Increased <i>Lrp6</i> expression in basal-like human breast cancer.

<p>The relative expression of <i>Lrp6</i> in breast cancer samples analyzed by Affymetrix and organized into defined subgroups. Each dot represents the relative expression of <i>Lrp6</i> in one tumor sample. (A) Expression data was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005813#pone.0005813-Richardson1" target="_blank">[30]</a>. <i>Normal</i> includes samples obtained from normal breast tissue. (B) Expression data was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005813#pone.0005813-Herschkowitz1" target="_blank">[31]</a>. <i>Normal Breast-like</i> includes samples obtained from normal and cancerous breast tissue that exhibited expression profiles similar to that of normal breast tissue. A subset of breast cancers within the basal-like subgroup of both studies exhibit increased expression of <i>Lrp6</i>.</p

Expression pattern of <i>Lrp6</i> in the mammary gland.

<p>β<i>-gal</i> expression directed from the <i>Lrp6</i> promoter in <i>Lrp6^+/−</i> mice was used as a surrogate marker for <i>Lrp6</i> expression. We used the β<i>-gal</i> substrates X-gal and DDAOG to identify cells that express <i>Lrp6</i>. Shown in (A-B) are X-gal-stained mammary whole mounts and 5-µm sections from 2-day-old (A) and 12-week-old (B) female mice. (A) <i>Lrp6</i> promoter-driven β<i>-gal</i> expression is visible as blue staining in both the basal and luminal mammary epithelium and in the mammary fat pad of newborn mice. (B) In older mice, β<i>-gal</i>-expressing cells are primarily identified within the basal epithelial cell layer and in the mammary fat pad. The <i>arrows</i> indicate typical cells that stained blue with X-gal. No staining was detected in <i>Lrp6^+/+</i> mammary glands, which were used as negative controls for X-gal staining. (C-F) Representative FACS results of DDAOG-stained and CD24/CD49f antibody–labeled mammary epithelial cells. The β<i>-gal</i>-cleaved product of DDAOG has far-red fluorescence and was used to detect cells with <i>Lrp6</i> promoter–driven β<i>-gal</i> expression. (C) The luminal and basal cell compartments are marked by pink and blue dashed lines, respectively. (D) 80% of the DDAOG-positive cells are found within the basal epithelial cell compartment. (E-F) DDAOG gating strategy. The DDAOG gate is indicated by the black line. The <i>Lrp6^+/−</i> sample (E) has 0.33% DDAOG-positive cells; the <i>Lrp6^+/+</i> negative control sample (F) has 0.01% DDAOG-positive cells.</p

Haploinsufficiency for <i>Lrp6</i> in postnatal mammary development.

<p>(A) Representative mammary whole-mount preparations are shown for juvenile (5-week-old) mice. The <i>arrows</i> indicate typical terminal end buds; <i>LN</i>, lymph node. (B) The result of morphometric analysis of the average number of TEBs at 5 weeks and (C) of branches per gland at 11 weeks. At least 10 animals of each genotype were analyzed for each time point. In the absence of one copy of <i>Lrp6</i>, the number of TEBs is reduced by 33% (<i>p</i> = 1.3×10⁻⁶, 2-tailed <i>t</i> test assuming unequal variances), and the number of branches per gland is reduced by 17% (<i>p</i> = 8.4×10⁻³, 2-tailed <i>t</i> test assuming unequal variances) compared with <i>Lrp6</i>^+/+ littermate controls. (D) Mammary whole mounts containing ductal colonization originating from transplanted <i>Lrp6^+/+</i> or <i>Lrp6^+/−</i> mammary epithelial cells. Also shown is a transplantation control whose inguinal fat pads were cleared of endogenous epithelium but not injected with mammary cells. (E) The inguinal mammary gland from an adult <i>Lrp6^+/−;Lrp5^−/−</i> female. The mammary gland contains a fat pad and a nipple with associated epithelium but lacks a ductal tree. <i>Box</i> indicates the nipple epithelium.</p

Wnt1-induced mammary tumorigenesis in <i>Lrp6^+/−</i> females.

<p>Shown in (A-B) are representative carmine stained skin pads and mammary whole mounts. (A) Skin pads collected from E18.5 <i>MMTV-Wnt1;Lrp6^+/+</i> and <i>MMTV-Wnt1;Lrp6^−/−</i> embryos. (B) Mammary whole mounts collected from adult <i>MMTV-Wnt1;Lrp6^+/+</i> and <i>MMTV-Wnt1;Lrp6^+/−</i> females. (C) The percentages of <i>MMTV-Wnt1;Lrp6^+/+</i> (<i>n</i> = 12) and <i>MMTV-Wnt1;Lrp6^+/−</i> mice (<i>n</i> = 13) that were tumor-free (as determined by weekly visual inspection and/or palpation) were plotted against the age when tumors were found. (D) Standard histopathological evaluation showed that all <i>Lrp6^+/+</i> and <i>Lrp6^+/− MMTV-Wnt1</i> tumors are moderately differentiated alveolar mammary adenocarcinomas.</p

<i>Lrp6</i> is required for embryonic mammary development.

<p>(A) Carmine-stained skin pads of inguinal mammary glands at E18.5. While in the <i>Lrp6^+/+</i> mammary gland the nipple, rudimentary ductal tree, and fat pad are all normally developed, the <i>Lrp6^−/−</i> mammary gland contains a small nipple, a single ductal out-growth, and an abnormally small fat pad. <i>Dashed lines</i> indicate inguinal epithelium. (B) Oil red O staining of mammary fat pads from <i>Lrp6^+/+</i> and <i>Lrp6^−/−</i> embryos. The <i>Lrp6^−/−</i> fat pad is abnormally small compared to that of the littermate control. <i>Scale bar</i>: 0.5 mm. (C-D) X-gal-stained <i>BAT-gal</i> embryo whole mounts (C) and histology sections of mammary placode (D) at E12.5. Cells expressing BAT-gal are stained blue. (C) X-gal stains the mammary placodes of <i>BAT-gal;Lrp6^+/+</i> embryos dark blue. <i>Arrow heads</i> indicate mammary placodes number 2, 3, 4, and 5. Mammary placodes are not readily visible on X-gal-stained <i>BAT-gal;Lrp6^−/−</i> embryo whole mounts. (D) On the histological level, the mammary placodes of <i>BAT-gal;Lrp6^−/−</i> embryos are significantly smaller and exhibit fewer cells with <i>BAT-gal</i> expression than the mammary placodes of littermate controls. <i>Dashed lines</i> indicate inguinal placodes.</p

Additional file 1 of Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial

Additional file 1: Table S1. Cell lines and mice models established. A total of 96 patients enrolled onto the NMTRC009 MGT trial had at least one tumor cell line generated in the laboratory setting. Since many subjects underwent multiple tumor biopsies and/or bone marrow biopsies, subjects may have >1 unique cell line, either from the same tumor obtained from different biopsy dates or from a different disease site (bone marrow). 47 subjects’ tumors underwent successful implantation into a NOD-SCID mouse to generate at least one PDX model. A total of 56 unique PDX models were generated