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>

<ol>
	<li>Kuehl, W. M., Bergsagel, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+myeloma:+evolving+genetic+events+and+host+interactions.">Multiple myeloma: evolving genetic events and host interactions.</a> Nat.Rev.Cancer. 2 (3), 175-187 (2002).</li><li>Kumar, S. K., Gertz, 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=Risk+adapted+therapy+for+multiple+myeloma:+back+to+basics.">Risk adapted therapy for multiple myeloma: back to basics.</a> Leuk.Lymphoma. , (2014).</li><li>Prideaux, S. M., Conway, O. E., Chevassut, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+architecture+of+multiple+myeloma.">The genetic architecture of multiple myeloma.</a> Adv.Hematol. 2014, 864058 (2014).</li><li>Chauhan, D., 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=A+novel+orally+active+proteasome+inhibitor+ONX+0912+triggers+in+vitro+and+in+vivo+cytotoxicity+in+multiple+myeloma.">A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma.</a> Blood. 116 (23), 4906-4915 (2010).</li><li>Kuehl, 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=Mouse+models+can+predict+cancer+therapy.">Mouse models can predict cancer therapy.</a> Blood. 120 (2), 238-240 (2012).</li><li>Nefedova, Y., Gabrilovich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+clinical+prospects+of+Notch+inhibitors+in+the+therapy+of+hematological+malignancies.">Mechanisms and clinical prospects of Notch inhibitors in the therapy of hematological malignancies.</a> Drug Resist.Updat. 11 (6), 210-218 (2008).</li><li>Yaccoby, S., Barlogie, B., Epstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+myeloma+cells+growing+in+SCID-hu+mice:+a+model+for+studying+the+biology+and+treatment+of+myeloma+and+its+manifestations.">Primary myeloma cells growing in SCID-hu mice: a model for studying the biology and treatment of myeloma and its manifestations.</a> Blood. 92 (8), 2908-2913 (1998).</li><li>Dazzi, F., 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=Normal+and+chronic+phase+CML+hematopoietic+cells+repopulate+NOD/SCID+bone+marrow+with+different+kinetics+and+cell+lineage+representation.">Normal and chronic phase CML hematopoietic cells repopulate NOD/SCID bone marrow with different kinetics and cell lineage representation.</a> Hematol.J. 1 (5), 307-315 (2000).</li><li>Lock, R. 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=The+nonobese+diabetic/severe+combined+immunodeficient+(NOD/SCID)+mouse+model+of+childhood+acute+lymphoblastic+leukemia+reveals+intrinsic+differences+in+biologic+characteristics+at+diagnosis+and+relapse.">The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse.</a> Blood. 99 (11), 4100-4108 (2002).</li><li>Feuring-Buske, 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=Improved+engraftment+of+human+acute+myeloid+leukemia+progenitor+cells+in+beta+2-microglobulin-deficient+NOD/SCID+mice+and+in+NOD/SCID+mice+transgenic+for+human+growth+factors.">Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors.</a> Leukemia. 17 (4), 760-763 (2003).</li><li>Pearce, D. 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=AML+engraftment+in+the+NOD/SCID+assay+reflects+the+outcome+of+AML:+implications+for+our+understanding+of+the+heterogeneity+of+AML.">AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML.</a> Blood. 107 (3), 1166-1173 (2006).</li><li>Li, 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=Combined+inhibition+of+Notch+signaling+and+Bcl-2/Bcl-xL+results+in+synergistic+antimyeloma+effect.">Combined inhibition of Notch signaling and Bcl-2/Bcl-xL results in synergistic antimyeloma effect.</a> Mol.Cancer Ther. 9 (12), 3200-3209 (2010).</li><li>Tassone, P., 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=A+clinically+relevant+SCID-hu+in+vivo+model+of+human+multiple+myeloma.">A clinically relevant SCID-hu in vivo model of human multiple myeloma.</a> Blood. 106 (2), 713-716 (2005).</li><li>Ranga, A., Gjorevski, N., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+through+stem+cell-based+organoid+models.">Drug discovery through stem cell-based organoid models.</a> Adv.Drug Deliv.Rev. 69-70, 19-28 (2014).</li><li>Pereira, R. F., Barrias, C. C., Granja, P. L., Bartolo, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+biofabrication+strategies+for+skin+regeneration+and+repair.">Advanced biofabrication strategies for skin regeneration and repair.</a> Nanomedicine.(Lond). 8 (4), 603-621 (2013).</li><li>Fischbach, 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=Engineering+tumors+with+3D+scaffolds).">Engineering tumors with 3D scaffolds).</a> Nat.Methods. 4 (10), 855-860 (2007).</li><li>Boulland, J. L., Halasi, G., Kasumacic, N., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+human+stem+cells+into+the+chicken+embryo.">Xenotransplantation of human stem cells into the chicken embryo.</a> J.Vis.Exp. (41), (2010).</li><li>Deryugina, E. I., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2.+Chick+embryo+chorioallantoic+membrane+models+to+quantify+angiogenesis+induced+by+inflammatory+and+tumor+cells+or+purified+effector+molecules.">Chapter 2. Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules.</a> Methods Enzymol. 444, 21-41 (2008).</li><li>Deryugina, E. I., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+model+systems+to+study+and+visualize+human+tumor+cell+metastasis.">Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis.</a> Histochem.Cell Biol. 130 (6), 1119-1130 (2008).</li><li>Kern, 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=GRP-78+secreted+by+tumor+cells+blocks+the+antiangiogenic+activity+of+bortezomib.">GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib.</a> Blood. 114 (18), 3960-3967 (2009).</li><li>Ribatti, D., Nico, B., Vacca, A., Presta, 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+gelatin+sponge-chorioallantoic+membrane+assay.">The gelatin sponge-chorioallantoic membrane assay.</a> Nat.Protoc. 1 (1), 85-91 (2006).</li><li>Ribatti, D. <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+embryo+chorioallantoic+membrane+as+a+model+for+tumor+biology.">The chick embryo chorioallantoic membrane as a model for tumor biology.</a> Exp.Cell Res. , (2014).</li><li>Dohle, D. 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=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+how+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: how it really works.</a> J.Vis.Exp. (33), (2009).</li><li>Kobayashi, 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=A+chick+embryo+model+for+metastatic+human+prostate+cancer.">A chick embryo model for metastatic human prostate cancer.</a> Eur.Urol. 34 (2), 154-160 (1998).</li><li>Koop, 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=Fate+of+melanoma+cells+entering+the+microcirculation:+over+80%+survive+and+extravasate.">Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate.</a> Cancer Res. 55 (12), 2520-2523 (1995).</li><li>Martowicz, A., Spizzo, G., Gastl, G., Untergasser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype-dependent+effects+of+EpCAM+expression+on+growth+and+invasion+of+human+breast+cancer+cell+lines.">Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines.</a> BMC.Cancer. 12, 501 (2012).</li><li>Martowicz, 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=EpCAM+overexpression+prolongs+proliferative+capacity+of+primary+human+breast+epithelial+cells+and+supports+hyperplastic+growth.">EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth.</a> Mol.Cancer. 12, 56 (2013).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> 6, 34 (2006).</li><li>Farnoushi, 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=Rapid+in+vivo+testing+of+drug+response+in+multiple+myeloma+made+possible+by+xenograft+to+turkey+embryos.">Rapid in vivo testing of drug response in multiple myeloma made possible by xenograft to turkey embryos.</a> Br.J.Cancer. 105 (11), 1708-1718 (2011).</li><li>Wunderlich, M., Krejci, O., Wei, J., Mulloy, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD34++cells+expressing+the+inv(16)+fusion+protein+exhibit+a+myelomonocytic+phenotype+with+greatly+enhanced+proliferative+ability.">Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability.</a> Blood. 108 (5), 1690-1697 (2006).</li><li>Hideshima, T., Mitsiades, C., Tonon, G., Richardson, P. G., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+multiple+myeloma+pathogenesis+in+the+bone+marrow+to+identify+new+therapeutic+targets.">Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets.</a> Nat.Rev.Cancer. 7 (8), 585-598 (2007).</li><li>Parikh, M. R., Belch, A. R., Pilarski, L. M., Kirshner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+tissue+culture+model+to+study+primary+human+bone+marrow+and+its+malignancies.">A three-dimensional tissue culture model to study primary human bone marrow and its malignancies.</a> J.Vis.Exp. (85), (2014).</li><li>Schuler, J., Ewerth, D., Waldschmidt, J., Wasch, R., Engelhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+multiple+myeloma:+a+critical+appraisal.">Preclinical models of multiple myeloma: a critical appraisal.</a> Expert.Opin.Biol.Ther. 13, S111-S123 (2013).</li><li>Kirshner, 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=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112 (7), 2935-2945 (2008).</li><li>Jilani, S. 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=Selective+binding+of+lectins+to+embryonic+chicken+vasculature.">Selective binding of lectins to embryonic chicken vasculature.</a> J.Histochem.Cytochem. 51 (5), 597-604 (2003).</li></ol>

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><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&amp;nbsp;</td><td>PromoCell</td><td>PC-C-12974</td><td></td></tr><tr><td>HEK293FT cells&amp;nbsp;</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&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp; 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>SPF-white hen eggs</td><td>Charles River</td><td></td><td>Fertilized&amp;nbsp; white Leghorn&amp;nbsp; chicken eggs</td></tr><tr><td>Plastic weighing boats</td><td>neoLab</td><td>Art.Nr. 1-1125</td><td>for ex-ovo culture</td></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>Roche</td><td>11 836 170 001</td><td></td></tr><tr><td>Complete Mini EDTA-free</td><td></td><td></td><td></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&amp;nbsp; 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>SuperFrost Microscope Slides</td><td>R. Langenbrinck&amp;nbsp;</td><td>Art.-Nr.</td><td></td></tr><tr><td>Labor- u. Medizintechnik</td><td>03-0060</td><td></td><td></td></tr><tr><td>DakoCytomation Wash Buffer 10x</td><td>DakoCytomation</td><td>Code-Nr.</td><td></td></tr><tr><td>S 3006</td><td></td><td></td><td></td></tr><tr><td>Target Retrieval Solution (10x)&amp;nbsp; pH 6,1</td><td>DAKO</td><td>Code-Nr.</td><td></td></tr><tr><td>S 1699</td><td></td><td></td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</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>&amp;nbsp;M0760</td><td></td></tr><tr><td>m-a-hu Ki67&amp;nbsp; clone MIB-1&amp;nbsp;&amp;nbsp;</td><td>DAKO</td><td>&amp;nbsp;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&amp;rsquo;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>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&amp;nbsp;</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&amp;nbsp;</td><td>Olympus, SZX10</td><td></td><td></td></tr><tr><td>Ultra Turrax&amp;nbsp;</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 ...

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Research

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Medicine

13.0K 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.

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

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Cancer Research

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

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 ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Developmental Biology

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...