Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes
Summary
This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.
Abstract
La capacidad de las células malignas para evadir el sistema inmune, caracterizado por el escape del tumor de ambos respuestas inmunitarias innatas y adaptativas, se acepta ahora como un sello importante de cáncer. Nuestra investigación sobre el cáncer de mama se centra en el papel activo que desempeñan linfocitos infiltrantes de tumores en la progresión tumoral y la evolución del paciente. Hacia este objetivo, hemos desarrollado una metodología para el aislamiento rápido de células linfoides intactos de tejidos normales y anormales en un esfuerzo para evaluar los próximo a su estado nativo. Los homogeneizados preparados usando un espectáculo disociador mecánica tanto una mayor recuperación de la viabilidad celular y preservando al mismo tiempo la expresión receptor de superficie en comparación tejidos digeridos con enzima para. Por otra parte, la digestión enzimática del material insoluble restante no se recuperó células CD45 + adicionales que indican que las mediciones cuantitativas y cualitativas en el homogeneizado primaria probablemente reflejen realmente la infiltración de las subpoblaciones en la Fragm tejidoent. Las células linfoides en estos homogeneizados pueden ser fácilmente caracterizado usando inmunológica (fenotipo, la proliferación, etc.) o molecular (ADN, ARN y / o proteína) enfoques. Células CD45 + también pueden ser utilizados para la purificación subpoblación, la expansión in vitro o crioconservación. Un beneficio adicional de este enfoque es que el sobrenadante tejido primario a partir de los homogeneizados se puede utilizar para caracterizar y comparar citocinas, quimiocinas, inmunoglobulinas y antígenos presentes en tejidos normales y malignos. Este funciones de protocolo extremadamente bien para los tejidos de mama humanos y deberían ser aplicables a una amplia variedad de tejidos normales y anormales.
Introduction
The tumor microenvironment is composed of various cell types with numerous studies showing they each play distinct and important roles in tumorigenesis1,2. These include, but are not limited to, infiltrating immune cells, stromal cells, endothelial cells and tumor cells3. Ex vivo studies of tumor infiltrating lymphocytes (TIL; CD45+ cells or leukocytes, which are predominantly lymphocytes in breast tumors) from fresh human tissue samples is made difficult by their low frequency, the small sample sizes often available for research and the potential for loss of viability during extraction. Because immune cells infiltrating tumors are usually present as passengers rather than permanent residents in general they are easier to release from the tissue matrix.
Dissociating tumor tissue while maintaining cellular integrity is technically challenging and has traditionally been performed using a combination of mechanical and enzymatic steps to prepare single cell suspensions4-8. This approach involves lengthy incubation periods and is associated with a significant reduction in cell viability as well as the loss of cell surface receptors by enzymatic cleavage. High quality flow cytometric studies characterizing TIL in the tumor microenvironment as well as clean purifications of CD45+ subpopulations by flow cytometry or antibody-coated beads are more difficult to achieve from enzyme-digested tumor tissue. In addition, the supernatant (SN) from the resulting tumor homogenate is not amenable to further analysis including quantification of secreted proteins (cytokines, chemokines, immunoglobulins or tumor antigens) or experimental treatment of normal cells, because of the potential for protein degradation in the enzymatic digests.
In our search for a method to prepare single cell homogenates from breast tissues [including tumor, non-adjacent non-tumor (NANT) and normal (from mammary reductions) breast tissues] without enzymatic digestion, we tested a variety of mechanical homogenization techniques. Homogenates prepared using a mechanical dissociator had increased cell viability (2-fold) and total cell recovery (2-fold) while preserving surface receptor expression. Enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells suggesting they were all released in the initial homogenate. Thus, this rapid and simple approach allows both qualitative and quantitative assessment of the CD45+ subpopulations present in various normal and malignant human tissues. An added advantage of this approach is that the SN from the initial homogenate (primary tissue SN) can be collected and stored for further analysis or experimentation.
Protocol
Representative Results
Discussion
Este estudio describe un método optimizado para la rápida preparación de los homogeneizados de tejidos normales y malignos de mama sin digestión enzimática para la clasificación posterior de células, la extracción, la criopreservación y / o análisis fenotípico de CD45 + subpoblaciones. El objetivo de este enfoque experimental es para producir imágenes de la TIL que refleja de cerca su estado in vivo y compararlos con los tejidos normales con mínima manipulación de los tejidos frescos de…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Este trabajo fue apoyado por becas fromthe Fondo Belga para la Investigación Científica (FNRS), Les Amis de l'Institut Bordet, FNRS-Opération Télévie, Cáncer del Plan de Bélgica, Fonds Lambeau-Marteaux, Fonds JC Heuson y Fonds Barsy.
Materials
| Equipment | Company | Catalog Number | Comments/Description |
| GentleMacs Dissociator | Miltenyi Biotec | 130-093-235 | BD Medimachine is somewhat equivalent |
| Centrifuge 5810 R | Eppendorf | N/A | or other standard table top centrifuge |
| Centrifuge 5417 R | Eppendorf | N/A | or other standard microcentrifuge |
| Esco Class II A2 Biosafety Cabinet | ESCO global | N/A | or other standard BSL2 hood |
| Inverted Microscope | Nikon eclipse TS100 | N/A | or other microscope compatible for a hemacytometer |
| Bürker Chamber | Marienfield | 640210 | or other standard hemacytometer |
| Navios Flow Cytometer | Beckman Coulter | N/A | or other flow cytometer (8-10 color recommended) |
| Materials | Company | Catalog Number | Comments/Description |
| GentleMacs C-Tube | Miltenyi Biotec | 130-096-344 | BD Medimachine uses Filcon |
| Cell Culture Dish | Sarstedt | 72,710 | or other non-pyrogenic plasticware |
| Disposable Scalpel | Swann-Morton | 0510 | or standard single use sterile scalpel |
| BD Cell Strainer 40µm | Becton Dickinson | 734-0002 | or other non-pyrogenic plasticware |
| BD Falcon Tube 50mL | Becton Dickinson | 352070 | or other non-pyrogenic plasticware |
| BD Falcon Tube 15mL | Becton Dickinson | 352097 | or other non-pyrogenic plasticware |
| BD FACS Tube 5mL | Becton Dickinson | 352008 | or other non-pyrogenic plasticware |
| Sterile Pasteur Pipette 5 mL | VWR | 612-1685 | or other non-pyrogenic plasticware |
| Microfuge Tube 1.5 mL | Eppendorf | 7805-00 | or other non-pyrogenic plasticware |
| Reagents | Company | Catalog Number | Comments/Description |
| X-Vivo 20 | Lonza | BE04-448Q | serum-free medium recommended |
| Phosphate buffered saline | Lonza | BE17-516F | standard physiological PBS |
| Trypan blue | VWR | 17942E | or other vital stain |
| VersaLyse | Beckman Coulter | A09777 | for flow cytometry experiments |
| Fixable viability Dye eFluor 780 | eBioscience | 65-0865-14 | for flow cytometry experiments |
| anti-CD3 FITC | BD Biosciences | 345763 | for flow cytometry experiments |
| anti-CD3 Vio Blue | Miltenyi Biotec | 130-094-363 | for flow cytometry experiments |
| anti-CD4 PE | BD Biosciences | 345769 | for flow cytometry experiments |
| anti-CD4 APC | Miltenyi Biotec | 130-091-232 | for flow cytometry experiments |
| anti-CD8 ECD | Beckman Coulter | 737659 | for flow cytometry experiments |
| anti-CD8 PerCP | BD Biosciences | 345774 | for flow cytometry experiments |
| anti-CD19 APC-Vio770 | Miltenyi Biotec | 130-096-643 | for flow cytometry experiments |
| anti-CD45 VioGreen | Miltenyi Biotec | 130-096-906 | for flow cytometry experiments |
References
- Chen, D. S., Mellman, I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 39 (1), 1-10 (2013).
- Boudreau, A., van’t Veer, J. L., Bissell, M. J. An ‘elite hacker’: breast tumors exploit the normal microenvironment program to instruct their progression and biological diversity. Cell Adh Migr. 6 (3), 236-248 (2012).
- Gajewski, T. F., Schreiber, H., Fu, Y. X. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 14 (10), 1014-1022 (2013).
- Quezada, S. A., et al. Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma. J Exp Med. 205 (9), 2125-2138 (2008).
- Grange, C., et al. Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation. J Immunol Methods. 372 (1-2), 119-126 (2011).
- McCauley, H. A., Guasch, G. Serial orthotopic transplantation of epithelial tumors in single-cell suspension. Methods Mol Biol. 1035, 231-245 (2013).
- Gros, A., et al. Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma. Clin Cancer Res. 18 (19), 5212-5223 (2012).
- Zirakzadeh, A. A., Marits, P., Sherif, A., Winqvist, O. Multiplex B cell characterization in blood, lymph nodes, and tumors from patients with malignancies. J Immunol. 190 (11), 5847-5855 (2013).
- Gu-Trantien, C., et al. CD4(+) follicular helper T cell infiltration predicts breast cancer survival. J Clin Invest. 123 (7), 2873-2892 (2013).
- Buisseret, L., et al. Lymphocytes Infiltrating Breast Cancer : Density, Composition And Organization. Annals of Oncology. 25 (1), 17 (2014).
- Garaud, S., et al. Characterization of B Cells Infiltrating Human Breast Cancer. Annals of Oncology. 25 (1), 18 (2014).
- Gu-Trantien, C., et al. Cxcl13-Producing Follicular Helper T Cells In Human Breast Cancer. Annals of Oncology. 25 (1), 17 (2014).
- Yee, C. The use of endogenous T cells for adoptive transfer. Immunol Rev. 257 (1), 250-263 (2014).
- Butler, M. O., et al. Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help. PLoS One. 7 (1), 30229 (2012).
- Ye, Q., et al. Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes. J Transl Med. 9, 131 (2011).
