Adenosquamous carcinoma (ASC) can be an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes

Adenosquamous carcinoma (ASC) can be an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. Physique 5 is shown for 5 ATCC cell lines derived from AC, ASC, and SCC tumors (see Table S4). The flow analysis of H596 and H647 double stained for CK5 α-Estradiol and CK7 are shown in B.(TIF) pone.0079456.s003.tif (6.0M) GUID:?A43DB5C9-E557-443C-BC08-D6AB7FF0D424 Physique S4: Analysis of CK5 and CK7 protein expression by flow cytometry. CK5 and CK7 protein expression was further analyzed in lung cancer derived cells by flow analysis of permeabilized LUCA22 monolayer cells double stained for CK5 and CK7 (2D). For comparison, human tumor cells isolated from subcutaneous (SQ) or sub-renal (SRC) implanted LUCA22 cells after 20 weeks in vivo are shown. Xenografts were dispersed to single cells for analysis and contain both stromal (CK7-/CK5-) and tumor cells.(TIF) pone.0079456.s004.tif (1.1M) GUID:?13ADF1EA-AD08-463E-B786-233916F05983 Figure S5: Flow analysis of cell surface proteins in 5 clones. Five randomly selected LUCA 22 clones were analyzed for expression of cell surface proteins using tagged antibodies and flow. These were compared to the parental LUCA22 line (A). The biggest variability was seen in SSEA4 expression shown as individual histograms in B. Double stain of 4 clones for CD24 and CD44 are shown in C. The diagrams in D show staining for CD117 (& side scatter) after 1 bulk sort and after the 4th successive sort of the population. The CD117 remains a minority population.(TIF) pone.0079456.s005.tif (2.3M) GUID:?4D570829-1267-4393-80C5-D639907C85C4 Physique S6: The LUCA35 cells and xenografts express multiple lung cell markers. Cells were grown in the usual medium (LUCA35 2D, F-H top) in Matrigel with differentiation medium (LUCA35 3D, F-H bottom) or in vivo as xenografts (9 panels in I). Frozen sections were stained as indicated. All isotype control stains on adjacent sections were unfavorable Rabbit polyclonal to LACE1 (see CK20 and isotype control section, bottom center for an example).(TIF) pone.0079456.s006.tif (7.4M) GUID:?6227EADE-E575-464B-B5D6-46A240F1AEAF Methods S1: CSLC selection and expansion from NSLC tumor tissue. LUCA CSLC characterization.Differentiation in vitro. (DOC) pone.0079456.s007.doc (73K) GUID:?D6FAE13B-0B68-466E-BD8A-B364A6448609 Results S1: Cytokeratin staining of ATCC control cell lines. α-Estradiol Analysis of LUCA22 clones. Use of double stains for SC and AC.(DOCX) pone.0079456.s008.docx (168K) GUID:?FF0658E2-2EC7-461C-B402-728885B25B55 Table S1: Media supplements for selective growth and differentiation of lung tumor derived cells. (DOCX) pone.0079456.s009.docx (30K) GUID:?5EC9C799-A30C-44CE-BE34-AB0EB94D5198 Table S2: STR analysis of lung cancer-derived cell lines. (DOCX) pone.0079456.s010.docx (150K) GUID:?E07C88F7-A9A1-46D8-9706-9573AC7075F6 Table S3: Characteristics of Lung Tumor-derived cell lines. (DOCX) pone.0079456.s011.docx (93K) GUID:?FCB02FD6-E715-4F82-B154-D31C447E91D4 Table S4: Characteristics of ATCC lung cancer cell lines. (DOCX) pone.0079456.s012.docx (31K) GUID:?4BA56D1E-0903-4C2A-AD65-1CBFBA59B0AD Table S5: RT2 qPCR Primer Assay probe catalog numbers. (DOCX) pone.0079456.s013.docx (113K) GUID:?66BECFC3-1B9C-40B3-B355-3119DB61E2AC Table S6: Tumorigenicity of LUCA22 and clones in SRC model. (DOCX) pone.0079456.s014.docx (107K) GUID:?3C16591B-66CE-40CE-8BEB-64B26FEF2F7D Abstract There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a α-Estradiol stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is usually unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded and the properties of stromal cells. See Methods S1 and Table S1 for detailed methods for isolation of cells from tumors; and defined media and supplement concentrations for selection, expansion, cloning and differentiation of the ASC-CSLC; and expansion of stromal cells. The conditions for the differentiation of lung stem cells in 3 dimensional MatrigelTM cultures were modified from Delgado, et al as described in the Methods S1. The tumor samples and isolated CSLC lines were commercially characterized by their unique Short Tandem Repeat patterns using 16 STR regions. This analysis is usually described in Methods S1 and.