Fig 1.
Validation of MIND model and detection of different receptors’ status in various human and mouse breast cancer cell lines.
(A) Schematic representation of mouse mammary intraductal injection through cleaved nipples. Mvt-1 or 4T1 cells were re-suspended in 0.04% trypan blue and injected through 4th inguinal mammary gland cleaved nipple. The experimental protocols are depicted in the left two panels. (B) Representative images of α-SMA and laminin-1 immunostaining in human biopsy samples (n = 26) and MCF-DCIS-MIND xenograft samples (n = 5). The images illustrate the similarity of the Human breast and MIND xenograft architecture. White arrows indicate staining (green) of laminin and α-SMA and Red arrows indicate possible localized death of MECs. Scale bar, 50μm. (C) Representative Western blot analysis of ER-α, PR and HER2 expressions in the cellular extracts of SKBR3, MCF-7, MDA-MB-231, Mvt-1, and 4T1 cell lines. SKBR3 cell extract was used as positive control for HER2, and MCF-7 cell extract was used as positive control for ER-α and PR. (n = three replicates). All photographs are cropped from original figures.
Fig 2.
Development of primary tumors in the mammary ducts of female FVB/N mice following Mvt-1 cells injection through MIND method.
(A) Representative photograph of whole mount of 4th inguinal mammary glands of female mice (FVB/N) injected with Mvt-1 cells for one week (n = 5). LN (arrow); Lymph node. (B-C) Representative H&E staining in serial sections of the mammary glands of female FVB/N mice injected with Mvt-1 cells for one week (n = 5). Images illustrate normal ducts and lobules (B-C) in the mammary glands. Scale bars represent 100–200 μm. (D-E) Representative photograph of lungs of FVB/N mice injected with Mvt-1 cell for one week (n = 5). The images illustrate no outgrowth of metastasis in the lungs. (F-G) Representative H&E staining serial sections of lungs of female FVB/N mice injected with Mvt-1 cells for one week (n = 5). The images show no detectable microscopic metastasis in the lungs. Scale bars, 100–200 μm. (H) Representative whole mount of 4th inguinal mammary gland of female FVB/N mice injected with Mvt-1 cells for two weeks (n = 5). Image shows palpable tumors in mammary gland. Arrow (T) indicates tumor, LN; Lymph node. (I-J) Representative H&E staining sections of mammary glands collected from the female FVB/N mice injected with Mvt-1 cells for two weeks (n = 5). The photographs illustrate DCIS-like structure (I) and micro-invasion (J).; T; Tumor. Black arrows indicate DCIS-like structures, and yellow arrow indicates microinvasion. Scale bar, 100–200μm. (K-L) Representative photograph of lungs of female FVB/N mice injected with Mvt-1 cell for two weeks (n = 5). The images illustrate no metastatic outgrowth in the lungs. (M-N) Representative H&E staining serial sections of lungs of female FVB/N mice injected with Mvt-1 cells for two weeks (n = 5). The images show no detectable microscopic metastasis in the lungs. Scale bars, 100–200 μm.
Fig 3.
Primary mammary tumor growth, progression and outgrowth of metastasis in the lungs of female FVB/N mice following Mvt-1 cell injection through MIND method.
(A) Representative photograph of the 4th inguinal mammary gland with tumor (T) of female mice (FVB/N) injected with Mvt-1 cells for three weeks (n = 5). The photograph illustrates tumor growth in the mammary ducts. (B-C) Representative H&E staining sections of the mammary gland of female FVB/N mice three weeks after the injection of Mvt-1 cells (n = 5). Images illustrate local invasion. Scale bars, 50–200 μm. (D-E) Representative photograph of lungs of female FVB/N mice injected with Mvt-1 cell for three weeks (n = 5). Photographs illustrate small outgrowth of metastasis (black) in the lungs (arrows). (F-G) Representative H&E staining sections of the lungs of female FVB/N mice injected with Mvt-1 cells for three weeks (n = 5). Arrows indicate microscopic metastatic growth in the lungs. Scale bars, 100–200 μm. (H) Representative photograph of the 4th inguinal mammary glands with tumor (T) of female mice (FVB/N) injected with Mvt-1 cells for four weeks (n = 5). (I-J) Representative H&E staining sections of the mammary gland of female FVB/N mice four weeks after the injection of Mvt-1 cells (n = 5). Images illustrate local invasion. Scale bars, 100–200 μm. (K-L) Representative photograph of lungs of female FVB/N mice injected with Mvt-1 cells for four weeks (n = 5). Photographs illustrate large outgrowths of metastasis (white) in the lungs (arrows). (M-N) Representative H&E staining lung sections with metastasis from female FVB/N mice injected with Mvt-1 cells after four weeks (n = 5). Scale bars, 100–200 μm. (O) The schematic diagram illustrates the summary of the duration of the sequential growth of lung metastasis from preneoplastic lesions following injection of Mvt-1 cells into the ducts through MIND method.
Fig 4.
Quantitative evaluation of tumor development in FVB mouse fourth inguinal mammary gland injected with Mvt-1 cells.
(A) Graph depicts the number of mice bearing normal ducts, DCIS and IC/Metastasis (n = 5). (B) Representation of the count of available normal mammary ducts, DCIS and IC/metastatic tumors in individual mice. (DCIS: Ductal carcinoma in-situ and IC: Invasive carcinoma). Data presents Mean ±SD. *indicates p<0.001 in an Anova analysis with Bonferroni post hoc test.
Fig 5.
In vivo imaging of GFP-signal in tumor-bearing mice.
(A-E) Representative whole-body imaging of female FVB/N mice after injecting GFP-labeled Mvt-1 cells. Mice were imaged at Day 0 showing no signal (A &B), day 7 exhibiting signal in the primary tumors (C) and Day 21 illustrating signals in both primary tumors and lung metastasis. (n = 5 mice). The right panel shows the scale of the intensity.
Fig 6.
Immunohistochemical characterization of primary tumors and lung metastasis.
(A-C) Representative photographs of H&E staining, α-SMA and PCNA immunohistochemical staining sections of normal ducts and lobules of the mammary gland of female FVB/N mouse injected with Mvt-1 cells for one week (n = 5). The ductal architecture demarcated by α-SMA (B) and the presence of Mvt-1 cells inside duct detected by PCNA immune-staining (C). Scale bar, 20–50 μm. Arrow indicates α-SMA-positive myoepithelial layer. (D-F) Representative photographs of H&E staining, α-SMA and PCNA immunohistochemical staining sections of an early stage of a DCIS-like structure formed in the mammary glands of a female FVB/N mouse injected with Mvt-1 cells for two weeks (n = 5). α-SMA immunostained the myoepithelial layer around the DCIS (E), and PCNA immunostained the highly proliferative Mvt-1 cells inside DCIS structure (F). Scale bar, 20–50 μm. Arrow indicates α-SMA-positive myoepithelial layer. (G-I) Representative photographs of H&E staining, α-SMA and PCNA immunohistochemical staining sections of an invasive area of the mammary glands of a female FVB/N mouse injected with Mvt-1 cells for three weeks (n = 5). Scale bar, 50μm. (J-L) Representative photographs of H&E staining, α-SMA and PCNA immunohistochemical staining in the blood vessels of a female FVB/N mouse injected with Mvt-1 cells for four weeks (n = 5). In this model, the preferred route of cell migration toward other organs to form a secondary tumor is via blood vessels. This staining shows a clear indication of cells delineating from adjacent tumors inside blood vessels. Scale bar,100 μm. (M-O) Representative photographs of H&E staining, α-SMA and PCNA immunohistochemical staining sections of lung metastasis of a female FVB/N mouse injected with Mvt-1 cells for four weeks (n = 5). Scale bar, 50μm.
Fig 7.
Tumor progression and metastasis in the mammary ducts of female BALB/c mice following injection of 4T1 cells through MIND method.
(A) Representative photograph of the whole mount of 4th inguinal mammary gland after the first week of 4T1 cell injection into the mammary ducts of female BALB/c wild-type mouse (n = 5). Note, no palpable tumors are apparent. LN, lymph node. (B) Representative photograph of the whole mount of 4th inguinal mammary gland after the second week of 4T1 cell injection into the mammary ducts of female BALB/c wild-type mouse (n = 5). Note, tumors are palpable (arrow). T, tumor, and LN, lymph node. (C-D) Representative photograph of 4th inguinal mammary glands of female BALB/c mouse injected with 4T1 cells for three weeks and four weeks, respectively (n = 5). The photograph illustrates large tumor growth in the mammary ducts (yellow lines). (E-H) Representative photographs of H&E-staining sections illustrate the sequential progression of tumor growths from DCIS to the invasion in the mammary ducts of a female BALB/c mice injected with 4T1 cells (n = 5). Scale bar, 20–100 μm. (I-K) The representative photograph of lungs of 4T1-tumor-bearing female BALB/c mice illustrate no metastatic growth macroscopically (I) or microscopically (J) (n = 5). However, a small outgrowth in the lungs (arrow) was detected by the third week, indicating initiation of metastasis to lungs (K). Scale bar, 200 μm. (L-N) Representative H&E staining sections of lymph nodes (L and M) and blood vessels (N) illustrate intravasation of 4T1 cells after three weeks of injection (n = 5). Box indicates the enlarge margination of Fig L. Arrow heads indicate 4T1 cells in the lymph node and blood vessels. Scale bars, 20–200 μm.
Fig 8.
Detection of EMT markers in the non-invasive and invasive tumor in TNBC-MIND model.
(A) Representative photographs of Cytokeratin 19 and β-catenin immunostaining serial sections of mammary glands of FVB/N mice injected with Mvt-1 cells for various times (n = 5). Scale bar, 25–200 μm. (B) Representative photographs of Twist and Vimentin immunostaining serial sections of mammary glands of female FVB/N mice injected with Mvt-1 cells for various times (n = 5). Scale bar, 20–200 μm. (C) Representative photographs of Cytokeratin 19 and β-catenin immunostaining sections of lungs illustrating no expression of epithelial markers in metastatic tumors generated by Mvt-1 cells in lungs (n = 5). Scale bar, 50–200 μm. (D) Representative photographs of Twist and Vimentin immunostaining sections of lungs illustrating elevated levels of mesenchymal markers in metastatic tumors generated by Mvt-1 cells in the lungs (n = 5). Scale bar, 100–200 μm.
Fig 9.
Schematic representation of the timescale of the progression of the TNBC from DCIS to metastatic growth in lungs in syngeneic mouse MIND model.