Depression and family support in breast cancer patients

MTS, migration and invasion assays in DCIS.COM cells that were previously transduced with scrambled control (Control) or BCL9 KD shRNA. The control cells and BCL9 KD cells were re-transduced with empty vector (EV), BCL9 overexpression (BCL9-OE) and BCL9 KD. BCL9-OE was achieved by transduction using the PCDH-BCL9 (BCL9-OE) acquired from Dr. Carrasco [11]. A Western blot analysis was performed using anti-BCL9, anti-vimentin, anti-E-cadherin antibodies, and anti-β-actin as a loading control. B MTS assay on control cells transduced with EV (control + EV), or BCL9-OE (control + BCL9-OE), BCL9-KD transduced with EV (BCL9 KD + EV), and BCL9-KD transduced with BCL9-OE (BCL9 KD + BCL9-OE). Bar graphs represent mean absorbance at 490 nm normalized to control ± standard error of the mean (SEM) (n = 6). C, D Representative images of the migration and invasion assays. Bar graph represents percent area of cells migrated (left) and invaded (right) under the membrane after 24 h. Invasion and migration were determined by ImageJ analysis of microscopic images per sample, the data are mean values normalized to control ± SEM (n = 3). E TopFlash and FopFlash reporter activity in DCIS.COM transduced as above that were either treated with Wnt3A or control conditioned medium (CM). Data represent mean ± SEM (n = 3, letters indicate statistically significant difference). (PDF 964 kb

MIND model for triple-negative breast cancer in syngeneic mice for quick and sequential progression analysis of lung metastasis

<div><p>Mouse models of breast cancer with specific molecular subtypes (e.g., ER or HER2 positive) in an immunocompetent or an immunocompromised environment significantly contribute to our understanding of cancer biology, despite some limitations, and they give insight into targeted therapies. However, an ideal triple-negative breast cancer (TNBC) mouse model is lacking. What has been missing in the TNBC mouse model is a sequential progression of the disease in an essential native microenvironment. This notion inspired us to develop a TNBC-model in syngeneic mice using a mammary intraductal (MIND) method. To achieve this goal, Mvt-1and 4T1 TNBC mouse cell lines were injected into the mammary ducts via nipples of FVB/N mice and BALB/c wild-type immunocompetent mice, respectively. We established that the TNBC-MIND model in syngeneic mice could epitomize all breast cancer progression stages and metastasis into the lungs via lymphatic or hematogenous dissemination within four weeks. Collectively, the syngeneic mouse-TNBC-MIND model may serve as a unique platform for further investigation of the underlying mechanisms of TNBC growth and therapies.</p></div

Schematic representation of the timescale of the progression of the TNBC from DCIS to metastatic growth in lungs in syngeneic mouse MIND model.

<p>Schematic representation of the timescale of the progression of the TNBC from DCIS to metastatic growth in lungs in syngeneic mouse MIND model.</p

Tumor progression and metastasis in the mammary ducts of female BALB/c mice following injection of 4T1 cells through MIND method.

<p><b>(A)</b> Representative photograph of the whole mount of 4^th 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>(B)</b> Representative photograph of the whole mount of 4^th 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. <b>(C-D)</b> 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). <b>(E-H)</b> 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. <b>(I-K)</b> 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. <b>(L-N)</b> 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.</p

In vivo imaging of GFP-signal in tumor-bearing mice.

<p><b>(A-E)</b> 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.</p

Quantitative evaluation of tumor development in FVB mouse fourth inguinal mammary gland injected with Mvt-1 cells.

<p><b>(A)</b> Graph depicts the number of mice bearing normal ducts, DCIS and IC/Metastasis (n = 5). <b>(B)</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.</p

Detection of EMT markers in the non-invasive and invasive tumor in TNBC-MIND model.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. (<b>D)</b> 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.</p

Validation of MIND model and detection of different receptors’ status in various human and mouse breast cancer cell lines.

<p><b>(A)</b> 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 4^th inguinal mammary gland cleaved nipple. The experimental protocols are depicted in the left two panels. <b>(B)</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. <b>(C)</b> 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.</p

Development of primary tumors in the mammary ducts of female FVB/N mice following Mvt-1 cells injection through MIND method.

<p><b>(A)</b> Representative photograph of whole mount of 4^th inguinal mammary glands of female mice (FVB/N) injected with Mvt-1 cells for one week (n = 5). LN (arrow); Lymph node. <b>(B-C)</b> 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. <b>(D-E)</b> 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. <b>(F-G)</b> 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. <b>(H)</b> 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. <b>(I-J)</b> 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. <b>(K-L)</b> 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. <b>(M-N)</b> 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.</p

Immunohistochemical characterization of primary tumors and lung metastasis.

<p><b>(A-C)</b> 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. <b>(D-F)</b> 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. <b>(G-I)</b> 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. <b>(J-L)</b> 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. <b>(M-O)</b> 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.</p