Abstract
Background: The high incidence of vascular and lymphatic metastasis is closely associated with poor prognosis and mortality in cancer. Finding effective inhibitors to prevent pathological angiogenesis and lymphangiogenesis relies on appropriate in vivo models. The chick embryo chorioallantoic membrane (CAM) is formed by the fusion of the chorion and allantois during embryonic development. Summary: In this context, we primarily summarize the changes in vascular and lymphatic vessel formation in tumors under the action of drugs using this model, providing a preclinical model basis for effective tumor inhibitors. Key Messages: Due to natural immunological defects, chick embryos accept various tissue and species transplants without immune response. The CAM model has been widely used in studying angiogenesis, antiangiogenesis, tumor growth, tumor metastasis, and drug efficacy. This review describes the use of CAM assays as a valuable method for testing the in vivo effects of drugs on vascular and lymphatic vessel formation before further investigating the effects of drugs on tumor vessels and lymphatic vessels in animal models.
Introduction
Angiogenesis and lymphangiogenesis play significant roles in tumor metastasis and spread [1]. Drugs targeting angiogenetic cascades have been developed, approved, and become standard treatment for certain diseases [1]. For example, bevacizumab, targeting vascular endothelial growth factor (VEGF)-A and inhibiting angiogenesis, is approved for treating advanced stages of colorectal, ovarian, and lung cancers [2]. Clinical evidence shows tumor cell dissemination through lymphatic vessels is three to five times more likely than through blood vessels [3]. Hence, radical lymphatic vessels and lymph node resection have become standard in cancer treatment [4]. However, few therapeutic strategies exist to inhibit lymphangiogenesis (such as lymphatic-target therapeutics) in cancer or to modulate the lymphatics in chronic inflammation [5].
Establishing appropriate in vivo models is crucial for studying tumor-derived blood and lymphatic vessel growth and identifying new antiangiogenic or anti-lymphangiogenic drugs. Most preclinical models are expensive, time-consuming, require surgical expertise, and need ethics committee approval [6, 7]. The 3Rs principle (Russell and Burch’s principle) in animal experimentation recommends replacing, reducing, and refining methods to minimize suffering [8, 9]. In this context, the chick embryo chorioallantoic membrane (CAM) assay has been introduced as an alternative in vivo model. It has been used to investigate endothelial cell proliferation, migration, and differentiation [10, 11], extracellular matrix remodeling [12], tumor-induced angiogenesis and metastasis [3‒15], and pharmacological compound effects [13, 14]. The CAM model offers advantages like short experimental period, rapid growth, low cost, ease of handling and housing, and natural immune deficiency of chick embryos [15, 16]. Its transparent membrane provides real-time visualization of embryonic development and vessel growth [15, 16]. The extensive vascularization and accessibility of the CAM model make it an ideal platform for studying antiangiogenic or anti-lymphangiogenic molecules. Existing research shows its value in both angiogenesis and lymphangiogenesis studies [9, 17‒19]. This review discusses the morphological structure and physiology of the CAM model, vascular and lymphatic-related research, and outlines its potential for future research in oncology, plastic surgery, and tissue engineering.
Structure and the Development of the CAM Model
The CAM consists of three layers: chorionic epithelium (ectoderm), mesenchyme (mesoderm), and allantoic epithelium (endoderm) [18, 20] (Fig. 1). The chorionic epithelium, composed of ectoderm and attached to the shell, primarily facilitates gas exchange [16]. The mesoderm, composed of vascular and stromal elements, serves as the foundation for the subsequent formation of the capillary plexus of the allantoic capillary network [16]. At embryonic day 3.5 (ED3.5), allantois development begins, and at ED4, the endodermal hind pushes out of the embryo’s body and enters the embryo’s outer body [16]. During ED4–10, the allantoic vesicle grows rapidly. During this time, the mesoderm of the allantoic and the mesodermal layer of the chorion begin to combine to form the CAM [16].
Structural diagram of chick embryo allantoic membrane model as a platform for cancer research.
Structural diagram of chick embryo allantoic membrane model as a platform for cancer research.
This vascularized allantois and avascular chorion combination causes the CAM surface to rapidly expand from 6 cm2 on ED6 to 65 cm2 on ED14, providing a large surface for in vivo experimentation [21]. Chick embryos typically incubate for 21 days, but experimental research is often conducted before ED17, as they are not considered living animals and do not require ethical approval in most countries [22].
The CAMs rich vascularization provides nutrients for transplanted tumors and fosters tumor growth. Immature blood vessels begin sprouting on ED1 and grow rapidly before ED8, generating capillary tufts and facilitating gas exchange with the external environment [23]. Capillary density continues to proliferate rapidly until ED8–11, after which the proliferation decreases [23]. During ED14, the density of blood vessels remains constant and angiogenesis ceases [10, 12, 24]. At ED18, the chick development is nearly concluded and blood and lymphatic vessels of the CAM begin to degenerate [12].
The lymphatics on the CAM developed more slowly than the vascular vessels. Experiments with quail-chicken chimeras demonstrated that allantoic mesoderm differentiate into lymphatic vessels [18]. Lymphatic vessels are associated with veins, and the larger veins are surrounded by lymphatic plexus [25]. The lymphatics of CAM have dual origins. The lymphatic system is primarily endogenous, with portions originating from posterior lymph sacs [18]. The lymphatics on the CAM drain via the umbilicus lymphatic trunks, whereas the lymphatic vessels within the embryo drain via the thoracic duct to the subclavian vein [18]. The deep lymphatics (located near the large veins) appear at ED4–5, whereas the lymphangiogenesis on the CAM begins at ED5–9 [26]. The formation of lymphatic vessels is accompanied by the expansion of blood vessels and blind-ended sprouts [27]. Initially, isolated lymphatic endothelial progenitor cells gather at the tips of developing blood vessels. The lymphatics then expand and divide, followed by the expansion of the blood vessels [18]. At that time, the polarized mural progenitor cells express alpha-smooth muscle actin [27]. At ED15, the immune system of the CAM begins to develop. Although the immune system responds to the tumor through the infiltration of monocytes and inflammatory cells (avian heterophages), the immune response will not be activated before ED18, allowing the transplantation of xenografts or cells without the risk of rejection [28].
In the CAM model, the molecular expression is comparable to that of mammals. Vascular endothelial growth factor receptor (VEGFR)-2 and -3 and prospero homeobox protein 1 (Prox-1) positively express on lymphatic cells [17]. VEGFR-2 is expressed in both lymphatic and blood vessels, whereas VEGR-3 is specifically expressed in lymphatic vessels [17]. The presence of vascular endothelial growth factor C (VEGF-C) is associated with lymphangiogenesis; it exists in two forms, immature and mature, with the mature form capable of binding to VEGFR-2 in the vascular blood, lymphatic vessels, and differentiated chorioallantoic membrane [29]. It can stimulate lymphatic endothelial cell proliferation and the formation of enlarged lymphatic sinuses [30]. VEGF-A is also present in the CAM, with its secretion peaking twice between ED8–9 and ED11–12 [31, 32]. VEGF-A/hepatocyte growth factor can stimulate the expression of Prox-1 in lymphatic endothelial cells [33]. After 2 weeks of incubation, most growth factors on the CAM reach a peak [33]. Fibroblast growth factor-2 is another endogenous cytokine that promotes angiogenesis, appearing at ED6–18 and peaking at ED10–14 during the chick development [34]. Hypoxia-inducible factor 1 alpha, VEGF, and VEGFR-2 have the highest expression on ED11, which is also associated with the peak in angiogenesis [35, 36].
Angiogenesis within the CAM Model
Angiogenesis is essential for tumor growth, facilitating the supply of nutrients and oxygen, transportation of metabolic waste, and migration to distant sites. The structure of the CAM fosters a microenvironment for tumor development [37]. During the first few days after a potential xenograft transplantation, the transplanted cells will be nourished by diffusion from the CAM [38]. Tumor growth might become visible without magnification after 2–3 days following transplantation [15]. The detailed process of xenograft transplantation is described in Figure 2.
Various compounds and growth factors can stimulate or inhibit angiogenesis on the CAM, and transplanted tumors undergo approximately 72 h of angiogenesis before tumor invasion [16]. The vascular reaction related to the graft occurred 72–96 h after transplantation. Due to the increased density of blood vessels surrounding the implant, the vascular reaction appears as blood vessels converge toward the implant’s center in a “spoke-wheel” pattern [16]. During the development of blood vessels, the mass and volume of the tumor grow rapidly, and necrosis of the tumor center will occur if the new blood vessels cannot support the maturation of the tumor [39]. Tumor blood vessels exhibit abnormal structures, with dilation, chaotic and uneven distribution, and irregular branching, in contrast to normal vessels [40]. Scaffolds such as collagen, Matrigel, and fibrin have been widely used in the CAM assay [41‒43]. Transplanting tumor cells together with the matrix into the CAM is essential for rapid tumor formation because of the vascularization of the scaffold on the CAM membrane.
The CAM model shows practicality in assessing the impact of drugs or tissues on angiogenesis. A series of representative examples exploring angiogenesis using the CAM model is illustrated in Table 1. Furthermore, the CAM model has been used to determine the effect of methylated resveratrol derivatives on angiogenesis, and consistent results are shown regarding the impact on angiogenesis in both the rat aortic ring and the CAM model [44]. The CAM model was also utilized to assess the efficacy of natural curcumin and curcumin-capped copper nanoparticles as potential anticancer and antiangiogenic agents [45]. The relationship between angiogenesis and the tumor microenvironment is studied by Kern et al. [46] on the CAM model. They certified the angiogenesis-inhibiting effect of bortezomib could be antagonized by soluble GRP-78 secreted by certain tumors and demonstrated that solid tumor cells can secrete GRP-78 into the microenvironment of the tumor on the CAM model [46]. The angiogenic potential of tumor tissues can also be validated on the CAM model. Vacca et al. confirmed the potential of lipoma tissue angiogenesis on the CAM model, and the effect of promoting angiogenesis was equivalent to that of fibroblast growth factor-2, confirming that the CAM model is a useful model for this type of research [47]. The CAM model is widely used to investigate the effect of growth factors on antiangiogenesis. The artemisinin derivative FO-ARS-123 acts as a novel VEGFR-2 inhibitor suppressing angiogenesis on the CAM [48]. Rao et al. [49] demonstrated that dihydroartemisinin inhibits angiogenesis in breast cancer on the CAM by regulating VEGF and MMP-2/-9. Methods for quantifying angiogenesis on the CAM primarily include the following techniques [23, 50]:
- 1.
Morphometric analysis to measure vascular diameter and length.
- 2.
Observation of vascular density using a fluorescent confocal microscope.
- 3.
Recording of capillary length, diameter, and density under a scanning electron microscope (Fig. 3).
- 4.
Calculation of branch points per square millimeter.
- 5.
Observation of blood vessel density on tissue slices.
Key publications of angiogenesis related research on the CAM model
Author, year . | Inoculation, observation time . | Tumor type, compounds . | Methods . |
---|---|---|---|
Subauste [42] (2009) | ED10, ED12.7–ED13.5 | Human SW620 colon carcinoma cell line, type I rat tail collagen | The number of grids containing angiogenic blood vessels |
Zhou et al. [51] (2015) | ED8, ED10 | Cervical cancer (HeLa), cell suspension | The number of second and third-order vessels |
Ferician [52] (2015) | ED7, ED14 | Human renal tumor, tissue fragments | IHC to test angiogenic growth factors and vascular network |
Patil [53] (2016) | ED5, ED7 | Human γδT gallbladder cancer cell line, cell suspension | The number of vessels’ branching points |
Valiulytė et al. [54] (2019) | ED7, ED12 | Human U87 MG glioblastoma cell line, type I rat tail collagen | The number of blood vessels bigger than 10 µm |
Author, year . | Inoculation, observation time . | Tumor type, compounds . | Methods . |
---|---|---|---|
Subauste [42] (2009) | ED10, ED12.7–ED13.5 | Human SW620 colon carcinoma cell line, type I rat tail collagen | The number of grids containing angiogenic blood vessels |
Zhou et al. [51] (2015) | ED8, ED10 | Cervical cancer (HeLa), cell suspension | The number of second and third-order vessels |
Ferician [52] (2015) | ED7, ED14 | Human renal tumor, tissue fragments | IHC to test angiogenic growth factors and vascular network |
Patil [53] (2016) | ED5, ED7 | Human γδT gallbladder cancer cell line, cell suspension | The number of vessels’ branching points |
Valiulytė et al. [54] (2019) | ED7, ED12 | Human U87 MG glioblastoma cell line, type I rat tail collagen | The number of blood vessels bigger than 10 µm |
In vivo CAM images and in silico CAM capillary network contours. Validate and quantify the effects of control, VEGF 50 ng, and VEGF 100 ng on angiogenesis. Images adapted from Guerra et al. [50] (permission by Elsevier and Copyright Clearance Center [5761730824132]).
In vivo CAM images and in silico CAM capillary network contours. Validate and quantify the effects of control, VEGF 50 ng, and VEGF 100 ng on angiogenesis. Images adapted from Guerra et al. [50] (permission by Elsevier and Copyright Clearance Center [5761730824132]).
Additionally, scientific approaches for quantifying angiogenesis include counting the number of capillaries and the number of blood vessels growing into the collagen matrix [55]. The use of μCT for three-dimensional vascular remodeling is another method employed [56, 57]. The ex oxo and in ovo three-dimensional vascular remodeling picture are depicted in Figure 4.
3D imaging of the vascular network within egg embryos using μCT. a Chick embryo with intravascular injection of barium contrast agent. Surface (b), depth (c), and depth 3D reconstructed (d) images by μCT. e Clinical digital mammographic unit. f–h 2D digital microangiography views. i Microscopic view of H&E staining picture. V: major CAM blood vessel; small red arrows: chorion capillaries. Images adapted from Chen et al. [57] (permission by Elsevier and Copyright Clearance Center [5761670236733]).
3D imaging of the vascular network within egg embryos using μCT. a Chick embryo with intravascular injection of barium contrast agent. Surface (b), depth (c), and depth 3D reconstructed (d) images by μCT. e Clinical digital mammographic unit. f–h 2D digital microangiography views. i Microscopic view of H&E staining picture. V: major CAM blood vessel; small red arrows: chorion capillaries. Images adapted from Chen et al. [57] (permission by Elsevier and Copyright Clearance Center [5761670236733]).
CAM from nontumoral tissue can also be utilized for chemical studies, such as the determination of DNA, protein, and collagen content, as well as reverse transcription-polymerase chain reaction analysis of gene expression in transplanting cells [58]. Histological examinations, including hematoxylin and eosin staining and immunohistochemical staining with CD31 antibody or von Willebrand factor, are common analyses employed to identify sprouting or de novo-generated vessels [51, 59].
Lymphangiogenesis Based on the CAM Model
Lymphangiogenesis, or its absence, is associated with numerous pathological conditions such as cancer metastasis, lymphedema, and organ transplant rejection [3, 60‒62]. The high incidence of lymphatic metastasis is closely linked to poor prognosis and high mortality in cancer patients [3]. Effective inhibitors are urgently needed to prevent pathological lymphangiogenesis and lymphatic cancerous spread. Clinical evidence suggests that lymphatic vessels contribute to cancer cell dissemination 3–5 times more than blood vessels [63]. This phenomenon can be attributed to lymphatic vessel dilation, which facilitates both the infiltration and extravasation of tumor cells, thereby promoting their survival [63].
Multiple in vitro and in vivo techniques have been employed to study the pharmacological regulation of lymphangiogenesis [64]. In vitro culture systems have been developed for the formation of lymphatic vessels, including in vitro culture of lymphatic endothelial cells obtained from Freund’s adjuvant, thoracic duct fragment explants, embryoid bodies, and immune-purified primary or immortalized human dermal cells [65]. The aortic ring assay is used for testing the molecular mechanisms of angiogenesis [66], investigating genetically modification of mouse lines [67], and evaluating the effect of pro- and antiangiogenic agents [68]. Further, the aortic ring assay has been used as a platform for the lymphatic capillary-like sprouting that can be quantified via supplementary imaging of lymphangiogenesis [65]. While these models are useful for studying cell proliferation and migration, their ability to simulate lymphangiogenesis is limited due to the inability to accurately replicate the complex system of growth factors, stabilizing niches, and regulatory factors that contribute to lymphoid growth [65, 69].
Various preclinical models have been developed to investigate lymphangiogenesis and lymphatic dysfunction, providing a better microenvironment for multifactorial diseases [70, 71]. In vivo models of lymphangiogenesis mainly include overexpression of VEGF-C in tumor cells or transgenic mice to induce lymphangiogenesis [72, 73], intraperitoneal injection Freund’s adjuvant reagent to promote lymphangiogenesis in murine models [74, 75], and resection or microsurgery to cutoff mouse lymphatic vessels in the tail [76]. The mouse tail model was developed further to test the efficacy of tacrolimus and identify potential mechanisms for treating lymphedema [77]. Doh et al. [78] developed a fluorescent reporter transgenic mouse model for live in vivo imaging of lymphangiogenesis. A standard model of lower limb lymphedema in rats was established, certifying that the combination of inguinal lymph node dissection, peripheral skin, and subcutaneous tissue resection is translationally sufficient, and no radiotherapy is necessary [79]. Another lymphedema model in rabbits was established by microsurgically transferring lymph nodes without transferring lymph vessels or cells, validating this model as a reliable tool for refining microsurgical techniques for lymphedema surgeries [80]. However, a limitation of using these models is the inflammatory response, making it difficult to distinguish whether the formation of lymphatic vessels is directly affected by lymphatic endothelial cells or indirectly affected by the production of inflammatory mediators [65].
The CAM model is also a valuable preclinical model for lymphangiogenesis experiments, particularly those involving immune cell trafficking, transplantation, therapeutic responses, and metastasis [30, 81‒84]. However, many studies examining lymphangiogenesis using the CAM model remain highly limited. Table 2 summarizes representative tumor metastasis experimentation on the CAM model.
Key publications of lymphangiogenesis related research on the CAM model
Author, year . | Inoculation, observation time . | Tumor type, compounds . | Methods . |
---|---|---|---|
Papoutsi et al. [85] (2001) | ED8 and ED10, ED14–17 | Rat C6 glioma and 10AS pancreatic carcinoma cells, cell suspension | 1. Protein expression of lymphatic marker (immunohistochemistry for α-SMA) |
2. Gene expression of lymphatic vessel growth regulator (Northern blot analysis of VEGF-C and -D) | |||
3. Lymphatics within tumor (VEGFR-3/Quek2 in situ hybridization) | |||
Deryugina [86] (2005) | ED10, ED11–16 | Human HT-1080 fibrosarcoma cell line, cell suspension injection | Lymphatic metastasize potential from the primary tumor (assessing tumor invasiveness via metastasis from chorionic vessels to lungs by Alu PCR, Immunohistochemistry for murine mAb 29-7) |
Subauste et al. [42] (2009) | ED12, ED15 | Human SW620 colon carcinoma cell line and SW480 cell variant, cell suspension injection | 1. Live cell imaging for cell extravasation from CAM vessels (cell tracker CMFDA) |
2. Assessing cell colonization in CAM and visceral organs (liver, spleen, lungs) by Alu qPCR | |||
3. Colon cancer cell colonization on the CAM (immunohistochemistry for antihuman pan-cytokeratin monoclonal antibodies) | |||
Lokman et al. [43] (2012) | ED11, ED14 | Human OVCAR-3, SKOV-3, and OV-90 ovarian cancer cell lines, Matrigel | Ovarian cancer cell invasion on the CAM (immunohistochemistry for antihuman cytokeratin clone AE1/AE3) |
Author, year . | Inoculation, observation time . | Tumor type, compounds . | Methods . |
---|---|---|---|
Papoutsi et al. [85] (2001) | ED8 and ED10, ED14–17 | Rat C6 glioma and 10AS pancreatic carcinoma cells, cell suspension | 1. Protein expression of lymphatic marker (immunohistochemistry for α-SMA) |
2. Gene expression of lymphatic vessel growth regulator (Northern blot analysis of VEGF-C and -D) | |||
3. Lymphatics within tumor (VEGFR-3/Quek2 in situ hybridization) | |||
Deryugina [86] (2005) | ED10, ED11–16 | Human HT-1080 fibrosarcoma cell line, cell suspension injection | Lymphatic metastasize potential from the primary tumor (assessing tumor invasiveness via metastasis from chorionic vessels to lungs by Alu PCR, Immunohistochemistry for murine mAb 29-7) |
Subauste et al. [42] (2009) | ED12, ED15 | Human SW620 colon carcinoma cell line and SW480 cell variant, cell suspension injection | 1. Live cell imaging for cell extravasation from CAM vessels (cell tracker CMFDA) |
2. Assessing cell colonization in CAM and visceral organs (liver, spleen, lungs) by Alu qPCR | |||
3. Colon cancer cell colonization on the CAM (immunohistochemistry for antihuman pan-cytokeratin monoclonal antibodies) | |||
Lokman et al. [43] (2012) | ED11, ED14 | Human OVCAR-3, SKOV-3, and OV-90 ovarian cancer cell lines, Matrigel | Ovarian cancer cell invasion on the CAM (immunohistochemistry for antihuman cytokeratin clone AE1/AE3) |
α-SMA, alpha-smooth muscle actin.
Oh et al. [25] described the structure of lymphatic vessels on the CAM model and confirmed that VEGF and VEGF-C can promote angiogenesis and lymphangiogenesis in the chick embryo. In a further translational step, the mechanisms underlying melanoma metastasis and tumor lymphatic vessel formation were comprehensively characterized [87]. The VEGF-C/flt4 interaction, crucial for tumor-associated lymphatic vessel formation, has been confirmed in the CAM model by Papoutsi et al. [88]. Others examined C6 glioma (low expression of VEGF-C) and 10AS pancreatic carcinoma (high expression of VEGF-C) in this model and found that 10AS can stimulate lymphangiogenesis [85]. This demonstrated that the lymphatic spreading of cancer is an interspecies conserved pathomechanisms and the in vivo lymphangiogenesis in the CAM of tumoral xenograft-derived lymphangiogenic factors [85]. Cimpean et al. [32] demonstrated that the angiogenesis-promoting cytokines VEGF-A and hepatocyte growth factor not only promote the expression of blood vessels in the CAM but also the expression of Prox-1-positive lymphatic endothelial cells. The dual effects of everolimus on lymphatic vessels and the potential effects of the drug on lymphangiogenesis were also confirmed in this model [89]. Klingenberg used the CAM model as the xenograft model of Burkitt lymphoma to study real-time lymphatic metastasis, confirming that CAM can be used as a tool for studying real-time lymphatic metastasis [90].
Like in angiogenesis, the CAM model can also serve as a valuable model for studying lymphangiogenesis. However, the full potential of in vivo studies of lymphangiogenesis in this model has not been realized due to the lack of specific antibodies against chick embryo antigenic determinants.
Conclusion
The formation of blood vessels and lymphatic vessels is a dynamic process that depends not only on the regulation of endothelial cells of the microvasculature and perivascular lymphatic cells but also on hemodynamics, the microenvironment, and growth factors [4]. The CAM model has been effectively utilized to investigate the proliferation, migration, and differentiation of endothelial cells, as well as the observation and analysis of tumor-induced angiogenesis and metastasis [82]. It has also been valuable for studying the provascular or anti-vascular effects of pharmacological compounds and for the development of translational tissue engineering [23]. However, current research on lymphatic vessels in CAM models, such as immune cell trafficking, graft rejection, and metastasis of tumor through the lymphatic vessels, is still limited. With extensive research on lymphatic vessels, we anticipate a great ability to test the inhibitory effect of related drugs on blood vessels and lymphatic vessels via the allantoic membrane of chicken embryos or to use this model to hinder the blood and lymphatic metastasis of related tumors.
Compared to conventional animal models, the CAM model requires only 2–5 days following cell suspension to observe tumor growth that can be monitored throughout the entire embryological development of the chick [15, 30]. It offers the advantages of a short cycle time, simple operation, and low cost [23]. However, CAM models still have limitations. There are few specific antibodies against antigenic determinants of chicken lymphatic vessels, and it is difficult to distinguish between the angiogenesis and lymphangiogenesis secondary to the graft and the blood vessels and lymphatic vessels of the model itself [85]. Obtaining experimental results is hampered by the growth and development of chicken embryos, the short time window for observation and treatment, and species-specific differences. For instance, oxygen diffuses into the blood vessels and surrounding tissues on the CAM surface, whereas in vivo mammalian tumor models and clinical cases frequently experience severe hypoxia, which can promote metastatic progression. In in vivo mammalian models, tumors generally arise within the tissue or organ of the host, providing an environment that more closely mimics the native tumor microenvironment found in humans. Nevertheless, CAM models are the most accessible and reliable preclinical platforms for studying angiogenesis and lymphangiogenesis related to various oncological conditions [82, 54].
The CAM model exhibits remarkable advantages in detecting the effects of drugs on tumor lymphatic and blood vessel formation [20]. It can serve as a bridge between in vitro experiments and mammalian studies, facilitating rapid and high-throughput screening of drug efficacy. In the future, with the development of chicken-specific antibodies, the CAM is a poised preclinical model to play a greater role in researching angiogenesis and lymphangiogenesis.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
No funding was received for this research.
Author Contributions
Z.Z.W. drafted the manuscript, created figures and tables, and edited the manuscript. P.A.W. reviewed the manuscript and made important intellectual contributions. C.H., F.F., and A.D. approved the final version of the manuscript.