저자들은 애리조나 대학교의 병리과에 재정적 지원에 대해 인정하고자합니다. 프로젝트는 WZ에 Mark and Jane Gibson 기부금으로 부분적으로 지원됩니다.

본 연구를 수행 한 애리조나 대학교 (University of Arizona)에서 Institutional Review Board 승인을 받았습니다. 정보에 입각 한 환자 동의서가 획득되었습니다. 1. 환자 선택 참고 : 애리조나 대학교 (University of Arizona)에서 Institutional Review Board 승인을 받았습니다. 총 38 명의 환자가 모집되었다. 환자의 나이는 32 세에서 86 세 사이 였고 평균 55 세였으며 그 중 26 명이 폐경기 였고 12 명은 폐경기 전이었다. <ol><li> 각 환자의 정보에 입각 한 동의를 얻으십시오. </li></ol> 2. 나팔관 세포 수집 절차 참고 : 현재 연구에 포함 된 환자는 모두 예방 적 위험 감소 또는 악성 종양에 대해 총 자궁 적출술 및 양측 난소 난소 절제술을 예정했다. 수술의 세부 사항은 세포학 연구를 수행 한 병리학 자에게는 알려지지 않았다. <ol><li> 수술 직후양측 난소, 양측 난관을 포함한 신생 조직 검체를 신중히 검사하여 양측 난관을 확인하십시오. </li><li> 혈액 공급을 고정시키는 30 분 이내에 방광 용액 (표 2 참조) 유리 병에 난관 세포를 모으고 배치하십시오. 권장 방부제는 메탄올을 기본으로 한 완충 용액입니다. 참고 : 관 세포 수집 (다음 단계)에는 부드러운 터치 브러시를 사용하십시오. 드문 경우를 제외하고는 난관을 자궁에서 분리 한 채로 난관 결장에서 세포를 모으기 위해 브러시 헤드를 삽입하면 나팔관이 자궁에 부착 될 때 발생합니다. <ol><li> 작은 포셉의 도움으로, 필라테스 끝을 통해 나팔관의 루멘에 브러시 ( 재료 표 참조)를 삽입하십시오. 천천히 시계 방향으로 돌리고 브러시를 가을의 우발적 인 부분을 통해 앞으로 움직이십시오opian 관, 그리고 그것이 협부에 도달 할 때까지 ampulla. </li><li> 천천히 브러시를 반 시계 방향으로 돌려서 브러시를 꺼냅니다. 루멘 내에서 브러시의 최대 속도가 1cm / s보다 느린 지 확인하십시오. </li><li> 솔루션에서 브러시를 10 번 돌리거나 회전시켜 방부제 용액 (바이알)에 브러시를 헹굽니다. </li><li> 바이알 캡을 조입니다. </li><li> 환자의 정보와 표본의 측면을 기록하십시오. </li><li> 유리 병을 4 ° C 냉장고 (최대 6 개월)에 보관하십시오. </li></ol></li></ol> 3. Tubal 세포학 슬라이드 준비 <ol><li> 슬라이드 준비를 위해 샘플 병을 자동 프로세서에로드하십시오. 참고 : 우리는 슬라이드 준비에 자동화 된 프로세서를 사용합니다. 샘플 바이알을 프로세서에 넣은 후 다음 단계가 자동으로 수행됩니다. (i) 샘플과 테스트 필터의 회전을 통해 셀과 파편이 분리되어 분산됩니다유리 병. (ii) Tubal 세포는 부드러운 진공에 의해 필터의 외부 표면에서 수집됩니다. (iii) 슬라이드 내에서 원형 영역에 세포가 부착되도록 필터를 뒤집어서 슬라이드에 대하여 가압한다. (iv) 슬라이드는 세포 고정 배지에 추가로 고정되고 염색 및 세포학 분석을 위해 준비된다. 불행히도 비슷한 품질의 결과를 얻을 수있는 대체 수동 방법은 없습니다. </li></ol> 4. 수정 된 파파 니콜라 우 염색 프로토콜 <ol><li> 표 1 에 나와있는 것처럼 자동화 된 비 gyn 얼룩 절차를 실행하여 단계 3.1에서 만든 슬라이드를 얼룩. 참고 : 염색법은 우리가 비 부인과 검체에 일상적으로 사용하는 Papanicolaou 염색법의 변형을 나타냅니다. 자동화 된 티슈 프로세서를 사용하여 일부 슬라이드를 얼룩지게합니다. 우리는이 연구에서 Gyn 염색 과정에 대해 Non-Gyn 염색법을 선택했다. Eosin은 EA-65 대신에 EA-65를 사용합니다.부인과 검체 (자궁 경부 세포진 검사). 또한 편평 세포 염색을 주로하는 Gyn 염색법의 OG-6는이 연구에서 편평 세포가 관찰되지 않기 때문에 염색법에 포함되지 않았다. EA65 (Eosin Azure)는 에오신 Y, 패스트 그린 FCF 및 비스마르크 브라운 Y의 3 가지 염료로 구성된 카운터 얼룩 용액입니다. 절차는 표 1에 설명되어 있습니다. </li><li> 다음과 같이 수동으로 Pap 얼룩 절차를 수행하십시오. <ol><li> 3.1 단계에서 만든 슬라이드를 95 % 에탄올로 10 회 정도 담그고 각 딥은 약 1 초가 걸립니다. </li><li> 슬라이드를 H 2 O에 10 번 담그고, 각 딥은 약 1 초가 걸린다. </li><li> 헤 마톡 실린에서 10 - 60 초 동안 슬라이드를 나 타냅니다. </li><li> 슬라이드를 H 2 O에 10 번 담그고, 각 딥은 약 1 초가 걸린다. </li><li> 슬라이드를 95 % 에탄올로 10 회 딥 (dip)하고, 각 딥은 약 1 초가 걸린다. </li><li> EA65에서 1 ~ 3 분 동안 슬라이드를 나 타냅니다. </li><li> 슬라이드 딥핑 intO 2 O, 10 회, 각 딥이 약 1 초 걸린다. </li><li> 슬라이드를 95 % 에탄올로 10 회 딥 (dip)하고, 각 딥은 약 1 초가 걸린다. </li><li> 슬라이드가 투명해질 때까지 크실렌으로 슬라이드를 담그십시오. 참고 : 이것은 수정 된 Papanicolaou 염색 절차입니다. 이 방법은 현장의 미세 바늘 흡인 및 STAT 표본과 같이 시간이나 공간이 제한된 경우보다 신속한 염색 방법으로 사용됩니다. 이 절차는 자동화 된 Non-Gyn 염색 절차와 유사한 품질의 염색을 제공합니다. 절차는 표 2에 설명되어 있습니다. </li></ol></li></ol> 5. 슬라이드에 셀 스핀 참고 : 각 시편에 대해 세 개의 cytospin 슬라이드가 만들어집니다. 한 슬라이드는 세포학 슬라이드와 형태학 비교를 위해 스테인드 H &amp; E입니다. 미래의 IHC 염색 연구를 위해 2 개의 염색되지 않은 슬라이드를 만들었습니다. 이 섹션의 세부 단계는이 백서의 초점이 아닙니다. 그러므로, 우리는 연장 된 d에서 그들을 논하지 않을 것이다.etails. <ol><li> 검사 할 각 샘플에 대해 라벨 슬라이드, 표본 깔대기 및 필터가있는 cytocentrifuge를 준비합니다. </li><li> 200 X g에서 5 분 동안 원심 분리하여 세포를 농축합니다. </li><li> 뜨는의 약 절반을 제거하고 부드럽게 pipetting하여 세포를 다시 일시 중지합니다. </li><li> 각 세포 현탁액 200 μL를 표본 깔때기에 넣으십시오. </li><li> 250 xg에서 5 분간 스핀. 조심스럽게 cytocentrifuge에서 슬라이드를 제거하고 고정을 위해 95 % 에탄올로 옮기십시오. </li><li> 염색하기 전에 공기를 건조시키고 장기간 보관하십시오. 참고 : 최종 슬라이드의 셀 밀도에 따라 5.2 및 5.3 단계를 건너 뛰거나 조정할 수 있습니다. 이 연구에서는 적절한 세포 밀도를 위해 고전력보기 당 적어도 2 개의 셀 클러스터가 필요합니다. </li></ol> 6. 첨단 끝 (SEE-FIM) 프로토콜의 섹션 설정 및 광범위 검토 <ol><li> 전체 표본을 수정하십시오 (자궁, 양측 난관 및 난소 포함)10 % 포르말린을 사용하여 각질 제거를 최소화합니다. </li><li> 부드럽게 조심스럽게 작은 집게와 가위를 사용하여 협부에서 자궁으로부터 난관을 분리하십시오. 접착력이있는 경우 작은 포셉과 가위를 사용하여 frightriated 끝을 난소 표면에서 조심스럽게 해부합니다. 이러한 단계는 친밀한 근접성으로 인해 난소와 난관 사이의 종양 교차 오염을 최소화하는 데 중요합니다. </li><li> Infundibulum과 fimbriae 세그먼트 (나팔관의 원위 1.0 - 2.0cm)를 나머지 표본에서 절개하십시오. </li><li> 2 mm 간격으로 종아리와 림프절을 절개하십시오. 조직 검사를 위해 toto의 모든 섹션 을 제출하십시오. </li><li> 지협과 협부를 2 - 3 mm 간격으로 절개하여 전적으로 제출하십시오. </li></ol>

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저자는 경쟁적인 금전적 이해 관계가 없다고 선언합니다.

예방 양안 난관 절제술 또는 난소 난소 절제술은 BRCA 유전자 돌연변이 및 강력한 가족 암 발병이있는 여성과 같은 고위험군에서 HGSC를 개발할 위험을 감소시키는 것으로 나타났습니다. 그러나 수술로 인한 불임 및 수술 폐경은 심각한 결과이며 특히 가임기 여성에게 어려운 결정을 내립니다. 이전의 연구는 난관 세포학과 조직학 17 사이의 훌륭한 상관 관계를 보여 주었다. 우리?...

난소 암 (HGSC)은 가장 흔하고 치명적인 유형의 난소 암이며 공중 보건에 중요한 위협이됩니다. 지난 10 년, 연구자들은 HGSC 대부분의 경우는 나팔관 대신 난소 자체가 1, 2, 3, 4, 5, 6에서 발생하는 것을 제안했다. Serous tubal intraepithelial carcinoma (STIC)는 HGSC 1 , 7 , 8 , 9 , 10 , 11 의 전조로 널리 받아 들여지고 있습니다. 지금까지 난소 암 조기 발견은 여전히 ​​어려운 일입니다. 암 항원 125 (CA-125)와 질식 초음파를 병용 한 현재 스크리닝 프로토콜환자 사망률에 거의 영향을 미치지 않는다 12 , 13 . 난소 암 검출 내막 샘플링은 매우 낮은 감도 14을 도시하고있다. 두 선구자 (15, 16)은 양성 나팔관 복강경 다이렉트 샘플링의 사용을 탐구 양성 나팔관 상피 세포 학적 기능을 특징으로하는 연구. 우리는 종양이 이미징에 의해 시각화되지 않은 초기 단계에서 나팔관으로부터의 직접 샘플링이 또한 STIC 또는 HGSC를 검출하는 실제적이고 직접적인 방법 일 수 있다고 믿습니다. 궁극적 인 목표는 고위험 인자가있는 환자에서 최소 침습적 복강경 검사의 유용성이지만, 현재 연구의 즉각적인 목표는 다음과 같습니다. 1) 양성 난관 상피 세포의 기본 세포 학적 특징을 확립합니다. 2) 난소 췌관 조영술에서 난관 세포 診의 민감도와 특이도를 검사한다.ers 및 암 전구체. 따라서 우리는 HGSC 및 그 전구체 STIC 17의 검출에서이 프로토콜의 효과를 시험하기 위해 다음과 같은 기본 tubal cytology 방법을 개발했습니다. 이 연구에서, 우리는 정기적으로받은 외과 표본의 큰 볼륨을 활용하고 외과 절제 관의 직접적인 샘플링을 수행 루틴 총 수익을 올리는 절차 이외에. 우리의 최근의 연구 (17)는 악성 또는 악성 종양에 대한 의심의 세포 학적 진단이 매우 HGSC (100 %)의 조직 학적 진단과 상관 관계가 있음을 보여 주었다. 또한, 난관 세포학적인 평가는이 연구에 포함 된 조직 학적으로 확증 된 두 가지의 STIC 경우에서만 표피 내 신 생물이 확인되었다. 우리는 난관 세포학이 조기 발견에 큰 가능성을 가지고 있으며 환자 관리에 귀중한 정보를 제공 할 것이라고 믿습니다.

<table>
	<tbody>
		<tr>
			<td>Cytobrush plus GT gentle touch of Medscand sample collection kit</td>
			<td>CooperSurgical</td>
			<td>C0112</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep preservCyt solution</td>
			<td>HOLOGIC</td>
			<td>234005</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep 2000 Processor</td>
			<td>HOLOGIC</td>
			<td>70031-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papanicolaou Stain, EA 65</td>
			<td>sigma aldrich</td>
			<td>HT40432</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>sigma aldrich</td>
			<td>652261</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>sigma aldrich</td>
			<td>493538</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>sigma aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>sigma aldrich</td>
			<td>H9627</td>
			<td></td>
		</tr>
		<tr>
			<td>Sakura Tissue Tek 2000 tissue processor</td>
			<td>SAKURA</td>
			<td>TISSUE-TEK VIP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo Dissecting Scissors,5.5 straight</td>
			<td>Pilgrim medical equiment</td>
			<td>FA710-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemware Forceps</td>
			<td>United state plastic corp</td>
			<td>76122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades,short</td>
			<td>SAKURA</td>
			<td>4785</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades handle,short</td>
			<td>SAKURA</td>
			<td>4786</td>
			<td></td>
		</tr>
	</tbody>
</table>

tubal cytology of the fallopian tube as a promising tool for ovarian cancer early detection

현재, 대부분의 난소의 고급 장 액성 암종 (HGSC)이 난관에서 비롯된 것으로 널리 알려져 있습니다. 그러나, 난소 암 식별을위한 마커 또는 도구의 부족으로 인해 HGSC의 조기 발견은 여전히 ​​어려움을 겪고 있습니다. 나팔관을 직접 샘플링하면 종양이 현저하게 보이지 않을 때 신생 세포의 감수성을 높일 수 있습니다. 우리는 신선하게받은 외과 견본에서 직접 난관 세포를 수집하는 절차를 개발했으며, 이는 조직 학적 발견과 우수한 상관 관계가 있음을 보여줍니다. 이 접근법은 위험도가 높은 환자 집단에서 최소 침습 복강경 검사의 미래 유용성을위한 기반을 마련합니다.

우리의 이전 연구는 부드러운 터치 브러시를 사용하여 절개 된 나팔관에서 직접 샘플링하는 것이 추후의 세포 학적 평가를 위해 양적으로 그리고 질적으로 적절한 표본을 산출 할 수 있음을 보여주었습니다. 또한 자동 슬라이드 준비와 수정 된 Papanicolaou 염색의 조합은 병리학자가 정확한 진단을 내리기 위해 세포 학적 세부 사항을 더 잘 시각화 할 수있게 해줍니다. 양성...

Watch this Scientific Journal Video about Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection at JoVE.com

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

저자들은 난소 암 조기 발견 도구로서 난관을 직접 수술 후 절제 한 후 난관 조영술을 시행하여 난소 세포학적인 방법을 연구 하였다. 여기, 우리는 새로받은 수술 표본에서 난관 세포를 수집하는 프로토콜을 제시한다.

난소 암 조기 발견을위한 유망한 도구로서의 난관 조영 세포학

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

tubal-cytology-fallopian-tube-as-promising-tool-for-ovarian-cancer

Research

JoVE Journal

Medicine

12.2K 조회수.  University of Arizona College of Medicine.  저자들은 난소 암 조기 발견 도구로서 난관을 직접 수술 후 절제 한 후 난관 조영술을 시행하여 난소 세포학적인 방법을 연구 하였다. 여기, 우리는 새로받은 수술 표본에서 난관 세포를 수집하는 프로토콜을 제시한다. 

비디오: Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

체외 Mesothelial 정리 분석에서는 그것이 난소 암 전이의 초기 단계 모델

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

연구

의학

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. 
Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-&gamma; mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9.
For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

We describe an ex vivo infection model for visualisation of direct interactions from bacterial pathogens with human fallopian tube cells. The whole organ tissue model was established to investigate C. trachomatis induced pathology to the female fallopian tube under "life-like" conditions.

조사를위한 인적 나팔관 모델<em&gt; C. trachomatis</em&gt; 감염

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

3 차원 Heterotypic<em&gt; 체외에서</em난소 암 개시 및 진행하는 동안 Stromal - 상피 조직의 상호 작용&gt; 모델링

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

암 치료 모니터링을위한 Cerenkov 발광 이미징 (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; OVO에서</em육종을위한 Xenograftmodel으로&gt; CAM-분석

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

고체 시편에서 기본 난소 암 세포를 얻는 방법

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

효능 평가<em&gt; H. 파이로리</em동소 이식 쥐 모델에서 방광암의 치료를위한 치료 도구로&gt; 단백질 HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

복강 내 난소 암 전이의 정량

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

인간의 복강의의 Organotypic 문화 난소 암 보급의 초기 단계 모델링

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...