Generation af Organ aircondition Medier og applikationer for at studere Organspecifikke påvirkninger på brystkræft Metastatisk Behavior
Summary
This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.
Abstract
Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.
Introduction
Brystkræft er den hyppigst diagnosticerede kræft hos kvinder og den anden hyppigste årsag til kræft-dødsfald 1. Brystkræft høje dødelighed skyldes primært svigt af konventionel behandling for at mindske og eliminere metastatisk sygdom; ca. 90% af kræftrelaterede dødsfald skyldes metastaser 2. Forståelse af de underliggende molekylære mekanismer i den metastatiske kaskade er altafgørende for udviklingen af terapeutiske effektive i både tidlige og sene brystkræft.
Tidligere forskning har hjulpet belyse flertrins karakter af brystkræft metastase og det antages, at resultatet af både cancer progression og metastase er i vid udstrækning afhængig interaktioner mellem cancerceller og værtsmiljøet 3. Kliniske observationer indikerer, at mange kræftformer vise orgel tropisme, dvs.., Tendensen til fortrinsvis metastaserer til specifikke organs.In den case af brystcancer, en patients sygdom typisk spreder eller metastaserer til 5 vigtigste steder, herunder knogle, lunger, lymfeknuder, lever og hjerne 4-6. Mange teorier er blevet udviklet for at forklare denne proces, men kun få har klaret testen af tid. Ewing teori om metastaser, foreslog i 1920'erne, hypotese thatthe distribution af metastaser var strengt på grund af mekaniske faktorer; hvorved tumorceller bæres hele kroppen ved normale definerede fysiologisk blod strømningsmønstre og simpelthen standse i den første kapillære leje de støder 7. I modsætning hertil Stephen Pagets 1889 "frø og jord" hypotese foreslog, at yderligere molekylære interaktioner var ansvarlige for overlevelse og vækst af metastaser, hvorved cancerceller ( "frø") kun kan etablere sig og proliferatein orgel mikromiljøer, der producerer passende molekylære faktorer ( "jord ") 8. Næsten et århundrede senere, Leonard Weiss undertog en meta-analyse af tidligere offentliggjorte Obduktionsdata og bekræftede Ewings forudsigelse om, at mange metastatiske tumorer detekteret ved obduktion blev fundet i de forventede forhold, som kunne forventes, hvis metastatisk orgel tropisme blev bestemt ved blod strømningsmønstre alene. Men i manyinstances der var, færre eller flere metastaser dannet på bestemte steder så ville forventes af Ewing foreslåede mekaniske faktorer 9. Disse konti og teorier tyder på, at specifikke orgel mikromiljøer spiller en kritisk rolle i formidling mønstre og efterfølgende vækst og overlevelse af mange kræftformer, herunder brystkræft.
Tidligere forskningsindsats har hovedsagelig fokuseret på tumor-celle afledte faktorer og deres bidrag til orglet tropisme observeret i brystkræft metastaser 10-12, dog lidt forskning har udforsket faktorer afledt af orgel mikromiljø, der kan give en gunstig niche for etableringaf brystkræft metastaser. Dette skyldes i høj grad de tekniske udfordringer ved at studere dele af orglet mikromiljø in vitro.
Den aktuelle artikel beskriver en omfattende ex vivo modelsystem til undersøgelse af indflydelsen af opløselige bestanddele af lymfeknude, knogle, lunge og hjerne på den metastatiske adfærd af humane brystcancerceller. Tidligere undersøgelser har valideret dette modelsystem ved at demonstrere, at forskellige brystcancercellelinier vise cellelinje specifik og organspecifik malign adfærd som reaktion på organ-konditionerede medier, der svarer til deres in vivo metastatiske potentiale 13. Denne model kan anvendes til at identificere og vurdere specifikke organ-afledte opløselige faktorer, der kan spille en rolle i den metastatiske adfærd bryst- og andre typer af cancerceller, herunder påvirkninger af væksten, migration, stængel-lignende opførsel, og genekspression, samt identificering afpotentielle nye terapeutiske mål for kræft. Dette er den første ex vivo-model, der kan bruges til at studere organspecifik metastatisk adfærd i detaljer og at vurdere betydningen af organ-afledte opløselige faktorer i drive processen af cancer metastase.
Protocol
Representative Results
Discussion
Metastase er en kompleks proces, hvor en række cellulære begivenheder er i sidste ende ansvarlig for vævs- invasion og fjernt tumor etablering 4,30,31. Den ex vivo modelsystem præsenteres her kan bruges til at studere to vigtige aspekter af metastatisk progression: kræft celle målsøgende eller migrering til et specifikt organ ( "at komme der") og vækst i dette organ ( "vokser der"). Mange undersøgelser har tidligere fokuseret på at identificere vigtige molekylære egensk…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master’s program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.
Materials
| 50 ml conical tubes | Thermo Scientific (Nunc) | 339652 | Keep sterile |
| 1X Phosphate-buffered saline | ThermoFisher Scientific | 10010-023 | Keep sterile |
| Nude mice | Harlan Laboratories | Hsd:Athymic Nude-Foxn1nu | Use at 6-12 weeks of age |
| Polystyrene foam pad | N/A | N/A | The discarded lid (~4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol. |
| Forceps | Fine Science Tools | 11050-10 | Keep sterile |
| Scissors | Fine Science Tools | 14058-11 | Keep sterile |
| Gauze pads | Fisher Scientific | 22-246069 | Keep sterile |
| 60 mm2 glass petri dishes | Sigma-Aldrich | CLS7016560 | Keep sterile |
| Scalpel blades | Fisher Scientific | S95937A | Keep sterile |
| DMEM:F12 | Life Technologies | 21331-020 | Warm in 37 °C water bath before use, keep sterile |
| 1 x Mito+ Serum Extender | BD Biosciences | 355006 | Referred to as "concentrated mitogen supplement" in the manuscript. Keep sterile |
| Penicillin-Streptomycin (10,000 U/mL) | Life Technologies | 15140-122 | Keep sterile |
| Rosewell Park Memorial Institute 1640 (RPMI 1640) | Life Technologies | 11875-093 | Warm in 37 °C water bath before use, keep sterile |
| Fetal Bovine Serum | Sigma-Aldrich | F1051-500ML | Keep sterile |
| Trypsin/EDTA solution | ThermoFisher Scientific | R-001-100 | Warm in 37 °C water bath before use, keep sterile |
| 6-well tissue culture plates | Thermo Scientific (Nunc) | 140675 | Keep sterile |
| 0.22 μm syringe filters | Sigma-Aldrich | Z359904 | Keep sterile |
| T75 tissue culture flasks | Thermo Scientific (Nunc) | 178905 | Keep sterile |
| Transwells | Sigma-Aldrich | CLS3464 | Keep sterile, use for migration assays |
| Anti-mouse Sca-1 | R&D Systems | FAB1226P | use at 10 µl/106 cells |
| Anti-mouse CD105 | R&D Systems | FAB1320P | use at 10 µl/106 cells |
| Anti-mouse CD29 | R&D Systems | FAB2405P-025 | use at 10 µl/106 cells |
| Anti-mouse CD73 | R&D Systems | FAB4488P | use at 10 µl/106 cells |
| Anti-mouse CD44 | R&D Systems | MAB6127-SP | use at 0.25 µg/106 cells |
| Anti-mouse CD45 | eBioscience | 11-0451-81 | use at 5 µl/106 cells |
| Anti-mouse gp38 | eBioscience | 12-5381-80 | use at 10 µl/106 cells |
| β-mercaptoethanol | Sigma-Aldrich | M6250 | Keep sterile |
| Protein arrays | RayBiotech Inc. | AAM-BLM-1-2 | Use 1 array per media condition (including negative control), in triplicate |
References
- Siegel, R. L., Miller, K. D., Jemal, A. Cancer statistics, 2015. CA Cancer J Clin. 65 (1), 5-29 (2015).
- Fidler, I. J. The organ microenvironment and cancer metastasis. Differentiation. 70 (9-10), 498-505 (2002).
- Chambers, A. F., Groom, A. C., MacDonald, I. C. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2 (8), 563-572 (2002).
- Kennecke, H., et al. Metastatic behavior of breast cancer subtypes. J Clin Oncol. 28 (20), 3271-3277 (2010).
- Nguyen, D. X., Bos, P. D., Massague, J. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer. 9 (4), 274-284 (2009).
- Ewing, J. Neoplastic Diseases: A Treatise on Tumors. Am J Med Sci. 176 (2), 278 (1928).
- Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 8 (2), 98-101 (1989).
- Weiss, L. Comments on hematogenous metastatic patterns in humans as revealed by autopsy. Clin Exp Metastasis. 10 (3), 191-199 (1992).
- Bos, P. D., et al. Genes that mediate breast cancer metastasis to the brain. Nature. 459 (7249), 1005-1009 (2009).
- Kang, Y., et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell. 3 (6), 537-549 (2003).
- Minn, A. J., et al. Genes that mediate breast cancer metastasis to lung. Nature. 436 (7050), 518-524 (2005).
- Chu, J. E., et al. Lung-Derived Factors Mediate Breast Cancer Cell Migration through CD44 Receptor-Ligand Interactions in a Novel Ex Vivo System for Analysis of Organ-Specific Soluble Proteins. Neoplasia. 16 (2), (2014).
- Deepak, S., et al. Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes. Curr Genomics. 8 (4), 234-251 (2007).
- Hammerschmidt, S. I., et al. Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo. J Exp Med. 205 (11), 2483-2490 (2008).
- Dominici, M., et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8 (4), 315-317 (2006).
- Baddoo, M., et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem. 89 (6), 1235-1249 (2003).
- Furger, K. A., Menon, R. K., Tuck, A. B., Bramwell, V. H., Chambers, A. F. The functional and clinical roles of osteopontin in cancer and metastasis. Curr Mol Med. 1 (5), 621-632 (2001).
- Radisky, E. S., Radisky, D. C. Matrix metalloproteinases as breast cancer drivers and therapeutic targets. Front Biosci (Landmark Ed). 20, 1144-1163 (2015).
- Radisky, E. S., Radisky, D. C. Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer. J Mammary Gland Biol Neoplasia. 15 (2), 201-212 (2010).
- Radisky, E. S., Radisky, D. C. Stromal induction of breast cancer: inflammation and invasion. Rev Endocr Metab Disord. 8 (3), 279-287 (2007).
- Kakinuma, T., Hwang, S. T. Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol. 79 (4), 639-651 (2006).
- Zlotnik, A. Chemokines and cancer. Int J Cancer. 119 (9), 2026-2029 (2006).
- Schlesinger, M., Bendas, G. Vascular cell adhesion molecule-1 (VCAM-1)–an increasing insight into its role in tumorigenicity and metastasis. Int J Cancer. 136 (11), 2504-2514 (2015).
- Cook, K. L., Shajahan, A. N., Clarke, R. Autophagy and endocrine resistance in breast cancer. Expert Rev Anticancer Ther. 11 (8), 1283-1294 (2011).
- Singh, P., Alex, J. M., Bast, F. Insulin receptor (IR) and insulin-like growth factor receptor 1 (IGF-1R) signaling systems: novel treatment strategies for cancer. Med Oncol. 31 (1), 805 (2014).
- Lee, S. H., Jeong, D., Han, Y. S., Baek, M. J. Pivotal role of vascular endothelial growth factor pathway in tumor angiogenesis. Ann Surg Treat Res. 89 (1), 1-8 (2015).
- Erdmann, R. B., Gartner, J. G., Leonard, W. J., Ellison, C. A. Lack of functional TSLP receptors mitigates Th2 polarization and the establishment and growth of 4T1 primary breast tumours but has different effects on tumour quantities in the lung and brain. Scand J Immunol. 78 (5), 408-418 (2013).
- Chambers, A. F., et al. Steps in tumor metastasis: new concepts from intravital videomicroscopy. Cancer Metastasis Rev. 14 (4), 279-301 (1995).
- Chiang, A. C., Massague, J. Molecular basis of metastasis. N Engl J Med. 359 (26), 2814-2823 (2008).
- Gupta, G. P., et al. Identifying site-specific metastasis genes and functions. Cold Spring Harb Symp Quant Biol. 70, 149-158 (2005).
- Frantz, C., Stewart, K. M., Weaver, V. M. The extracellular matrix at a glance. J Cell Sci. 123, 4195-4200 (2010).
- Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng Part A. 16 (8), 2581-2591 (2010).
- Bonnans, C., Chou, J., Werb, Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol. 15 (12), 786-801 (2014).
- Hynes, R. O. The extracellular matrix: not just pretty fibrils. Science. 326 (5957), 1216-1219 (2009).
- Psaila, B., Lyden, D. The metastatic niche: adapting the foreign soil. Nat Rev Cancer. 9 (4), 285-293 (2009).
- Lee, R. H., Oh, J. Y., Choi, H., Bazhanov, N. Therapeutic factors secreted by mesenchymal stromal cells and tissue repair. J Cell Biochem. 112 (11), 3073-3078 (2011).
