The work was supported by VEGA 2/0042/21 and APVV 20-0129. The contribution of V. Hunto&#353;ov&#225; is the result of the project implementation: Open scientific community for modern interdisciplinary research in medicine (Acronym: OPENMED), ITMS2014+: 313011V455 supported by the Operational Program Integrated Infrastructure, funded by the ERDF.

The research was performed in compliance with institutional guidelines. All equipment and reagents must be autoclaved or sterilized with 70% ethanol or UV light.
1. Egg incubation
<ol>
	<li>Store fertilized quail eggs at 10-15 &#176;C for a maximum of 4-5 days before starting incubation. Use only clean and undamaged eggs.</li>
	<li>Incubate the eggs in a forced draught incubator for ~ 53-54 h. Lay the eggs horizontally with the egg rotation switched off, at 50%-60% humidity and 37.5 &#176;C incubation temperature.</li>
</ol>
2. Ex ovo culture preparation
NOTE: After initial incubation, the eggs are suitable for starting the ex ovo cultivation.
<ol>
	<li>Disinfect the egg surface with 70% ethanol, without rotating the egg.</li>
	<li>In a sterile laminar-flow cabinet, open the eggshell using small sterile surgical scissors and transfer the contents into a 6-well culture plate. If properly done, the embryo will lie on top of an undamaged yolk. After each egg, disinfect the scissors with 70% ethanol.</li>
	<li>Add approximately 5 mL of sterile water to the gaps in the 6-well plate, as humidity is essential to prevent the CAM from drying out.</li>
	<li>Place the embryos in an incubator until further experiments, while maintaining a temperature of 37 &#176;C and 80%-90% humidity.</li>
	<li>When the CAM is fully developed (from ED7), place a sterilized silicone ring (6 mm in diameter and approximately 1.5 mm in thickness) on the CAM surface, along the small capillaries. Avoid major blood vessels when placing the ring. 
	NOTE: The ring defines the workspace, helps mark the place of substance application, and prevents the liquid contents from leaking out.</li>
	<li>Terminate the cultivation of the embryos according to the country&#39;s legislation.</li>
	<li>Importantly, wear gloves or keep hands disinfected with 70% ethanol during work. Aspirate improperly tipped embryos or unfertilized eggs with a vacuum aspirator.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63422/63422fig01.jpg" /> 
Figure 1: Ex ovo culture preparation. (A) Japanese quail eggs as they are stored and incubated. (B) Egg surface disinfection with ethanol. (C) Eggshell is cut with scissors. (D) The content of the egg is emptied into the well. (E) Properly prepared 3-day old embryo, with developing CAM vasculature. (F) Culture plates stored in an incubator. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig01large.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/63422/63422fig02.jpg" /> 
Figure 2: 6-well culture plate with embryos and silicone rings placed on top. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Inoculation of tumor cells
NOTE: All procedures require the use of a sterile laminar flow cabinet.
<ol>
	<li>Culture different types of adherent cells in flasks according to respective culture protocols.
	<ol>
		<li>Remove the old medium from the flasks and rinse the cell layer with sterile phosphate-buffered saline (PBS) to remove traces of the medium.</li>
		<li>After trypsinization, inhibit the trypsin by adding the culture medium and harvest the cells into centrifuge tubes. Centrifuge the cells and resuspend the cell pellet in a fresh culture medium. Count the cells and resuspend them in a cell culture medium at the desired concentration. 
		NOTE: It is also possible to produce 3D cell cultures, i.e., spheroids using, for example, the hanging drop method21.</li>
	</ol>
	</li>
	<li>Implant 1 x 105-1 x 106 tumor cells or 5-15 spheroids (per CAM) in 30 &#181;L of cell medium solution into the silicone ring on top of the CAM. 
	NOTE: The volume depends on the size of the silicone ring used. If a silicone ring with a larger diameter is used, a larger volume is needed to cover the entire area. Depending on the cell type, or for different types of experiments, various cell concentrations may be used. Sometimes, fine scraping of CAM is used to improve the adhesion of the embedded cells.</li>
	<li>Return the CAMs with inoculated cells to the incubator (37 &#176;C and 80%-90% humidity).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63422/63422fig03.jpg" /> 
Figure 3: Inoculation of CAM with tumors. (A) Aspiration of spheroids with a pipette, and (B) implantation on the CAM surface. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Application of photosensitizer
<ol>
	<li>Application of hypericin
	<ol>
		<li>Under dim lighting, prepare a 2 mM stock solution of hypericin by dissolving it in 100% dimethyl sulfoxide (DMSO). Prepare a 79 &#181;M working solution of hypericin shortly before the experiment with sterile PBS or saline solution. Ensure the final concentration of DMSO in all solutions is always under 0.2%, which does not affect the development of the embryo.</li>
		<li>Under sterile conditions, apply an appropriate volume of hypericin solution to the silicone ring. A volume of 30 &#181;L is sufficient to fill a ring with a diameter of 6 mm.</li>
		<li>Keep embryos in an incubator at 37 &#176;C and 80%-90% humidity. Hypericin in an aqueous solution is photoactive after its monomerization in the tissue, approximately 3 h after its application on the CAM.</li>
	</ol>
	</li>
	<li>Application of&#160;hypericin with LDL, HDL, or nanoparticles as transport systems 
	NOTE: These transport systems are used to improve the penetration of hypericin into cells and cell structures.
	<ol>
		<li>Due to the photosensitivity, perform all the steps, including the preparation of solutions, under dim lighting and store the solutions in the dark.</li>
		<li>Prepare the hypericin-LDL and hypericin-HDL formulations by mixing appropriate volumes of lipoproteins and hypericin stock solutions in PBS according to reference15. The concentration of LDL or HDL to hypericin is 20:115.</li>
		<li>Synthesize polymer nanoparticles by living cationic ring-opening polymerization according to references12,13. Adjust the concentration of hypericin in polymer nanoparticles with PBS to 10&#160;&#956;M12,13.</li>
	</ol>
	</li>
</ol>
5. PDD and PDT
<ol>
	<li>Conduct PDD
	<ol>
		<li>Under sterile conditions, apply photosensitizer (hypericin, hypericin-loaded polymeric nanoparticles, or hypericin with lipoprotein carrier) on the CAM surface, with or without tumor cells (ED 7-9).</li>
		<li>Illuminate the CAM using violet excitation light (custom-made circular LED light of 405 nm wavelength) and record the fluorescence of hypericin in CAM tissue and tumor cells with a digital camera at time intervals of 0, 1, 3, 5, 24, and 48 h after hypericin administration.</li>
		<li>If the research requires an image of the CAM in white light, record the CAM before hypericin administration and at the end of the experiment, shortly before the tissue fixation, as the light affects the photoactivation of hypericin.</li>
		<li>Evaluate the fluorescence intensity using an image processing and analysis program (e.g., ImageJ22).
		<ol>
			<li>Split RGB image channels into separate red, green, and blue images. Analyze the red intensity image in 8-bit form. Evaluate the red intensity from the area inside the ring and approximate it as the hypericin fluorescence. Obtain whole image profile plots (using ImageJ&#39;s Profile Plot plugin) at different time intervals after hypericin administration (i.e., from 0 h, right after administration, up to 48 h).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Conduct PDT
	<ol>
		<li>Perform the PDT at least 3 h after hypericin application.</li>
		<li>Place the optical fiber above the surface of the CAM for the laser ray to cover the entire area within the silicone ring.</li>
		<li>Perform in vivo irradiation (1-2 min) with a 405 nm laser light at a fluence rate of 285 mW/cm2.</li>
		<li>Record CAM using white light and 405 nm fluorescent light before and after photodynamic treatment (24 h and 48 h).</li>
		<li>Detect photodamage 24 h and 48 h after PDT from the images taken in white light. Evaluate vascular damage by a semiquantitative score16: 1&#160;-&#160;no&#160;destruction;&#160;2&#160;-&#160;partial closure of capillaries (diameter &#8804;10 &#181;m) with no destruction of medium or larger (diameter &#8805;50 &#181;m) and smaller vessels (diameter 10-50 &#181;m); 3 - partial closure of smaller vessels with capillaries vanishing; 4 - partial closure of larger vessels with smaller vessels and capillaries vanishing; and 5 - total photo thrombosis with most of the vessels vanishing.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63422/63422fig04.jpg" /> 
Figure 4: Treatment of CAM with laser light. This picture was taken for illustrative purposes. For PDD or PDT, the room must be dark. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Preparing CAM for further evaluation
<ol>
	<li>Paraffin embedding
	<ol>
		<li>Fix the CAM tissue in a cultivation plate with 4% paraformaldehyde (PFA) in PBS for a minimum of 2&#160;h and a maximum of overnight.</li>
		<li>Remove the PFA and carefully cut out from the CAM a part of the tissue within the silicone ring.</li>
		<li>Immediately dehydrate the CAM tissue in ascending alcohol series as follows. Place the CAM tissue in 70% ethanol for 3 min, eosin solution for 2 min (for easier location of the tissue in the paraffin block), 96% ethanol for 3 x 5 min (always in a new Petri dish), and 100% ethanol for 5 min and xylene for 2 x 10 min.</li>
		<li>Using a spatula or thin brush, transfer the samples as quickly as possible to the dissolved paraffin in Petri dishes (59 &#176;C). After 24 h, place the tissue in histological mold, fill it with paraffin embedding medium, and allow it to solidify in the refrigerator. Cut the solidified CAM from the embedding medium, turn it in the tray by 90&#176;, fill it again with the embedding medium, and let it solidify.</li>
		<li>Prepare 5-10 &#181;m sections on a microtome for histopathological analysis to determine PDT-induced damage.</li>
	</ol>
	</li>
	<li>Preparation of frozen CAM sections for histology 
	NOTE: For some methodologies including histology, frozen sections prepared on cryostat microtome are more suitable. Follow the below steps for preparing the frozen CAM sections.
	<ol>
		<li>Carefully mount native or 4% paraformaldehyde-fixed CAM on the glass slide.</li>
		<li>Fill embedding mold to half with optimal cutting temperature compound (OCT) and freeze in liquid nitrogen or a mixture of dry ice and ethanol.</li>
		<li>After freezing, carefully tilt and slide the CAM from the glass slide onto the top of the frozen OCT medium. Place again in the mold, cover with OCT medium, and freeze as in Step 6.2.2.</li>
	</ol>
	</li>
	<li>Fractal analysis of quail CAM 
	NOTE: Vasculature changes caused by PDT can be assessed by calculating the fractal dimension coefficient19.
	<ol>
		<li>In the laminar flow cabinet, overflow CAM in a cultivation plate with a pre-warmed (37 &#176;C) fixation solution of 4% paraformaldehyde and 2% glutaraldehyde in PBS.</li>
		<li>Remove the fixation solution after 48 h. Carefully separate the CAM from the embryo with micro-scissors and a fine brush and wash it in PBS.</li>
		<li>Mount the washed CAM on a glass slide and let it slowly dry.</li>
		<li>Photograph the slide using a digital camera and a transilluminator as a source of homogenous white light.</li>
		<li>Process digital images using ImageJ software22. For the analysis, select a square region (512 &#215; 512 pixels) from the area with distal arterial branches. Binarize and skeletonize the images and calculate the fractal dimension coefficient (Df) following the procedures described in reference31.</li>
	</ol>
	</li>
	<li>Molecular analysis of CAM tissue
	<ol>
		<li>For molecular analysis, carefully separate native CAM, freeze in liquid nitrogen, and store at -80 &#176;C.</li>
		<li>Determine gene expression according to standard protocols for RNA isolation23, reverse transcription into cDNA, and quantitative PCR24.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63422/63422fig05.jpg" /> 
Figure 5: CAM tissue for fractal analysis. After PDT, CAM is fixed, mounted on a slide, and dried for fractal analysis. The picture was taken in white light using a transilluminator. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Nowak-Sliwinska, P., van Beijnum, J. R., van Berkel, M., vanden Bergh, H., Grif&#64257;oen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+regrowth+following+photodynamic+therapy+in+the+chicken+embryo+chorioallantoic+membrane.">Vascular regrowth following photodynamic therapy in the chicken embryo chorioallantoic membrane.</a> Angiogenesis. 13 (4), 281-292 (2010).</li><li>van Leengoed, H. L. L. M., vander Veen, N., Versteeg, A. A. C., Ouellet, R., van Lier, J. E., Star, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+photodynamic+effects+of+phthalocyanines+in+a+skin-fold+observation+chamber+model:+role+of+central+metal+ion+and+degree+of+sulfonation.">In-vivo photodynamic effects of phthalocyanines in a skin-fold observation chamber model: role of central metal ion and degree of sulfonation.</a> Photochemistry Photobiology. 58 (4), 575-580 (1993).</li><li>Ainsworth, S. J., Stanley, R. L., Evans, D. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+stages+of+the+Japanese+quail.">Developmental stages of the Japanese quail.</a> Journal of Anatomy. 216 (1), 3 (2010).</li><li>De Fouw, D. O., Rizzo, V. J., Steinfeld, R., Feinberg, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+microcirculation+in+the+chick+chorioallantoic+membrane+during+normal+angiogenesis.">Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesis.</a> Microvascular Research. 38 (2), 136-147 (1989).</li><li>Sandau, K., Kurz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+vascular+growth+processes:+a+stochastic+biophysical+approach+to+embryonic+angiogenesis.">Modelling of vascular growth processes: a stochastic biophysical approach to embryonic angiogenesis.</a> Journal of Microscopy. 175 (3), 205-213 (1994).</li><li>Kurz, H., Ambrosy, S., Wilting, J., Marm&#233;, D., Christ, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+pattern+of+capillary+endothelial+cells+in+chorioallantoic+membrane+development+indicates+local+growth+control,+which+is+counteracted+by+vascular+endothelial+growth+factor+application.">Proliferation pattern of capillary endothelial cells in chorioallantoic membrane development indicates local growth control, which is counteracted by vascular endothelial growth factor application.</a> Developmental Dynamics. 203 (2), 174-186 (1995).</li><li>Huss, D., Poynter, G., Lansford, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Japanese+quail+(Coturnix+japonica)+as+laboratory+animal+model.">Japanese quail (Coturnix japonica) as laboratory animal model.</a> Lab Animal. 37 (11), 513-519 (2008).</li><li>Gottfried, V., Lindenbaum, E. S., Kimel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+chorioallantoic+membrane+(CAM)+as+an+in-vivo+model+for+photodynamic+therapy.">The chick chorioallantoic membrane (CAM) as an in-vivo model for photodynamic therapy.</a> Journal of Photochemistry and Photobiology, B: Biology. 12 (2), 204-207 (1992).</li><li>Mi&#353;kovsk&#253;, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypericin+-+a+new+antiviral+and+antitumor+photosensitizer:+mechanism+of+action+and+interaction+with+biological+molecules.">Hypericin - a new antiviral and antitumor photosensitizer: mechanism of action and interaction with biological molecules.</a> Current Drug Targets. 3 (1), 55-84 (2002).</li><li>&#268;avarga, I., 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=Photodynamic+effect+of+hypericin+after+topical+application+in+the+ex+ovo+quail+chorioallantoic+membrane+model.">Photodynamic effect of hypericin after topical application in the ex ovo quail chorioallantoic membrane model.</a> Planta Medica. 80 (1), 56-62 (2014).</li><li>Martinez-Poveda, B., Quesada, A. R., Medina, 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=Hypericin+in+the+dark+inhibits+key+steps+of+angiogenesis+in+vitro.">Hypericin in the dark inhibits key steps of angiogenesis in vitro.</a> Europan Journal of Pharmacology. 516 (2), 97-103 (2005).</li><li>Datta, 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=Unravelling+the+excellent+chemical+stability+and+bioavailability+of+solvent+responsive+curcumin-loaded+2-ethyl-2-oxazoline-grad-2-(4-dodecyloxyphenyl)-+2-oxazoline+copolymer+nanoparticles+for+drug+delivery.">Unravelling the excellent chemical stability and bioavailability of solvent responsive curcumin-loaded 2-ethyl-2-oxazoline-grad-2-(4-dodecyloxyphenyl)- 2-oxazoline copolymer nanoparticles for drug delivery.</a> Biomacromolecules. 19 (7), 2459-2471 (2018).</li><li>Hunto&#353;ov&#225;, V., 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=Alkyl+Chain+length+in+poly(2-oxazoline)-based+amphiphilic+gradient+copolymers+regulates+the+delivery+of+hydrophobic+molecules:+a+case+of+the+biodistribution+and+the+photodynamic+activity+of+the+photosensitizer+hypericin.">Alkyl Chain length in poly(2-oxazoline)-based amphiphilic gradient copolymers regulates the delivery of hydrophobic molecules: a case of the biodistribution and the photodynamic activity of the photosensitizer hypericin.</a> Biomacromolecules. 22 (10), 4199-4216 (2021).</li><li>Bur&#237;kov&#225;, 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=Hypericin+fluorescence+kinetics+in+the+presence+of+low+density+lipoproteins:+study+on+quail+CAM+assay+for+topical+delivery.">Hypericin fluorescence kinetics in the presence of low density lipoproteins: study on quail CAM assay for topical delivery.</a> General Physiology and Biophysic. 35 (4), 459-468 (2016).</li><li>Lenkavska, 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=Benefits+of+hypericin+transport+and+delivery+by+low-+and+high-density+lipoproteins+to+cancer+cells:+From+in+vitro+to+ex+ovo.">Benefits of hypericin transport and delivery by low- and high-density lipoproteins to cancer cells: From in vitro to ex ovo.</a> Photodiagnosis and Photodynamic Therapy. 25, 214-224 (2019).</li><li>R&#252;ck, A., B&#246;hmler, A., Steiner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDT+with+TOOKAD+studied+in+the+chorioallantoic+membrane+of+fertilized+eggs.">PDT with TOOKAD studied in the chorioallantoic membrane of fertilized eggs.</a> Photodiagnosis and Photodynamic Therapy. 2 (1), 79-90 (2005).</li><li>Gottfried, V., Davidi, R., Averbuj, C., Kimel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+damage+to+chorioallantoic+membrane+blood+vessels+by+porphycene-induced+photodynamic+therapy.">In vivo damage to chorioallantoic membrane blood vessels by porphycene-induced photodynamic therapy.</a> Journal of Photochemistry and Photobiology, B: Biology. 30 (2-3), 115-121 (1995).</li><li>Buzz&#225;, H. H., Silva, L. V., Moriyama, L. T., Bagnato, V. S., Kurachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+vascular+effect+of+Photodynamic+Therapy+in+chorioallantoic+membrane+using+different+photosensitizers.">Evaluation of vascular effect of Photodynamic Therapy in chorioallantoic membrane using different photosensitizers.</a> Journal of Photochemistry and Photobiology B: Biology. 138, 1-7 (2014).</li><li>Dougherty, T. J., 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=Photodynamic+therapy.">Photodynamic therapy.</a> Journal of the National Cancer Institute. 90, 889-905 (1998).</li><li>Xiang, 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=Real-time+optoacoustic+monitoring+of+vascular+damage+during+photodynamic+therapy+treatment+of+tumor.">Real-time optoacoustic monitoring of vascular damage during photodynamic therapy treatment of tumor.</a> Journal of Biomedical Optics. 12 (1), 01400-01408 (2007).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> Journal of Visualized Experiments. (51), 2720 (2011).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 36-42 (2004).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.</a> Analytical Biochemistry. 162 (1), 156-159 (1987).</li><li>M&#225;&#269;ajov&#225;, M., &#268;avarga, I., S&#253;korov&#225;, M., Valachovi&#269;, M., Novotn&#225;, V., Bil&#269;&#237;k, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+angiogenesis+by+topical+application+of+leptin+and+high+and+low+molecular+heparin+using+the+Japanese+quail+chorioallantoic+membrane+model.">Modulation of angiogenesis by topical application of leptin and high and low molecular heparin using the Japanese quail chorioallantoic membrane model.</a> Saudi Journal of Biological Sciences. 27 (6), 1488-1493 (2020).</li><li>Mangir, N., Dikici, S., Claeyssens, F., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Ex+Ovo+chick+chorioallantoic+membrane+(CAM)+assay+to+evaluate+the+biocompatibility+and+angiogenic+response+to+biomaterials.">Using Ex Ovo chick chorioallantoic membrane (CAM) assay to evaluate the biocompatibility and angiogenic response to biomaterials.</a> ACS Biomaterials Science Engineering. 5 (7), 3190-3200 (2019).</li><li>Marshall, K. M., Kanczler, J. M., Oreffo, R. O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+applications+of+the+egg:+chorioallantoic+membrane+assay+and+ex+vivo+organotypic+culture+of+materials+for+bone+tissue+engineering.">Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering.</a> Journal of Tissue Engineering. 11, 1-25 (2020).</li><li>Merlos Rodrigo, M. A., 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=Extending+the+applicability+of+in+ovo+and+ex+ovo+chicken+chorioallantoic+membrane+assays+to+study+cytostatic+activity+in+neuroblastoma+cells.">Extending the applicability of in ovo and ex ovo chicken chorioallantoic membrane assays to study cytostatic activity in neuroblastoma cells.</a> Frontiers in Oncology. 11, 1-10 (2021).</li><li>Meta, M., Kundekov&#225;, B., Bil&#269;&#237;k, B., M&#225;&#269;ajov&#225;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+silicone+ring+application+on+CAM+vasculature+in+Japanese+Quail+(Coturnix+japonica).">The effect of silicone ring application on CAM vasculature in Japanese Quail (Coturnix japonica).</a> Proceedings of the Student Scientific Conference Faculty of Natural Sciences of Comenius University, Bratislava, Slovakia. , 385-390 (2019).</li><li>Kohli, N., 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=Pre-screening+the+intrinsic+angiogenic+capacity+of+biomaterials+in+an+optimised+ex+ovo+chorioallantoic+membrane+model.">Pre-screening the intrinsic angiogenic capacity of biomaterials in an optimised ex ovo chorioallantoic membrane model.</a> Journal of Tissue Engineering. 11, 1-15 (2020).</li><li>Kundekov&#225;, B., M&#225;&#269;ajov&#225;, M., Meta, M., &#268;avarga, I., Bil&#269;&#237;k, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioallantoic+membrane+models+of+various+avian+species+differences+and+applications.">Chorioallantoic membrane models of various avian species differences and applications.</a> Biology-Basel. 10 (4), 301 (2021).</li><li>Parsons-Wingerter, P., Elliott, K. E., Clark, J. I., Farr, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+selectively+stimulates+angiogenesis+of+small+vessels+in+arterial+tree.">Fibroblast growth factor-2 selectively stimulates angiogenesis of small vessels in arterial tree.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 20 (5), 1250-1256 (2000).</li><li>Buzz&#225;, H. H., Zangirolami, A. C., Davis, A., G&#243;mez-Garc&#237;a, P. B., Kurachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+analysis+of+a+tumor+model+in+the+chorioallantoic+membrane+used+for+the+evaluation+of+different+photosensitizers+for+photodynamic+therapy.">Fluorescence analysis of a tumor model in the chorioallantoic membrane used for the evaluation of different photosensitizers for photodynamic therapy.</a> Photodiagnosis and Photodynamic Therapy. 19, 78-83 (2017).</li></ol>

The authors have no conflicts of interest.

For successful ex ovo cultivation, it is important to follow the protocol above. Moreover, if the eggs are not opened carefully enough or there is insufficient humidity during the cultivation, the yolk sack sticks to the shell and often ruptures. The start of an ex ovo cultivation at the time of about 60 h of egg incubation ensures the high survival rate of the embryos, as they are already large enough to survive the handling. At the later developmental stages, the CAM becomes thinner and adheres to the...

The chicken chorioallantoic membrane (CAM) model is well known and widely used in various areas of research. It is a richly vascularized extraembryonic organ that provides gas exchange and mineral transport1. Due to the transparency and accessibility of this membrane, individual blood vessels and their structural changes can be observed in real time2. Despite the advantages, chick CAM also has some limitations (e.g., larger breeding facilities, egg production, and feed consumption) that could be avoided by using other avian species. In this protocol, an alternative ex ovo CAM model using Japanese quail (Coturnix japonica) embryo is described. Due to its small size, it allows the use of a much larger number of experimental individuals than chicken CAM. Moreover, the shorter 16-day embryonic development of quail embryos is another advantage. The first larger vessels on quail CAM appear on embryonic day (ED) 7. This can be directly compared with chick embryo development (stages 4-35); however, the later stages of development are no longer comparable and require less time for the quail embryo3. Of interest is the regular occurrence of microvascular branching similar to that of chicken CAMs4,5,6. Rapid sexual maturation, high egg production, and low-cost breeding are other examples that favor the use of this experimental model7.
An avian CAM model is often used in photodynamic therapy (PDT) studies8. PDT is used to treat several forms of cancer (small localized tumors) and other non-oncological diseases. Its principle is in the delivery of a fluorescent drug, a photosensitizer (PS), to the damaged tissue and its activation with light of the appropriate wavelength. One prospective PS used in research is hypericin, originally isolated from the medicinal plant St. John&#39;s wort (Hypericum perforatum)9. The strong photosensitizing effects of this compound are based on its photochemical and photophysical properties. These are characterized by multiple fluorescence excitation peaks in the 400-600 nm range, which induce the emission of fluorescence at about 600 nm. The absorption maxima of hypericin within the spectral band are in the 540-590 nm range, and the fluorescence maxima are in the 590-640 nm range9. To achieve these photosensitizing effects, hypericin is excited by laser light at a wavelength of 405 nm after local administration10. In the presence of light, hypericin can exhibit virucidal, antiproliferative, and cytotoxic effects11, while there is no systemic toxicity, and it is rapidly released from the organism. Hypericin is a lipophilic substance that forms water-insoluble, non-fluorescent aggregates, which is why several types of nanocarriers, such as polymeric nanoparticles12,13 or high- and low-density lipoproteins (HDL, LDL)14,15, are used to help its delivery and penetration into the cells. Since CAM is a naturally immunodeficient system, tumor cells can be implanted directly on the membrane surface. The model is also well suited for recording the extent of PDT-induced vascular damage according to a defined score16,17. Light of lower intensity compared to PDT can be used for photodynamic diagnosis (PDD). Monitoring the tissue under violet excitation LED light also leads to photoactivation of photosensitizers18,19,20 that results in an emission of fluorescent light, yet it does not provide enough energy to start a PDT reaction and damage the cells. It makes it a good tool for tumor visualization and diagnosis or monitoring the pharmacokinetics of used PSs14,15.
This article describes the preparation of the quail ex ovo CAM assay with survival rates over 80%. This ex ovo culture was successfully applied in a large number of experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-Well Cell Culture Plate</td>
			<td>Sarstedt</td>
			<td>83.392</td>
			<td>Transparent polystyrene, sterile</td>
		</tr>
		<tr>
			<td>CO2 Incubator ESCO CCL-0508</td>
			<td>ESCO, Singapore</td>
			<td>CCL-050B-8</td>
			<td>CO2 cell culture incubator</td>
		</tr>
		<tr>
			<td>cryocut Leica CM 1800</td>
			<td>Reichert-Jung, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>digital camera Canon EOS 6D II</td>
			<td>Canon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>diode laser 405 nm</td>
			<td>Ocean Optics, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>67-68-5</td>
			<td>dimethyl sulfoxid</td>
		</tr>
		<tr>
			<td>eosin</td>
			<td>Sigma-Aldrich</td>
			<td>15086-94-9</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>fine brush size 2</td>
			<td>Faber-Castell</td>
			<td>281802</td>
			<td>brush for CAM separation and manipulation</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>111-30-8</td>
			<td></td>
		</tr>
		<tr>
			<td>hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>517-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>hypericin</td>
			<td>Sigma-Aldrich</td>
			<td>84082-80-4</td>
			<td></td>
		</tr>
		<tr>
			<td>incubator Bios Midi</td>
			<td>Bios Sedl<img alt="Equation 1" src="/files/ftp_upload/63422/63422eq01.jpg" style="margin: auto;" />any, Czech Republic</td>
			<td></td>
			<td>Forced draught incubator for initial incubation</td>
		</tr>
		<tr>
			<td>incubator Memmert IF160</td>
			<td>Memmert, Germany</td>
			<td></td>
			<td>Forced air circulation incubator for CAM incubation</td>
		</tr>
		<tr>
			<td>Kaiser slimlite plano, LED light box</td>
			<td>Kaiser, Germany</td>
			<td>2453</td>
			<td>Transilluminator</td>
		</tr>
		<tr>
			<td>LED light 405 nm</td>
			<td></td>
			<td></td>
			<td>custom made circular LED light</td>
		</tr>
		<tr>
			<td>macro lens Canon MP- E 65 mm f/2.8</td>
			<td>Canon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microscope Kapa 2000</td>
			<td>Kvant, Slovakia</td>
			<td></td>
			<td>optical microscope</td>
		</tr>
		<tr>
			<td>microtome Auxilab 508</td>
			<td>Auxilab, Spain</td>
			<td></td>
			<td>manual rotary microtome</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast Plus</td>
			<td>Sigma-Aldrich</td>
			<td>P3683</td>
			<td>parafin medium for tissue embedding</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>Phosphate saline buffer</td>
		</tr>
		<tr>
			<td>scissors Castroviejo</td>
			<td>Orimed</td>
			<td>&#160;OR66-108</td>
			<td>micro scissors for CAM separation</td>
		</tr>
		<tr>
			<td>software ImageJ 1.53</td>
			<td>public domain</td>
			<td></td>
			<td>image processing and analysis program</td>
		</tr>
		<tr>
			<td>stock solution HDL</td>
			<td>Sigma-Aldrich</td>
			<td>437641-10MG</td>
			<td>high density lipoproteins</td>
		</tr>
		<tr>
			<td>stock solution LDL</td>
			<td>Sigma-Aldrich</td>
			<td>437644-10MG</td>
			<td>low density lipoproteins</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td>Optimal Cutting Temperature Compound 118 mL squeeze bottles</td>
		</tr>
	</tbody>
</table>

quail chorioallantoic membrane - a tool for photodynamic diagnosis and therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The localization&#160;of the tumor on the CAM surface is difficult in white light. Photosensitizer (here, hypericin) used in PDD is expected to be taken up selectively by the tumor and helps visualize the tumor. The addition of hypericin and the use of fluorescent light (e.g., 405 nm) showed the tumor (squamous cell carcinoma TE1) position very well (Figure 6A). Histological analysis showed vital tumor cells invading healthy tissues. Concentric structures of abnormal squamous cells, often de...

Watch this Scientific Journal Video about Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy at JoVE.com

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

quail-chorioallantoic-membrane-tool-for-photodynamic-diagnosis

Research

JoVE Journal

Cancer Research

2.6K Views.  IABG, Bratislava, Slovakia. The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

Video: Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

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 ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...