['1726', 'vitro', 'if', 'grown', 'in', 'normal', 'concentrations', 'of', 'Ca2+.', 'MCFI0A', 'cells', 'spontaneously', 'arose', 'from', 'MCF10M', 'after', '849', 'days', 'in', 'culture', 'and', 'do', 'not', 'senesce', '(3).', 'MCF10AneoT', 'was', 'derived', 'following', 'transfection', 'of', 'MCFIOA', 'cells', 'with', 'the', 'mutated', 'T-24', 'HRAS', 'gene', 'and', 'has', 'a', 'transformed', 'phenotype', 'in', 'vitro', '(4,5).', 'MCFi0AneoT', 'cells', 'were', 'reported', 'to', 'be', 'tumorigenic', 'in', 'nude', 'mice', '(5),', 'but', 'animals', 'in', 'that', 'study', 'were', 'killed', 'only', '2-4', 'weeks', 'after', 'cell', 'inoculation.', 'However,', 'MCFIOA', 'cells', 'also', 'form', 'palpable', 'masses', 'that', 'are', 'transient', 'but', 'persist', 'for', 'up', 'to', '5', 'weeks', 'prior', 'to', 'regression', '(6).', 'In', 'the', 'present', 'study,', 'we', 'have', 'examined', 'the', 'long-term', 'tumorigenic', 'potential', 'of', 'MCF10AneoT', 'cells', 'transplanted', 'into', 'the', 'dorsal', 'flank', 'of', 'nude/beige', 'mice', '(CS7/BALB/c', 'nu/nu', 'bg/bg)', 'and', 'maintained', 'for', 'more', 'than', 'a', 'year.', 'We', 'report', 'that', 'these', 'cells', 'give', 'rise', 'to', 'a', 'sequence', 'of', 'histologic', 'changes', 'that', 'resemble', 'proliferative', 'breast', 'disease', 'in', 'humans', 'and', 'that', 'include', 'the', 'development', 'of', 'carcinomas.', 'In', 'contrast,', 'neither', 'MCF10A', 'nor', 'MCF1I0AneoN', 'forms', 'persistent', 'lesions,', 'and', 'neither', 'gives', 'rise', 'to', 'proliferative', 'breast', 'disease-like', 'histology.', 'Materials', 'and', 'Methods', 'Mice.', 'Five-week-old', 'male', 'nude/beige', 'mice', '(C57/BALB/c', 'nu/nu', 'bg/bg)', 'weighing', 'approximately', '20', 'g', 'were', 'used.', 'They', 'were', 'obtained', 'from', 'Life', 'Sciences,', 'St.', 'Petersburg,', 'Fla.', 'Mice', 'were', 'housed', 'three', 'to', 'a', 'cage', 'and', 'fed', 'ad', 'libitum.', 'They', 'were', 'maintained', 'in', 'accordance', 'with', 'guidelines', 'of', 'the', 'National', 'Institutes', 'of', 'Health', 'Guide', 'for', 'the', 'Care', 'and', 'Use', 'of', 'Laboratory', 'Animals', 'at', 'the', 'Michigan', 'Cancer', 'Foundation', 'Animal', 'Facility,', 'which', 'is', 'fally', 'accredited', 'by', 'the', 'American', 'Association', 'for', 'Accreditation', 'of', 'Laboratory', 'Animal', 'Care.', 'Cell', 'lines.', 'The', 'derivation', 'of', 'the', 'MCFIOA', 'cell', 'line', 'from', 'the', 'mortal', 'cell', 'line', 'MCFIOM', 'established', 'from', 'a', 'subcutaneous', 'mastectomy', 'was', 'described', 'previously', '(3).', 'Cell', 'lines', 'MCF1OAneoN', 'and', 'MCF10AneoT', '(S)', 'were', 'obtained', 'from', 'Dr.', 'Jose', 'Russo', '(Fox', 'Chase', 'Cancer', 'Center,', 'Philadelphia,', 'Pa.),', 'who', 'transfected', 'at', 'passage', '113', 'MCFIOA', 'cells', 'with', 'the', 'neomycin-resistance', 'gene', 'and', 'the', 'normal', 'HRAS', 'gene', 'or', 'with', 'the', 'neomycin-resistance', 'gene', 'and', 'the', 'mutated', 'T-24', 'HRAS', 'gene', '(7),', 'respectively.', 'The', 'MCF10AneoN,', 'MCFI0AneoT,', 'and', 'the', 'transplant', 'generation', '|', 'MCF10AneoT.TG|', 'cells', 'were', 'maintained', 'in', "Dulbecco's", 'modified', 'Eagle', 'medium/F-12', 'medium', 'with', '5%', 'equine', 'serum,', '0.t', 'jag/mL', 'cholera', 'toxin,', '10', 'g/mL', 'insulin,', '160', '[U/mL', 'penicillin,', '100', 'g/mL', 'streptomycin,', '0.5', 'g/mL', 'amphotericin', 'B,', '0.5', 'pe/mL', 'hydrocortisone,', 'and', '0.02', 'g/mL', 'epidermal', 'growth', 'factor.', 'Human', 'breast', 'epithelial', 'cell', 'lines', 'MCF12M', 'and', 'MCFI2A', 'were', 'obtained', 'from', 'a', 'reduction', 'mammoplasty', '(6),', 'and', 'tines', '184A]', 'and', '184B5', '(8)', 'were', 'obtained', 'from', 'the', 'American', 'Type', 'Culture', 'Collection', '(ATCC),', 'Rockville,', 'Md..', 'at', 'passages', '33', 'and', '23,', 'respectively;', 'all', 'were', 'cultured', 'under', 'the', 'same', 'conditions', 'as', 'MCFIGA', 'cells.', 'Human', 'breast', 'tumor', 'cell', 'lines', 'BT20', 'and', 'MCF7', 'were', 'cultured', 'as', 'previously', 'reported', '(9).', 'The', 'T47D', 'cell', 'line', 'was', 'obtained', 'from', 'ATCC', 'and', 'was', 'cultured', 'under', 'ATCC-recommended', 'conditions.', 'Growth', 'in', 'immunodeficient', 'mice.', 'Cells', 'were', 'suspended', 'after', 'treatment', 'with', '0.025%', 'edetate', 'sodium', '(Versene:', 'Dow,', 'Midland,', 'Mich.)', 'and', '0.05%', 'trypsin', 'in', 'a', 'Ca?+-', 'and', 'Mg?+-free', 'balanced', 'salt', 'solution,', 'and', '1', 'x', '107', 'cells', 'in', '0.1', 'mL', 'Matrigel', '(Collaborative', 'Research,', 'Inc..', 'Bedford,', 'Mass.)', 'were', 'injected', 'subcutaneously', 'into', 'the', 'dorsal', 'flank', 'of', 'S-week-old', 'male', 'nude/beige', 'mice.', 'Groups', 'of', '12', 'mice', 'were', 'given', 'injections', 'of', '|', 'x', '10?', 'cells', 'per', 'mouse', 'of', 'MCFIGA,', 'MCF{0AneoN,', 'MCFIQAneoT,', 'and', 'MCF10AneoT.TG1.', 'We', 'elected', 'to', 'inject', 'cells', 'in', 'Matrigel', 'because', 'it', 'has', 'been', 'reported', 'that', 'the', 'tumorigenic', 'potential', 'of', 'human', 'cells', 'is', 'markedly', 'increased', 'if', 'cells', 'are', 'suspended', 'in', 'Matrigel', 'for', 'injection', 'into', 'immunodeficient', 'mice', '(/0).', 'Intra-', 'fatpad', 'injections', '(orthotopic', 'transplantation)', 'were', 'not', 'possible', 'because', 'of', 't)', 'the', 'inoculum', 'volume', 'required', 'to', 'inject', '1', 'X', '107', 'cells', 'in', 'Matrigel', 'and', '2)', 'the', 'large', 'needle', 'gauge', 'required', 'to', 'inject', 'the', 'viscous', 'Matrigel.', 'To', 'assess', 'formation', 'and', 'persistence', 'of', 'or', 'growth', 'of', 'palpable', 'nodules,', 'we', 'periodically', 'inspected', 'animals', 'by', 'measuring', 'wo', 'perpendicular', 'diameters', 'with', 'vernier', 'calipers.', 'One', 'mouse', 'per', 'group', 'was', 'killed', '1', 'week', 'after', 'injection', 'of', 'cells:', 'ARTICLES', ' ', ' ', 'thereafter,', 'mice', 'were', 'observed', 'as', 'long', 'as', 'possible.', 'Xenografts', 'were', 'obtained', 'from', 'mice', 'killed', 'because', 'of', 'ill', 'health', 'unrelated', 'to', 'the', 'xenograft,', 'such', 'as', 'weight', 'loss', 'or', 'the', 'appearance', 'of', 'facial', 'lesions.', 'Recovery', 'of', 'ceils', 'from', 'lesions.', 'Cells', 'were', 'recovered', 'from', 'three', 'palpable', 'lesions', '{one', 'lesion', 'each,', '100', 'and', '287', 'days', 'after', 'injection', 'of', 'MCFIQAneoT', 'cells', 'in', 'Matrigel', 'and', '218', 'days', 'after', 'injection', 'of', 'celis', 'derived', 'from', 'the', 'day-100', 'lesion)', 'that', 'developed', 'in', 'nude/beige', 'mice', 'by', 'enzymatic', 'dissociation', 'of', 'the', 'lesions', 'with', 'callagenase', 'for', '6-72', 'hours', 'in', 'tightly', 'capped', 'T25', 'flasks', 'on', 'a', 'rocking', 'platform', 'at', '37', '°C.', 'The', 'digestion', 'mixture', 'consisted', 'of', '125', 'U', 'of', 'collagenase', '(Sigma', 'Chemical', 'Co.,', 'St.', 'Louis,', 'Mo.)', 'in', '70%', 'minimal', 'essential', 'medium', "{Hanks'),", '20%', 'minimal', 'essential', 'medium', '(Earle’s),', 'and', '7.5%', 'equine', 'serum', 'and', 'was', 'supplemented', 'with', '10', 'we/mL', 'insulin,', '100', 'TU/mL', 'penicillin,', '100', 'we/mL', 'streptomycin,', 'and', '0.5', 've/mL', 'amphotericin', 'B.', 'As', 'clumps', 'of', 'cells', 'were', 'released,', 'they', 'were', 'removed,', 'washed', 'by', 'centrifugation,', 'and', 'plated', 'onto', 'plastic', 'to', 're-establish', 'cells', 'in', 'tissue', 'culture.', 'Digestion', 'mixture', 'was', 'added', 'to', 'the', 'undigested', 'Portion,', 'and', 'the', 'process', 'was', 'repeated.', 'Fibroblasts', 'were', 'removed', 'from', 'Primary', 'cultures', 'by', 'brief', 'rinses', 'with', 'trypsin', 'and', 'edetate', 'sodium.', 'DNA', 'isolation', 'and', 'Southern', 'blot', 'analysis,', 'High-molecular-weight', 'DNA', 'was', 'isolated', 'from', 'cells', 'or', 'tissue', 'using', 'a', 'sodium', 'dodecyl', 'sulfate-', 'proteinase', 'K', 'digestion', 'and', 'phenol-—chloroform', 'extraction', 'method', '(//),', 'DNAs', 'were', 'digested', 'to', 'completion,', 'size', 'fractionated', 'by', 'agarose', 'gel', 'electrophoresis,', 'and', 'blotted', 'to', 'GenePrint', 'Light', '(Promega', 'Corp.,', 'Madison,', 'Wis.)', 'or', 'Gene', 'Screen', 'Plus', '{Du', 'Pont', 'NEN', 'Research', 'Products,', 'Boston,', 'Mass.)', 'membranes', 'for', 'hybridization', 'with', 'chemiluminescent', 'or', 'radiolabeled', 'Probes,', 'respectively.', 'D2S44', 'locus', 'Hae', 'If', 'fragments', 'were', 'identified', 'by', 'hybridization', 'with', 'an', 'alkaline', 'phosphatase-conjugated', 'YNH24', 'oligonucieotide', 'probe', 'and', 'visualized', 'with', 'the', 'GenePrint', 'Light', 'Chem-', 'iluminescent', 'Detection', 'System', 'as', 'per', 'the', "vendor's", 'specifications', '(Promega', 'Corp.).', 'Other', 'probes', 'included', 'an', 'Hae', 'IIT', 'fragment', '(Promega', 'Corp.)', 'from', 'the', 'pYNH24', 'clone', 'for', 'the', 'D2844', 'locus', '(22)', 'and,', 'for', 'HRAS,', 'a', '2.8-kilobase', '(kb)', 'Sac', 'I', 'fragment', 'that', 'contains', 'exons', '1', 'through', '4', 'of', 'the', 'HRAS', 'gene', '(/3)', 'and', 'a', '2.7-kb', 'Sac', 'I-BamHI', 'fragment', 'that', 'contains', 'VNTR', '(variable', 'number', 'of', 'tandem', 'repeal}', 'sequences', '3‘', 'to', 'HRAS', '{23),', 'respectively.', 'These', 'probes', 'were', 'radiolabeled', 'by', 'random', 'priming', '(3,6).', 'The', 'mouse', 'mammary', 'tumor', 'virus', '(MTY)', 'long', 'terminal', 'repeat', '(LTR)', 'clone', 'p201', '(ATCC', '45004)', 'was', 'fadiolabeled', 'by', 'nick', 'translation', '(3).', 'Hybridizations', 'and', 'washing,', 'under', 'stringent', 'conditions,', 'and', 'autoradiography', 'were', 'as', 'described', '(/1,/4).', 'Cytogenetic', 'analysis.', 'Methods', 'for', 'harvest', 'were', 'detailed', 'in', 'the', 'original', 'report', 'on', 'the', 'MCFIQA', 'cell', 'tine', '(3).', 'Cytogenetic', 'nomenclature', 'is', 'according', 'to', 'the', 'Intemational', 'System', 'for', 'Human', 'Cytogenetic', 'Nomenclature', '(/5)', 'supplemented', 'by', 'Guidelines', 'for', 'Cancer', 'Cytogenetics', '(16).', 'Results', 'Growth', 'In', 'Vivo', 'MCFIOA', 'ceils', 'form', 'transient,', 'small', 'palpable', 'nodules', 'in', 'nude', 'mice.', 'These', 'nodules', 'disappear', 'within', '4-5', 'weeks.', 'For', 'example,', 'in', 'one', 'experiment,', '12', 'of', '12', 'mice', 'had', 'palpable', 'Jesions', '(median', 'diameter', 'of', '5.5', 'mm)', '1', 'weck', 'after', 'the', 'subcutaneous', 'injection', 'of', '1', 'X', '107', 'MCFIOA', 'cells', 'in', 'Matrigel;', 'five', 'of', '10', 'mice', 'still', 'had', 'palpable', 'lesions', '(median', 'diameter', 'of', '3', 'mm)', 'after', 'week', '3.', 'By', '5', 'weeks', 'after', 'injection,', 'none', 'of', 'the', 'lesions', 'were', 'palpable,', 'and', 'none', 'reappeared', 'during', 'the', 'next', '22', 'weeks', 'of', 'observation.', 'Thus,', 'the', 'addition', 'of', 'Matrigel', 'did', 'not', 'potentiate', 'the', 'tumorigenic', 'potential', 'of', 'these', 'cells', 'compared', 'with', 'growth', 'in', 'nude', 'mice', 'of', 'MCF10A', 'cells', 'without', 'Matrigel', 'as', 'previously', 'reported', '(3,6).', 'MCF10AneoN', 'cells', 'behaved', 'similarly', 'in', 'nude/beige', 'mice.', 'For', 'example,', '|', 'week', 'after', '12', 'mice', 'had', 'been', 'given', 'a', 'subcutaneous', 'injection', 'of', '1', 'X', '10?', 'MCFIOAneoN', 'cells', 'in', 'Matrigel,', '11', 'of', 'these', 'mice', 'had', 'palpable', 'lesions', '(median', 'diameter', 'of', '4.5', 'mm).', 'Although', 'five', 'of', '11', 'mice', 'had', 'palpable', 'lesions', '(median', 'diameter', 'of', '3', 'mm)', '4', 'weeks', 'after', 'injection,', 'all', 'nodules', 'regressed', 'by', '5', 'weeks', 'and', 'none', 'reappeared', 'for', 'up', 'Journal', 'of', 'the', 'National', 'Cancer', 'Institute,', 'Vol.', '85,', 'No.', '21,', 'November', '3,', '1993']