Tumorgraft response to carboplatin and paclitaxel with or without targeted therapy.

<p>(A) Kaplan-Meier Curve showing animal survival (log-rank test p = 0.8456). (B) There was no difference in final tumor weight between groups (one-way ANOVA p = >0.05 each comparison), which was the primary endpoint. (C) Western blot of PAR in tumor treated with ABT-888. (D) Western blot of total and phospho-AKT in tumors after treatment. Abbreviations: Carboplatin and paclitaxel (C/P), EVRI (pan-HER and VEGF inhibitor BMS 690514), IGF-1Ri (BMS 754807), PARPi (ABT-888).</p

Dosing algorithms for dose finding portion of study.

<p>*Starting dose level</p><p>Dosing algorithms for dose finding portion of study.</p

Tumorgraft response to treatment in vivo.

<p>Mice were randomized to receive (n = 13 each group) either saline, carboplatin and paclitaxel (C/P), or ifosfamide and paclitaxel (I/P). (A) Kaplan-Meier Curve demonstrates survival. The C/P and I/P curves were significantly different, log-rank test p = 0.0027. (B) Tumor palpation score over time reflects the change in tumor size with treatment. (C) Tumor weights in each cohort were normalized against the final mean control weight obtained at necropsy on day 7 (which represents the maximum-allowed tumor burden). The difference between C/P and I/P relative tumor mass was significant (two-tailed t test, p <0.0001).</p

Potential targets of novel therapies.

<p>(A) Receptor tyrosine kinase (RTK) dot blot array. Goat IgG and phosphate buffered saline (PBS) are background controls. Each RTK is blotted in duplicate. (B) The proportion of signal contributed by select RTKs in serum free media (SFM), complete media (CM), and in tissues harvested after engraftment in a mouse (<i>in vivo</i>). (C) Microarray analysis for expression of EGFR, VEGFC, and IRS1 in model PH003 (red shape) and 35 other ovarian patient-derived tumorgraft models (black shapes). (D) Array comparative genomic hybridization showing chromosome 7. Red dots represent probes from the tumor genome and Green dots represent probes from the patient’s germline DNA. A balance of Red and Green probes signifies equal representation of tumor and germline DNA in the q arm. The p arm shows an unbalanced representation of tumor genomic DNA (log2 ratio 0.399 compared to germline). The bold vertical jagged line shows the average log2 ratio of tumor and germline DNA along chromosome 7.</p

Hematoxylin and Eosin staining of PH003.

<p>Patient (A & B) and tumorgraft (C & D) tissues are shown. Images were captured with a 40x objective.</p

Correlation of abdominal palpation score and tumor weight at necropsy.

<p>(A) The palpation score was based on tumor size relative to the mouse hemi-pelvis. (B) Palpation score prior to necropsy correlated with tumor weight at necropsy (Pearson r = 0.8965, two-tailed p <0.0001).</p

IRS isoforms mediate distinct gene expression profiles, functional pathways, and breast cancer subtype association.

<p>(A) Venn diagrams depicting four distinct IRS isoform gene signatures were derived from overlapping and differential global gene expression patterns in response to IGF-I. (B) Target gene validation confirms both distinct and overlapping patterns of IRS-regulated gene expression. Gene expression was normalized to RPLP0 and is presented as fold-change of treatment (black bars) vs. serum-free (white bars) conditions. Error bars represent standard deviation and all results are representative of at least three independent replicates. (C) IRS gene signature enrichment in breast tumor subtypes in the UNC337 cohort. Median expression values are represented here in graphical format with p-values included for each of the IRS gene signatures.</p

IRS proteins regulate TGFβ2 mRNA expression and breast cancer cell motility.

<p>(A) Expression of TGFβ1 and TGFβ2 by qPCR in T47D-YA-IRS-1 (#10 and #20) and T47D-YA-IRS-2 (#1 and #6). (B) IGF-induced TGFβ2 expression in MCF10A, MCF-7L, MCF-7 ATCC, MDA-231 and F11 cells. For A & B, all cells were exposed to 5nm IGF-I for 4 hours prior to harvesting mRNA. Gene expression was normalized to RPLP0 and is presented as fold-change of treatment (black bars) vs. serum-free (white bars) conditions. (C) TGFβ2 expression was assessed by qPCR in an IRS-gene deletion mouse models (left) and IRS-overexpressing SH-EP neuroblastoma cells (right). (D) IRS-1, IRS-2 and TGFβ2 expression in a panel of patient breast tumors. Arrows indicate invasive breast carcinoma. Yellow bars signify high gene expression, blue bars signify low gene expression. E) pSMAD2 was examined by immunoblot at the indicated time points in MCF-7 cells. (F) Cell motility was examined by modified Boyden chamber assay. MCF-7 cells were incubated in the presence of neutralizing antibodies to either TGFβ1 or TGFβ2 and IGF-induced motility assessed. Error bars represent standard deviation and all results are representative of at least three independent replicates.</p

Gene set enrichment analysis for each IRS gene signature was performed using DAVID (Database of Annotation, Visualization and Integrated Discovery, v6.7).

<p>P-value indicates modified Fisher’s exact Probability Value and a high E-Scores (Enrichment Scores) indicates significant gene enrichment in the annotation cluster.</p

Multivariate Cox regression analysis of RFS & OS in Luminal B breast cancer tumors.

<p>Multivariate Cox regression analysis of RFS & OS in Luminal B breast cancer tumors.</p