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International Journal of Molecular Sciences
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  • Review
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9 January 2024

Experimental Tumor Induction and Evaluation of Its Treatment in the Chicken Embryo Chorioallantoic Membrane Model: A Systematic Review

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1
Institute of Biopathology and Regenerative Medicine (IBIMER), Biomedical Research Center (CIBM), 18100 Granada, Spain
2
Instituto de Investigación Biosanitaria de Granada (ibs.GRANADA), 18012 Granada, Spain
3
Department of Anatomy and Embryology, University of Granada, 18071 Granada, Spain
4
Service of Medical Oncology, Hospital Virgen de las Nieves, 18014 Granada, Spain
Int. J. Mol. Sci.2024, 25(2), 837;https://doi.org/10.3390/ijms25020837
This article belongs to the Special Issue Targeted Therapies and Molecular Methods in Cancer, 2nd Edition
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Abstract

The chorioallantoic membrane (CAM) model, generated during avian development, can be used in cancer research as an alternative in vivo model to perform tumorigenesis in ovo due to advantages such as simplicity, low cost, rapid growth, and being naturally immunodeficient. The aim of this systematic review has been to compile and analyze all studies that use the CAM assay as a tumor induction model. For that, a systematic search was carried out in four different databases: PubMed, Scopus, Cochrane, and WOS. After eliminating duplicates and following the established inclusion and exclusion criteria, a total of 74 articles were included. Of these, 62% use the in ovo technique, 13% use the ex ovo technique, 9% study the formation of metastasis, and 16% induce tumors from patient biopsies. Regarding the methodology followed, the main species used is chicken (95%), although some studies use quail eggs (4%), and one article uses ostrich eggs. Therefore, the CAM assay is a revolutionary technique that allows a simple and effective way to induce tumors, test the effectiveness of treatments, carry out metastasis studies, perform biopsy grafts of patients, and carry out personalized medicine. However, unification of the methodology used is necessary.
Keywords:
CAM; tumor; xenograft; in ovo; ex ovo

1. Introduction

Constant progress in understanding the mechanisms underlying tumor formation, as well as their migration and invasion into other tissues, is essential for the development of effective strategies in both cancer prevention and treatment. Traditional in vivo mice models have allowed us to obtain valuable information in this field, although they also present multiple limitations due to specific restrictions such as large cohorts of animals [1], slow tumor development, or ethical considerations [2]. In this context, egg models have emerged as promising tools that offer unique advantages not only in terms of accessibility, cost, and experimental manipulation [3] but also in terms of immune responses, as they are naturally immunodeficient hosts because until the late stages of incubation, the lymphoid system is not fully developed [4]. Because of that, in most countries, there is no need for ethical committees to approve this type of research if it ends on day 14 [5], making this another advantage of this type of model.
During the development of the embryo within the egg, the mesodermal layer of the chorion, together with the allantois, fuses to generate the chorioallantoic membrane (CAM), thus connecting the embryonic circulation to the CAM, generating a large vascular network [4]. Since the CAM is not innervated, the embryo cannot feel pain [6]. The presence of the CAM provides nutrition for developing xenograft models due to its supportive environment surrounded by vessels that allows cell extravasation, ultimately leading to metastatic foci [3]. The CAM model can be used in cancer research as an alternative in vivo model to perform angiogenesis [7], tumorigenesis of solid tumors or cell suspensions [8], tumor chemosensitivity [9], and metastasis assays [10], among others.
The two main egg models in tumorigenesis are the in ovo and ex ovo models, which are carried out inside and outside the egg, respectively. Both use the CAM for tumor development, although they have different advantages. The in ovo model allows for obtaining information on the extravasation of tumor cells and is mainly used for the study of metastasis [11], while the ex ovo model is mainly used in angiogenesis studies since it allows easier observation of the CAM [12].
Therefore, the aim of this systematic review is to search and discuss the literature over time on the use of the CAM model in cancer for tumorigenic assays in vitro or as a xenograft model and analyze the type of assay performed and the methodology used by the authors and relate it to the results obtained.

2. Materials and Methods

This systematic review was previously registered in the OSF database on 10 November 2023 (https://doi.org/10.17605/OSF.IO/BN58M).

2.1. Study Eligibility and Data Sources

The present systematic review has been developed through a bibliographic search in four different databases: Cochrane, PubMed, SCOPUS, and Web of Science. To perform the search in PubMed, the following “MeSH” terms were used: “Chorioallantoic membrane” and “neoplasms”, with the formula obtained: “ovo”[All Fields] AND (“chorioallantoic membrane”[MeSH Terms] OR (“chorioallantoic”[All Fields] AND “membrane”[All Fields]) OR “chorioallantoic membrane”[All Fields]) AND (“analysis”[MeSH Subheading] OR “analysis”[All Fields] OR “assay”[All Fields] OR “biological assay”[MeSH Terms] OR (“biological”[All Fields] AND “assay”[All Fields]) OR “biological assay”[All Fields] OR “assays”[All Fields] OR “assayed”[All Fields] OR “assaying”[All Fields] OR “assays”[All Fields]) AND (“cancer s”[All Fields] OR “cancerated”[All Fields] OR “canceration”[All Fields] OR “cancerization”[All Fields] OR “cancerized”[All Fields] OR “cancerous”[All Fields] OR “neoplasms”[MeSH Terms] OR “neoplasms”[All Fields] OR “cancer”[All Fields] OR “cancers”[All Fields] OR “tumor” [All Fields]). In the case of the other databases, this formula was adopted. Moreover, this systematic review has followed the PRISMA guide to guarantee its correct execution [13].

2.2. Inclusion Criteria

Because in ovo tumor induction has been gaining great relevance in recent years, the literature search has not been restricted by publication date. Two types of studies were included: articles that treated these tumors and those that only generated tumors and studied their growth. Likewise, articles that have carried out studies on different species of birds have been included.
To reduce the possible risk of bias, after reviewing the bibliography of the articles included in the systematic review, those that met the inclusion and exclusion criteria were also added to this systematic review.

2.3. Exclusion Criteria

The main exclusion criteria were studies in which tumors were not induced, such as studies where they only used eggs to carry out angiogenic studies without tumor induction. Similarly, articles that did not specify the in ovo tumor induction methodology were excluded, although those that referenced previous works were included. Those whose methodology was incomplete but detailed the tumor induction process were also included. Articles that only used the egg to test biocompatible materials or scaffolds, without inducing tumors, were also excluded. Likewise, articles that were not research and were a protocol were also excluded.
Regarding language, articles that were written in a language other than Spanish, English, or French were excluded.

2.4. Study Selection

G.P. and C.M. (Cristina Mesas) carried out the first bibliographic search independently and agreed on the search formula for each database, obtaining 357 articles. Once the articles were obtained, those that were not original articles, were not freely accessible, and were repeated, were excluded, obtaining a total of 273 articles.
In the second step of the procedure, independently, M.A.C. and C.M. (Cristina Mesas) carried out a detailed reading of the articles, excluding those that did not meet the inclusion and exclusion criteria, obtaining a total of 74 articles, which were the ones that were finally analyzed in this systematic review (Figure 1).
Figure 1. Flow diagram illustrating the search and selection process for articles included in this systematic review.

2.5. Data Extraction

Following the procedure described, M.A.C. and C.M. (Cristina Mesas) carried out the procedure independently. According to Cohen’s kappa statistic test [14], there was a good correlation between M.A.C. and C.M. (Cristina Mesas) Any disagreements were resolved through discussion until a consensus was reached. Otherwise, a third experienced author made the final decision. Finally, each article was subjected to a quality test independently by M.A.C. and C.M. (Cristina Mesas). This quality test has two parts: the first consists of general filters on cell lines and patient biopsies (score ≥ 5). Articles that did not reach this score were excluded. The second phase consisted of questions about CAM methodology, results, discussion, and conclusions. The articles were classified according to the score obtained: low quality (score 0–5), medium quality (score 6–15), and high quality (score 16–20). Following this quality study, 6 articles were excluded (Figure 1), and 74 articles were included and are analyzed in Table 1, Table 2, Table 3, Table 4 and Table 5. The tables contain the references of the articles, the summarized CAM methodology, specifying the days on which the interventions on the eggs were carried out, and the techniques applied to study induced tumors. They also include the cell lines or patient biopsies used, as well as the main results obtained.
Table 1. The CAM assay as a methodology for tumor induction in ovo from cell lines.
Table 2. CAM assay as a methodology used to evaluate the efficacy of antitumor treatments on tumors induced from cell lines.
Table 3. The CAM assay as a methodology used for tumor induction ex ovo from cell lines.
Table 4. The CAM assay as a methodology used to carry out a study on the formation of metastases.
Table 5. The CAM assay as a patient-derived xenograft model.

3. Results

After the analysis described above, 74 articles have been analyzed in the systematic review (Figure 1). Tumor induction in the CAM has been gaining great interest in recent years. Figure 2A corroborates this with a graphical representation of the articles published per year using this technique, showing exponential growth. It is noteworthy that a substantial number of articles using this methodology began to be published only in 2018, reaching 15 annual publications within the last three years.
Figure 2. Graphic representation of (A) the number of scientific publications over the years considering the methodology used and (B) the methodologies used in tumor induction.
If we analyze the type of study that can be carried out on eggs to induce tumors, 62% of the articles induce tumors from cell lines using the in ovo technique, while 13% perform the ex ovo technique (Figure 2B). Likewise, it is worth noting that 16% of the studies carried out a xenograft methodology based on patient biopsies, with these studies being the most recent; they were conducted mainly in the last three years (Figure 2A). However, the study of in ovo metastasis is the least used technique (9%), although it has been performed in recent years (Figure 2B).
After applying the inclusion and exclusion criteria, all articles were included regardless of the poultry species used to perform the CAM assay. As shown in Figure 3A, 95% of the articles used chicken as the bird species for the study. However, there is one article that uses ostrich eggs and three articles that use quail eggs.
Figure 3. Graphic representation of (A) bird species used for in ovo experimentation, (B) articles that present the ethics committee’s approval for animal experimentation or not, (C) end point day on which embryo euthanasia is performed, and (D) methodology used to perform euthanasia on the embryo. W: with ethics committee approval; WO: without ethics committee approval.
Another key aspect of the use of fertilized eggs for experimentation is the ethics committee’s approval for animal experimentation. For fertilized eggs, according to different laws, they do not require ethics committee approval for their use in investigation. In fact, as seen in Figure 3B, 84% of the studies do not have this approval. Although 16% do have approval from the ethics committee, most articles specify that it is not necessary. The studies that use patient biopsies stand out as being the ones that provide the most approval from the ethics committee (Figure 3B).
There is a discrepancy between the end point of the experiment and, therefore, when the euthanasia of the embryo occurs. As can be seen in Figure 3C, most studies end the experimentation on day 14 (27%), followed by day 17 (16%), day 16 (15%), and day 18 (14%), highlighting that 10% of the articles do not specify the exact day of the end point. Furthermore, most articles do not specify how embryo euthanasia was carried out (81%). Among those that specify the procedure, 9% carry out euthanasia of the embryo by decapitation, 6% by freezing at −20 °C, 3% by intravenous injection of pentobarbital, and 1% by an incision in the vitelline artery (Figure 3D).

3.1. Establishment and Tumor Formation in the CAM from Cell Lines following the In Ovo Methodology

Among the 74 articles included in this systematic review, 48 induced tumors in the CAM from cell lines using the in ovo technique. Of these, 22 studied tumor genesis without treating them [15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36]. Successful results have been obtained in most studies, achieving high efficacy in tumor induction in the vast majority of tumor lines in which work has been carried out. There is great diversity in the types of cancer that have been tested for tumor induction in the CAM, with breast cancer cell lines being predominant (26%), followed by retinoblastoma (18%), glioblastoma (13%), and ovarian cancer (9%) (Figure 4A).
Figure 4. Graphic representation of the tumor induction procedure from cell lines in the CAM following the in ovo methodology, specifying (A) cell lines used to induce the tumor in the CAM, (B) the days in which the hole in the eggshell was drilled, (C) the day in which tumors were induced in the CAM, (D) the matrix used to induce the tumor, and (E) studies that were used in the study and characterization of induced tumors.
A key aspect in carrying out this methodology is the day on which the hole in the eggshell is drilled. As can be seen in Figure 4B, there is much disparity in the day that it drills the hole. A total of 35% of the studies drilled the hole in the eggshell on day 3 of development, and 18% drilled it on day 4, highlighting that 13% of the studies did not specify the exact day. Similarly, this discrepancy was observed on the day of tumor induction, with 31% of studies inoculating tumor cells into the CAM on day 7 of development, followed by 26% on day 10 and 13% on day 8 (Figure 4C).
On one hand, the inoculation method for tumor cells is also not clearly established. In half of the studies analyzed (55%), tumor establishment was established with Matrigel, although culture medium (18%) and PBS (9%) were also used. However, other matrices are also used to establish tumor growth, as in the case of Gečys et al. (2023), who used rat tail collagen as the matrix for cell inoculation [16], or Yart et al. (2022), who used Geltrex [22]. On the other hand, it should be noted that only 9% of the studies used a silicone ring in the CAM to infiltrate the tumor cells into the CAM and facilitate its establishment at a fixed point, preventing its spread through the CAM (Figure 4D).
To assess the viability of the tumor induction assay and what this entails for the embryo, numerous techniques can be applied to provide information depending on the objective of the study. The most commonly used technique, in 31% of studies, was histology (Figure 4E), as in the case of Jaworski et al. (2013), who compared tumor formation in two different glioblastoma cell lines, where they observed histological differences in tumors formed between both lines using hematoxylin–eosin staining (HE) [34]. Another widely used technique is immunohistochemistry (IHC) in 21% of the cases (Figure 4E). Buschmann et al. (2022), performed immunohistochemical staining of HIF-1α to determine the hypoxic phenotype of tumors generated from lung cancer and colon cancer cells and also studied the positive values of the cell proliferation marker ki-67 [18].
In addition to conventional techniques for the study of tumors generated in vivo, complementary techniques, such as ultrasound, can also be used in the egg. Eckrich et al. (2020) obtained the tumor volume induced in the CAM from liver cancer cell lines by ultrasound and correlated it with other histological techniques, like hematoxylin and eosin (HE) stain [26].
Although the vast majority of studies were carried out in chicken embryos, there are experiences carried out in other types of embryos, as is the case of Gečys et al. (2023), who used ostrich embryos to carry out their studies on breast cancer, in which, based on known models of tumor generation in chicken embryos, they managed to establish the number of cells suitable for tumor induction in the ostrich CAM, and they also performed histological techniques that allowed them to determine the high proliferative activity of the tumor cells that they worked with [16] (Table 1).
Finally, studies using stem cells as a treatment for tumors, such as Gečys et al., who used adipose tissue-derived mesenchymal stem cell EVs [16], and Waltera et al., who used bone marrow-derived mesenchymal stromal cells [17], were analyzed. In both, stem cells were used to reduce tumor size and malignancy (Table 1).

3.2. Tumor Induction Model in the CAM Using the In Ovo Technique to Determine the Effectiveness of Treatments

Of the 48 articles that carried out in ovo tumor induction studies, 26 of them test different treatments on these induced tumors [37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62]. The most studied tumor types in this modality were breast cancer (28%), followed by colorectal, lung, and pancreatic cancer with 9% each, and prostate cancer, glioblastoma, melanoma, neuroblastoma, and ovarian cancer with 6% each. Finally, the least studied are cervical, hepatocellular, head and neck, and rhabdomyosarcoma cancer in 3% of the articles (Figure 5A). It should be noted that tumor formation was not successfully achieved in all the lines tested. Lung cancer cell lines SW1573, H1299, and H292 were not used to test the treatment because of the great irregularity of the tumors formed. In fact, only the A549 cell line formed solid tumors, while H460 formed tumors but was less compact [39].
Figure 5. Graphical representation of tumor induction studies and in ovo treatment, highlighting (A) the type of cancer studied, (B) the day of embryonic development when the hole was drilled in the eggshell, (C) studies with and without the use of a ring, (D) studies that induce tumor formation by means of matrices or a culture medium, (E) route of administration of drugs, and (F) techniques applied in resected tumors.
The analysis of the methodology that followed the drilling of the hole in the eggshell was heterogeneous. In fact, the day of development on which the hole was opened varied among the studies. The preferred day by most studies was days 3, 8, and 10 (15% of studies in each case), followed by days 7 (12%), 6 (12%), 4 (11%), 9 (8%), and 3.5 (4%), and 8% of the articles did not specify the day on which the egg hole was opened (Figure 5B). Notably, in 15 of the 26 articles, tumor induction was performed on the same day in which the hole was opened [37,38,39,40,41,42,43,44,45,46,47,48,50,53,56,59].
On one hand, as shown in Figure 5C, 35% of the articles directly deposited the cells on the CAM. However, the majority of the articles (65%) deposited the CAM on a ring in which the cells were inoculated, acting as a barrier to prevent the spread of the cells through the CAM. On the other hand, many studies used cellular matrices containing the cells for tumor induction, such as Matrigel and Geltrex in 61% and 4% of the cases. In 31% of the studies, the cells were inoculated in a culture medium, and 4% of the published articles did not specify where the tumor cells were inoculated for the CAM (Figure 5D).
The in ovo tumors were developed for the purpose of testing treatments. Generally, one or two days after tumor induction, the tumors were treated. However, there are studies that induced tumors with previously treated cells, such as Mariho et al. (2018), who treated SKOV3 ovarian cancer cells with ApoA1 and CDDP, and later that day, deposited it in the CAM, continuing with the treatment to induce tumor formation [61]. Many studies employed chemotherapies such as Vincristine [37], cisplatin [38,39,43,49,50,53,61], 5-Fu [48], or Doxorubicin [48,56,59]. In addition, new therapies, such as plant extracts [51,55] or nanoparticles [38,49,58], have been tested. Treatments were administered directly on the tumor surface, that is, topically, in 50% of the cases (Figure 5E). However, there are studies, such as Waschkies et al. (2020), who used topical treatment on the tumor surface by means of air and carbogen flows directed to the tumor through plastic tubes [57]. Only 19% of the analyzed studies used injected treatments. While most of these treatments were injected into blood vessels, only one article performed intratumoral injections [59]. A total of 31% of them did not specify the route of drug administration (Figure 5E).
Once the tumor was resected, a wide variety of techniques were used for its analysis (Figure 5F). The most commonly applied technique was histology by HE staining (29%), followed by immunohistochemistry (19%), PCR and fluorescence (7%), and bioluminescence (5%). Other techniques were used to a lesser extent, such as metabolic and lipid analysis, HPLC, ELISA, lipid peroxidation assays, SOD activity, GSH levels, and MRI. A total of 14% of the articles did not employ any alternative technique for tumor study other than microscopy imaging (Figure 5F).
In all included studies, the treatments were effective, and a significant decrease in the tumor volume induced in the CAM could be observed with respect to the untreated controls. In fact, it was also possible to corroborate, in the case of the study carried out by Bohm et al. (2019), that treated tumors also showed a greater capacity for CAM invasion, a higher degree of vascularization, and a more aggressive phenotype [62] (Table 2).

3.3. Effectiveness of Tumor Induction following the Ex Ovo Methodology

Eleven articles included in this systematic review [38,53,63,64,65,66,67,68,69,70,71] carried out tumor induction in the CAM using the ex ovo model by transferring the contents of the egg to a plate. The most common day on which studies break the egg and transfer its contents to a plate is day 3 (64%), followed by day 2 (9%), although 27% of the studies do not specify the day (Figure 6A).
Figure 6. Graphic representation of tumor induction in the CAM following the ex ovo methodology including (A) the day that they break the egg and transfer its contents to a plate, (B) cell lines used to induce the tumor, (C) the day on which tumors are induced in the CAM, (D) information on treatment received and tumor induction methodology, and (E) studies used to study the tumors generated. W: with ethics committee approval; WO: without ethics committee approval.
The most commonly used cancer cell lines in ex ovo experimentation in this model were osteosarcoma and glioblastoma (both at 19%), followed by breast cancer and myeloma (both at 13%) (Figure 6B).
There is greater variability in terms of the day of tumor induction, including day 9 of development in 37% of the cases, followed by day 7 (27%) and day 10 (18%) (Figure 6C).
The most commonly used method for induction was direct treatment by injection, although there is the possibility of using a silicone ring or Matrigel for localized induction of the tumor (Figure 6D).
Treatment of tumor cells is usually performed on the same day or after tumor cell implantation, although there are two articles in which the cells implanted in the CAM have been previously treated with the drug [53,64]. In the study by Merlos et al. (2021), the treatment of the cells did not refer to the use of a drug, but cell culture was performed beforehand in the presence or absence of Matrigel to evaluate the difference in tumor growth and development in both cases [53].
Most assays performed to study tumor formation were histology and immunohistochemistry with 36% and 27%, respectively, although qRT-PCR, Western blotting, proteasomal assays, ELISA, cytotoxicity, and fluorescence assays had also been performed (Figure 6E).
All articles that used the ex ovo methodology obtained favorable results, both in correct tumor induction and in its reduction after treatment. There are studies in which they treat the cells before inducing the tumor [64,66], while others perform the treatment once the tumor is established in the CAM [38,53,63,69].

3.4. Use of the CAM Assay as a Model for Metastatic Induction and to Study the Effectiveness of Treatments

In ovo studies have been used as metastatic models, verifying their effectiveness in the formation of metastases in different organs of the embryo; they have also been used to test different treatments and verify their effectiveness in tumor migration. In this systematic review, six articles used the CAM assay to induce metastasis formation in ovo [53,62,72,73,74,75]. In these studies, the main day on which the hole in the eggshell is opened was days 8 and 10 (33% each one), followed by days 3 and 7 (17% each one) (Figure 7A). Colorectal cancer is the most used cell line for study in ovo metastasis (33%), although pancreatic cancer, neuroblastoma, prostate cancer, and bone marrow have been tested (Figure 7B). During tumor induction, only two articles used a silicon ring, while three articles embedded the tumor in Matrigel to ameliorate tumor induction. In addition, to study the formation of metastasis both in the CAM and the embryo, studies have been carried out on the effectiveness of treatments in reducing metastasis in two articles (Figure 7C). An example of this is Pawlikowska et al., who carried out assays with non-small-cell lung cancer and prostate cancer tumor cell lines. They performed metastasis assays in several lines of both tumors, which were analyzed using macroscopic fluorescence and 3D imaging studies. In addition, they tested chemotherapy drugs, like cisplatin and docetaxel, and their effect on metastasis. The results obtained showed a decrease in metastasis of the CAM and the chick embryo after pharmacological treatment [75]. Merlos et al. carried out an in ovo study to determine the metastatic capacity of two antitumor drugs, cisplatin and ellipticine. They used the UKF-NB-4, a human neuroblastoma tumor cell line to induce tumors in fertilized chicken eggs. Six days after induction, they performed a qPCR with human-specific Alu sequence primers, both in the CAM and in different organs such as the liver, brain, and lungs. In their results, they obtained a significant reduction in the extravasation of cancer cells to the distal organs, although the authors consider that this may be due to the antitumor effect of the drug itself and not a specific antimetastatic effect [53]. Although only two articles test direct treatments in ovo, there are also articles that perform prior treatments on cell lines before inducing the formation of metastasis. This is the case of a study that used Alu sequences to demonstrate that by inhibiting miR-21, the metastatic capacity of tumor cells from the LS174T colon cancer cell line is statistically decreased in an in ovo model. To perform this, they transfected the tumor cells with LNA-anti-miR-21, thus silencing the expression of this miRNA. Metastasis was not reduced in those groups in which miR-21 was not silenced, which could be a future target in tumor treatment [74] (Table 4).
Figure 7. Graphic representation of the use of the CAM assay for the study of in ovo metastasis formation, analyzing (A) the day in which the hole in the eggshell was drilled, (B) the cell lines used to induce the tumor, (C) the methodology carried out to induce the tumor in the CAM, (D) the main organs where metastases have been observed, and (E) the applied studies that can be used to characterize metastasis or tumor induction. W: with ethics committee approval; WO: without ethics committee approval.
The effectiveness of the trial has been demonstrated through the analysis of metastases, with the organs with the most prevalence in the formation of metastases being the liver (43%), followed by the distal CAM in 29% of the studies and the lungs and brain (14% each) (Figure 7D).
Metastases produced in eggs can be detected by different methodologies, such as qRT-PCR (28%) and immunohistochemistry (27%) (Figure 7E). Herrman et al. carried out different types of in ovo studies, concluding that MRI was capable of being a sensitive technique for detecting metastasis deposits of at least 12 cells. They performed this with two approaches, one of them by injecting hypoxic fluorescent tumor cells SK-N-AS (GFP SK-N-AS) directly into the brain of white leghorn chicken embryos and the second one by inducing a tumor in the CAM through SK-N-AS fluorescent cells loaded with micron-sized iron particles (GFP MPIO-labeled SK-N-AS) [73].

3.5. Efficacy of the Patient-Derived Xenograft Model and Its Use to Predict Chemotherapeutic Drug Sensitivity/Resistance

In the last 3 years, publications using the CAM assay for tumor induction from patient biopsies have increased significantly (Figure 2A). Of the 14 articles included in the systematic review about this method [1,71,76,77,78,79,80,81,82,83,84,85,86,87], 29% used a biopsy from colorectal cancer, followed by ovarian cancer (12%) and breast cancer (11%) (Figure 8A). Regarding methodologies, only two articles followed ex ovo methodology [71,83], while the rest of the articles used in ovo assays (Figure 8B). In the rest of the results, there has been also a discrepancy regarding the opening day of the hole in the eggshell, and day 9 was the day in which the majority drill the hole (25%), followed by days 4 and 7 (17% in both). It should be noted that 17% of the articles do not specify the exact day (Figure 8C).
Figure 8. Graphic representation of the procedure to induce CAM tumors from patient biopsies, analyzing (A) the type of tumor of origin from which the biopsy is obtained, (B) the type of methodology used in the CAM test, (C) the day on which the hole in the eggshell, in the case of the in ovo method, is drilled, (D) the number of articles that treated the tumor and used a ring and Matrigel to ameliorate the induction, and (E) the main studies that can be applied to analyze the formed tumor. W: with ethics committee approval; WO: without ethics committee approval.
Regarding tumor induction, seven articles used a silicon ring in the CAM during tumor induction, and only six articles mixed a fragment of biopsy with Matrigel before induction in the CAM (Figure 8D). Tsimpaki et al. (2023) used the CAM assay as a xenograft model of biopsies from patients with uveal melanoma, analyzing different implantation methodologies. For this, different methodologies were carried out: (i) direct implantation, (ii) implantation on a drop of Matrigel previously placed in the CAM on a lacerated blood vessel, or (iii) implantation on a drop of Matrigel placed in the center of the ring. Tumors were successfully induced in all groups, although there were differences in tumor dissemination, depending on whether they were in the ring or not. In addition, they carried out numerous studies (ultrasound, scans, fluorescein angiography) that allowed them to obtain deeper analyses of tumor formation [78]. Although detailed studies can be carried out, 43% of the analyzed studies performed histology analysis, and 36% of them carried out immunohistochemistry studies (Figure 8E).
The results in all articles showed good implantation of the biopsy fragments; in addition, when treated with the chemotherapy used in the clinic, a predictive response correlation was observed between the egg and the patient’s response. Charbonneau et al. (2023) conducted a study in which they biopsied 60 patients with glioma who were being treated with CB and TMZ. Biopsy fragments were implanted into the CAM, and treatments were injected IV into the CAM vasculature. After analyzing the results obtained, 98.3% of the biopsies were successfully established in the CAM, in addition to maintaining the histopathological and molecular characteristics of the original tumor. Additionally, there was a correlation between the patient’s response to chemotherapy and the in ovo response [71].
Another aspect to highlight is that primary tumors grafted onto the CAM as ground homogenates adopted a different morphological phenotype compared to the original tumors, while fresh tumors grow well and show similar histology to the original tumor. Moreover, ki-67 expression was better retained in tumors grafted from frozen samples compared to fresh ones [86].

4. Discussion

For hundreds of years, the use of fertilized chicken eggs has been crucial for the investigation of embryonic development. Thanks to these studies, very important concepts, such as the neural tube or the germ layers, have been described [88]. Currently, the study of the chicken egg and, in particular, its CAM, is gaining great importance in various fields. In biomedicine, as discussed in this systematic review, its application in tumor induction studies and testing of antitumor treatments is widespread [37]. In addition, many studies focus on the search for antiangiogenic drugs using the CAM vascular network [89]. Recently, the CAM has started to be used in tissue engineering for organoid implantation. Moeinvaziri et al. (2021) implanted an otic organoid generated from pluripotent stem cells, mesenchymal stem cells, and endothelial cells into the CAM. Its implantation in the CAM stimulated the maturation of cells similar to human hair cells [90]. Within regenerative biomedicine, the CAM can be used as a bioreactor to culture and study human living bone regeneration [91]. It is even very useful for the biological characterization of materials through irritability testing. Chen et al., 2018, implanted bioactive collagen–bioglass scaffolds and did not detect inflammation or necrosis in the membrane, demonstrating its biological compatibility [92]. On the other hand, since the use of the chick embryo has the advantage of developing outside the mother, there has been great interest in its application for studies in the field of epigenetics [93]. Of particular note is the application of the CAM in the field of microbiology, where the virulence, invasiveness, and pathogenicity of bacteria and yeasts are studied [94].
Nowadays, the CAM assay has become increasingly relevant for the study of various cancer processes, including tumor induction. This assay has several advantages over mouse experimentation, one of them being the high vascularization and immature immune system, which allows experimentation with cell lines or tissues from different species without immune response [88,95]. In addition, the chorioallantoic membrane model is considered an in vivo model that fits the 3R principle of animal experimentation, replacement, reduction, and refinement [96]. Another advantage of this model over the use of mice for experimentation is that no ethical approval is required for its use, as chick embryos do not develop the nervous system until day 17 of ontogenesis [97], so experiments are terminated before the development of the regions associated with pain. However, The National Institute of Health and the Institutional Animal Care and Use Committee (IACUC) [98] state that chick embryos can be used without any ethical restrictions until day 14 of gestation, as they lack pain perception. There are studies that confirm that pain sensation in chicken embryos is impossible up to incubation day 7, but there are no specific time points defined from which the chicken embryo is able to develop the nociception and pain sensation [99]. In our systematic review, there is a disparity in the days on which researchers euthanize chick embryos, although in 27% of studies, euthanasia occurs on day 14, followed by 16% of the studies in which it occurs on day 17 (Figure 3C). This disparity may be due to the lack of a clear protocol regarding the end point of the methodology. Most authors use day 14 or earlier since approval by the ethics committee is not required until day 14, although other factors, such as the type of sample used in the study and the type of study performed (in vivo or ex vivo), also affect these differences. However, it would be desirable to unify criteria and establish a single end point day for chick embryo research.
The results analyzed from the articles included in the present systematic review show the effectiveness of inducing tumors in the egg CAM from different tumor types. Tumors have been successfully induced from in vitro cell lines of different tumor types, and the most studied are breast cancer, colorectal cancer, lung cancer, glioblastoma, and pancreatic cancer cell lines. These lines have been used in both in ovo and ex ovo, which are the types of cancer with the highest incidence and mortality all over the world [100]. In metastasis formation studies, pancreatic cancer lines are the most used, which is understandable since this tumor, despite not having a high prevalence, presents a high mortality rate [101]. For tumor induction from patient biopsies, the most studied tumors are those whose biopsies are easy to acquire, such as colorectal cancer, retinoblastoma, and ovarian cancer. Seeing the effectiveness of tumor induction on the egg CAM, studies should be carried out with other cell lines, such as liver or stomach cancer, which also have a high prevalence and mortality in the population.
There is a large discrepancy in the day on which the hole is opened in the eggshell. It is generally opened on day 3, although in studies of metastasis formation or tumor induction from patient biopsies, it was opened on day 9 or 10. Perhaps this discrepancy is due to the fact that in the case of tumor formation from cell lines, 6 days are necessary to consolidate tumor formation, while in the case of patient biopsies, these are already consolidated and can be implanted in later days of embryonic development. The same would occur in the case of metastatic studies where the cells are injected IV. Specific studies would be necessary to report on the risks of opening the window in the eggshell on different days. It should be noted that although tumor induction was effective without any matrix, the use of Matrigel is used among all studies in all types of assays. The extracellular matrix (ECM) forms the non-cellular physical support for the cellular constituents of all tissues and organs. The components of the ECM encompass cellular and biomechanical signals that maintain morphogenesis, differentiation, tissue homeostasis, integrity, and elasticity [102]. The use of Matrigel promotes correct tumor formation since it provides an ideal tumor microenvironment for its growth and differentiation [103,104].
Nowadays, there are studies aimed at demonstrating the potential of the CAM as a study model for precision medicine. Currently, mice are used for such studies, which has some drawbacks, such as the time required, the high cost, or the requirement for approval of the experiment by the ethics committee [37]. Also, the use of the CAM allows direct observation of the evolution of the tumor mass. It has been observed that tumor cells engrafted in the CAM behave similarly to the patient’s tumors, such as angiogenesis, metastasis, or matrix interaction. In addition, the implantation of tumor biopsies allows the recovery of some characteristics of the primary tumor, such as cellular polymorphism [97]. For the drug effect, these appear to behave the same in mice and the CAM, but the sub-toxic dose limit is lower in the chicken model, causing the death of the embryo [48]. On the other hand, the dose supported by the mouse is higher, and the toxicity in this model is reflected by a reduction in weight. In addition to the reduced toxicity threshold, the CAM study has certain limitations that are overcome by the use of humanized mice, such as the use of genetic modifications of HLA or cytokines [97]. Even so, the CAM assay allows a first study to estimate the sub-toxic dose that can be used in the mouse and make an approximation of the effect of the drug, avoiding the need for a first experiment with the mice [48].
The limitations found while developing this systematic review were focused on the lack of information in the articles’ methodologies. Many of them did not present complete methodological information regarding the day of egg opening, tumor induction, or end point day, which are very relevant pieces of information when it comes to standardizing processes. This causes a lot of variability to occur between the analyzed studies. Likewise, a gap has been found regarding the method used for euthanasia of the embryos. Although approval by the ethics committee of these processes is not necessary as long as they are carried out before the 14th, some rules should be established for their completion, such as specifying the end point day and the euthanasia method.

5. Conclusions

In this systematic review, the utility of the chicken egg CAM assay method in biomedical research has been comprehensively explored, with a specific focus on tumor induction and the evaluation of antitumor treatments. Over the years, the use of the CAM has evolved from its fundamental role in the study of embryonic development to become a valuable tool for investigating a wide range of biological and medical processes. The results reveal the effectiveness of the CAM model in inducing tumors from various cell lines, from high-incidence cancers, such as breast and colorectal, to less common types. The versatility of the model has allowed not only the successful induction of tumors in ovo and ex ovo but also the evaluation of antitumor treatments with promising results. This review highlights the importance of unifying criteria in the methodology, such as the day of egg opening, the method of tumor induction, and the end point of the experiment. Furthermore, the relevance of the extracellular matrix, especially the use of Matrigel, in the adequate formation of tumors is highlighted, providing an ideal microenvironment for tumor growth and differentiation. The CAM is presented as a valuable alternative to traditional mouse models, offering important advantages, such as high vascularization, an immature immune system, and the absence of the need for ethical approval for studies up to day 14 of gestation. Furthermore, its application in precision medicine seems promising, providing the opportunity to directly observe the evolution of tumor masses and recover characteristics of primary tumors through the implantation of biopsies. Despite the important advances and contributions of the CAM model, this review highlights the need for greater standardization and transparency in the presentation of methodological data in the scientific literature. The lack of detailed information in some studies analyzed represents a challenge in the comparison and synthesis of results. This systematic review highlights the crucial role of the CAM in biomedical research, particularly in the field of oncology. Opportunities to improve methodological coherence are identified, and the importance of continuing to explore the potential of this model in various areas of scientific research is highlighted.

Author Contributions

Conceptualization, C.M. (Cristina Mesas), G.P. and C.M.; methodology, C.M. (Cristina Mesas), M.A.C. and G.P.; formal analysis, C.M. (Cristina Mesas) and G.P.; investigation, C.M. (Cristina Mesas), M.A.C., G.P. and K.D.; data curation, P.L., J.M. and K.D.; writing—original draft preparation, C.M. (Cristina Mesas), M.A.C. and G.P.; writing—review and editing, C.M. and J.P; visualization, C.M. (Cristina Mesas) and G.P.; supervision, C.M. (Consolación Melguizo) and J.P.; funding acquisition, C.M. (Consolación Melguizo) and J.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Instituto de Salud Carlos III (PI19/ 01478-FEDER). In addition, this work was partially supported by project PMPTA22/00136 funded by the Instituto de Salud Carlos III-FEDER and the CPP2022-009967 and CPP2022-010017 Project from the Spanish Ministry of Science and Innovation (FEDER).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the main article.

Acknowledgments

M.A.C. would like to acknowledge FPU2019 grants (ref. FPU19/04967) from the Ministerio de Educación, Ciencia y Deporte y Competitividad (Spain).

Conflicts of Interest

The authors declare no conflicts of interest.

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