Cell lines The mouse breast carcinoma cell lines 4T1 selleck chemicals llc and EMT6 were pur chased from the American Type Culture Collection and maintained in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum and 1% antibiotics at 37 C in a humidified 5% CO2 atmosphere. IL 6 e pressing EMT6 cells were established by transfection with the pcDNA3. 1 IL 6 construct using PromoFectin, according to the manufacturers instructions. Control cells were transfected with the pcDNA3. 1 vector only. Stably transfected clones were established by selection with G418 at a concentration of 500 ug ml for three weeks. IL 6 e pressing EMT6 clones were selected by ELISA. IL 6 knockdown 4T1 cells and Stat3 knockdown 4T1 cells were established using the lenti viral vectors containing the shRNA.

Cells were infected with the shIL 6 or shSTAT3 virus and cultured in the presence of puromycin, and IL 6 knockdown 4T1 and Stat3 knockdown 4T1 clones were selected by ELISA and Western blotting, respectively. Tumor models 4T1 and EMT6 cells were injected into the mammary fat pads of BALB c mice. Tumor growth was monitored every other day thereafter. At 26 or 29 days after injection, the mice were euthanized and primary tumor masses, livers and lungs were fi ed in 4% parafor maldehyde for 24 hours and embedded in para ffin. Sections were stained with H E for histopathological analysis. Numbers of tumor masses in the liver and lungs were determined under a dissecting microscope before fi ing with 4% PFA. To deplete MDSCs, mice were intraperitoneally injected with 100 ug of anti Gr 1 antibody or control Rat immunoglobulin G 1 twice a week, starting three days after 4T1 cell injection.

To block IL 6 trans signaling in the afferent pathways of metastasis, 4T1 cells were injected into the mammary fat pads and gp130 Fc was administered continuously using an osmotic mini pump. To block IL 6 trans signaling in the efferent pathways of metastasis, 4T1 cells were injected intrave nously into BALB c mice and mice were intravenously injected with gp130 Fc 4 four days after cell injection. Flow cytometry and MDSC isolation To analyze MDSCs, mice were sacrificed 19 or 21 days after cancer cell injection. Mononuclear cells from the liver, lungs and primary tumor masses were isolated by Percoll gradient centrifugation. Tissues were digested with collagenase D and DNase I for one hour at 37 C.

Cells were suspended in 30% Percoll, layered onto the top of a 70% Percoll gradient and centrifuged. The interface was retained. Ne t, the cells were incubated with mAbs to mouse CD11b, Ly6C and Gr 1 that were conjugated to phycoerythrin, PerCP Cy5. 5, or allophycocyanin. They were then analyzed using a FACSCalibur Anacetrapib flow cytometer and FlowJo software. To isolate splenic MDSCs from na ve or tumor bearing mice, splenocytes were prepared and labeled with phycoerythrin conjugated anti CD11b mAb and allophycocyanin conjugated anti Gr 1 mAb.