以原位肝腫瘤模式探討細胞素基因治療引發淋巴細胞之移行作用對治療效果之影響

Abstract

至今，在許多動物及臨床實驗中，GM-CSF 已廣泛的應用於數種不同的腫瘤治療模式中，並且皆可觀察到有效的治療結果。因此，本研究中以反轉錄病毒載體將GM-CSF基因轉殖至大鼠肝腫瘤細胞 GP7TB，並將此具有分泌 GM-CSF 特性之肝腫瘤細胞作為腫瘤疫苗，並以 rat F344大鼠之原位肝腫瘤及皮下腫瘤作為動物模式，探討應用腫瘤疫苗引發之腫瘤抗原專一性免疫反應於控制原位肝腫瘤生長之效果。在實驗結果中發現，將輻射處理過後之腫瘤疫苗注射於大鼠皮下，所引發之腫瘤抗原專一性免疫反應，只能夠抑制皮下腫瘤，卻無法抑制肝腫瘤之生長。此外亦發現，將腫瘤疫苗施打於大鼠之皮下，在肝腫瘤中浸潤的免疫性細胞（effector cell）數目隨著時間而顯著減少，反之，在皮下腫瘤中則可觀察到，這些細胞數目隨著時間而明顯增加。相較於皮下腫瘤中浸潤之CD8 T細胞發生細胞凋亡之數目，肝腫瘤中浸潤之CD8 T細胞發生細胞凋亡之數目並無明顯增加。此外，將腫瘤疫苗施打於大鼠之皮下或肝臟，並將免疫過後之大鼠脾臟細胞取出，與輻射處理過之腫瘤細胞 GP7TB經過試管內活化 (in vitro activation)，大多數的腫瘤抗原專一性的 T 細胞皆表現 CD45RC-CD25+ 以及CD44hiCD62Llo 之effector/ memory 細胞標誌。此外，經皮下腫瘤疫苗免疫過後之大鼠 T 細胞可以表現 CLA+或者 CCR4+ skin-homing 標誌，但是不能表現

In this study, tumor cells were engineered to secrete GM-CSF to create systemic antitumor immunity and cause tumor regression in several cases. The efficacy of subcutaneous and liver immunization of GM-CSF-expressing tumor cell vaccines on the growth of subcutaneous or orthotopic liver tumors were compared. The experimental results showed that two doses of subcutaneous irradiated tumor cell vaccine significantly controlled the growth of subcutaneous tumors, but was ineffective against orthotopic liver tumors. Effector cell infiltration in liver tumors was markedly decreased compared with subcutaneous tumors. After in vitro activation, the majority of tumor antigen-specific T cells obtained from either the liver or the subcutaneous immunization, expressed CD25+, CD45RC-CD3+, and CD44hiCD62Llo effector/memory phenotype. T cells derived from subcutaneous immunization expressed CLA+ or CCR4+ skin-homing markers, but did not express the α4β 7 gut-homing marker. Conversely, liver immunization induced T cells expressed neither the skin-homing marker nor the gut-homing marker. The results of adoptive transfer revealed that T cells derived from subcutaneous immunization preferred to migrate to tumors implanted at subcutaneous sites but did not migrate to liver tumor sites. By contrast, liver immunization yielded significantly better therapeutic effects on the liver tumors than on the subcutaneous tumors. The in vitro activated spleen cells from liver immunized animals, when adoptively transferred, preferentially migrated to liver tumor sites. Moreover, the microarray analytical results showed that higher levels of CCR4 transcripts were expressed in the T cells from the subcutaneously immunized animals than those from the liver immunized animals, whereas the expression patterns of CXCR4 and CXCR3 transcripts were just opposite. These results indicate that different immunization routes induce distinct T cell populations. Thus, the homing behavior of T cells depends on the immunization route and is an important factor in determining the efficacy of immunotherapy for regional tumors.