Supplementary tables from Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in Triple-Negative Breast Cancer

Supplementary Tables. Supplementary Tables (including statistical analysis of relationships between one-electron reductase activity, HR status and chemosensitivity) and supplementary references related to this material</p

Supplementary Methods, Tables 1-5, Figures 1-6 from The 2-Nitroimidazole EF5 Is a Biomarker for Oxidoreductases That Activate the Bioreductive Prodrug CEN-209 under Hypoxia

PDF file - 370K</p

Supplementary Figures from Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in Triple-Negative Breast Cancer

Supplementary Figures. Supplementary Figures including correlations of HAP sensitivity between cell lines, development and characterisation of doxycycline-inducible shRNA cell lines, and effect of BRCA2 knockout on radiation sensitivity of DLD-1 tumor xenografts</p

CCR Translation for This Article from The 2-Nitroimidazole EF5 Is a Biomarker for Oxidoreductases That Activate the Bioreductive Prodrug CEN-209 under Hypoxia

CCR Translation for This Article from The 2-Nitroimidazole EF5 Is a Biomarker for Oxidoreductases That Activate the Bioreductive Prodrug CEN-209 under Hypoxi

Table_1_Bystander Effects of Hypoxia-Activated Prodrugs: Agent-Based Modeling Using Three Dimensional Cell Cultures.pdf

Intra-tumor heterogeneity represents a major barrier to anti-cancer therapies. One strategy to minimize this limitation relies on bystander effects via diffusion of cytotoxins from targeted cells. Hypoxia-activated prodrugs (HAPs) have the potential to exploit hypoxia in this way, but robust methods for measuring bystander effects are lacking. The objective of this study is to develop experimental models (monolayer, multilayer, and multicellular spheroid co-cultures) comprising ‘activator’ cells with high expression of prodrug-activating reductases and reductase-deficient ‘target’ cells, and to couple these with agent-based models (ABMs) that describe diffusion and reaction of prodrugs and their active metabolites, and killing probability for each cell. HCT116 cells were engineered as activators by overexpressing P450 oxidoreductase (POR) and as targets by knockout of POR, with fluorescent protein and antibiotic resistance markers to enable their quantitation in co-cultures. We investigated two HAPs with very different pharmacology: SN30000 is metabolized to DNA-breaking free radicals under hypoxia, while the dinitrobenzamide PR104A generates DNA-crosslinking nitrogen mustard metabolites. In anoxic spheroid co-cultures, increasing the proportion of activator cells decreased killing of both activators and targets by SN30000. An ABM parameterized by measuring SN30000 cytotoxicity in monolayers and diffusion-reaction in multilayers accurately predicted SN30000 activity in spheroids, demonstrating the lack of bystander effects and that rapid metabolic consumption of SN30000 inhibited prodrug penetration. In contrast, killing of targets by PR104A in anoxic spheroids was markedly increased by activators, demonstrating that a bystander effect more than compensates any penetration limitation. However, the ABM based on the well-studied hydroxylamine and amine metabolites of PR104A did not fit the cell survival data, indicating a need to reassess its cellular pharmacology. Characterization of extracellular metabolites of PR104A in anoxic cultures identified more stable, lipophilic, activated dichloro mustards with greater tissue diffusion distances. Including these metabolites explicitly in the ABM provided a good description of activator and target cell killing by PR104A in spheroids. This study represents the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the power of combining mathematical modeling of pharmacokinetics/pharmacodynamics with multicellular culture models to dissect bystander effects of targeted drug carriers.</p

The demographic and clinical characteristics of PE and control groups.

<p>The demographic and clinical characteristics of PE and control groups.</p

Expression on <i>SOX2</i> and <i>SOX2OT</i> in breast cancer cell lines grown in suspension culture.

<p>A and B) Expression of <i>SOX2</i> and <i>SOX2OT</i> in MDA-MB-231 (A) and MCF-7 (B) breast cancer cell lines cultured in suspension relative to the cells grown as monolayer was measured by qRT-PCR. Three consecutive passages of cells were grown in suspension as S-p1 to S-p3 respectively. M represents the expression in monolayer culture. Error bars represent the standard error of the mean of three biological replicates. C) Western blot analysis showing expression of SOX2 in MCF-7 and MDA-MB-231 cells grown as monolayers and in suspension. D) Expression of <i>SOX2</i> and <i>SOX2OT</i> in TamC3 and TamR3 derivatives of MCF-7 grown in suspension relative to MCF-7 monolayer culture measured by qRT-PCR. Error bars represent standard deviations of three technical replicates. E) Expression of <i>SOX2</i> and <i>SOX2OT</i> in monolayer MDA-MB-231-luc-D3H2LN cells (<i>in vitro</i>) and the same cells grown as orthotopic xenografts (<i>in vivo</i>) were measure by qRT-PCR. The error bars are standard error of the mean of six individual tumors. *, **, and *** represent <i>p</i> values of &lt;0.05, 0.01 and 0.001 respectively.</p

Supplementary Figures 1-3 from Selective Tumor Hypoxia Targeting by Hypoxia-Activated Prodrug TH-302 Inhibits Tumor Growth in Preclinical Models of Cancer

PDF file - 2.7MB</p

Schematic genomic organization of <i>SOX2</i> and <i>SOX2OT</i> and their expression patterns in breast cancer.

<p>A) The genomic context of <i>SOX2</i> and its upstream region. The gray boxes show the proposed promoter and enhancer regions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102140#pone.0102140-Miyagi1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102140#pone.0102140-Kondo1" target="_blank">[27]</a>. B) The genomic context of <i>SOX2</i> and <i>SOX2OT</i>, derived from the UCSC browser. <i>SOX2</i> is located in an intron of <i>SOX2OT</i>. The triangles above each gene show the location of primers used in qRT-PCR. The <i>SOX2</i> region is enlarged, and the direction of gene transcription shown with arrows. The phylogenetic conservation of each region is shown below the gene diagram (Mammal Cons). Vertical rectangular shading shows the conserved exonic regions of <i>SOX2OT</i>. The locations of these genes are adopted from UCSC genome browser March 2006 (NCBI36/hg18). C) Heat map showing the expression of <i>SOX2</i> and <i>SOX2OT</i> in breast cancer samples analyzed by TCGA. It covers the expression of <i>SOX2OT</i>; chromosome 3: 182810845–182941699. <i>SOX2</i> region is shown by an arrow. The samples are classified based on the estrogen receptor status. D and E) Expression of <i>SOX2</i> and <i>SOX2OT</i> respectively in ER+ (n = 595) and ER− (n = 176) breast cancer samples (data derived from TCGA). Mann-Whitney rank sum test showed that these genes were expressed differently according to estrogen status: <i>p</i> values of &lt;0.05 and &lt;0.005 were calculated for D and E respectively.</p

Expression of SOX2 and <i>SOX2OT</i> in different breast cancer cell lines.

<p>A) The expression of <i>SOX2</i> and <i>SOX2OT</i> relative to <i>HPRT</i> and <i>GAPDH</i> in 18 breast cell lines measured by qRT-PCR. Scatter plot showing the expression of <i>SOX2</i> and <i>SOX2OT</i>. Black and white circles represent ER− and ER+ cell lines. B) Box plot indicating expression of <i>SOX2</i> and <i>SOX2OT</i> in ER+ and ER− breast cancer cell lines. * represents <i>p</i> value &lt;0.05. C) Expression of <i>SOX2</i> and <i>SOX2OT</i> in five MCF-7 sub-lines relative to the MCF-7 parental line was measured by qRT-PCR. Error bars represent standard deviations of three technical replicates.</p