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Figure S3 - PDF file 26K, Figure S3. AD-01 is not cytotoxic. No change in surviving fraction following AD-01 treatment in both MDA-231 and MCF-7 cells was observed.

Table S2 - PDF file 86K, A: Clinical information and the average MFE for every solid primary breast tumour received; tumours of similar grade and status were combined and the average MFE calculated. B: Tumour initiation data following implantation of MCF-7 cells and AD-01 or PBS treatment from day 1 of implantation

Figure S6 - PDF file 42K, Figure S6. High FKBPL and low Sox2 expression shows a trend towards better patient survival. Samples with opposing FKBPL and Sox2 expression (high FKBPL/low Sox2 and low FKBPL/high Sox2) were analyzed using microarray data from publically available data set (GSE7390). Samples were stratified based on median expression values of Sox2 and FKBPL. Kaplan-Meier survival curves of breast cancer patients (n=104) presented

Figure S4 - PDF file 73K, Figure S4. (A) Protein levels of Nanog and Oct4 in MDA-231 cells following treatment with HA or AD-01, alone or in combination. (B) Protein levels of Nanog and Oct4 in MDA-231 cells following transfection with FKBPL siRNA (siFKBPL) or NT siRNA (siNT) evaluated by immunofluorescence

Figure S1 - PDF file 49K, Figure S1. (A) Mammosphere formation efficiency (MFE) in MDA-231 cells exposed to varying concentrations of AD-01. (B) Primary (1), secondary (2) and tertiary (3) mammosphere formation in the MDA-231 cell line following 1 nM AD-01 treatment (black bar - control; grey bar - AD-01 treatment). See Fig. 1 for further description of the methods. (C) 1 and 2 mammosphere formation in the MCF-7 cell line versus MCF-7 cells stably overexpressing FKBPL (D2). Data points are mean plus-minus SEM. n greater than or equal to 3. * p

Supplementary Data Legends - PDF file 63K, Data legends for supplemental figs S1 to S7

Table S1 - PDF file 66K, Percentage ESA+/CD44+/CD24- or ALDH+ cells gated using flow cytometry (n=3)

Figure S7 - PDF file 47K, Figure S7. High FKBPL and low Oct4 expression shows a trend towards better patient survival. Samples with opposing FKBPL and Oct4 expression (high FKBPL/low Oct4 and low FKBPL/high Oct4) were analyzed using microarray data from publically available data set (GSE7390). Samples were stratified based on median expression values of Oct4 and FKBPL. Kaplan-Meier survival curves of breast cancer patients (n=104) presented

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC).Experimental Design: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti–stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence.Results: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA+/CD44+/CD24− or aldehyde dehydrogenase (ALDH)+ cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01–mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling.Conclusions: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial. Clin Cancer Res; 19(14); 3881–93. ©2013 AACR.

Figure S5 - PDF file 48K, Figure S5. High FKBPL and low Nanog expression correlates with better DMFS and shows a trend towards better RFS. Samples with opposing FKBPL and Nanog expression (high FKBPL/low Nanog and low FKBPL/high Nanog) were analysed using microarray data from publically available data set (GSE7390). Samples were stratified based on median expression values of NANOG and FKBPL. Kaplan-Meier survival curves of breast cancer patients (n=94) presented