Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis
Since their introduction almost a century ago, chick embryo model systems involving the technique of chorioallantoic grafting have proved invaluable in the in vivo studies of tumor development and angiogenesis and tumor cell dissemination. The ability of the chick embryo’s chorioallantoic membrane (CAM) to efficiently support the growth of inoculated xenogenic tumor cells greatly facilitates analysis of human tumor cell metastasis. During spontaneous metastasis, the highly vascularized CAM sustains rapid tumor formation within several days following cell grafting. The dense capillary network of the CAM also serves as a repository of aggressive tumor cells that escaped from the primary tumor and intravasated into the host vasculature. This spontaneous metastasis setting provides a unique experimental model to study in vivo the intravasation step of the metastatic cascade. During experimental metastasis when tumor cells are inoculated intravenously, the CAM capillary system serves as a place for initial arrest and then, for tumor cell extravasation and colonization. The tissue composition and accessibility of the CAM for experimental interventions makes chick embryo CAM systems attractive models to follow the fate and visualize microscopically the behavior of grafted tumor cells in both spontaneous and experimental metastasis settings.
KeywordsChick embryo CAM models Tumor cell metastasis Intravasation Angiogenesis Live cell imaging
Complementing murine models for tumor cells dissemination, chick embryo model systems offer a number of unique advantages to study the complex, multistep process of tumor cell metastasis. Since the lymphoid system is not fully developed till late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. Different chick embryo model systems allow for comprehensive analysis of specific stages and aspects of cancer cell dissemination such as tumor cell intravasation in the spontaneous metastasis model, tumor cell colonization in the experimental metastasis model or tumor-induced angiogenesis in the collagen onplant model. The core of these model systems is the use of a specialized tissue, i.e., chorioallantoic membrane (CAM), which provides a uniquely supportive environment for primary tumor formation and a source of angiogenic blood vessels. In addition to nurturing developing xenografts, the CAM blood vessel network provides conduits for tumor cell intravasation, dissemination, and vascular arrest and finally, a repository where arrested cells extravasate to form micro metastatic foci. Due to the ease of repetitive experimental manipulations before or after tumor cell grafting, the CAM models allow one to identify individual steps of the metastatic cascade where specific molecules or biochemical processes manifest their functional involvement. This review will emphasize the experimental approaches based on CAM models and also highlight histological analyses of human tumor cells, which have been successfully employed in our laboratory to mechanistically address the functionality of several metastasis-related molecules.
CAM development, structure, and imaging
During chick embryo incubation, the CAM is formed between days 5 and 6 by partial fusion of chorion and allantois (Melkonian et al. 2002; Romanoff 1960). The CAM, functionally serving as lungs of the embryo, develops fast and surrounds the whole embryo by day 12 of incubation. Histologically, the CAM contains three major layers, i.e., the ectoderm attached to the shell membrane, the mesoderm enriched in blood vessels and stromal components, and the endoderm facing the allantoic cavity. By day 10 of incubation, the CAM also comprises the fully developed ectoderm capillary plexus, which represents a network of tiny capillaries connecting the arterial and venous blood vessel networks.
If the chick embryo is injected with fluorescent-tagged lectin such as Lens culinaris agglutinin (LCA), the intact vascular network of the CAM can be visualized by a top planar view of whole-mount preparations in a fluorescent microscope (Fig. 1b). Complementing conventional histology, the immunofluorescence evaluation provides a more discernable radiation of terminal arterial and venous capillaries forming a dense capillary network of the ectodermal plexus. This plexus comprises capillaries of so small diameter (approximately that of one erythrocyte) and such high density that the whole vascular network generates a honey comb structure, allowing for maximal surface and, therefore maximal levels of gas and nutrient exchanges.
Highlighting the vasculature with Sambuco negro agglutinin (SNA), which specifically binds to chicken endothelium, results in a better discrimination of the ectoderm capillary plexus and blood vessels within CAM mesoderm (Fig. 1c). Analysis under higher magnification (Fig. 1d) demonstrates tiny capillaries stained brown with SNA and appearing embedded into the thin layer of ectoderm cells counterstained blue with hematoxylin. Structural organization of the CAM can be also appreciated by DIC microscopy of the CAM stretched out on the glass slides. Since the CAM is so thin, differential focusing allows for detailed visualization of different planes: the ectoderm surface, the capillary plexus (Fig. 1e), the vasculature network (Fig. 1f), and the endoderm.
The ectoderm capillary plexus is one of the most important histological features of the CAM pertinent to chick embryo metastasis and angiogenesis models since it efficiently supports tissue grafting via rapid neovascularization and also serves as a repository of experimentally inoculated or spontaneously intravasated tumor cells.
CAM model for spontaneous tumor cell intravasation and metastasis
The highly vascularized nature of the CAM greatly promotes the efficiency of tumor cell grafting (Armstrong et al. 1982; Murphy 1913; Ossowski and Reich 1980). Tumor cells are grafted in ovo in 20–30 μl cell inoculums introduced through a small window made in the shell above the lowered (i.e., “dropped”) CAM. The CAM “dropping” is performed by making an air pocket between separated shell membrane and the CAM. Depending on the tissue origin, number of tumor cells and their proliferation capacity, primary CAM tumors can reach up to 500–600 mg in 6–7 days after cell inoculation. Remarkably, within 5–7 days not only do aggressive tumor cells develop sizable tumors, but they can escape the primary site, invade surrounding stroma, intravasate into blood vessels, and reach distal portions of the CAM and internal organs, where disseminated cells extravasate and form micro metastasis foci. Therefore, similar to the murine model, all steps of the multistep metastatic cascade are recapitulated in the chick embryo spontaneous metastasis model but, importantly, in a very short period of time.
Immunohistochemical detection makes it possible to localize human tumor cells within the chick embryo tissue background and therefore identify specific places where aggressive tumor cells manifest their differential ability to spontaneously metastasize. Such analysis was initially performed by employing the high and low disseminating variants of human HT-1080 fibrosarcoma (HT-hi/diss and HT-lo/diss, respectively), which were generated in our laboratory by in vivo selection for a 50–100-fold difference in their ability to intravasate and disseminate during spontaneous metastasis in the chick embryo (Deryugina et al. 2005). Importantly, these fibrosarcoma dissemination variants give rise to primary tumors of similar sizes, thus excluding inefficiency in the grafting or in vivo proliferation as the reasons responsible for the lack of intravasation in HT-lo/diss cells.
The use of fluorescent-tagged tumor cells provides additional advantages allowing their visualization in live, non-fixed CAM with the vasculature differentially highlighted by fluorescence-tagged LCA. When GFP-labeled HT-1080 cells were used in the spontaneous metastasis assay, few HT-lo/diss cells were found escaping primary tumor sites and those that escaped appeared fragmented and randomly scattered among blood vessels (Fig. 2C1). Interestingly, primary HT-hi/diss tumors presented intra-tumoral vasculature that appeared distorted and dilated and sometimes encompassing intravascular tumor foci. In accordance with initial observations indicating vasculotropism of HT-hi/diss cells, tumor cells escaping the primary site were found along blood vessels proximal to the tumor border (Fig. 2C2). Quantification of tumor cells localized in a close proximity to blood vessels demonstrated a sixfold differential between HT-hi/diss over HT-lo/diss intravasation variants (Fig. 2d).
Actual levels of intravasation and vascular dissemination of human tumor cells in the chick embryo could be determined by a very sensitive quantitative PCR analysis based on amplification of Alu DNA repeats characteristic of the primate genome (Schmid and Jelinek 1982). This Alu qPCR analysis confirmed very low levels of HT-lo/diss dissemination and indicated the onset of intravasation of HT-hi/diss cells between days 3 and 4 after tumor cell grafting and steadily increasing levels of intravasation occurring during the next 3–4 days (Deryugina et al. 2005).
CAM models for analysis of tumor vasculotropism in vivo
Twelve-hour video microscopy demonstrated that both HT-hi/diss and HT-lo/diss manifested high locomotion activity within tumors and at tumor-stroma border. However, while HT-lo/diss cells after leaving the microtumor appeared to be drawn back to the primary site, some of HT-hi/diss were persistent in their directional migration away from the tumor (Fig. 5, right panels). Notably, this directional migration of HT-hi/diss cells was associated with locomotion towards or along CAM blood vessels, therefore manifesting cell behavior that has been defined by the above-introduced term “vasculotropism”.
Inflammatory cell influx into CAM primary tumors
Inflammatory cells constitute an important cellular component of the primary tumor microenvironment, which paradoxically can perform anti-tumor surveillance in parallel with facilitating tumor progression (Balkwill et al. 2005; Condeelis and Pollard 2006; de Visser et al. 2006; van Kempen et al. 2006). To analyze whether in addition to the differential in intravasation capacity, our HT-1080 variants exhibited differential in the capacity to induce inflammatory cell influx, we performed immunohistochemical analysis of inflammatory cells infiltrating primary CAM tumors.
Circulating human tumor cells in the vasculature of chick embryos during spontaneous metastasis
Although being rare due to rapid vascular arrest, circulating tumor cells can be identified in the peripheral blood of cancer patients especially at the late stages of disease (Gallagher et al. 2008; Hasselmann et al. 2001; He et al. 2008; Maheswaran et al. 2008). The size of aggressive tumors and time of their development also correlate with the appearance and increasing frequency of tumor cells in the circulation in mice (Allan et al. 2005; Scatton et al. 2006). High levels of intravasation exhibited by HT-hi/diss cells prompted us to attempt detection of circulating human tumor cells during spontaneous metastasis in the chick embryo. This was accomplished by employing a newly-developed fiber-optic array scanning technology (FAST), capable of detecting circulating human tumor cells in cancer patients with exceptional sensitivity (Krivacic et al. 2004).
The unambiguous detection of human cells that actually are present in the host circulation provides strong evidence that dissemination of tumor cells from the primary CAM tumors indeed involves intravasation, i.e., that HT-hi/diss cells actively enter the vasculature of the chick embryo. It also emphasizes that the spontaneous human tumor/chick embryo model truly recapitulates this clinically established aspect of the metastatic cascade.
Tumor cell extravasation and colonization in the chick embryo experimental metastasis model
Close histological evaluation pointed to increased blood vessel density around SW620 micro foci, therefore suggesting that SW620 cells might have higher angiogenic potential as compared with SW480 cells. This suggestion was first confirmed by direct comparison of angiogenic potential of the two cell lines in our collagen onplant angiogenesis assay (Deryugina and Quigley 2008) and then, by inhibiting experimental metastasis of SW620 cells by function-blocking mAb specifically targeting human VEGF (unpublished data). In view of the known correlation between high VEGF production by metastatic colon carcinomas, including SW620 cells, and high levels of angiogenesis in colon carcinoma metastases, our findings represent a pertinent example of successful use of the chick embryo model system to study specific pathways involved human tumor cell dissemination.
Our recent study of the role of CDCP1 in tumor cell vascular dissemination represents an example of the power of histological examination in identifying the physiological mechanism underlying the functionality of metastasis-related molecules. CDCP1 is a relatively new member of CUB domain-containing proteins, discovered in our laboratory during the course of generating metastasis-blocking antibodies by a process of subtractive immunization (Zijlstra et al. 2003). CDCP1 was identified as an antigen precipitated by a subtractive immunization mAb 41-2 (Hooper et al. 2003). Although it is widely expressed among different normal tissues, including stem cells and cell progenitors of hematopoietic origin, CDCP1 expression has been shown elevated in a number of carcinomas (Perry et al. 2007; Scherl-Mostageer et al. 2001; Uekita et al. 2008). CDCP1 has been implicated in tumor cell functions associated with the capacity of tumor cells to withstand anoikis (Uekita et al. 2007), however, the precise role of CDCP1 in metastasis has not been elucidated in sufficient detail.
Altogether, these findings illustrate that histological analysis effectively complement CAM metastasis model systems and provide insightful views on the functional mechanisms of individual metastasis-related molecules as well as specific behavioral patterns of tumor cells during individual stages of metastatic dissemination.
The authors would like to acknowledge Dr. Tatyana Kupriyanova, Dr. Nicole Lazarus, Dr. Veronica Ardi and Ms. Juneth Partridge for their expertise in preparation of histological samples and live imaging of cells.
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