br Sections were deparaffinized rehydrated
20. Sections were deparaffinized, rehydrated, and stained with 0.2% eosin (Sigma Aldrich) in H2O and Meyers HTX Plus (Histolab) for 5 min each. Metasta-ses b50 μm in diameter were regarded as micro-metastases and N50 μm as macro-metastases. Imag-ing was performed using an Olympus BX63 micro-scope, DP80 camera, and cellSens Dimension v 1.12 software (Olympus Cooperation).
For the cancer stem cell analysis, a piece from every tumor (10 per mouse) was collected in MACS Tissue Storage Solution (Miltenyi Biotec), tumor cells were dissociated utilizing the Tumor Dissociation Kit (Miltenyi Biotec), red blood cell were depleted with Red Blood Cell Lysis Solution (Miltenyi Biotec), protein epitopes were blocked using the FcR Blocking Reagent (Miltenyi Biotec), and 1 × 106 cells incubated with a mix of primary LDN-193189 (Table S1) and the same amount of cells incubated with a mix of isotype control antibodies. Finally, samples were analyzed with Cytoflex flow cytometer (Beckman).
Cancer stem cell characteristics are induced in COMP-expressing cells
To examine whether COMP expression alters cancer stem cell characteristics of breast cancer cell lines we
injected 3 different serial dilutions of MDA-MB-231 COMP-expressing cells and the corresponding control cells into the mammary fat pad of NSG mice (Fig. 1A). COMP-expressing cells yielded higher tumor positive mice compared to the control cells. Moreover, the theoretical frequency of cancer stem cells was 169 times higher in MDA-MB-231 COMP-expressing cells (χ2 = 28.9, p = 7.8 × 10−8) compared to the control cells, calculated with the ELDA software  (Fig. 1B, C). In addition, we evaluated the tumorsphere formation ability of MDA-MB-231 and BT-20 cells in vitro. COMP-transfected cells formed larger spheres compared to control cells, a hallmark of an increased cancer stem cell population (Fig. 1D). The increase in tumorsphere size was blocked by DAPT inhibitor or when Jagged1 binding to Notch3 was inhibited by a neutralizing antibody against the extracellular part of Jagged1 (Fig. 1D, E). Subsequently, we assessed aldehyde dehydrogenase (ALDH) enzyme activity (Fig. 2A), a well-established marker of cancer stem cells , and observed that COMP-expressing cells had significantly more ALDH activity compared with the control cells (Fig. 2B, C). When the activation of the Notch pathway was blocked using DAPT (a γ-secretase inhibitor, which blocks signaling from Notch receptors) in two different breast cancer cell lines engineered to express COMP (Fig. 2D), we observed a significant reduction of ALDH activity (Fig. 2B, C). The expression of the established stem cell marker CD133  was increased in COMP-expressing BT-20 cells (Fig. 2E), and the CD133 mRNA levels showed a corresponding down-regulation in the presence of DAPT (Fig. 2F). MDA-MB-231 cells lacked expression of CD133, as assessed by qPCR and flow cytometry (data not shown).
The Notch3 receptor is activated by COMP expression
In a previous study, we observed Notch pathway upregulation by COMP expression in a breast cancer xenograft model (Fig. S1) . Additionally, previous reports have indicated that Notch pathway activation can lead to cancer stem cell induction [10,11]. To confirm this, we isolated RNA from MDA-MB-231 COMP-expressing cells and evaluated Notch pathway related gene expression by a qPCR array. Several Notch-related genes were up- or down-regulated (Fig. 3A). Three genes were statistically significantly down-regulated in COMP-expressing cells compared to control cells: ADAM17 (p = 0.041), c-Cbl (p = 0.036), and β-Catenin (p = 0.021). To further confirm that Notch is activated in the presence of COMP, we performed a luciferase reporter assay on both cell lines. As expected, breast cancer cells expressing COMP had increased Notch activity com-pared to control cells (Fig. 3B). Among the Notch receptors, we only observed specific activation of Notch3 in COMP-expressing MDA-MB-231 cells,
112 COMP leads to activation of Notch3
compared to control cells (Fig. 3C), with similar data observed in the BT-20 cells (Fig. S2A). Moreover, MDA-MB-231 cells expressing COMP showed in-
creased Notch3 mRNA expression (Fig. 3D) while levels of Notch1 and Notch2 were not affected by COMP expression (Fig. S2B).
Fig. 2. Cancer stem cell population is enriched in breast cancer cell lines expressing COMP. (A) Representative flow cytometry dot plots of ALDH stained COMP expressing cells, control cells, and COMP expressing cells after addition of DAPT. (B) The population of ALDH positive cells is higher in COMP-expressing MDA-MB-231 cells and this population is reduced when Notch activation is inhibited by DAPT, (C) the same phenomenon is observed in BT-20 cells. (D) Western blot analysis of Notch3 activation in the presence of 10 mM of DAPT inhibitor. (E) Representative flow cytometry histograms of BT-20 cells labelled with CD133 specific antibody or corresponding isotype control. CD133 cancer stem cell marker is upregulated in BT-20 cells expressing COMP compared with the control cells. (F) The CD133 mRNA expression in BT-20 cells expressing COMP is reduced in presence of DAPT. One-way ANOVA, Bonferroni's multiple comparisons test was used when comparing 3 or more groups, (*b0.05, **b0.01, ***b0.001, ****b0.0001). NCID: Notch Intracellular Domain, NEXT: Notch Extracellular Truncated domain.