Over an 18 month period, discarded tissue from 3 chemo-naive SCLC patients undergoing therapeutic bronchoscopy for acute bronchial obstruction was obtained fresh and transported to the laboratory in 1X PBS at 4°C. All samples were anonymized, and obtained in accordance with the Johns Hopkins University Institutional Review Board. Due to the small amount of material available, the entire sample was used to generate a xenograft. Under aseptic conditions, tumor samples were finely minced with razor blades, vigorously triturated in 1XPBS, passed through a 60μm filter, centrifuged and then resuspended in 500μl of Matrigel (BD Biosciences) at 4°C. Cells were then injected subcutaneously in the flanks of 5 NOD/SCID mice that were monitored for tumor growth. When the P0 tumors reached 1cm in diameter, the mouse was sacrificed and the tumor divided into sections for snap freezing, frozen section, formalin fixation, conventional cell culture or serial passage. All animal studies were performed in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee.
Serial passage in vivo was performed by disaggregating the tumor as described above. Aliquots of cells were then injected into the flanks of athymic nude mice in Matrigel, or cryopreserved in 90% RPMI (Invitrogen)/10% DMSO (Sigma). Conventional cell lines were established by seeding an aliquot of disaggregated cells in culture with Advanced RPMI (Invitrogen)/1% Bovine Calf Serum (Invitrogen). Cell lines were passaged and cryopreserved in standard fashion for SCLC cultures. Publically available SCLC cell lines were obtained from ATCC and cultured in Advanced RPMI (Invitrogen)/1% Bovine Calf Serum. Xenografts derived from these conventional cell lines were grown in the flanks of nude mice as described above. Orthotopic xenografts were generated by dorsoscapular, transcutaneous injection of cells suspended in Matrigel into the right lung of nude mice, essentially as described (12 (link)).
Serial passage in vivo was performed by disaggregating the tumor as described above. Aliquots of cells were then injected into the flanks of athymic nude mice in Matrigel, or cryopreserved in 90% RPMI (Invitrogen)/10% DMSO (Sigma). Conventional cell lines were established by seeding an aliquot of disaggregated cells in culture with Advanced RPMI (Invitrogen)/1% Bovine Calf Serum (Invitrogen). Cell lines were passaged and cryopreserved in standard fashion for SCLC cultures. Publically available SCLC cell lines were obtained from ATCC and cultured in Advanced RPMI (Invitrogen)/1% Bovine Calf Serum. Xenografts derived from these conventional cell lines were grown in the flanks of nude mice as described above. Orthotopic xenografts were generated by dorsoscapular, transcutaneous injection of cells suspended in Matrigel into the right lung of nude mice, essentially as described (12 (link)).
