100 μm

Fig. 6

Functional presentation of miR-126a-3p. A TEXs and miR-126a-3p mimics promote HUVECs migration (n = 4). Scale bar: 100 μm. B Representative images of TEXs and miR-126a-3p mimics promoting HUVECs tube formation, (n = 3). Scale bar: 100 μm. C Quantitative analysis of the migration. D Quantitative analysis of the proliferation. E Quantitative analysis of the total points. **P < 0.01 or ***P < 0.001

Fig. 7

TEXs accelerates wound healing in diabetic mice. A Flow chart of animal assay model. B The representative images of wounds healing on days 1, 3, 10, 18, 21. The diameter of the metal ring is 1.5 cm (n = 8). C Histograms showing the wound closure rate. D Serum glucose trend during wound healing. E The change trend of mouse avoirdupois. F H&E and and Masson staining observation on the 21st day of wound healing in the TEXs and control groups. Scale bar: 500 μm. G Quantitative analysis of the intensity of collagen per field in Masson stain. Data were presented as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 8

In vivo immunohistochemistry and immunofluorescent staining results. A Immunohistochemical examination of the PCNA levels. Scale bar: 100 μm. B Quantitative analysis of immunohistochemistry results (n = 3). C Immunofluorescence examination of the CD31 levels. Scale bar: 100 μm. D Quantitative analysis of immunofluorescence results (n = 3). *P < 0.05; ***P < 0.001

Fig. 9

TEXs do not show tumorigenicity. A Nude mouse tumorigenicity assay performed to evaluate tumorigenic capacity of TEXs, HEXs and 4T1 cells. B The serum monitoring of tumor markers