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We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.
Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.
Mice have served as the classic model organism for the study of human diseases, including malignancy. As mammals, they share many similarities with humans. Their high degree of genetic similarity has permitted transgenic manipulation of the mouse genome to provide enormous insight into the genetic control of human diseases1. Extensive experience in the handling of and experimentation with mice has resulted in their being the model of choice for biomedical research. However, in addition to the ethical and scientific concerns regarding murine models, they can also be quite costly and time consuming2,
All of the experiments presented herein were reviewed and approved by the appropriate ethical committees at the University of California, Los Angeles (UCLA). The use of deidentified, primary human tumors has been approved by the UCLA Institutional Review Board (Protocol numbers 17-000037, 17-001169, and 11-001363). At UCLA, Animal Research Committee review is not required for experiments using chicken embryos; protocol approval is only required when the eggs will be hatched. However, best practices, such as the AVMA Guid.......
Thus far, we have found this method of implantation to be successful for ovarian, kidney, prostate, and bladder cancers. Each was optimized to identify specific conditions for implantation, although there may be flexibility. Of the tested tumor types, ovarian cancer growth was much less pronounced and typically not visible without the assistance of bioluminescence imaging (Figure 1). However, a stiffening of the CAM could be felt with forceps in the area of i.......
Tumor expansion and engraftment using the CAM model permits more rapid and directly observable tumor growth than existing in vivo animal models. In addition, costs are significantly lower once the initial purchase of equipment is complete, especially when compared to the cost of immunocompromised mice. The initial, immunocompromised state of chicken embryos readily permits engraftment of human and murine tissue. Even with these strengths, the CAM model does have limitations. The short time that can be a benefit could als.......
The authors wish to thank Dr. Fuyuhiko Tamanoi and Binh Vu for the initial training on this method. Discussions with Dr. Eva Koziolek have been instrumental in optimizing this approach and have been very much appreciated. This work would not have been possible without funding from the following sources: the Tobacco-Related Disease Research Program Postdoctoral Fellowship (27FT-0023, to ACS), the Department of Defense (DoD) Ovarian Cancer Research Program (W81XWH-17-1-0160), NCI/NIH (1R21CA216770), Tobacco-Related Disease Research Program High Impact Pilot Award (27IR-0016), and UCLA institutional support, including a JCCC Seed Grant (NCI/NIH P30CA016042) and a 3R Gran....
Name | Company | Catalog Number | Comments |
-010 Teflon (PTFE) White 55 Duro Shore D O-Rings | The O-Ring Store | TEF010 | Nonstick ring for cell seeding. 1/4"ID X 3/8"OD X 1/16"CS Polytetrafluoroethylene (PTFE). |
C4-2 | ATCC | CRL-3314 | Human prostate cancer cell line. |
CWR22Rv1 | CWR cells were the kind gift of Dr. David Agus (Keck Medicine of University of Southern California) | ||
Cytokeratin 8/18 Antibody (C-51) | Novus Biologicals | NBP2-44929-0.02mg | Used at a dilution of 1:100 for immunohistochemical analysis of human ovarian CAM tumors. |
D-Luciferin Firefly, potassium salt | Goldbio | LUCK-1G | |
Delicate Operating Scissors; Curved; Sharp-Sharp; 30mm Blade Length; 4-3/4 in. Overall Length | Roboz Surgical | RS6703 | This is provided as an example. Any similar curved scissors would work as well. |
Dremel 8050-N/18 Micro 8V Max Tool Kit | Dremel | 8050-N/18 | This kit contains all necessary tools. |
Fertilized chicken eggs (Rhode Island Red - Brown, Lab Grade) | AA Lab Eggs Inc. | N/A | A local egg supplier would need to be identified, as this supplier only delivers regionally. |
HT-1376 | ATCC | CRL-1472 | Human bladder cancer cell line. |
Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit | Incubator Warehouse | HB1588D-NONE-1102-1588-1357 | Other egg incubators may be used, but their reliability would need to be verified. After implantation, a cell incubator with the CO2 disabled may also be used. |
ID8 | Not commercially available, please see PMID: 10753190. | ||
Incu-Bright Cool Light Egg Candler | Incubator Warehouse | 1102 | Other candlers may be used; however, this is preferred among those that we have tested. This candler is included in the aforementioned incubator kit. |
Iris Forceps, 10cm, Curved, Serrated, 0.8mm tips | World Precision Instrument | 15915 | This is provided as an example. Any similar curved forceps would work as well. Multiple brands have been used for this method. |
Isoflurane | Clipper Distributing | 0010250 | |
IVIS Lumina II In Vivo Imaging System | Perkin Elmer | ||
Matrigel Membrane Matrix HC; LDEV-Free | Corning | 354248 | Extracellular matrix solution |
MyC-CaP | ATCC | CRL-3255 | Murine prostate cancer cell line. |
Portable Pipet-Aid XP Pipette Controller | Drummond Scientific | 4-000-101 | Any similar pipet controller would be appropriate. |
PrecisionGlide Hypodermic Needles | BD | 305196 | This is provided as an example. Any 18G needle would work similarly. |
RENCA | ATCC | CRL-2947 | |
Semken Forceps | Fine Science Tools | 11008-13 | This is provided as an example. Any similar forceps or another style that suits researcher preference would be appropriate. |
SKOV3 | ATCC | HTB-77 | Human ovarian cancer cell line. |
Specimen forceps | Electron Microscopy Sciences | 72914 | This is provided as an example. The forceps used for pulling away the shell for bioluminescence imaging are approximately 12.8 cm long with 3 mm-wide tips. |
Sterile Cotton Balls | Fisherbrand | 22-456-885 | This is provided as an example. Any sterile cotton balls would suffice. |
Stirring Rods with Rubber Policeman; 5mm diameter, 6 in. length | United Scientific Supplies | GRPL06 | This is provided as an example. Any similar glass stir rods would work as well. |
T24 | ATCC | HTB-4 | Human bladder cancer cell line. |
Tegaderm Transparent Dressing Original Frame Style 2 3/8" x 2 3/4" | Moore Medical | 21272 | |
Tissue Culture Dishes, 10 cm diameter | Corning | 353803 | This is provided as an example. Any similar, sterile 10-cm dish may be used. Tissue culture treatment is not necessary. |
Tygon Clear Laboratory Tubing - 1/4 x 3/8 x 1/16 wall (50 feet) | Tygon | AACUN017 | This is provided as an example. Any similarly sized tubing would work as well. |
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