Name: establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis
Uploaded: 2015-05-01T13:00:00.000+00:00
Duration: 10 min 4 s
Description: 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

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).

실험 동물 복지의 hatching.The NIH 사무실이이 지역 (HTTP 작성 지침을 제공 할 때까지 오스트리아 법, 미국 공중 보건 서비스 조류 배아의 실험 동물 복지 사무소에 따르면 라이브 척추 동물로 간주되지 않습니다 // www.grants.nih.gov/grants/olaw/references/ilar91.htm 및 NIH 출판 번호 : 06-4515). 1. 세포 배양 및 렌티 바이러스 형질 <ol><li> 문화 MM 세포주 OPM-2, RPMI-8226 및 RPMI1640 배지에서 골수로부터 인간 중간 엽 줄기 세포의 존재에서 10 % 소 태아 혈청, 100 IU / ml의 페니실린, 100 μg의 / ㎖ 스트렙토 마이신 및 2 mM의 글루타민으로 보충 37 ℃에서 5 % CO 2. </li><li> 형질 감염 5 × 30 μL 리포좀 형질 감염 시약과 형질 매체 (10 mL)을 이용하여 바이러스 포장 믹스 (9 μg의) 및 3 μg의 DNA pLenti6 / V5 DEST eGFP는 벡터 6 HEK 293FT 세포. 12 시간 후 형질 매체를 제거하고10 % 소 혈청 및 1 % 비 필수 아미노산 (NEAA)과 10 ml의 DMEM 배지를 추가한다. </li><li> 오일 후, 수영 세포 (1,000 XG, 5 분)의 원심 분리 후 HEK293FT 세포의 상층 액을 수집합니다. 다른 28 설명 된대로 실시간 PCR에 의해 바이러스 역가를 결정합니다. </li><li> 완전한 성장 배지에서 24 웰 플레이트에 eGFP는 렌티 바이러스 입자 (1 × 10 5 입자) 1 × 10 6 MM 세포를 형질. 삼일 후, 2 μg의가 / ml로 배양 배지에 첨가하여 블라스트 선택 프로세스를 시작한다. 시중에서 판매하는 eGFP는의 렌티 바이러스를 들어, 500 μg의 / ㎖ 네오 마이신을 사용합니다. </li><li> 선택 2 주 후, MM 세포를 표현 eGFP는 클러스터가 나타납니다; 원심 분리 (1,000 XG, 5 분)에 의해 세포를 수집하고 실험 영업 이익률-2 eGFP는 및 RPMI-8226 eGFP는의 서브 라인으로 그들을 확장 (제 2, 3). </li></ol> 2. 3D-다발성 골수종 회전 타원체 모델 <ol><li> 침착 콜라겐 타입 I 용액 및 10 × DMEM 오N 얼음. </li><li> 콜라겐 매트릭스로 10 × DMEM 배지 1/10 볼륨 믹스; 7.4의 pH 값에 콜라겐 산성 용액을 중화시키기에 NaOH (0.2 N)을 추가; 저장 콜라겐 / 얼음 매체 솔루션입니다. </li><li> 형질 전환 MM 세포주 (영업 이익률-2 eGFP는 또는 RPMI-8226 eGFP는을, 회전 타원체 당 25 만) 혼합 인간 중간 엽 세포 (50,000 세포 / 회전 타원체, 즉, 30 μL 드롭)에 있습니다. </li><li> 15ml의 튜브 (1000 XG, 5 분)에서 원심 분리 세포 혼합물, 세포 펠렛을 차가운 제조 콜라겐 혼합물 (1 ㎖)를 첨가하고, (1000 μL 팁으로) 잘 섞는다. </li><li> 즉시 멸균 파라핀 필름에 24 웰 플레이트 (100 ㎕의 팁)와 콜라겐 / 세포 혼합액 30 μL 피펫 세포 / 콜라겐 혼합물을 37 ℃ (도 1a 참조)에서 30 분 동안 중합 할 수있다. </li><li> 1, 10 및 100 nM의 볼테 조미 브를 포함하는 1 ㎖의 배지와 오버레이 MM의 스페 로이드 (도 1b 참조). </li><li> 37 ° C에서 배양 72 시간 후, 문서 회전 타원체로형광 실체 현미경 (도 1C 참조). </li><li> GFP의 측정에 반응 튜브 내의 넓은 플랫 턱 겸자를 이용하여 각각의 회전 타원체로 이동 (도 1d 참조). </li></ol> CAM 3. 3D 다발성 골수종 이종 이식 모델 <ol><li> 3 일 동안 특별한 37 ° C에서 조류 계란 인큐베이터와 70 %의 습도에서 계란을 품어. </li><li> 그 후, 열려있는 계란과 전송 배아는 세포 배양 접시의 뚜껑 에탄올, 광장, 10-CM 플라스틱 무게 보트와 함께 소독하고 캠 (그림 2A 참조) 개발할 수 있도록, 더 육일은 &quot;전 비켜&quot;를 품어. </li><li> 침착 콜라겐 타입 I 용액 및 얼음에 10 × DMEM 7.4의 pH 값으로 산성 콜라겐 용액을 중화 (N 0.2) NaOH를 추가 콜라겐 매트릭스로 10 × DMEM 배지의 1/10 양을 혼합; 저장 콜라겐 / 얼음 매체 솔루션입니다. </li><li> 형질 전환 MM 세포주 (영업 이익률-2 eGFP는 또는 혼합RPMI-8226 eGFP는; 인간 중간 엽 세포와 회전 타원체) 당 250,000 (50,000 세포 / 회전 타원체). </li><li> 15ml의 튜브에서 원심 셀 (1000 XG, 5 분, 각 시험 화합물 1 바이알 용) 세포 펠릿을, (목표 작업 농도) 약물을 1 ㎖를 차가운 준비된 콜라겐 혼합물을 넣고 잘 섞어 (1000 μL의 끝). </li><li> 37 ° C에서 세포 외 기질을 중합 할 수 있도록 30 분 동안 잘 페이트 6 콜라겐 방울 파라 필름에 (30 μL 각)을 놓는다. </li><li> (2cm 떨어진 배아에서) 9 일 된 닭 배아의 캠의 치료 표면에 집게의 사용과 단계 3.6에서 전송 &quot;onplants&quot;(각 닭 배아 4 onplants, 그림 2B 참조) </li><li> 형광 실체 현미경에 의해 생체 내 37 ° C에서 계란 인큐베이터에서 성장과 70 %의 습도, 문서 이종 이식 5 일 후 (그림 2C 참조). 5 시간 동안 냉장고에 4 ° C에서 저체온증으로 닭 배아를 안락사. </li><li> 안과 가위에 의해 바로 아래 CAM 조직과 이종 이식을 제거하고 넓은 평면 턱과 포셉. GFP의 측정을 사용합니다 (제 4 장, 2D 그림 참조) 또는 혈관 및 / 또는 침입 종양 세포 (섹션 5)의 면역 조직 화학 분석. </li></ol> ELISA에 의해 eGFP는 단백질의 정량 4. <ol><li> 0.5 ㎖의 RIPA 버퍼가 200 μg의 / ㎖ 프로테아제 억제제를 포함에 각 MM은 회전 타원체 또는 절제 이종 이식을 전송합니다. </li><li> 얼음에 조직 균질로 회전 타원체 / 이종 이식 균질화. </li><li> 액체 질소에 세 동결 / 해동 사이클 및 37 ° C의 물을 욕조를 수행합니다. </li><li> 20 분 (12,000g) 및 저장 상층 액을 4 ℃에서 원심 분리기 균질. </li><li> 샘플을 ELISA 키트의 분석 버퍼 (200 μL)에 1:20 희석. 제조사의 프로토콜에 따라, 비 오티 닐화 항 GFP 항체를 이용한 ELISA 키트 상업적 GFP-GFP에 의해 레벨을 측정한다. </li></ol> 5. Immunohi혈관에 침입 종양 세포의 stochemical 분석 <ol><li> 4 ° C에서 / 4 % 파라 포름 알데히드 O에서 N을 CAM 지역과 절제 이종 이식을 수정합니다. </li><li> 매립 카세트에 고정 이종 놓고 증가 등급 알코올 시리즈 (50 %, 70 %, 80 %, 95 % 에탄올, 크 실롤 파라핀 각각 단계 60 분)으로 매립 조직 스테이션에 전송할. </li><li> 벤치 탑 로터리 마이크로톰의 사용에 의해 섹션 이종 이식 (5 μm의). 56 ° C에서 유리 슬라이드의 O / N에 파라핀 섹션 굽는다. </li><li> 이중 증류수 감소 등급 알코올 시리즈 의한 Deparaffinize 섹션 (크 실롤, 95 %, 80 %, 70 %, 50 % 에탄올, 이중 증류수, 각 단계에서 10 분). </li><li> 항원 검색 용액 수조 (95 ° C, 20 분간)에 항원 검색을 수행하여 (버퍼 citrate-하여 pH 7.0, 100 μL 부피 참조). </li><li> 30 분 동안 100 ㎕의 3 % H 2 O 2 / 메탄올 내인성 퍼 옥시 데이즈 활성을 차단. </li><li> PBS를 함유하는 차단 부45 분 (부피 100 μL) 10 % 소 태아 혈청. </li><li> PBS는 RT에서 1 % 소 태아 혈청을 함유하는 희석 차 항체 (1μg / ㎖) 100 ㎕와 함께 1 시간 동안 얼룩. </li><li> PBS로 3 회 세척 후, PBS는 RT에서 1 % 소 태아 혈청을 함유하는 비오틴 이차 항체 (0.1 μg의 / mL)로 1 시간 동안 배양. </li><li> PBS로 3 회 세척 한 후 제조자의 지시에 따라 아비딘 / 비오틴 - 복합체 (ABC) 및 디아 미노 벤지딘 (DAB) 기질 용액에 의해 발색 반응을 수행한다. </li><li> 5.11. 헤 마톡 실린으로 두 번 증류수, Counterstain과에 섹션을 전송하여 반응을 중지하고 합성 설치 매체 섹션을 탑재합니다. </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>

저자는 더 경쟁 금융 이익이 없다

내화성 MM에 대한 새로운 치료제의 개발은 약물에 대한 인간 MM 세포 민감도를 평가하는 데 시간이 덜 소요되고 비용이 생체 시스템을 필요로한다. 지금까지, 단 몇 생체 시스템은 새로운 항 골수종 치료의 임상 평가에 사용할 수 있습니다. 그들 모두는 화합물 라이브러리 (29)의 대규모 스크리닝에 대한 자신의 한계를 가지고있다. 인간의 MM 셀에 대한 가장 좋은 현재의 모...

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

다발성 골수종 (MM), 악성 형질 세포 질환, 난치성 유지 및 신규 약물은 환자의 예후를 개선하기 위해 요구된다. 인해 골 미세 자동 / 분비 성장 인자의 부족으로 인간 MM 세포는 배양하기 어렵다. 따라서, 인간의 MM 세포에 새로운 치료제의 작용을 연구하기 위해 시험 관내 및 생체 내 배양 시스템에서 적절한 확립이 절실히 요구되고있다. 여기서 우리는 시험 관내 및 생체 내에서 복잡한 3D 환경에서 인간 다발성 골수종 세포를 성장 모델을 제시한다. MM 세포주 OPM-2 및 RPMI-8226를 형질 전환 유전자 GFP를 발현하는 형질 감염시키고 인간 중간 엽 세포와 콜라겐의 존재 하에서 배양 하였다 타입 I 입체 타원체로 매트릭스합니다. 더하여, 구 상체는 닭 배아 융모 막 (CAM)에 그래프트 된 종양 성장은 스테레오 형광 현미경에 의해 모니터 하였다. 두 모델 모두 새로운 치료 DRU의 연구를 허용복잡한 3D 환경 및 트랜스 특정 GFP-ELISA에서의 균질화 이식 후 종양 세포 질량의 정량화에 GS. 또한, 직하 숙주 조직으로 호스트 및 종양 세포의 침윤 혈관 신생 반응을 입체 현미경으로 매일 모니터링 및 인간 종양 세포 (기-67, CD138, 멘틴) 또는 호스트 벽화 세포 덮는 혈관에 대해 면역 조직 화학 염색에 의해 분석 될 수있다 (최근 desmin / ASMA). 결론적 onplant 시스템은 복잡한 3 차원 환경에서 MM 세포 성장 및 혈관 신생을 연구 할 수 있으며 MM 세포의 생존과 증식을 치료 표적 신규 화합물에 대한 스크리닝을 가능하게한다.

3D 다발성 골수종 타원체 내 분석 대상 화합물의 시험 관내 분석에서 때문에 체외에서 일차 인간 MM 세포 배양의 한계로는 골수에서 세포 성장 매트릭스 및지지 차 인간 중간 엽 세포 이용하게 인간 MM 세포 라인에 대한 새로운 3D 체외 배양 모델을 확립 (도 1a를, B). EGFP 유전자 MM 셀 라인은 회전 타원체의 3D 성장 후 시각화 및 MM 종?...

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

인간 다발성 골수종 (MM) 세포는 간엽 세포의 생존과 증식을위한 세포 외 매트릭스 성분의지지 미세 환경을 필요로한다. 우리는 종양의 성장, 침윤 및 혈관 신생에 항암제의 효과를 연구하기 위해 접목 인간 골수종 및 중간 엽 세포와 생체 내 닭 배아 모델을 설립했다.

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

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

13.0K 조회수.  Innsbruck Medical University.  인간 다발성 골수종 (MM) 세포는 간엽 세포의 생존과 증식을위한 세포 외 매트릭스 성분의지지 미세 환경을 필요로한다. 우리는 종양의 성장, 침윤 및 혈관 신생에 항암제의 효과를 연구하기 위해 접목 인간 골수종 및 중간 엽 세포와 생체 내 닭 배아 모델을 설립했다. 

비디오: 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.

조류 엠브료 모델을 사용하여 암 전이의 정량 분석

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

연구

의학

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.

초음파의 이용 가이드 생물학 관련 전이성 종양 Xenografts의 설립을 위한 세포 이식 조직 감독

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

암 연구학

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.

위암 환자 유래 이종이식 모델 및 1차 세포주 구축

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.

마우스 신장과 닭 융모알란투암 막에 설립된 전이성 맑은 세포 신장 세포 암종 모델 비교

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.

비보 모델에서 닭 융모난토암 막을 사용하여 부인과 및 비뇨기과 암을 연구

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.

면역 결핍 마우스의 피하 및 간내 인간의 간세포 암 이종 이식의 생성

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.

쥐과 동 소성 방광 이종 이식의 최소 침습 설립

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

<meta charset="utf8"></head><body>골육종 PDX 마우스 모델을 위한 프로토콜: 성장 및 약물 내성 연구

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.

<meta charset="utf8"></head><body>저자 스포트라이트: 암 연구를 위한 면역결핍 마우스의 인간 골육종 진행 복제

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

인간 골육종 조직을 이용한 환자 유래 이종이식 마우스 모델 구축

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

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

요약

더 많은 비디오 탐색

이 비디오의 챕터

조류 엠브료 모델을 사용하여 암 전이의 정량 분석

초음파의 이용 가이드 생물학 관련 전이성 종양 Xenografts의 설립을 위한 세포 이식 조직 감독

위암 환자 유래 이종이식 모델 및 1차 세포주 구축

마우스 신장과 닭 융모알란투암 막에 설립된 전이성 맑은 세포 신장 세포 암종 모델 비교

비보 모델에서 닭 융모난토암 막을 사용하여 부인과 및 비뇨기과 암을 연구

면역 결핍 마우스의 피하 및 간내 인간의 간세포 암 이종 이식의 생성

쥐과 동 소성 방광 이종 이식의 최소 침습 설립

인간 골육종 조직을 이용한 환자 유래 이종이식 마우스 모델 구축

인간 골육종 조직을 이용한 환자 유래 이종이식 마우스 모델 구축

인간 골육종 조직을 이용한 환자 유래 이종이식 마우스 모델 구축