br Bar graphs indicate mean and standard error across
Bar graphs indicate mean and standard error across biological re-plicates (n ≥ 3), unless otherwise indicated. n in all Figure legends corresponds to biological replicates. Significance was assessed using two-tailed t-tests, and a p value of ≤ 0.05 was considered as statistically significant. For immunofluorescence intensity and Heparin quan-titation, bars indicate median and interquartile range within a single experiment; two-tailed t-tests for these experiments were performed on medians from n ≥ 3 biological replicates. For Kaplan Meier plots, sta-tistical significance was assessed using a log rank test. Plots were gen-erated using R through R Studio (v1.1.383), using R package ggplot2 (v2.2.1) .
4.7. Data availability
The datasets generated and analysed in this study are included within the manuscript (and Supplementary) or have been deposited in the Gene Expression Omnibus (GEO, for RNA-Seq and ChIP-Seq data) under accession number GSE103794. All other data are available upon reasonable request.
Assays for phenotypes (clonogenic, proliferation, anoikis), Immunofluorescence, RNA-Seq, qPCR, ChIP-Seq are described in Supplementary Methods.
The authors thank Simon Andrews and Felix Krueger (Babraham Institute) for help and guidance with ChIP Seq analysis; Tapas Kundu, Simon Andrews, Colin Jamora, Aswin S.N. Seshasayee, Sasikala P.S. for insights and discussions; C-CAMP and Agrigenome labs for Illumina sequencing.
Conception and design: C. Rodrigues, S. Krishna, P. Varga-Weisz, C. Pattabiraman, D. Notani
Development of methodology: C. Rodrigues, C. Pattabiraman
Acquisition of data: C. Rodrigues, C. Pattabiraman, A. Vijaykumar, R. Arora
Clinical biopsy data: C. Rodrigues, S. Sheshadri, R.V. Kumar
Analysis and interpretation of data: C. Rodrigues, S. Krishna, P. Varga-Weisz, D. Notani, R. Arora
Writing, review and/or revision of the manuscript: C. Rodrigues, S. Krishna
Study supervision: S. Krishna, P. Varga-Weisz, D. Notani, R.V. Kumar
This work was supported by grants from DBT, NCBS-TIFR, and travel support from ICGEB, EMBL CPP, DBT, Infosys Foundation, Boehringer Ingelheim Fonds, SOUND consortium, and EACR (C. Rodrigues). Dr. Reety Arora was supported by the Wellcome Trust/DBT India Alliance (Early Career Award IA/E/14/1/501773).
Conflicts of interest statement
Appendix A. Supporting information
C. Rodrigues, et al.
A Syngeneic Mouse Model of Epithelial Ovarian Cancer Port Site Metastases
Ivy Wilkinson-Ryan* †, Melissa M. Pham*, Petra Sergent†, Laura J. Tafe*,† and Brent L. Berwin†
* Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA; †Geisel School of Medicine at Dartmouth, One Medical Center Drive, Lebanon, NH 03756, USA
Epithelial ovarian cancer (EOC) is a deadly gynecologic malignancy, but animal models for the study of EOC pathophysiology and drug efficacy are limited. Based on the finding that women with EOC are at risk for metastasis at a trocar site after laparoscopy, we developed a syngeneic murine model of port-site metastasis of EOC. We leveraged the ID8 murine EOC cell line to induce intra-peritoneal tumors in mice. Once durable intraperitoneal tumor was confirmed with bioluminescence imaging, intra-abdominal wall tumors were induced by abdominal wall puncture with a hollow bore needle. This resulted in a robust system in which C57BL/6 mice developed metastatic deposits at a rate of 66.7% ± 10.77; no intra-abdominal wall metastases were seen in control samples (P = .0003, CI 41.16–90.84). Immunodeficient NOD SCID gamma mice developed puncture site metastases in 70% ± 10.0 of mice and also had no metastases documented in control sites (P = .002, CI 42.24–97.76). In addition we were able to demonstrate the presence of immune infiltrates within the metastatic deposits of C57BL/6 mice via IHC. Therefore, in this study we demonstrate the predictable development of invasive abdominal wall metastases in a syngeneic mouse model of EOC. This model enables studies of the metastatic process and provides a novel system in which to test the effect of therapies on a clinically-relevant model in an immune competent mouse.