The CAM-Delam assay to evaluate the metastatic capacity of cancer cells is relatively fast, easy, and cheap. The method can be used for unraveling the molecular mechanisms regulating metastasis formation and for drug screening. An optimized assay for analyzing human tumor samples could be a clinical method for personalized cancer treatment.
The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form secondary tumors). Delamination, defined as the degradation of the basal lamina and basement membrane, is the initial process that facilitates the transmigration and spread of cancer cells to other tissues and organs. Scoring the delamination capacity of cancer cells would indicate the metastatic potential of these cells.
We have developed a standardized method, the ex ovo CAM-Delam assay, to visualize and quantify the ability of cancer cells to delaminate and invade, thereby being able to assess metastatic aggressiveness. Briefly, the CAM-Delam method includes seeding cancer cells in silicone rings on the chick chorioallantoic membrane (CAM) at embryonic day 10, followed by incubation from hours to a few days. The CAM-Delam assay includes the use of an internal humidified chamber during chick embryo incubation. This novel approach increased embryo survival from 10%-50% to 80%-90%, which resolved previous technical problems with low embryo survival rates in different CAM assays.
Next, the CAM samples with associated cancer cell clusters were isolated, fixed, and frozen. Finally, cryostat-sectioned samples were visualized and analyzed for basement membrane damage and cancer cell invasion using immunohistochemistry. By evaluating various known metastatic and non-metastatic cancer cell lines designed to express green fluorescent protein (GFP), the CAM-Delam quantitative results showed that the delamination capacity patterns reflect metastatic aggressiveness and can be scored into four categories. Future use of this assay, apart from quantifying delamination capacity as an indication of metastatic aggressiveness, is to unravel the molecular mechanisms that control delamination, invasion, the formation of micrometastases, and changes in the tumor microenvironment.
Approximately 90% of mortality in cancer patients is caused by the consequences of cancer metastasis, which is the formation of secondary tumors in other parts of the body from where the cancer originally originated1. It is, therefore, of importance to identify metastatic-related mechanisms to find potential targets to suppress the formation of tumor metastases. Subsequently, there is a need for model systems in which the metastatic process can be evaluated.
During metastasis, cancer cells undergo epithelial-to-mesenchymal transition (EMT), a normal cellular process in which epithelial cells lose their adherence and polarity properties and instead acquire an invasive mesenchymal character2. Delamination is part of the EMT process and involves the degradation of laminin in the basement membrane, which is a prerequisite for cancer cells to leave the primary tumor and invade other tissues. The major factors that are upregulated during metastasis formation include matrix metalloproteinases (MMPs), ADAM (a disintergin and metalloproteinase), ADAMTS (ADAM with thrombospondin motifs), and membrane-type MMPs (MT-MMPs)3,4. These factors degrade molecules such as laminin, which is expressed in all basement membranes, to facilitate cell migration and invasion.
The chorioallantoic membrane (CAM) of a fertilized chick egg is a type of basement membrane. Fertilized chick eggs have been used as metastatic models, in which cancer cells have been seeded on the extraembryonic CAM and later metastasis formation observed in the chick embryos5. Moreover, in vivo mouse metastatic models are frequently used, in which cancer cells are implanted in the mice and metastases in various organs are analyzed6. This approach is time-consuming, expensive, and may cause discomfort for the animals. To address this, we have developed the CAM-Delam assay, a faster and cheaper model to evaluate the metastatic aggressiveness of cancer cells. In this model, the ability of cancer cells to degrade the chick CAM (e.g., the delamination capacity) is combined with potential cancer cell invasion into the mesenchyme and used as a measurement of metastatic aggressiveness.
The present article, based on a previous publication7, describes the CAM-Delam assay in detail, from fertilized chick egg handling, cancer cell culture and seeding, dissection, and analyses of CAM samples, to the scoring of the delamination capacity of cancer cells into four categories: intact, altered, damaged, and invasion. We also give examples of how this assay can be used to determine the molecular mechanisms regulating the delamination process.
In brief, Figure 1 summarizes the overall steps in the CAM-Delam assay. The below protocol is based on 30 cultured fertilized chicken eggs and the use of two different cancer cell lines seeded separately in three rings/egg and analyzed at four time points.
1. Egg incubation
2. Preparation of weighing boat, plastic boxes, and dissection instruments
3. Opening the eggs and transfer to the internal humidified chamber
NOTE: Use gloves and a face mask to avoid contamination.
4. Preparation of silicone rings
5. Preparation of cancer cells
NOTE: Solutions such as cell culture medium, trypsin, and 1x PBS are stored at 4 °C and should be heated to 37 °C in a water bath before adding to the cells. After heating, rinse the bottles in 70% ethanol and dry before use.
6. Seeding the cancer cells on the CAM
NOTE: Use gloves and a face mask to avoid contamination.
7. Isolation of the CAM with associated cancer cells
8. Sectioning CAM-Delam samples
9. Immunohistochemistry staining
10. Microscopy imaging and delamination scoring
Figure 1 presents key steps in the CAM-Delam assay7. The use of internal humidified chambers (Figure 1C) significantly improved the survival rate of the chick embryos from <50% up to 90% at incubation day 10 and from ~15% up to 80% at incubation day 13 (Figure 2).
Figure 1: Key steps of the CAM-Delam assay. (A) Incubate fertilized chicken eggs horizontally without rotation. (B,C) On day 3 of incubation, crack the eggs and place them in sterile weighing boats (B), and position the boats in an internal humidified chamber for further incubation (C). (D) Prepare silicone rings. (E) On day 10 of incubation, place the silicone rings on the CAM, and seed 1 x 106 cancer cells inside the rings using a pipette. (F,G) At different time points (hours to days), dissect the CAM with attached cancer cells, (F) fix in PFA, treat with sucrose, position in frozen section medium, (G) and freeze in −80 °C. (H) An example of a sectioned CAM with associated GFP+ cancer cells (green) and laminin immunohistochemistry staining (red). Scale bars = 2 mm (E,F), 1 mm (G), and 100 µm (H). This figure is from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; PFA = paraformaldehyde; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
Figure 2: Chicken embryo survival in relation to incubation method. (A,B) On incubation day 10, chicken embryo survival in internal HCs doubled (mean value 89.33; N = 105) compared with incubation in Petri dishes (A; mean value 40; N = 64) and non-humidified chambers (B; mean value 48.67; N = 46). (C,D) On incubation day 13, a high survival rate was still observed using HC (mean value 81.67; N = 105), whereas a major decrease in embryo survival was noticed using PD (C; mean value 11.33; N = 64), and N-HC (D; mean value 18.33; N = 46). Statistical significance was tested using an unpaired two-tailed t-test. The error bars indicate the standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001. This figure was modified from Palaniappan et al.7. Abbreviations: HC = humidified chamber; PD = Petri dishes; N-HC = non-humidified chamber; E X = Incubation day X. Please click here to view a larger version of this figure.
Analyses of different cancer cell lines expressing GFP (U251 glioblastoma, PC-3U prostate, SW620 colon, and A549 lung) using this protocol were reported previously by Palaniappan et al.7. The results from the CAM-Delam assay include differences in morphology of the basal lamina, detected by laminin, and cancer cell invasion, defined as cells that have crossed the chick basal lamina layer into the chick mesodermal layer (Figure 3). These results show that the capacity of cancer cells to degrade basal lamina and invade the mesenchyme can be scored into one of four categories: 1) intact basal lamina without visible alterations (Figure 3B), 2) altered but undamaged basal lamina (Figure 3C), 3) damaged basal lamina without cell invasion (Figure 3D), and 4) damaged basal lamina with cell invasion (Figure 3E). Another observation was that, when cancer cells caused an altered or damaged basal lamina, the CAM was also thickened with an increase of blood vessel formation, defined by antibody staining against von Willebrand's Factor, which is synthesized in blood vessels8 (Figure 4C–H). These two phenotypes, a thickened CAM and increased formation of blood vessels, were not observed when the CAM was intact (Figure 4A,B).
Figure 3: CAM-Delam scoring. (A–E) A CAM-Delam scoring example based on the integrity of the CAM basal lamina, visualized by anti-laminin (red), and potential invasion of GFP-expressing cancer cells (green). (A) Control CAM without cancer cells. (B–E) In the responses to cancer cells, four categories describing the morphology of the basal lamina can be scored: (B) intact laminin, (C) altered but undamaged laminin (indicated by an asterisk), (D) damaged laminin but without cancer cell invasion (arrows), damaged laminin with cell invasion (arrowheads). Scale bar = 100 µm (A–E). This figure is from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
Figure 4: Evaluation of CAM thickening and blood vessel formation. (A–H) An example of visualization of CAM thickening and blood vessel formation, detected by anti-Von Willebrand Factor (red), in response to the lamina of various cancer cell types. (A,B) Control CAM without cancer cells. (C,D) In response to a non-metastatic cancer cell line (scored as Intact), no evident thickening of the mesenchyme or increased blood vessel formation was detected. (E–H) In response to metastatic cancer cells resulting in altered or damaged Laminin, the mesenchyme was thickened (indicated by double arrowheads) and increased blood vessel formation was observed (indicated by arrows). Scale bar = 100 µm. This figure was modified from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
U251 glioblastoma and PC-3U prostate cancer cells are two examples of cancer cell lines with completely different delamination capacities (Figure 5). PC-3U cells induced damaged laminin after 1.5 days, with clear invasion after 2.5 days (Figure 5A). In contrast, U251 cells only induced minor alterations of laminin after 1.5-3.5 days but never caused any visible damage to laminin (Figure 5B).
Figure 5: Visualization of the delamination capacity of prostate (PC-3U) and glioblastoma (U251) cancer cells. (A) PC-3U cells induced minor alteration of laminin after 14 h, damage of laminin after 1.5 days (arrow), and the initiation of invasion after 2.5 days, which was increased after 3.5 days (arrowheads). (B) U251 cells caused minor alteration of laminin after 1.5-3.5 days. The right panels show graphs representing the CAM-Delam scoring. The y-axis indicates the number of samples, and the x-axis indicates the time points of culture. Scale bar = 100 µm (A,B). This figure was modified from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein. Please click here to view a larger version of this figure.
The CAM-Delam assay can be used to define molecular mechanisms that regulate the delamination process. One example is the use of CoCl2 treatment to induce hypoxia with or without the combination of inhibiting matrix metalloproteinases (MMP) using the broad MMP inhibitor GM6001 (Figure 6). After CoCl2 treatment, U251 non-metastatic cancer cells acquired the ability to induce delamination and invasive cells, which was suppressed when CoCl2 treatment was combined with the MMP inhibitor GM6001 (Figure 6). Thus, the CAM-Delam assay can be useful when defining molecules and molecular pathways that affect the delamination process.
Figure 6: Delamination patterns in response to U251 cells exposed to CoCl2 alone or together with an MMP inhibitor. (A–C) U251 cells cultured for 3.5 days on the CAM during various conditions. (A)U251 cells cultured alone did not induce any damage to laminin. (B) Cultured U251 cells preexposed to CoCl2 (24 h) prior to washing and cell seeding on the CAM induced laminin damage and cell invasion. (C) Pretreatment with a broad-spectrum MMP inhibitor GM6001 (for 1 h), followed by CoCl2 exposure (24 h) before washing and seeding U251 cells on the CAM, suppressed the effect of the CoCl2 treatment, and no obvious laminin damage or cancer cell invasion was detected. Scale bar = 100 µm (A–C). Panels (B) and (C) are from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein; CoCl2 = cobalt chloride; MMP = matrix metalloproteinase. Please click here to view a larger version of this figure.
This paper describes the CAM-Delam assay to evaluate the metastatic aggressiveness of cancer cells, determined by scoring basal lamina modulations and potential cell invasion into the mesenchyme within a period of hours to a few days. A previous technical problem for various CAM assays has been the low survival of chick embryos. This issue was resolved by introducing the use of an internal humidified chamber during embryo incubation, which increased embryo survival from 10%-50% to 80%-90%7. The use of an internal humidified chamber may, therefore, be valuable in CAM-assays in general, as well as in other ex ovo chick experiments.
The presented scoring time points at 14 h, 1.5 days, 2.5 days, and 3.5 days after seeding 1 x 106 cancer cells on the CAM are based on rigorous method development using six different cancer cell lines and are sufficient to distinguish the range from non-delaminating to delaminating-with-invasion capacities of cancer cell lines. A minimum use of four eggs with three rings in each per time point and per cell line is suggested, and this should be repeated at least once or according to experimental designs and statistical requirements. One advantage of the CAM-Delam assay is obtaining informative results regarding the delamination capacity of cancer cells within a few days to estimate the aggressiveness of cancer cells and potential risk for metastasis formation. The rapid delivery of results is facilitated by monitoring the degradation of the basal lamina due to the invading cancer cells and the subsequent microtumors/tumor buds and organ metastases. Traditionally, CAM models have been used to analyze the formation of organ metastases, which takes around 2 weeks to be determined9. By using seven different cancer cell lines, we have previously verified7 that the delamination scoring is linked to the ability of cancer cells to form metastases in rodent models10,11,12,13,14, which supports the predictive value of the CAM-Delam assay. Moreover, mouse models require an even longer time, several weeks up to months, before metastases can be examined15,16. In brief, this developed CAM-Delam assay, focused on scoring delamination capacity and not on examining later tumor formation in the chick embryo, is, therefore, a good complement to existing chicken CAM invasion and mouse tumor models.
A limitation in the CAM-Delam assay may be the unclear visualization of the basal lamina if the cancer cells themselves express laminin. If so, other markers indicating the basal lamina, such as E-cadherin, could be used7. Other CAM invasion studies have used type IV collagen to visualize the CAM and pan-cytokeratin and vimentin to identify invading cancer cells and the formation of microtumors/tumor buds17,18.
Delamination is a normal cellular process, both during development and later in life, which makes it possible for cells to leave an epithelium and migrate to other tissues. Examples of delaminating cells during development are neural crest and olfactory pioneering neurons19,20; later in life, wound healing is dependent on delamination21. During cancer, this process is upregulated in the wrong cells and/or at the wrong time. Thus, the CAM-Delam method can be of use to unravel the molecular mechanisms that regulate delamination, which would be of importance for both basic biological and disease knowledge. Such delamination studies would include adding factors of interest to the cancer cells seeded on the CAM or studying genetically modified cancer cells. One example presented here is CoCl2 pretreatment of the non-metastatic cell line U251 to induce hypoxia, which leads to the induction of a metastatic aggressive capacity that could be suppressed by a broad-spectrum MMP inhibitor. Thus, finding key molecules that control delamination increases the possibility of designing inhibitors to suppress this process. In relation to this, another potential use for the CAM-Delam protocol is in drug screening for the suppression of delamination and cell invasion. Furthermore, in the clinic, the evaluation of cancer severity is a critical component for diagnosis, planning treatment, and care. Currently, the TNM staging system (T, tumor size; N, node-whether the cancer has spread to the lymph nodes; M, distant metastasis) is used to evaluate the severity of the cancer22. The CAM-Delam assay defines an innovative approach to evaluate the aggressiveness of cancer cells and potential risk for metastases formation and might be a useful complement to the TNM staging system. Notable is that TNM staging is based on the analyses of fixed tissue samples, whereas a potential clinical CAM-Delam approach would examine fresh or fresh-frozen tissue in combination with techniques to revive frozen cells23.
The authors have nothing to disclose.
We thank the following researchers at Umeå University for their help with relevant cancer cell lines and antibodies: L. Carlsson (von Willebrand Factor antibody), J. Gilthorpe (U251), and M. Landström (PC-3U). We also thank Hauke Holthusen in the Gilthorpe lab for the generation of the HEK293-TLR-AAVS1 stable cell line. Work in the Gunhaga laboratory was supported by the Swedish Cancer Foundation (18 0463), Umeå Biotech Incubator, Norrlands Cancerforskningsfond, the Swedish Research Council (2017-01430), and the Medical Faculty at Umeå University.
EQUIPMENT | |||
Centrifuge | Rotanta 480 R, Hettich zentrifugen | ||
Countess II FL Automated Cell Counter | Invitrogen | ||
Cryostat | HM 505 E, Microm | ||
Digital camera | Nikon DS-Ri1 | ||
Dissection Microscope | Leica M10 | For CAM-sample dissection and positioning in molds | |
Egg incubator | Fiem | Many other sources are available. Must be cleaned and sterilized with 70 % ethanol before each use | |
Epifluorescence microscope | Nikon Eclipse, E800 | Equiped with a digital camera, for scoring delamination and cell invasion. | |
Fine forceps | Many sources are available | Must be sterilized before use | |
Freezer -80 °C | Thermo Fisher Scientific | 8600 Series | Model 817CV |
Inverted microscope | Nikon Eclipse TS100 | For cell culture work | |
Scissors (small) | Many sources are available | Must be sterilized before use | |
MATERIALS | |||
anti-Laminin-111 | Sigma-Aldrich | L9393 | Primary anti-rabbit antibody (1:400) |
anti-rabbit Cy3 | Jackson Immuno Research | 111-165-003 | Secondary antibody (1:400) |
anti-von Willebrand Factor | DAKO | P0226 | Primary antibody (1:100) |
Cobalt(II) chloride |
Sigma-Aldrich | 232696-5G | CAUTION: moderate toxicity chemical. Handle with care only in fume hood. Follow manufacturers instructions |
DAPI | Sigma-Aldrich | D9542-10MG | 4',6-diamidino-2-phenylindole, dihydrochloride (1:400) |
Fertilized chicken eggs | Strömbäcks Ägg, Vännäs, Sweden | Any local egg supplyer | |
Fetal bovine serum | Life Technologies | 10500-064 | |
Fluorescence mounting medium | Allent Technologie | S302380-2 | Avoid bubble formation when mounting. Allow to dry at +4 °C |
Glass chambers for silicon rings | Many sources are available | We used 15 mL glass chambers. Around 20 silicon rings fit in one chamber. Avoid crowding, since the rings may stick together and aggravate work. | |
Glass coverslips | VWR International | 631-0165 | |
GM6001 MMP Inhibitor | Sigma-Aldrich | CC1010 | |
Microscope slides for immunohistochemistry | Fisher Scientific | 10149870 | |
NEG-50 frozen medium | Cellab | 6506 | |
Paraformaldehyde | Sigma-Aldrich | 30525-89-4 | CAUTION: highly toxic. Handle with care only in fume hood, follow manufacturers instructions. Use all protective clothing. |
Peel-A-Way Embedding Molds |
Polysciences | 18985 | |
Penicillin–streptomycin | Gibco | 15070063 | |
Petri dishes | Sarstedt | 83.3903 | 15 cm in diameter for cell culture |
Plastic box | Esclain | ||
PureCol EZ Gel Collagen | Cellsystems | 5074-35ML | 5 mg/mL. Gelatinous material. Pipette very slowly and carefully to avoid cells being lost in the bubble formations. |
RPMI medium | Thermo Fisher Scientific | 21875034 | |
Silicon rings | VWR International | 228-1580 | Inner/outer diameter: 4/5 mm. Should be sterilized before use. Avoid repeated autoclaving of unused rings. |
Trypan blue | Fisher Scientific | T10282 | |
Trypsin | Life Technologi | 15400054 | 0.50% |
Weighing boats | VWR International | 611-0094 | |
SOLUTIONS | |||
Collagen-RPMI media mixture (1 mL) | Compelete RPMI Media 750 µL |
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PureCol EZ Gel Collagen 250 µL |
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Mix and use immediately | |||
Complete RPMI media (500 mL) | RPMI Media 445 mL |
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FBS 50 mL |
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Penicillin–streptomycin 5mL |
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Store at 4 °C | |||
PB (0.2 M; 1 000 mL) | Na2HPO4 (MW 141.76) 21.9 g |
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NaH2PO4 (MW 137.99) 6.4 g |
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add deionized water up to final volume of 1000ml | |||
Store in RT | |||
PFA (4 %) in 0.1 M PB (100 mL) | Deionized water 50 mL |
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0.2 M PB 50 mL |
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Paraformaldehyde (PFA) 4g |
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heat to 60 °C in water bath | |||
add 5 M NaOH 25 µL |
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Stir to dissolve the PFA powder | |||
Store at 4 °C | |||
TBST (1 000 mL) | 50mM Tris pH 7,4 50 mL |
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150mM NaCI 30 mL |
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0,1% Triton X-100 10 mL |
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H2O (MQ) 900 mL |
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Trypsin (0.05 %; 10 mL) | 1x PBS 9 mL |
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10x Trypsin (0.5 %) 1 mL |
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10 mL in total, Store at 4 °C |