The authors want to thank Ms. Cornelia Heis for her excellent technical assistance in immunohistochemistry and preparation of chicken embryos. This work was supported by the Austrian Science Fund (FWF Grant No. P19552) and the European Union (EU FP7 project Optatio No: 278570).

According to the Austrian law, and the Office of Laboratory Animal Welfare of the US public health service avian embryos are not considered as live vertebrate animals until hatching.The NIH Office of Laboratory Animal Welfare has provided written guidance in this area (http://www.grants.nih.gov/grants/olaw/references/ilar91.htm and NIH Publication No.: 06-4515).
1. Cell Culture and Lentiviral Transfection
<ol>
	<li>Culture MM cell lines OPM-2, RPMI-8226 and human mesenchymal stem cells from bone marrow in RPMI1640 medium, supplemented with 10% bovine fetal calf serum and 100 IU/ml penicillin, 100 &#956;g/ml streptomycin and 2 mM glutamine in the presence of 5% CO2 at 37&#176;C.</li>
	<li>Transfect 5 x 106 HEK 293FT cells with viral packaging mix (9 &#181;g) DNA and 3 &#181;g pLenti6/V5 dest eGFP vector by the use of 30 &#181;l liposomal transfection reagent and transfection medium (10 ml). Remove transfection medium after 12 h and add 10 ml DMEM medium with 10% bovine calf serum and 1% non-essential amino acids (NEAA).</li>
	<li>After 5 days, collect supernatants of HEK293FT cells after centrifugation of swimming cells (1,000 x g, 5min). Determine viral titer by real time PCR as described elsewhere 28.</li>
	<li>Transfect 1 x 106 MM cells with eGFP lentiviral particles (1 x 105 particles) in a 24 well plate in complete growth medium. After 3 days, start selection process by adding 2 &#181;g/ml blasticidin to the culture medium. For the commercially available eGFP lentivirus, use 500 &#181;g/ml neomycin.</li>
	<li>After 2 weeks of selection, clusters of eGFP expressing MM cells will appear; collect cells by centrifugation (1,000 x g, 5 min) and expand them as OPM-2eGFP and RPMI-8226eGFP sublines for experiments (section 2 and 3).</li>
</ol>
2. 3D-Multiple Myeloma Spheroid Model
<ol>
	<li>Chill collagen type-I solution and 10 x DMEM on ice.</li>
	<li>Mix 1/10 Volume of 10 x DMEM medium into collagen matrix; add NaOH (0.2 N) to neutralize acidic collagen solution to a pH value of 7.4; store collagen/medium solution on ice.</li>
	<li>Mix transgenic MM cell lines (OPM-2eGFP or RPMI-8226 eGFP; 250,000 per spheroid,) with human mesenchymal cells (50,000 cells/ spheroid, i.e., 30 &#181;l drop).</li>
	<li>Centrifuge cell mixture in 15 ml tubes (1000 x g, 5min), add cold prepared collagen mixture (1mL) to the cell pellet and mix well (with 1,000 &#181;l tip).</li>
	<li>Immediately pipette 30 &#181;l of the collagen/cell mixture (with 100 &#181;l tip) in a 24 well plate on sterile paraffin film and allow cell/collagen mixture to polymerize for 30 min at 37&#176;C (see Figure 1A).</li>
	<li>Overlay MM spheroids with 1mL culture medium containing 1, 10 and 100 nM bortezomib (see Figure 1B).</li>
	<li>After 72 hr of incubation at 37&#176;C, document spheroids by fluorescence stereomicroscopy (see Figure 1C).</li>
	<li>Transfer each spheroid by the use of forceps with wide flat jaws in a reaction tube for measurement of GFP (see Figure 1D).</li>
</ol>
3. 3D Multiple Myeloma Xenograft Model in the CAM
<ol>
	<li>Incubate chicken eggs in a special incubator for avian eggs at 37&#176;C and 70% humidity for three days.</li>
	<li>Thereafter, open eggs and transfer embryos to sterilized with ethanol, square, 10-cm plastic weighing boats with cell culture plate lid and incubate &#8220;ex ovo&#8221; for further six days, so that the CAM is able to develop (see Figure 2A).</li>
	<li>Chill collagen type-I solution and 10 x DMEM on ice, mix 1/10 Volume of 10 x DMEM medium into collagen matrix, Add NaOH (0.2 N) to neutralize acidic collagen solution to a pH value of 7.4; store collagen/medium solution on ice.</li>
	<li>Mix transgenic MM cell lines (OPM-2eGFP or RPMI-8226eGFP; 250,000 per spheroid,) with human mesenchymal cells (50,000 cells/ spheroid).</li>
	<li>Centrifuge cells in 15 ml tubes (1,000 x g, 5min; for each test compound 1 vial), add 1mL cold prepared collagen mixture with drug (at desired working concentration) to the cell pellet and mix well (with 1,000 &#181;l tip).</li>
	<li>Place collagen drops (30 &#181;l each) on parafilm in a 6 well pate for 30 min to allow polymerization of the extracellular matrix at 37&#176;C.</li>
	<li>Transfer &#8220;onplants&#8221; from step 3.6 with the use of forceps to the untreated surface of the CAM (2 cm away from embryo) of 9-day-old chicken embryos (4 onplants for each chicken embryo, see Figure 2B)</li>
	<li>After 5 days of in vivo growth in an egg incubator at 37&#176;C and 70% humidity, document xenografts by fluorescence stereomicroscopy (see Figure 2C). Euthanize chicken embryos by hypothermia at 4&#176;C in the fridge for 5h.</li>
	<li>Remove xenografts with subjacent CAM tissue by an ophthalmic scissor and forceps with wide flat jaws. Use them for measurement of GFP (section 4, see Figure 2D) or for immunohistochemical analysis of blood vessels and/or invading tumor cells (section 5).</li>
</ol>
4. Quantification of eGFP Protein by ELISA
<ol>
	<li>Transfer each MM spheroid or excised xenograft into 0.5 ml RIPA Buffer containing 200 &#181;g/ml protease inhibitors.</li>
	<li>Homogenize spheroid/xenograft with a tissue homogenizer on ice.</li>
	<li>Perform three freezing/thawing-cycles in liquid nitrogen and 37&#176;C water bath.</li>
	<li>Centrifuge homogenate at 4&#176;C for 20 min (12,000 g) and store supernatants.</li>
	<li>Dilute samples 1:20 in assay buffer (200 &#181;l) of the ELISA kit. Measure GFP-levels by a commercial GFP ELISA Kit using biotinylated anti-GFP antibodies, according to the manufacturer&#8217;s protocol.</li>
</ol>
5. Immunohistochemical Analysis of Blood Vessels and Invading Tumor Cells
<ol>
	<li>Fix excised xenografts with CAM area in 4% paraformaldehyde O/N at 4&#176;C.</li>
	<li>Place fixed xenografts into embedding cassettes and transfer them into a tissue embedding station with an increasing graded alcohol series (50%, 70%, 80%, 95% ethanol, xylol and paraffin; each step 60 min).</li>
	<li>Section xenografts (5 &#181;m) by the use of a benchtop rotary microtome. Bake paraffin sections on glass slides O/N at 56&#176;C.</li>
	<li>Deparaffinize sections by a decreasing graded alcohol series to double-distilled water (xylol, 95%, 80%, 70%, 50% ethanol, double-distilled water; each step 10 min).</li>
	<li>Perform antigen retrieval in a water bath (95&#176;C, 20 min) with an antigen retrieval solution (citrate- buffer; pH 7.0; volume 100 &#181;l).</li>
	<li>Block endogenous peroxidase activity with 100 &#181;l 3% H2O2/methanol for 30 min.</li>
	<li>Block sections in PBS containing 10% fetal calf serum for 45 min (volume 100 &#181;l).</li>
	<li>Stain for 1 hr with 100 &#181;l&#160;of primary antibody (1&#181;g/ml) diluted in PBS containing 1% fetal calf serum at RT.</li>
	<li>After washing 3-times in PBS, incubate for 1h with biotinylated secondary antibody (0.1 &#181;g/ml) in PBS containing 1% fetal calf serum at RT.</li>
	<li>After washing 3-times in PBS perform color reaction by the avidin/biotin-complex (ABC) and the diaminobenzidine (DAB) substrate solution according to manufacturer&#8217;s instructions.</li>
	<li>5.11. Stop reaction by transferring sections to double-distilled water, counterstain with hematoxylin and mount sections with a synthetic mounting medium.</li>
</ol>

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The authors have no competing financial interests

The development of new therapeutic agents for refractory MM requires less time consuming and expensive in-vivo systems to evaluate sensitivity of human MM cells to drugs. Hitherto, only few in-vivo systems are available for the preclinical evaluation of new anti-myeloma therapies. All of them have their limitations for large scale screening of compound libraries 29.&#160; 
 
The best current models for human MM cells are highly immune-deficient mice 7,13,30 and turkey embryos 29...

Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency 1. Although new treatment options such as proteasome inhibitors (bortezomib) and immune modulatory drugs (pomalidomide and lenalidomide) are available, MM still remains an incurable malignancy with a grim prognosis 2. The bad prognosis might be explained by the extraordinary heterogeneity of MM cell clones that contributes to variable responses to therapy, in particular under long time treatment and selection pressure of MM clones 3.
Preclinical testing of new drugs and their combinations in vitro and in vivo is a critical and time-consuming step for future drug development. Thus, useful in-vivo models of MM are required to gain a better understanding of the biology of the disease and to enable the discovery of new drugs. Actually, the best xenotransplantation models for hematological malignancies and therapeutics are immune-deficient mice, such as the severe-combined immunodeficient (SCID) mice 4-7, the non-obese diabetic/SCID (NOD/SCID) mice 8,9 or the &#946;-microglobulin-knockout NOD/SCID mice 10,11.
Although murine models of human MM in some aspects can resemble the phenotype of human disease, immune-deficient mice are inbred, therefore simulate only one individual response to a drug and costs are very high. Due to immunosuppression animals require special maintenance conditions and the engraftment of human MM in mice requires 6 weeks to 2 months 9,12, unless cells are grafted directly to the bone marrow using a technically demanding procedure with lower rates of animal survival 7,13. Therefore, new methods using stem-cell based organoid models 14, tissue engineering 15 or sophisticated 3D cell culture models 16 have been established. They will compete in the near future with classical animal experiments for preclinical drug testing, but cannot replace systemic toxicity tests in living organisms.
The chicken embryo has been demonstrated before to be a suitable organism for xenotransplantation of human cells and tissues due to lack of adaptive immune response until hatching 17-19. Moreover, each chicken embryo reflects an individual reaction to applied drugs or tumor cells due to genetic diversity within the chicken population. The chorioallantoic membrane (CAM) is a well-established system to study tumor-dependent angiogenesis 20-22. When solid tumors are grafted to the CAM, they display many characteristics of cancers in vivo, including proliferation, invasion, angiogenesis and metastasis 23-27. 
Based on the previous experience of our group with CAM xenograft models20,26,27, a human MM model was established that combines the advantage of a human 3D culture system with the model of ex ovo developing chicken embryos. This MM model system allows real time monitoring of MM growth progression, quantification of cell mass and preclinical drug testing.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RPMI-8226 cells</td>
			<td>DSMZ</td>
			<td>ACC 9</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>OPM-2 cells</td>
			<td>DSMZ</td>
			<td>ACC 50</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells&#160;</td>
			<td>PromoCell</td>
			<td>PC-C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293FT cells&#160;</td>
			<td>Invitrogen</td>
			<td>R700-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160; HyClone</td>
			<td>ThermoScientific</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut- Pen- Strep solution</td>
			<td>Sigma</td>
			<td>G6784</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>31966</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Sigma Life Sciences</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Medium/Opti-MEM&#160;</td>
			<td>Gibco</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>eGFP lentiviral particles</td>
			<td>GeneCopoeia</td>
			<td>LPP-EGFP-LV105</td>
			<td>Ready to use viral particles</td>
		</tr>
		<tr>
			<td>pLenti6/V5Dest6 eGFP vector</td>
			<td>Invitrogen</td>
			<td>PN 35-1271</td>
			<td>from authors</td>
		</tr>
		<tr>
			<td>ViralpowerTM packaging mix&#160;</td>
			<td>Invitrogen</td>
			<td>P/N 35-1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent/ Lipofectamin 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invitrogen</td>
			<td>R210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Biochrom</td>
			<td>A2912</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-Type1&#160; Rat Tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Life Technologies</td>
			<td>Art.Nr. 10338582</td>
			<td></td>
		</tr>
		<tr>
			<td>plitidepsin</td>
			<td>Pharmamar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bortezomib</td>
			<td>LKT Lab., Inc.</td>
			<td>B5871</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">SPF-white hen eggs</td>
			<td rowspan="3">Charles River</td>
			<td rowspan="3"></td>
			<td rowspan="3">Fertilized&#160; white Leghorn&#160; chicken eggs</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Plastic weighing boats</td>
			<td rowspan="2">neoLab</td>
			<td rowspan="2">Art.Nr. 1-1125</td>
			<td rowspan="2">for ex-ovo culture</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Petridish square (Lids)</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td>for ex-ovo culture</td>
		</tr>
		<tr>
			<td>RIPA Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>#9806</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Tablets</td>
			<td rowspan="2">Roche</td>
			<td rowspan="2">11 836 170 001</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Complete Mini EDTA-free</td>
		</tr>
		<tr>
			<td>GFP ELISA</td>
			<td>Cell Biolabs, Inc.</td>
			<td>AKR-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocette II</td>
			<td>Simport</td>
			<td>M493-6</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160; 37%</td>
			<td>Roth</td>
			<td>7398.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolut</td>
			<td>Normapur</td>
			<td>20,821,321</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histol</td>
			<td>Roth</td>
			<td>Art.Nr.6640.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Sigma</td>
			<td>A6330</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">SuperFrost Microscope Slides</td>
			<td>R. Langenbrinck&#160;</td>
			<td>Art.-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Labor- u. Medizintechnik</td>
			<td>03-0060</td>
		</tr>
		<tr>
			<td rowspan="2">DakoCytomation Wash Buffer 10x</td>
			<td rowspan="2">DakoCytomation</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 3006</td>
		</tr>
		<tr>
			<td rowspan="2">Target Retrieval Solution (10x)&#160; pH 6,1</td>
			<td rowspan="2">DAKO</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 1699</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu ASMA clone 1A4</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu CD138 clone MI15</td>
			<td>DAKO</td>
			<td>M7228</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Vimentin clone V9</td>
			<td>DAKO</td>
			<td>M0725</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Desmin clone D33</td>
			<td>DAKO</td>
			<td>&#160;M0760</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Ki67&#160; clone MIB-1&#160;&#160;</td>
			<td>DAKO</td>
			<td>&#160;M7240</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat- anti-mouse IgG</td>
			<td>Vector Laboratories Inc.</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories Inc.</td>
			<td># PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST DAB Tablet Set.</td>
			<td>Sigma Biochemicals</td>
			<td># D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haemalaun solution</td>
			<td>Merck</td>
			<td>1,092,490,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti Histokitt</td>
			<td>Roth</td>
			<td>Art.Nr.6638.2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top rotary microtome</td>
			<td>Thermo Electron, Shandon Finesse ME+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding station</td>
			<td>Leica, TP1020</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg-Incubator</td>
			<td>Grumbach&#160;</td>
			<td>BSS160</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light&#160;</td>
			<td>Olympus, SZX10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Turrax&#160;</td>
			<td>IKA T10</td>
			<td></td>
			<td>Homogenizer</td>
		</tr>
	</tbody>
</table>

establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

In vitro analysis of target compounds in 3D multiple myeloma spheroid assays
Due to the limitation of culturing primary human MM cells in vitro we established new 3D in vitro culture models for human MM cell lines making use of an extracellular growth matrix and supportive primary human mesenchymal cells from bone marrow (Figure 1A,&#160;B). EGFP transgenic MM cell lines allow visualization and quantification of MM t...

Watch this Scientific Journal Video about Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis at JoVE.com

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

establishment-human-multiple-myeloma-xenograft-model-chicken-to-study

Research

JoVE Journal

Medicine

12.9K Views.  Innsbruck Medical University. Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.