e 634Mul/J) and actin-GFP mice B5Nagy/J) were purchased from Jackson laboratory and relevant licenses were purchased for these experiments. MMTV-PyMT and actin-GFP mice were bred, and 3-week-old female bitransgenic offspring were sacrificed for collection of tumors, as described. Focal, 0.5 1 mm hyperplastic tumors were microdissected with No. 15 scalpels and placed in HBSS solution. Tumors were then implanted into cleared No. 4 mammary fat pads of 3 week-old female FVB/n mice. Tumor measurements were made using electronic digital calipers and tumor volumes were Residual and Disseminated Tumors in MMTV-PyMT Immunohistochemistry Samples for histology were fixed overnight in 4% paraformaldehyde/PBS and paraffin embedded. Sections were cut, rehydrated, processed with citrate buffer antigen retrieval, and blocked with 5% BSA or serum. Primary antibodies were diluted in 0.56 blocking solution and incubated on slides overnight at 4uC. Primary antibodies used include 221244-14-0 web anti-Stat1, anti-p-Stat1, anti-F480, and anti-phospho-H2A.X. For immunofluorescence, sections were incubated 19239230 with biotinylated anti-rabbit secondary antibody and then streptavidin-Alexa 488 or Alexa 564. For immunohistochemistry, sections were incubated with biotinylated anti-rabbit 9726632 secondary antibody, streptavidin-HRP and then ABC signal amplification reagent, followed by DAB chromogenic detection. differentiated breast adenocarcinoma. Tumor-bearing mice were treated with 25 mg/kg docetaxel, 5 mg/kg doxorubicin, and 120 mg/kg cyclophosphamide every 21 days, similar to patients. Mice lost 810% body weight after each dose but recovered within 21 days of dosage. Tumors underwent significant regression within seven days after the first dose of chemotherapy. By day 10, tumors stabilized and started to grow once again. Within 21 days after the first dose, the tumors relapsed and grew nearly to the size of the original tumor. Subsequent administration of chemotherapy every 21 days led to similar patterns of tumor regression and relapse. We collected the untreated tumors, residual tumors at day 10, and relapsed tumors at day 80 for further analysis. GFP-positive tumor cells from these samples were FACS-sorted, and RNA was harvested 
for mRNA microarray profiling. RT-PCR mRNA quality was assessed with Agilent Bioanalyzer and quantified with the Nanodrop instrument. 0.251 mg mRNA was reverse-transcribed to cDNA for 48648 Fluidigm analysis, which was performed according to manufacturer’s instructions. Gene primers for RT-PCR were designed and validated by Fluidigm. All cDNA measurements were normalized to Gapdh expression. Independent sets of samples were used for RT-PCR, microarray profiling, and protein analysis. Enrichment of Jak/Stat, Notch, and Epigenetics Genes in Residual Tumors Microarray profiling of untreated, residual, and relapsed tumors revealed a number of signaling pathways and biomarkers enriched in residual tumors. These include the Jak/Stat pathway, DNA damage response/repair pathways, and Akt signaling pathways. Ingenuity pathway analysis of microarray data identified Stat1 as an important signaling node in residual tumors. Interestingly, several genes in the Ifnc/Jak/Stat pathway, such as Gbp1, Gbp3, Gbp4, Ifi47, Tgtp, and Stat1, were up-regulated in residual tumors relative to untreated and/or relapsed tumors. We validated the enrichment of these genes in residual tumors with Fluidigm RT-PCR. GFPpositive tumor cells were sorted from adenomas, carcinomas, residual tumors,
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