We thank the following researchers at Ume&#229; University for their help with relevant cancer cell lines and antibodies: L. Carlsson (von Willebrand Factor antibody), J. Gilthorpe (U251), and M. Landstr&#246;m (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), Ume&#229; Biotech Incubator, Norrlands Cancerforskningsfond, the Swedish Research Council (2017-01430), and the Medical Faculty at Ume&#229; University.

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
<ol>
	<li>Use fertilized chick eggs from a local hatchery, which guarantee over 90% fertilization 
	NOTE: The eggs should not have been stored for more than 4 days at room temperature (RT). In Sweden, ethical permission for the experimental use of embryonic chickens is only required from embryonic day (E) 14 and beyond.</li>
	<li>If needed, carefully mechanically wipe off any dirt or feathers on the eggs using a dry paper towel or wet in water.</li>
	<li>Clean and sterilize the egg incubator with 70% ethanol before use (every time).</li>
	<li>Place the desired number of eggs in egg trays and incubate the chick eggs horizontally in an egg incubator at 37.5-38 &#176;C with 70% humidity. Consider this as incubation day 0 (Figure 1A).</li>
</ol>
2. Preparation of weighing boat, plastic boxes, and dissection instruments
<ol>
	<li>Use 70% ethanol to sterilize 30 weighing boats and 30 small plastic boxes (~0.4 L) with transparent caps.</li>
	<li>Dry the weighing boats and boxes in a laminar hood overnight (ON) and store in a closed plastic box until further use on incubation day 3.</li>
	<li>Sterilize 2 L of distilled or deionized H2O per 30 incubated eggs.</li>
	<li>Sterilize two pairs of scissors and three pairs of forceps by spraying 70% ethanol. Air-dry the instruments and then store them in a sterilized box until incubation day 3. 
	&#8203;NOTE: These actions (Steps 2.1-2.4) can be done any day prior to incubation day 3. Use gloves to avoid contamination.</li>
</ol>
3. Opening the eggs and transfer to the internal humidified chamber
NOTE: Use gloves and a face mask to avoid contamination.
<ol>
	<li>Add approximately 50 mL of sterilized H2O to the sterilized plastic boxes. Keep the water-filled boxes with closed lids, to avoid contamination, at RT until use.</li>
	<li>On incubation day 3, crack the eggshell using the sharp part of scissors and cut a straight opening in the shell.</li>
	<li>Manually break open the eggshell over a weighing boat and collect the egg white, the yolk, and its attached healthy embryo in the weighing boat (Figure 1B). Look for an intact&#160;embryo with a beating heart, intact yolk, and developed blood vessels for the experiment (&#62;95% of the incubated eggs). Discard eggs with a malformed embryo, an embryo without a beating heart, a broken egg yolk, or damaged yolk blood vessels.</li>
	<li>Gently transfer the weighing boat into an internal humidified chamber. 
	NOTE: For Steps 3.2.-3.4., work as fast as possible to avoid contamination, and if possible, use a laminar hood.</li>
	<li>Incubate the internal humidified chamber in the egg incubator (Figure 1C).</li>
	<li>Culture the shell-less eggs from day 3 to day 10 in the egg incubator before being used (see Step 3.6.), and when needed, add water to the incubator to maintain 70% humidity.</li>
	<li>Check the internal humidified chambers through the transparent plastic lid for dead embryos or contaminated shell-less eggs every day, and remove them from the incubator.</li>
</ol>
4. Preparation of silicone rings
<ol>
	<li>To prepare silicone rings, cut a silicone tube with an inner and outer diameter of 4 mm and 5 mm, respectively, in ~1 mm thickness, preferably using a paper cutter (Figure 1D).</li>
	<li>Transfer the silicone rings to small glass bottles (Figure 1D), cover with metal foil, and sterilize them using an autoclave or similar. 
	NOTE: Avoid repeated autoclaving of unused silicone rings that might be contaminated, since such treatment decreases the capacity of the silicone rings to attach to the membrane, with subsequent leakage of the cancer cell/collagen/RPMI solution outside of the rings.</li>
	<li>Store the sterile silicone rings at RT. 
	&#8203;NOTE: This step can be done any day prior to incubation day 10.</li>
</ol>
5. Preparation of cancer cells
NOTE: Solutions such as cell culture medium, trypsin, and 1x PBS are stored at 4 &#176;C and should be heated to 37 &#176;C in a water bath before adding to the cells. After heating, rinse the bottles in 70% ethanol and dry before use.
<ol>
	<li>Culture the cancer cell lines of interest in the relevant culture medium in a cell culture incubator at 37 &#176;C in the presence of 5% (v/v) CO2. 
	NOTE: Here, U251-GFP glioblastoma and PC-3U-GFP prostate cancer cells were cultured in complete RPMI medium-RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS).</li>
	<li>Plan the culture of cancer cells so that approximately 50 &#215; 106 cancer cells are ready for harvesting on incubation day 10 of the CAM-Delam assay.</li>
	<li>Change the cell culture medium every 2-3 days, or when the medium color changes from pink to orange/yellow.</li>
	<li>Optional: Induce hypoxia by treating the cancer cells with CoCl2 to investigate the molecular mechanistic effects on delamination 1 day before cell harvesting. 
	CAUTION: CoCl2 has moderate toxicity. Handle with care in a fume hood, and follow the manufacturer&#39;s instructions.
	<ol>
		<li>Prepare fresh 20 mM CoCl2 stock solution by dissolving 0.0258 g in 10 mL of sterilized distilled H20 in a 15 mL conical tube wrapped with aluminum foil to protect from light.</li>
		<li>In a 50 mL conical tube, mix 250 &#181;L of the 20 mM CoCl2 stock solution in 25 mL of RPMI medium supplemented with 1% (v/v) PS (but without FBS) per 15 cm diameter cell culture dish. Vortex gently.</li>
		<li>Remove the complete RPMI medium from the cell culture dishes.</li>
		<li>Wash the cells with sterilized 1x PBS 2x.</li>
		<li>Add 25 mL of RPMI medium with 200 &#181;M CoCl2 cell trypsinization (see Step 5.6).</li>
	</ol>
	</li>
	<li>On egg incubation day 10, prepare 1 mL of a collagen/RPMI mix (ratio 1:3), containing 250 &#181;L of type I collagen (5 mg/mL) and 750 &#181;L of cell culture RPMI medium supplemented with 10% FBS and 1% (v/v) PS. Keep the collagen/RPMI-mix on ice.</li>
	<li>Trypsinize the cancer cells to isolate the cells by first removing the cell culture medium and wash 2x with 1x PBS.</li>
	<li>Add 3 mL of trypsin solution (0.05%) per 15 cm diameter cell culture dish and incubate for 2-3 min in a cell culture incubator until the cells detach. Use a table-top, inverted microscope to see the detached cells. If needed, gently tap the side of the flask to dislodge remaining clustered or attached cells.</li>
	<li>Inactivate trypsin by adding 5 mL of complete RPMI medium to each cell culture dish and collect the cell suspension from all cell culture dishes into a 50 mL tube.</li>
	<li>Count the cancer cells by the Trypan Blue exclusion method to distinguish live from dead cells using a cell counter.Add 10 &#181;L of the cell suspension to 10 &#181;L of 0.4% trypan blue stain. Mix the sample by pipetting up and down a few times, and then load 10 &#181;L of the cell mixture per chamber into the sample slide in the cell counter. Repeat cell counting 3x to verify the cell number/mL.</li>
	<li>Calculate and centrifuge the correct volume cell culture suspension containing 50 x 106 live cancer cells in a 50 mL tube at 500 &#215; g for 5 min.</li>
	<li>Discard the supernatant and mix the cell pellet with 1 mL of the collagen/RPMI mix. As collagen is a gelatinous material, mix the cells very slowly and carefully with a 1 mL pipette to avoid losing cells in the formation of bubbles.</li>
	<li>Keep the prepared cell suspension on ice and bring it to the egg working area. 
	&#8203;NOTE: Avoid keeping the prepared cancer cells on ice for too long. The prepared cancer cells must be placed on the CAM within 15-25 min.</li>
</ol>
6. Seeding the cancer cells on the CAM
NOTE: Use gloves and a face mask to avoid contamination.
<ol>
	<li>At incubation day 10, take out the internal humidified chambers with the incubated shell-less eggs from the incubator. 
	NOTE: Approximately 90% of the initial number of the incubated eggs can be used; see Figure 2.</li>
	<li>Open the internal humidified chamber and place up to six silicone rings on the CAM using sterilized forceps. 
	NOTE: Avoid placing the silicone rings near each other or close to bigger blood vessels.</li>
	<li>Mix the cancer cell suspension by pipetting to get an even distribution of cancer cells, and add 20 &#181;L (1 &#215; 106 cells) of the prepared cancer cell suspension inside a silicone ring (Figure 1E). When adding the cells, keep the pipette tip above the CAM to make sure not to damage the membrane. 
	NOTE: According to previous findings7, different cancer cells can be seeded in separate rings on the same CAM.</li>
	<li>Close the internal humidified chamber and put it in the egg incubator.</li>
</ol>
7. Isolation of the CAM with associated cancer cells
<ol>
	<li>After 14 h, 1.5 days, 2.5 days, and 3.5 days of incubation, take out approximately seven internal humidified chambers and open the lids one at a time.</li>
	<li>With a pair of scissors, dissect the cultured cancer cells attached to the CAM (so-called CAM-Delam samples) by cutting outside the silicone ring (Figure 1F).</li>
	<li>Immediately transfer the isolated CAM-Delam sample using forceps to 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) in a Petri dish for fixation of the tissue. Keep on ice or at 4 &#176;C for 1 h. 
	NOTE: Store 4% PFA solution at 4 &#176;C for a maximum of 5 days. CAUTION: PFA is toxic. When handling PFA powder and PFA solutions, use a fume hood and wear a face mask and gloves. Avoid inhaling powder or solution vapors. Follow the manufacturer&#39;s instructions.</li>
	<li>Decapitate the chicken embryos and discard as biological waste in specific bins.</li>
	<li>Remove the 4% PFA solution and add 30% sucrose in 0.1 M PB to the CAM-Delam samples and equilibrate at 4 &#176;C for 1 h.</li>
	<li>Under a dissection microscope, carefully remove the silicone ring using forceps. With a pair of scissors, cut the CAM-Delam sample in a rectangular shape with the cancer cells in the middle (Figure 1G).</li>
	<li>With a pair of forceps, transfer the CAM-Delam samples to frozen section medium in a Petri dish to remove excess sucrose and then to embedding molds in frozen section medium.</li>
	<li>Under a dissection microscope, position the CAM-Delam sample in a U-shape in a vertical direction in the embedding molds using any needle-like instrument (Figure 1G).</li>
	<li>Freeze and store the CAM-Delam samples at &#8722;80 &#176;C.</li>
</ol>
8. Sectioning CAM-Delam samples
<ol>
	<li>Section the frozen CAM-Delam samples at 10 &#181;m on 5-6 consecutive slides using cryosectioning.</li>
	<li>Store the slides with sections at &#8722;80 &#176;C or use directly for immunohistochemistry staining.</li>
</ol>
9. Immunohistochemistry staining
<ol>
	<li>Bring the slides from &#8722;80 &#176;C to RT for 5 min before starting the immunoprotocol.</li>
	<li>Make a line with a hydrophobic marker on the slides where the sections end. Let them dry for a few minutes.</li>
	<li>Place the slides in a humidified (H2O) chamber and cover the sections with ~200-500 &#181;L of blocking solution (10% fetal calf serum and 0.1% sodium azide in Tris-buffered saline with 0.1% Triton X-100 [TBST]) and incubate for 15-30 min. 
	NOTE: From this step onwards, do not let the slides dry at any time.</li>
	<li>Pour off the blocking solution and replace it with 100-150 &#181;L of the primary antibody of interest diluted in the blocking solution and incubate ON at 4 &#176;C. Preferably use rabbit anti-laminin-111 antibody to define the basal lamina together with a cancer cell marker, if not using GFP or other fluorescent-tagged cancer cell lines. 
	NOTE: Here, a rabbit anti-von Willebrand Factor was also used to detect blood vessels in the CAM.</li>
	<li>Prepare three glass cuvettes filled with TBST buffer.</li>
	<li>Pour off the primary antibody solution, transfer the slides to the glass cuvettes, and wash at least 3x for 5 min each in TBST.</li>
	<li>Remove excess TBST from the slide and from the hydrophobic barrier area with a soft paper tissue.</li>
	<li>Cover the slide with 100-150 &#181;L of a suitable secondary fluorescent antibody diluted in blocking solution combined with 4&#39;,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and incubate in the dark at RT for 1 h.</li>
	<li>Pour off the secondary antibody solution, transfer the slides to the glass cuvettes, and wash at least 3x for 5 min each in TBST.</li>
	<li>Remove excess TBST from the slide and from the hydrophobic barrier area with a soft paper tissue.</li>
	<li>Mount the slides by putting 1-2 drops of fluorescent mounting medium on the slide, and gently place a glass coverslip, avoiding air bubbles.</li>
	<li>Let the slides dry for at least 1 h at 4 &#176;C before analyzing, and store the slides 4 &#176;C.</li>
</ol>
10. Microscopy imaging and delamination scoring
<ol>
	<li>Photograph the sections using an epifluorescence microscope equipped with a digital camera, preferably at 10x magnification (Figure 1H).</li>
	<li>Analyze the sections using the following CAM-Delam scoring categories (Figure 3):
	<ol>
		<li>Look for intact basal lamina without visible alterations.</li>
		<li>Look for altered but undamaged basal lamina.</li>
		<li>Look for damaged basal lamina without cell invasion.</li>
		<li>Look for damaged basal lamina with cell invasion.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Vasantharajan, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epigenetic+landscape+of+circulating+tumour+cells.">The epigenetic landscape of circulating tumour cells.</a> Biochimica et Biophysica Acta - Reviews on Cancer. 1875 (2), 188514 (2021).</li><li>Acloque, H., Adams, M. S., Fishwick, K., Bronner-Fraser, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+the+importance+of+changing+cell+state+in+development+and+disease.">Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.</a> Journal of Clinical Investigation. 119 (6), 1438-1449 (2009).</li><li>Itoh, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-type+matrix+metalloproteinases:+their+functions+and+regulations.">Membrane-type matrix metalloproteinases: their functions and regulations.</a> Matrix Biology. 44-46, 207-223 (2015).</li><li>Przemyslaw, L., Boguslaw, H. A., Elzbieta, S., Malgorzata, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAM+and+ADAMTS+family+proteins+and+their+role+in+the+colorectal+cancer+etiopathogenesis.">ADAM and ADAMTS family proteins and their role in the colorectal cancer etiopathogenesis.</a> BMB Reports. 46 (3), 139-150 (2013).</li><li>Chu, P. Y., Koh, A. P., Antony, J., Huang, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+chick+chorioallantoic+membrane+as+an+alternative+model+for+cancer+studies.">Applications of the chick chorioallantoic membrane as an alternative model for cancer studies.</a> Cells Tissues Organs. 211 (2), 222-237 (2021).</li><li>MacDonald, I. C., Groom, A. C., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+spread+and+micrometastasis+development:+quantitative+approaches+for+in+vivo+models.">Cancer spread and micrometastasis development: quantitative approaches for in vivo models.</a> Bioessays. 24 (10), 885-893 (2002).</li><li>Palaniappan, T. K., Slekiene, L., Jonasson, A. K., Gilthorpe, J., Gunhaga, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAM-Delam:+an+in+vivo+approach+to+visualize+and+quantify+the+delamination+and+invasion+capacity+of+human+cancer+cells.">CAM-Delam: an in vivo approach to visualize and quantify the delamination and invasion capacity of human cancer cells.</a> Scientific Reports. 10 (1), 10472 (2020).</li><li>Carrillo, M., Kim, S., Rajpurohit, S. K., Kulkarni, V., Jagadeeswaran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+von+Willebrand+factor.">Zebrafish von Willebrand factor.</a> Blood Cells, Molecules and Diseases. 45 (4), 326-333 (2010).</li><li>Leupold, J. H., Patil, N., Allgayer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+egg+chorioallantoic+membrane+(CAM)+model+as+an+in+vivo+method+for+the+investigation+of+the+invasion+and+metastasis+cascade+of+malignant+tumor+cells.">The chicken egg chorioallantoic membrane (CAM) model as an in vivo method for the investigation of the invasion and metastasis cascade of malignant tumor cells.</a> Methods in Molecular Biology. 2294, 17-26 (2021).</li><li>Jia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+endostatin+endostar+inhibits+tumor+growth+and+metastasis+in+a+mouse+xenograft+model+of+colon+cancer.">Recombinant human endostatin endostar inhibits tumor growth and metastasis in a mouse xenograft model of colon cancer.</a> Pathology and Oncology Research. 18 (2), 315-323 (2012).</li><li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+inhibition+of+presenilin+2+inhibits+glioma+cell+growth+and+invasion+and+is+involved+in+the+regulation+of+Nrg1/ErbB+signaling.">RNAi-mediated inhibition of presenilin 2 inhibits glioma cell growth and invasion and is involved in the regulation of Nrg1/ErbB signaling.</a> Neuro-Oncology. 14 (8), 994-1006 (2012).</li><li>Qin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+superfamily+15+promotes+lymphatic+metastasis+via+upregulation+of+vascular+endothelial+growth+factor-C+in+a+mouse+model+of+lung+cancer.">Tumor necrosis factor superfamily 15 promotes lymphatic metastasis via upregulation of vascular endothelial growth factor-C in a mouse model of lung cancer.</a> Cancer Science. 109 (8), 2469-2478 (2018).</li><li>Ren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+metastatic+phenotype+of+a+panel+of+established+osteosarcoma+cells.">Characterization of the metastatic phenotype of a panel of established osteosarcoma cells.</a> Oncotarget. 6 (30), 29469-29481 (2015).</li><li>Zang, G., Mu, Y., Gao, L., Bergh, A., Landstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKCzeta+facilitates+lymphatic+metastatic+spread+of+prostate+cancer+cells+in+a+mice+xenograft+model.">PKCzeta facilitates lymphatic metastatic spread of prostate cancer cells in a mice xenograft model.</a> Oncogene. 38 (22), 4215-4231 (2019).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nature Reviews Cancer. 11 (2), 135-141 (2011).</li><li>Jung, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor+xenograft+models+for+preclinical+assessment+of+anticancer+drug+development.">Human tumor xenograft models for preclinical assessment of anticancer drug development.</a> Toxicology Research. 30 (1), 1-5 (2014).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferase+EZH2+mediates+tumor+progression+on+the+chick+chorioallantoic+membrane+assay,+a+novel+model+of+head+and+neck+squamous+cell+carcinoma.">The histone methyltransferase EZH2 mediates tumor progression on the chick chorioallantoic membrane assay, a novel model of head and neck squamous cell carcinoma.</a> Translational Oncology. 6 (3), 273-281 (2013).</li><li>Steinmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAPK1+loss+triggers+tumor+invasion+in+colorectal+tumor+cells.">DAPK1 loss triggers tumor invasion in colorectal tumor cells.</a> Cell Death &amp; Disease. 10 (12), 895 (2019).</li><li>Andrieu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP14+is+required+for+delamination+of+chick+neural+crest+cells+independently+of+its+catalytic+activity.">MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity.</a> Development. 147 (7), (2020).</li><li>Palaniappan, T. K., Slekiene, L., Gunhaga, L., Patthey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+apoptosis+during+the+formation+of+the+terminal+nerve+ganglion+by+olfactory+placode-derived+cells+with+distinct+molecular+markers.">Extensive apoptosis during the formation of the terminal nerve ganglion by olfactory placode-derived cells with distinct molecular markers.</a> Differentiation. 110, 8-16 (2019).</li><li>Thiery, J. P., Acloque, H., Huang, R. Y., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions+in+development+and+disease.">Epithelial-mesenchymal transitions in development and disease.</a> Cell. 139 (5), 871-890 (2009).</li><li>Pineros, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+TNM:+a+registry+tool+to+reduce+gaps+in+cancer+staging+information.">Essential TNM: a registry tool to reduce gaps in cancer staging information.</a> The Lancet Oncology. 20 (2), 103-111 (2019).</li><li>Walsh, A. J., Cook, R. S., Sanders, M. E., Arteaga, C. L., Skala, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+response+in+organoids+generated+from+frozen+primary+tumor+tissues.">Drug response in organoids generated from frozen primary tumor tissues.</a> Scientific Reports. 6, 18889 (2016).</li></ol>

The authors have no competing interests to disclose.

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.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Rotanta 480 R, Hettich zentrifugen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>HM 505 E, Microm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Nikon DS-Ri1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Leica M10</td>
			<td></td>
			<td>For CAM-sample dissection and positioning in molds</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Fiem</td>
			<td></td>
			<td>Many other sources are available. Must be cleaned and sterilized with 70 % ethanol before each use</td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Nikon Eclipse, E800</td>
			<td></td>
			<td>Equiped with a digital camera, for scoring delamination and cell invasion.</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Many sources are available</td>
			<td></td>
			<td>Must be sterilized before use</td>
		</tr>
		<tr>
			<td>Freezer -80 &#176;C</td>
			<td>Thermo Fisher Scientific</td>
			<td>8600 Series</td>
			<td>Model 817CV</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon Eclipse TS100</td>
			<td></td>
			<td>For cell culture work</td>
		</tr>
		<tr>
			<td>Scissors (small)</td>
			<td>Many sources are available</td>
			<td></td>
			<td>Must be sterilized before use</td>
		</tr>
		<tr>
			<td>MATERIALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Laminin-111</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td>Primary anti-rabbit antibody (1:400)</td>
		</tr>
		<tr>
			<td>anti-rabbit Cy3</td>
			<td>Jackson Immuno Research</td>
			<td>111-165-003</td>
			<td>Secondary antibody (1:400)</td>
		</tr>
		<tr>
			<td>anti-von Willebrand Factor</td>
			<td>DAKO</td>
			<td>P0226</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td> 
			Cobalt(II) chloride</td>
			<td>Sigma-Aldrich</td>
			<td>232696-5G</td>
			<td>CAUTION: moderate toxicity chemical. Handle with care only in fume hood. Follow manufacturers instructions</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-10MG</td>
			<td>4&#39;,6-diamidino-2-phenylindole, dihydrochloride (1:400)</td>
		</tr>
		<tr>
			<td>Fertilized chicken eggs</td>
			<td>Str&#246;mb&#228;cks &#196;gg, V&#228;nn&#228;s, Sweden</td>
			<td></td>
			<td>Any local egg supplyer</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence mounting medium</td>
			<td>Allent Technologie</td>
			<td>S302380-2</td>
			<td>Avoid bubble formation when mounting. Allow to dry at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Glass chambers for silicon rings</td>
			<td>Many sources are available</td>
			<td></td>
			<td>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.&#160;</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>VWR International</td>
			<td>631-0165</td>
			<td></td>
		</tr>
		<tr>
			<td>GM6001 MMP Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>CC1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides for immunohistochemistry</td>
			<td>Fisher Scientific</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>NEG-50 frozen medium</td>
			<td>Cellab</td>
			<td>6506</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td>CAUTION: highly toxic. Handle with care only in fume hood, follow manufacturers instructions. Use all protective clothing.</td>
		</tr>
		<tr>
			<td> 
			Peel-A-Way Embedding Molds</td>
			<td>Polysciences</td>
			<td>18985</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sarstedt</td>
			<td>83.3903</td>
			<td>15 cm in diameter for cell culture</td>
		</tr>
		<tr>
			<td>Plastic box</td>
			<td>Esclain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PureCol EZ Gel Collagen</td>
			<td>Cellsystems</td>
			<td>5074-35ML</td>
			<td>5 mg/mL. Gelatinous material. Pipette very slowly and carefully to avoid cells being lost in the bubble formations.</td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21875034</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon rings</td>
			<td>VWR International</td>
			<td>228-1580</td>
			<td>Inner/outer diameter: 4/5 mm. Should be sterilized before use. Avoid repeated autoclaving of unused rings.</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologi</td>
			<td>15400054</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Weighing boats</td>
			<td>VWR International</td>
			<td>611-0094</td>
			<td></td>
		</tr>
		<tr>
			<td>SOLUTIONS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-RPMI media mixture (1 mL)</td>
			<td></td>
			<td></td>
			<td>Compelete RPMI Media 
			750 &#181;L</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>PureCol EZ Gel Collagen 
			250 &#181;L</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Mix and use immediately</td>
		</tr>
		<tr>
			<td>Complete RPMI media (500 mL)</td>
			<td></td>
			<td></td>
			<td>RPMI Media 
			445 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>FBS 
			50 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Penicillin&#8211;streptomycin 
			5mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PB (0.2 M; 1 000 mL)</td>
			<td></td>
			<td></td>
			<td>Na2HPO4 (MW 141.76) 
			21.9 g</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>NaH2PO4 (MW 137.99) 
			6.4 g</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>add deionized water up to final volume of 1000ml</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Store in RT</td>
		</tr>
		<tr>
			<td>PFA (4 %) in 0.1 M PB (100 mL)</td>
			<td></td>
			<td></td>
			<td>Deionized water 
			50 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 M PB 
			50 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Paraformaldehyde (PFA) 
			4g</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>heat to 60 &#176;C in water bath</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>add 5 M NaOH 
			25 &#181;L</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Stir to dissolve the PFA powder</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>TBST (1 000 mL)</td>
			<td></td>
			<td></td>
			<td>50mM Tris pH 7,4 
			50 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>150mM NaCI 
			30 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0,1% Triton X-100 
			10 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>H2O (MQ) 
			900 mL</td>
		</tr>
		<tr>
			<td>Trypsin (0.05 %; 10 mL)</td>
			<td></td>
			<td></td>
			<td>1x PBS 
			9 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>10x Trypsin (0.5 %) 
			1 mL</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>10 mL in total, Store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

cam-delam assay to score metastatic properties by quantifying delamination and invasion capacity of cancer cells

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.

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 &#60;50% up to 90% at incubation day 10 and from ~15% up to 80% at incubation day 13 (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64025/64025fig1v2.jpg" /> 
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)&#160;Prepare silicone rings. (E)&#160;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 &#8722;80 &#176;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 &#181;m (H). This figure is from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; PFA = paraformaldehyde; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64025/64025fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64025/64025fig2v2.jpg" /> 
Figure 2: Chicken embryo survival in relation to incubation method. (A,B)&#160;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 signi&#64257;cance was tested using an unpaired two-tailed t-test. The error bars indicate the standard deviation. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 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. <a href="https://www.jove.com/files/ftp_upload/64025/64025fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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&#39;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).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64025/64025fig03.jpg" /> 
Figure 3: CAM-Delam scoring. (A-E)&#160;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 &#181;m (A-E). This figure is from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64025/64025fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64025/64025fig04.jpg" /> 
Figure 4: Evaluation of CAM thickening and blood vessel formation. (A-H)&#160;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 &#181;m. This figure was modified from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64025/64025fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64025/64025fig05.jpg" /> 
Figure 5: Visualization of the delamination capacity of prostate (PC-3U) and glioblastoma (U251) cancer cells. (A)&#160;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)&#160;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 &#181;m (A,B).&#160;This figure was modified from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64025/64025fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64025/64025fig06.jpg" /> 
Figure 6: Delamination patterns in response to U251 cells exposed to CoCl2 alone or together with an MMP inhibitor. (A-C)&#160;U251 cells cultured for 3.5 days on the CAM during various conditions. (A)U251 cells cultured alone&#160;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 &#181;m (A-C).&#160;Panels (B) and (C) are from Palaniappan et al.7. Abbreviations: CAM = chorioallantoic membrane; GFP = green fluorescent protein; CoCl2 = cobalt chloride; MMP = matrix metalloproteinase. <a href="https://www.jove.com/files/ftp_upload/64025/64025fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells at JoVE.com

CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells

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 ...

cam-delam-assay-to-score-metastatic-properties-quantifying

Research

JoVE Journal

Cancer Research

2.5K Views.  Umeå University. 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. 

Video: CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...