originating cell line: K-562 tumor type: chronic myelogenous (blast crisis) leukemia in vivo passage: P0
All cell lines were obtained from the Division of Cancer Treatment and Diagnosis Tumor Repository (DTP, FNLCR, Frederick, MD) or American Type Culture Collection (ATCC) (Manassas, Va). In addition, the identities of all cell lines used in this study were confirmed using Identifiler® STR genotyping (Applied Biosystems). All mice used in the study were obtained from the Animal Production Program (FNLCR, Frederick, MD). Tumor cells for inoculation were derived from the 4th in vitro passage from cryopreserved cell stocks. Cells (1 x 10^7 cells/0.1 ml/inoculation) were inoculated subcutaneously into mice (n=10 or more mice depending on the tumor cell line). For comparative purposes, 2-3 samples of in vitro cultivated cells at passage 4 post-cryopreservation were also prepared for microarray analysis (P0). When tumors at P1, P4 and P10 reached approximately 500 mg (10x10 mm), 5 tumors were harvested, cut into small pieces and flash frozen in liquid nitrogen using pre-chilled tubes. Samples were transferred to a -70°C freezer for holding prior to processing. Serial tumor passage was achieved by harvesting donor tumor material (n=2 to 5 donors) at each passage, mincing the tumors (2x2 mm fragments), pooling the samples and inoculating the next recipients subcutaneously using tumor implant trocars. Athymic nude mice (nu/nu NCr) served as the primary host for most xenograft studies. In several instances (particularly with human leukemias and lymphomas) it was difficult to establish and passage tumors in nu/nu NCr mice. In these instances, SCID.NCr or NOD.SCID/NCr mice were assessed for suitability. In general, female mice were used as they are less aggressive, easily group-housed and routinely used in xenograft experiments. Male mice were used for prostate tumors (PC-3, PC-3/M). Finally, in those instances where hormone effects on tumor growth are important (e.g., estradiol-dependent breast), mice were supplemented with estradiol.
Total RNA was isolated using RNeasy (Qiagen).
The RNA samples were submitted to Expression Analysis, Inc. (Durham, NC) for microarray analysis using the GeneChip® Human U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Samples were labeled according to the "Affymetrix Technical Manual" (www.expressionanalysis.com).
Samples were hybridized according to the "Affymetrix Technical Manual" (www.expressionanalysis.com).
Microarray images were captured using a GeneChip® 3000 Scanner.
K-562 P0 T1 Gene expression data from K-562 originating cell line.