The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

참고 : 환자 조직 조달이 제도적 검토위원회 (IRB)를 통해 권한이 부여 된 기념 슬로안 케터링 암 센터에서 Biospecimen 및 임상 프로토콜 (프로토콜 번호는 각각 09-121 및 11-041을) -approved. 1 조직 조달 <ol><li> 환자의 기본 종양 / 전이 조달 참고 : 현재까지,이 프로토콜은 수술로 제거 췌장, 위 및 유방 종양 유형뿐만 아니라, 같은 림프종 전이에 수행되었습니다. <ol><li> 멸균 유출 방지 컨테이너 내의 밀봉 및 살균 누수 방지 플라스틱 시편 가방에 병리학 부서에 택배 또는 공압 튜브 시스템에 의해 표본을 제공하기 위해 외과 팀을 직접. 더 포르말린 고정액으로 신선한 상태의 시료를 수송 할 수있는 외과 팀을 직접. </li><li> 어느 것이 Inc의 멸균 기술을 사용하여 신선한 표본을 채취하기 위해 병리학 자 또는 병리학 지원을 직접층류 후드 멸균 장갑과 악기의 ludes 사용. </li><li> 수술의 완성에서 30 분에서 엄격하게 유지된다 조달 시료의 채취 시간을 기록한다. 냉 허혈을 고려하여 효과를 고려,&gt; 30 분 (18)의 감기 허혈성 시간 절제술에서 샘플을 채취하지 않습니다. </li><li> 무균 환경을 유지하기 위해 층류 후드에서 1.0 cm (3)에 약 0.5 cm (3)의 일차 종양 표본을 제거하는 병리학 부서 담당자 직접. 가능하면, 전위 프랭크 중앙 괴사 (세포 사멸)를 피하기 위해 인덱스 병변의 주변부로부터 종양 조직을 선택한다. 참고 : 괴사 조직은 다음 기준 중 하나에 의해 심하게 인식 할 수 있습니다 : 색상이나 조직의 창백 손실; 괴사 조직 부드럽고 무른 인 강도의 손실; 괴사 및 실행 가능한 조직 사이에 뚜렷한 경계. </li><li> pathologi 직접성 또는 병리학 보조 진단 평가를위한 종양의 즉각적인 샘플링 초과 후 (수술 폐기)에 주변 조직을 제공합니다. 약 5 ㎖의 1 % 항생제 (페니실린과 스트렙토 마이신)를 포함하는 최소 필수 미디어 (MEM)의 포함 된 15 ㎖의 멸균 원뿔 원심 분리 관에 시료를 놓습니다. </li><li> 사용 가능한 (예를 들어, 유방 절제 표본의 겨드랑이 꼬리 림프절)은 같은 크기 (0.5 cm 3 cm 3 내지 1.0)의 조잡한 포지티브 관련된 림프절 표본을 조달 일차 종양의 약물 반응과 비교하면. 일차 종양 표본 마찬가지로, 1 % 항생제 (페니실린 스트렙토 마이신)를 포함하는 약 5 ㎖의 최소 필수 매체 (MEM)의 함유를 15 ㎖의 멸균 원추형 원심 분리 튜브에 연결된 림프절 표본을 배치. </li><li> 만약 즉 수술 표본 허가 (예를 들어, 유방 절제 표본)을, (주 종양에서 먼) 제거의 정상 조직 샘플 (크기,약 5 ml의 전송만을 목적으로 1 %의 항생제를 함유하는 최소 필수 매체 (MEM)의 함유를 15 ㎖의 멸균 원추형 원심 분리 관에서 통상 고밀도 / 섬유질 유방 실질)​​ 및 장소. 실험실 시설 이전에 따라,에 cryovial, &#39;스냅&#39;동결 및 저장에 &#39;정상&#39;표본을 전송 -80 C의 냉동고 ° 향후 분자 분석을 위해. </li><li> 젖은 얼음에 모든 시편을 놓고 즉시 생체 신선한 조직 절편에 대한 실험실 공간으로 운반 할 것. 하지 부에게 정상적인 시료를 수행 아니라 미래의 분자 분석을위한 C의 냉동고 °에 -80 저장합니다. 미래의 분자 분석을 위해 -80 ° C의 냉동고에 cryovial, &#39;스냅&#39;동결 및 저장에 대한 기본 종양의 부분과 관련된 전이을 전송합니다. </li></ol></li><li> 임상 연구에서 조직의 조달 (예, 선택적 코어 생검 바늘 (CNB)) 참고 : 우리는 환자의 종양 조직을 얻을치료 시작 전 액세스 종양 전이 CNB을 거쳐 그들의 동의를 임상 프로토콜에 등록. CNBs는 현재까지 유방암, 한계 영역 림프종, 맨틀 세포 림프종, 전이성 paraganglioma, 자궁 경부 (편평 상피 세포) 및 난소 암 환자에서 수행되었다. <ol><li> 중재 방사선 또는 관련 부서로부터 임상 인력 함유 멸균 원추형 원심 분리 관을 전처리 코어 생검을 수행하고 15 ml의 시료를 배치 유무 약 5 ㎖의 1 % 항생제 (페니실린 스트렙토 마이신)을 함유하는 최소 필수 매체 (MEM)의. </li><li> 젖은 얼음에 생검 표본을 놓고 즉시 생체 신선한 조직 절편에 대한 실험실 공간으로 운반 할 것. </li></ol></li></ol> 단면 및 Vibratome 설정 재료의 2 준비 <ol><li> 단면 용 재료의 제조 <ol><li> AGA 4 g을 첨가하여 4 % 아가로 오스 용액을 만들기PBS 100 ㎖로 상승했다. 오토 클레이브 (주기 설정 : 액체, 121 ° C, 30 분, 평방 인치 당 15파운드)를 처음 사용할 경우, 사용하기 전에 솔루션을 제공합니다. 이후 용도를 위해, 아가로 오스는 RT에서 굳은 것을 염두에 각각 사용하기 전에 (30 초 간격으로 높은 열을) 액화 적절 ~ 25 ° C (즉, 따뜻한 터치)으로 냉각 전자 레인지에서 4 % 아가로 오스를 배치합니다. </li><li> 비 영구적 인 조직 내장 매체로 4 % 아가로 오스를 사용합니다. 4 %의 아가 로스를 충분히 냉각하고 무균 집게 매립 금형 티슈 시편을 넣고 4 % 아가 로즈 부어와 액체 상태 여전히 때. </li><li> 매립 금형의 절삭면의 벽에 직접 주변 조직 시료 (~ 2mm의 거리)를 배치 아니지만. 4 % 아가로 신속 고화하기 때문에 적절히 빠르게 조직 시편을 위치시킨다. 단면 동안 솔루션을 유지하기위한 55 ° C의 물을 욕조에 4 % 아가로 오스를 유지합니다. </li><li> 단면 분석 24 웰 플레이트에 1 ㎖의 O 추가각 웰에 F 완전한 미디어 (즉, MEM + 10 % 소 태아 혈청 + 1 % 항생제). 단면의 기간에 걸쳐 RT (~ 25 ° C)에서 24 웰 플레이트를 유지합니다. </li></ol></li><li> Vibratome 설정의 준비 <ol><li> 진동 마이크로톰을 사용하여, (10)에 칼날이 시험편에 접근하는 속도 [10 (2.5 mm / 초) 0 (0.00 mm / 초)]와 블레이드 [0 (0 Hz에서)의 주파수를 설정 (100 Hz에서) ]. 조직 (즉, 밀도 / 탄력)의 질감에 따라 설정을 조정합니다. </li><li> 침윤성 암의 주요 유방 종양의 경우, 각각 6, 3, 속도 및 주파수 설정을 조정합니다. 밀도가 낮은 / 회사 종양 조직의 경우, 속도가 감소하고 그에 따라 주파수를 증가시킨다. </li><li> (- 999 μm의 1) 기기의 범위 내에서 부분의 두께를 설정합니다. 이러한 분석의 경우, 200 μm의에서 단면 두께를 설정합니다. </li><li> 시험편 디스크 그쪽에 액상 접착제의 얇은 층을 사용하여 아가로 포매 표본 마운트t는 젖은 얼음에 쌌다 미디어의 저수지에 잠겨있다. </li></ol></li></ol> 3 조직 단면, 처리 및 분석 <ol><li> 즉시 전체 미디어의 1 ml의 멀티 웰 플레이트에서 멸균 포셉과 장소를 사용하여 단면에 정밀 슬라이스 섹션을 제거합니다. 충분한 부분이 상호 반응 및 분석을 위해 획득 될 때, 표준 조직 배양 조건 설정 조직 배양 인큐베이터에서 치료 반응을 위해 멀티 웰 플레이트를 배치했다. </li><li> 배양 아티팩트 반대로 용량 의존적 반응을 확인하기위한 노력으로 처리 된 부분 책꽂이 다중 제어부를 타고. (- 4 시간 ~ 2) 18도 차가운 허혈 시간의 해로운 영향에서, - (40 분 ~ 30) 조직 빠르게 단면을 수행합니다. 단지 1 시간의 순응 기간 다음 섹션의 치료를 수행합니다. </li><li> 1 시간의 순응 기간에 따라, 용량 (치료 관련 증가 추가; <em.&gt; 예, 0 - 치료 기간에 대한 표준 조직 배양 조건에서 섹션과 집으로 약물의 10 μM 농도). 이 연구를 위해 24 및 / 또는 48 시간 평가 기간을 사용합니다. 및 96 시간 평가 기간까지의 기간 동안, 본 연구에서 시험 그러나 제어 (차량 전용) 조직 섹션은 가능한 유지됩니다. </li><li> 치료 후, RT O에서 10 % 포르말린과 장소를 포함하는 튜브에 조직 절편을 배치하여 멸균 조건 및 포르말린으로 고정 섹션이 모두 제거 / N 또는 4 % 파라 포름 알데히드를 함유하는 튜브 (유리 병에서 16 % 파라 포름 알데히드를 희석 2 시간 동안 RT에서 4 %의 최종 농도) 및 장소와 PBS. <ol><li> 큰 조직 섹션을 수정 (예. ~ 6mm X 4mm) 10 % 포르말린의 O에서 200 μm의 두께 / RT에서 N. 작은 부분을 수정 (예를 들면., 1mm X 1mm) 실온에서 2 시간 동안 4 % 파라 포름 알데히드에서 200 μm의 두께. NOTE : 정착액 (F)의 양을 효율적으로 침투ixative 조직의 부피를 20 배한다. 주어진 적절한 고정 사용하는 모든 조직의 크기에 정착 1 ㎖의 보증을위한 매우 작은 조직의 크기 (예를 들어, 6mm 3 조직 = 6 μL 볼륨),이 프로토콜의 결과. </li><li> 고정 후 조직 표본은 병리학 부서 담당자가 포함 된 파라핀있다. 충전 슬라이드 위에 파라핀 블록 표본 섹션 헤 마톡 실린 및 스테인드 에오신 및 평가 면역 조직 화학적 (예를 들면, 세포 사멸) 할 수 있습니다. </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+human+prostate+tissue+and+drug+development.">Ex vivo culture of human prostate tissue and drug development.</a> Nature Reviews Urology. 10, 483-487 (2013).</li><li>Jhaveri, K., 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=Heat+shock+protein+90+inhibitors+in+the+treatment+of+cancer:+current+status+and+future+directions.">Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions.</a> Expert Opinion on Investigational Drugs. 5, 611-628 (2014).</li><li>Gerecitano, J. 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=Using+124-PU-H71PET+imaging+to+predict+intratumoral+concentration+in+patients+on+a+phase+I+trial+of+PU-H71.">Using 124-PU-H71PET imaging to predict intratumoral concentration in patients on a phase I trial of PU-H71.</a> Journal of Clinical Oncology. 31, 11076-11 (2013).</li><li>Yildiz-Aktas, I., Dabbs, D. J., Bhargava, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cold+ischemic+time+on+the+immunohistochemical+evaluation+of+estrogen+receptor,+progesterone+receptor,+and+HER2+expression+in+invasive+breast+carcinoma.">The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.</a> Modern Pathology. 25, 1098-1105 (2012).</li></ol>

No conflicts of interest disclosed.

효과적인 치료 전략을 개발하려고 할 때 암 생물 학자들은 상당한 도전에 직면하고 있습니다. 확립 암 세포주에서 약물 개발을 테스트하는 것은 정확하게 생체 반응과 PDX 모델에서 생체 내 실험은 노동 집약적이고 비용이 매우 다를 수있다. 상기를 감안 기본 환자의 생체 기술의 애플리케이션 (15)은 현재 설정된 암 세포주와 환자 유래의 이종 이식 (PDX)의 분자량 분석 ...

효과적인 암 치료제를 개발하는 것이 매우 어려운 것으로 입증되었습니다. 암 세포주와 종양 이식편 -뿐만 아니라 이종 절반 위에 세기 1,2,3 대한 암 연구에서 사용되어왔다. 현재까지, 약물 감수성 및 내성 둘 확립 암 세포주 및 이종 이식 환자 유래의 분자량 분석 (PDX)이 필수적이다. 그러나, 확립 암 세포주에서의 시험 화합물들은 생체 내 효능을 예측할 수없고, 특히 PDX 모델에서, 동물의 생체 내 연구에 대응하는, 매우 많은 비용과 시간이 소요된다. 이 모델 시스템, 종양 진행 및 치료 전략에 응답하여 네이티브 미세의 영향에 알리는 즉 무능력의 한계는 이러한 분석을 보완하기위한 추가 방법을 개발하는 연구 분야를 주도하고있다. 최근의, 높아진 관심은 환자 다치의 생체 분석으로 지급되는암 치료 반응은 암세포의 내재적 분자 조성물 단독이 아니라 오히려 크게 기존 배양 방법에 의해 효과적으로 요약 할 수없는 종양 세포 미세 환경 (6, 7) 기능에 의해 영향을 받는다는 인해 큰 이해 또는 이식편 4, 5 / 또는 PDX. 상기 컨텍스트의 생체 외 분석 (예. 인접한 주위 종양 세포 미세 환경의 영향)보다는 셀룰러 균주 8, 9의 생체 외 분석을보다 실용적 일차 종양 / 전이 섹션의 평가를 의미한다. 우리는 모두 환자의 기본 종양 (정밀 분리 된 신선한 조직 섹션 즉.) 및 관련 전이 (즉, 림프절)을 충실하게 응답에 알립니다 (IC50), 오프 대상 효과 생체 기술에 여기에보고하고 분자 수 있습니다 저항과 피드백 메커니즘의 분석. 치료 적 레이저 각막 절제의 또한, 상관 관계 분석바이오 마커 및 유전자 발현 프로파일 대 utic 감도 / 저항 (즉. 높은 약물 반응이 특히 생물학적 프로필을 가진 환자와 일치) 관심 실험 약물에 반응 할 가능성 환자를 확인하기 위해 수행 될 수있다. 다중 매개 변수 방식으로 생체 기술과 평가의 적용은 환자의 선택과 임상 결과의 전반적인 개선을 향한 움직임이다. 생체 내 치료 반응 분석은 암 치료제의 전임상 및 임상 개발의 표준 도구가 될 수 있고, 치료 개발 전략에 맞춤 의학의 접근 방식으로 단계로 구상된다.

<table><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr></tbody></table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

이 연구를 위해 생체 외 기술은 열 충격 단백질 90 저해제 (Hsp90i)의 치료 감수성 / 내성 상관 분석에 사용 하였다. 이 Hsp90i, 유방암 일차 종양, ER + 침윤성 암종 (IDC), 및 관련된 림프절 전이의 임상 평가에 치료 반응을위한 생체 외 분석 하였다 &#160; (그림 1). 여러 200 μm의 시리얼 섹션 Hsp90i의 차량 만 증가 복용량 (0.25, 0.50, 1.0 및 2.5 μM)로 처리 하였다. 그?...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

설립 암 세포주 및 이종 이식편은 지난 수십 년간 암 연구의 주류이었다. 그러나, 최근의 증거는 치료 반응이 크게 종양 세포 미세 환경에 의해 영향을 받는다는 의미한다. 따라서, 우리는 약물 개발을 목적으로 일차 종양 시료의 생체 외 분석을 개발 하였다.

기본 종양 및 / 또는 전임상에 대한 관련 전이 및 치료제의 임상 개발의 전 생체 치료 응답

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

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JoVE Journal

Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.7K 조회수.  Memorial Sloan Kettering Cancer Center.  설립 암 세포주 및 이종 이식편은 지난 수십 년간 암 연구의 주류이었다. 그러나, 최근의 증거는 치료 반응이 크게 종양 세포 미세 환경에 의해 영향을 받는다는 의미한다. 따라서, 우리는 약물 개발을 목적으로 일차 종양 시료의 생체 외 분석을 개발 하였다. 

비디오: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

유방암의 마우스 모델에서 종양 Microenvironment에 약물 응답의 영상을 라​​이브

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

연구

의학

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

종양 침투 T 세포 독성의 정형 외 췌장 종양 및 Ex 생체 특성의 생성

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

암 연구학

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

꼬리 정맥 전이 검사의 결합된 사용 및 생체 내 실시간 생체 내 이미징을 결합하여 폐암 전이성 식민지화 및 폐의 부담을 정량화

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

오르가노티픽 종양 조직 슬라이스에서 실시간 약물 반응 측정

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

유방암을 위한 전임상 모형으로 유방 종양의 정형소 이식

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

환자 유래 종양은 환자의 약물 내성을 예측하기위한 "라이브"전임상 플랫폼으로 이월합니다.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

EGFR 돌연변이 NSCLC 환자 유래 오가노이드에서 표적 치료법에 대한 민감도 프로파일링

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

벌크 및 단일 오가노이드 분석을 위한 광시야 라이브 셀 이미징을 사용한 다중 파라미터 종양 오가노이드 약물 스크리닝

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

암 약물 과민 반응의 효과를 평가<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

기본 종양 및 / 또는 전임상에 대한 관련 전이 및 치료제의 임상 개발의 전 생체 치료 응답

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