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Patient data.

Control arms for Fig. 6 experiments AND Potential future approaches.

Purpose: Investigation of clonal heterogeneity may be key to understanding mechanisms of therapeutic failure in human cancer. However, little is known on the consequences of therapeutic intervention on the clonal composition of solid tumors.Experimental Design: Here, we used 33 single cell–derived subclones generated from five clinical glioblastoma specimens for exploring intra- and interindividual spectra of drug resistance profiles in vitro. In a personalized setting, we explored whether differences in pharmacologic sensitivity among subclones could be employed to predict drug-dependent changes to the clonal composition of tumors.Results: Subclones from individual tumors exhibited a remarkable heterogeneity of drug resistance to a library of potential antiglioblastoma compounds. A more comprehensive intratumoral analysis revealed that stable genetic and phenotypic characteristics of coexisting subclones could be correlated with distinct drug sensitivity profiles. The data obtained from differential drug response analysis could be employed to predict clonal population shifts within the naïve parental tumor in vitro and in orthotopic xenografts. Furthermore, the value of pharmacologic profiles could be shown for establishing rational strategies for individualized secondary lines of treatment.Conclusions: Our data provide a previously unrecognized strategy for revealing functional consequences of intratumor heterogeneity by enabling predictive modeling of treatment-related subclone dynamics in human glioblastoma. Clin Cancer Res; 23(2); 562–74. ©2016 AACR.

Legends for Supplementary Figs. S1-S8 and Supplementary Tables

Killer plate® compounds and screening data.

Supplementary figure legends for Supplementary Figures 1-3. In addition, supplementary Methods with detailed protocols and sequence information.

Extreme limiting dilution assay AND Expression of signature neural stem-like genes.

Confirmatory subclone selection experiments applying clinical sample BN035.

Glioma stem cells (GSC) and epithelial–mesenchymal transition (EMT) are strongly associated with therapy resistance and tumor recurrence, but the underlying mechanisms are incompletely understood. Here, we show that S100A4 is a novel biomarker of GSCs. S100A4+ cells in gliomas are enriched with cancer cells that have tumor-initiating and sphere-forming abilities, with the majority located in perivascular niches where GSCs are found. Selective ablation of S100A4-expressing cells was sufficient to block tumor growth in vitro and in vivo. We also identified S100A4 as a critical regulator of GSC self-renewal in mouse and patient-derived glioma tumorspheres. In contrast with previous reports of S100A4 as a reporter of EMT, we discovered that S100A4 is an upstream regulator of the master EMT regulators SNAIL2 and ZEB along with other mesenchymal transition regulators in glioblastoma. Overall, our results establish S100A4 as a central node in a molecular network that controls stemness and EMT in glioblastoma, suggesting S100A4 as a candidate therapeutic target. Cancer Res; 77(19); 5360–73. ©2017 AACR.

Box plots showing IC50 values from pilot data pharmacological profiling.

Patient data. AND Drug and compound details. AND Primer sequences for qRT-PCR.

Supplementary Table 2 listing significantly (q,0.05 and FC>2) in S100A4-KD cells (common to both siRNA compared to scrambled control siRNA).

Confirmatory subclone selection experiments applying clinical sample BN046.

Supplementary legend

Supplementary Table 4 showing IPA prediction of upstream regulators for downregulated genes in S100A4 knockdown cells

Niclosamide dose-response curves.

Cell cycle analysis of pGBM #046 after niclosamide exposure.

List of pGBMs and their respective passage numbers used for the various experimental paradigms in this study.

Supplementary Table 5 showing IPA prediction of upstream regulators for up-regulated genes in S100A4 knockdown cells

Maintenance of patient- and disease-specific genetic alterations ex vivo.

Full-length blots presented in this study.

Purpose: Glioblastoma is a highly malignant, invariably fatal brain tumor for which effective pharmacotherapy remains an unmet medical need.Experimental Design: Screening of a compound library of 160 synthetic and natural toxic substances identified the antihelmintic niclosamide as a previously unrecognized candidate for clinical development. Considering the cellular and interindividual heterogeneity of glioblastoma, a portfolio of short-term expanded primary human glioblastoma cells (pGBM; n = 21), common glioma lines (n = 5), and noncancer human control cells (n = 3) was applied as a discovery platform and for preclinical validation. Pharmacodynamic analysis, study of cell-cycle progression, apoptosis, cell migration, proliferation, and on the frequency of multipotent/self-renewing pGBM cells were conducted in vitro, and orthotopic xenotransplantation was used to confirm anticancer effects in vivo.Results: Niclosamide led to cytostatic, cytotoxic, and antimigratory effects, strongly reduced the frequencies of multipotent/self-renewing cells in vitro, and after exposure significantly diminished the pGBMs' malignant potential in vivo. Mechanism of action analysis revealed that niclosamide simultaneously inhibited intracellular WNT/CTNNB1-, NOTCH-, mTOR-, and NF-κB signaling cascades. Furthermore, combinatorial drug testing established that a heterozygous deletion of the NFKBIA locus in glioblastoma samples could serve as a genomic biomarker for predicting a synergistic activity of niclosamide with temozolomide, the current standard in glioblastoma therapy.Conclusions: Together, our data advocate the use of pGBMs for exploration of compound libraries to reveal unexpected leads, for example, niclosamide that might be suited for further development toward personalized clinical application. Clin Cancer Res; 19(15); 4124–36. ©2013 AACR.

Supplementary data showing expression and lontg-term self-renewal of S100A4+/GFP+ cells in spontaneous and transplanted tumors, including reactivation of S100A4 in S100A4-KD tumors. IPA and GSEA analyses showing pathways enriched in S100A4 expressing cells.

Supplementary Figure and Table Legends from Aldehyde Dehydrogenase–Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer

Supplementary Figure 1 from Aldehyde Dehydrogenase–Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer

Supplementary Table 1 from Aldehyde Dehydrogenase–Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer

Patients with chronic ulcerative colitis are at increased risk of developing colorectal cancer. Although current hypotheses suggest that sporadic colorectal cancer is due to inability to control cancer stem cells, the cancer stem cell hypothesis has not yet been validated in colitis-associated cancer. Furthermore, the identification of the colitis to cancer transition is challenging. We recently showed that epithelial cells with the increased expression of aldehyde dehydrogenase in sporadic colon cancer correlate closely with tumor-initiating ability. We sought to determine whether ALDH can be used as a marker to isolate tumor-initiating populations from patients with chronic ulcerative colitis. We used fluorescence-activated cell sorting to identify precursor colon cancer stem cells from colitis patients and report both their transition to cancerous stem cells in xenografting studies as well as their ability to generate spheres in vitro. Similar to sporadic colon cancer, these colitis-derived tumors were capable of propagation as sphere cultures. However, unlike the origins of sporadic colon cancer, the primary colitic tissues did not express any histologic evidence of dysplasia. To elucidate a potential mechanism for our findings, we compared the stroma of these different environments and determined that at least one paracrine factor is up-regulated in the inflammatory and malignant stroma compared with resting, normal stroma. These data link colitis and cancer identifying potential tumor-initiating cells from colitic patients, suggesting that sphere and/or xenograft formation will be useful to survey colitic patients at risk of developing cancer. [Cancer Res 2009;69(20):8208–15]

Supplementary Figure 2 from Aldehyde Dehydrogenase–Expressing Colon Stem Cells Contribute to Tumorigenesis in the Transition from Colitis to Cancer