Journal: Cancer Cell International

Article Title: The potential role of collagen type VII in breast cancer proliferation

doi: 10.1186/s12935-024-03449-4

Figure Lengend Snippet: Non-tumourigenic MCF-12 cells express higher Collagen type VII compared with tumourogenic MCF-7 breast cancer line. A. Gene expression analysis of laminins; LAMA1 , LAMA3 , LAMA5 , non-collagens; Fibronectin ( FN1 ), Nidogen-1 ( NID-1 ), Tenascin-C ( TNC ), collagens; COL1A1 , COL3A1 , COL4A1 , COL5A1 , COL6A1 , COL7A1 and integrins ( INTA6 and INTB4 ) in MCF-7 cells exposed to normoxia (N), hypoxia (H) or restored normoxia (rN). Data are expressed as means of triplicates and represent fold change relative to N (100) with colour coding (White = 100, Green > 100 and Red < 100). B. Left: Representative western blot against Collagen type VII (ColVII), Cyclin-D1 and β-Actin in MCF-7 cells exposed to normoxia (N) and hypoxia (H). Right: Quantification of Western blot bands (Densitometric units) showing relative expression of the target proteins to β-Actin. Data are expressed as means ± SEM of triplicates. P -values represent the t-tests N vs. H. C. Left: Representative Ki67 (Red) staining of MCF-7 and MCF-12 cultures. Nuclei are stained with DAPI (blue). Scale bar = 50 μm. Insets: Dotted areas´ magnifications. Right: Quantification in percentage of Ki67 positive MCF-7 and MCF-12 per total cells/area after 5 days of culture. Data are expressed as means ± SEM of four experiments. P -values represent the t-tests MCF-7 vs. MCF-12.. D. Left: Representative Collagen type VII (Red) staining of MCF-7 and MCF-12 cultures. Nuclei are stained with DAPI (blue). Scale bar = 50 μm. Insets: Dotted areas´ magnifications. Right: Collagen-VII signal quantification (Pixels) in MCF-7 and MCF-12 cultures per area after 5 days of culture. Data are expressed as means ± SEM of triplicates. P -values represent the t-tests between MCF-7 vs. MCF-12. E. Above: Representative western blot against Collagen type VII (ColVII), Cyclin-D1 and β-Actin in MCF-7 and MCF-12 cells after 5 days of culture. Below: Quantification of Western blot bands (Densitometric units) showing relative expression of the target proteins to β-Actin. Data are expressed as means ± SEM of triplicates. P -values represent t-tests MCF-7 vs. MCF-12

Article Snippet: Culture plates were coated with 2–20 ng/mL human recombinant ColVII (rhColVII; Origene, Rockville, MD, USA, #TP318470) in 0.5% Gelatin (Merck.

Techniques: Expressing, Western Blot, Staining

Journal: Cancer Cell International

Article Title: The potential role of collagen type VII in breast cancer proliferation

doi: 10.1186/s12935-024-03449-4

Figure Lengend Snippet: Spheroids of Collagen type VII high expressing AdMSCs reduce the proliferation of MCF-7 cells. A. Schematic picture of the experimental procedure to sort high and low ColVII expressing AdMSCs using CoCl 2 induced hypoxia and magnetic beads via anti Integrin-α6 antibody conjugated with APC. Both subpopulations were used to generate spheroids. After one day, 25 000 MCF-GFP cells were put in contact with the spheroid’s cultures for 24 h. MCF-7 attached to AdMSC cocultures were transferred into a new vial, the GFP signal measured and cultured for 5 days. The GFP signal was measured at day 1 and day 5 and the expression of ki67 measured at day 5. B. Left: Representative immunocytochemical staining of ColVII (red) in Integrin-6α + (Int-α6 + ) and Integrin-6α − (Intα-6 − ) spheroids after 2–3 days of culture following sorting. Nuclei are stained with DAPI (blue). Scale bar = 50 μm. Right: ColVII signal quantification (Pixels) per area of Int-α6 + and Int-α6 − spheroids 2–3 days of spheroid culture. Data are expressed as means ± SEM of triplicates. P -values were calculated by t-tests between Int-α6 + vs. Int-α6 − . C. Left: Representative immunocytochemical staining of GFP in Int-α6 + and Int -α6 − spheroids after one and five days of coculture with MCF-7-GFP. Scale bar = 50 μm. Right: GFP signal quantification at day one and day five. Data are expressed as means ± SEM of triplicates. P -values were calculated by t-tests between Int-α6 + vs. Int-α6 − . D. Above: Representative immunocytochemical staining of Ki67 (red) and GFP (green) in Int-α6 + and Int-α6 − spheroids after 5 days of coculture with MCF-7-GFP. Scale bar = 50 μm. Below: GFP and Ki67 signal quantification on day five. Data are expressed as means ± SEM of three independent experiments with different number of spheroids combined, with each data point representing one spheroid. Means have been grouped and p value has been calculated by t-test

Article Snippet: Culture plates were coated with 2–20 ng/mL human recombinant ColVII (rhColVII; Origene, Rockville, MD, USA, #TP318470) in 0.5% Gelatin (Merck.

Techniques: Expressing, Magnetic Beads, Cell Culture, Staining

Journal: Cancer Cell International

Article Title: The potential role of collagen type VII in breast cancer proliferation

doi: 10.1186/s12935-024-03449-4

Figure Lengend Snippet: Collagen type VII expression in human breast tissue tumour area A. Left: Representative immunohistochemical ColVII staining in human triple negative breast cancer (TNBC) and estrogen receptor (ER) positive breast cancer. Scale bar = 100 μm. Right: Graph showing each patient’s ( n = 15) percentage of positive ColVII-expression in tumour area stratified by hormone receptor status, either as ER-positive or TNBC. P -value was calculated by Mann-Whitney U test between ER-positive and TNBC. B. Above left: Representative immunohistochemical staining of CK5 in healthy breast terminal duct lobular unit of the mammary gland and ER-positive tumours. Above center: same histological site as above left showing representative ColVII staining and zoom in the selected area in right. Below left: Representative histological staining of CK5 in ER-positive breast tissue ducts. Below center: same histological site that below left showing a representative ColVII staining and zoom in the selected area in right. Scale bar = 100 μm

Article Snippet: Culture plates were coated with 2–20 ng/mL human recombinant ColVII (rhColVII; Origene, Rockville, MD, USA, #TP318470) in 0.5% Gelatin (Merck.

Techniques: Expressing, Immunohistochemical staining, Staining, MANN-WHITNEY