实验转移和CTL过继转移免疫小鼠模型
Summary
一个实验性肺转移和CTL免疫小鼠模型的肿瘤细胞- T细胞相互作用分析<em>在体内</em>。
Abstract
实验转移小鼠模型是一个简单的，但有关生理转移模型。肿瘤细胞静脉注射（四）为小鼠尾静脉和殖民，从而在肺部，类似肿瘤细胞自发转移的最后步骤：在流通领域，外渗及远端器官的殖民化的生存。从治疗的观点来看，实验转移模型是最简单和理想的模式，自疗法的目标往往是：建立在远端器官的转移瘤的转移终点。在此模型中，肿瘤细胞注射四到小鼠尾静脉，并允许在肺部进行殖民和增长。然后注入肿瘤特异性CTL转移轴承鼠标第四。肺转移的数量和大小，可以控制被注入肿瘤细胞的数量和肿瘤生长的时间。因此，各个阶段的转移，从最小的转移到全身广泛转移，可为蓝本。肺转移，从而允许更容易直观地观察和定量分析与油墨的通货膨胀。
Protocol
1。实验转移小鼠模型前一天的肿瘤细胞的注射剂，种子之一T75容量瓶中，用高达1 × 10 7 CMS4了10 mL的RPMI培养基中含有10％的血清，以获得快速增长的肿瘤细胞中的细胞。过夜孵育在37 ° C。 在注射当天，去除培养基，用PBS冲洗细胞一次，然后用0.05％胰酶EDTA收获肿瘤细胞在37 ° C为5分钟。停止与10 mL的RPMI培养基中含有10％血清中反应。细胞转移到锥形管。 分拆为3分钟，…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
由美国国立卫生研究院（CA133085吉隆坡）和美国癌症协会的资助（RSG – 09 – 209 – 01 – TBG的吉隆坡）支持。
Materials
Solutions:
India Ink Solution (17):
- Pour 150 ml of distilled water into a 250 ml flask.
- Add 4 drops ammonium hydroxide to the distilled water.
- Add 30 ml India Ink stock (i.e. Sanford Black Magic Waterproof Drawing Ink 4465 Item 44011) to the ammonia and water mixture.
- Top off with distilled water to a volume of 200 ml. Solution is ready for injection.
Fekete’s Solution (17):
Fekete’s solution is used to bleach India ink-inflated tumor-bearing lungs to distinguish white tumor nodules from the black background of normal tissues.
- Add 350ml 95% EtOH to 1L glass bottle.
- Add 150ml distilled water
- Add 50ml formaldehyde
- Add 25ml glacial acidic acid
Referencias
- Ryan, M. H., Bristol, J. A., McDuffie, E., Abrams, S. I. Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope. J Immunol. 167 (8), 4286-4292 (2001).
- Caldwell, S. A., Ryan, M. H., McDuffie, E., Abrams, S. I. The Fas/Fas ligand pathway is important for optimal tumor regression in a mouse model of CTL adoptive immunotherapy of experimental CMS4 lung metastases. J Immunol. 171 (5), 2402-2412 (2003).
- Liu, K., Caldwell, S. A., Greeneltch, K. M., Yang, D., Abrams, S. I. CTL Adoptive Immunotherapy Concurrently Mediates Tumor Regression and Tumor Escape. J Immunol. 176 (6), 3374-3382 (2006).
- Yang, D., Stewart, T. J., Smith, K. K., Georgi, D., Abrams, S. I., Liu, K. Downregulation of IFN-gammaR in association with loss of Fas function is linked to tumor progression. International journal of cancer. 122 (2), 350-362 (2008).
- Pages, F., Berger, A., Camus, M. Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med. 353 (25), 2654-2666 (2005).
- Galon, J., Costes, A., Sanchez-Cabo, F. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 313 (5795), 1960-194 (2006).
- Strater, J., Hinz, U., Hasel, C. Impaired CD95 expression predisposes for recurrence in curatively resected colon carcinoma: clinical evidence for immunoselection and CD95L mediated control of minimal residual disease. Gut. 54 (5), 661-665 (2005).
- Camus, M., Tosolini, M., Mlecnik, B. Coordination of intratumoral immune reaction and human colorectal cancer recurrence. Cancer research. 69 (6), 2685-2693 (2009).
- Dudley, M. E., Wunderlich, J. R., Yang, J. C. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol. 23 (10), 2346-2357 (2005).
- Srivastava, M. K., Sinha, P., Clements, V. K., Rodriguez, P., Ostrand-Rosenberg, S. Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer research. 70 (1), 68-77 (2010).
- Nagaraj, S., Gabrilovich, D. I. Tumor escape mechanism governed by myeloid-derived suppressor cells. Cancer research. 68 (8), 2561-2563 (2008).
- Nguyen, D. X., Bos, P. D., Massague, J. Metastasis: from dissemination to organ-specific colonization. Nature reviews. 9 (4), 274-284 (2009).
- Heijstek, M. W., Kranenburg, O., Rinkes, B. o. r. e. l., H, I. Mouse models of colorectal cancer and liver metastases. Digestive surgery. 22 (1-2), 1-2 (2005).
- Yang, D., Ud Din, N., Browning, D. D., Abrams, S. I., Liu, K. Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression. Clin Cancer Res. 13 (17), 5202-5210 (2007).
- Yang, D., Thangaraju, M., Browning, D. D. IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression. J Immunol. 179 (7), 4775-4782 (2007).
- Yang, D., Thangaraju, M., Greeneltch, K. Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells. Cancer research. 67 (7), 3301-3309 (2007).
- Wexler, H. Accurate identification of experimental pulmonary metastases. Journal of the National Cancer Institute. 36 (4), 641-645 (1966).
Tags
Experimental MetastasisMouse ModelCTL Adoptive Transfer ImmunotherapyTumor CellsIntravenous InjectionTail VeinsLungsMetastasis ModelTherapeutic Point Of ViewEstablished Metastatic TumorDistal OrganLung MetastasesTumor-specific CTLsInjectionColonization And GrowthNumber And Size ControlStages Of MetastasisMinimal MetastasisExtensive MetastasisInk Inflation Analysis
Citar este artículo
Zimmerman, M., Hu, X., Liu, K. Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model. J. Vis. Exp. (45), e2077, doi:10.3791/2077 (2010).