The authors would like to acknowledge the Department of Pathology, University of Arizona, for the financial support. The project is partially supported by the Mark and Jane Gibson endowment fund to WZ.

An Institutional Review Board approval was received from University of Arizona for conducting this study. Informed patient consent forms were obtained.
1. Patient Selection
NOTE: An Institutional Review Board approval was obtained from University of Arizona. A total of 38 patients were recruited. The patients&#39; ages ranged from 32 to 86 years old with a mean of 55 years, of whom 26 patients were post-menopausal and 12 patients were pre-menopausal.
<ol>
	<li>Obtain informed consent from each patient.</li>
</ol>
2. Fallopian Tube Cells Collection Procedure
NOTE: The patients included in the current study were all scheduled for total hysterectomy and bilateral salpingo-oophorectomy for either prophylactic risk reduction or malignancy. The details of surgery were unknown for pathologist who performed the cytology study.
<ol>
	<li>Immediately after surgical excision, examine the fresh tissue specimen including uterus, bilateral ovaries, and bilateral fallopian tubes carefully to identify the bilateral fallopian tube.</li>
	<li>Collect and place fallopian tube cells into the preservative solution (see Table of Materials) vial within 30 min of clamping the blood supply; the recommended preservative is a methanol-based, buffered preservative solution. 
	NOTE: Use a gentle touch brush for the tubal cell collection (upcoming steps). With the exception of rare cases in which the fallopian tube is submitted detached from the uterus, inserting a brush head to collect cells from the tubal fimbria occurs typically when the fallopian tube is attached to the uterus.
	<ol>
		<li>With the help of a small forceps, insert the brush (see Table of Materials) into the lumen of the fallopian tube through the fimbriae end. Slowly rotate clockwise and move the brush forward through the infundibular portion of the fallopian tube, then the ampulla until it reaches the isthmus.</li>
		<li>Slowly rotate and reverse the brush counter-clockwise to pull out the brush. Ensure that the maximal speed of the brush within the lumen is slower than 1 cm/s.</li>
		<li>Rinse the brush in the preservative solution (in a vial) by rotating and swirling the brush 10 times in the solution.</li>
		<li>Tighten the vial cap.</li>
		<li>Record the patient&#39;s information and the laterality of the specimen.</li>
		<li>Store the vial in a 4 &#176;C refrigerator (for up to) 6 months.</li>
	</ol>
	</li>
</ol>
3. Tubal Cytology Slide Preparation
<ol>
	<li>Load the sample vial into the automated processor for slide preparation. 
	NOTE: We use the automated processor for slide preparation. The following steps are performed automatically after the sample vial is placed into the processor: (i) The cells and debris are separated and dispersed through the rotation of the test filter within the sample vial. (ii) Tubal cells are collected from the exterior surface of the filter by a gentle vacuum. (iii) The filter is inverted and pressed against the slide to let the cells to adhere to the circular area within the slide. (iv) The slide is further fixed in a cell fixative bath and ready for staining and cytology analysis. Unfortunately, there is no alternative manual method, which can yield results with a similar quality.</li>
</ol>
4. Modified Papanicolaou Staining Protocol
<ol>
	<li>Stain the slides made from step 3.1 by running the automatized non-gyn stain procedure, as illustrated in Table 1. 
	NOTE: The staining method represents a modification of the Papanicolaou staining technique that we use routinely for non-gynecologic specimens. We use an automated tissue processor to stain some slides. We chose the Non-Gyn staining procedure over the Gyn staining procedure in this study. The Eosin used is EA-65, instead of the EA-50 used for gynecological specimens (Pap smear specimen). In addition, OG-6 in the Gyn staining procedure, which is mainly for squamous cell staining, is not included in the staining procedure since no squamous cells are expected to be seen in this study. EA65 (Eosin Azure) is a counter stain solution comprised of 3 dyes: Eosin Y, Fast green FCF, and Bismarck brown Y. The procedure is illustrated in Table 1.</li>
	<li>Perform a manual quick Pap stain procedure as follows.
	<ol>
		<li>Dip the slide made in step 3.1 into 95% ethanol, 10 times, with each dip taking about 1 s.</li>
		<li>Dip the slide into H2O, 10 times, with each dip taking about 1 s.</li>
		<li>Emerge the slide in hematoxylin for 10 - 60 s.</li>
		<li>Dip the slide into H2O, 10 times, with each dip taking about 1 s.</li>
		<li>Dip the slide into 95% ethanol, 10 times, with each dip taking about 1 s.</li>
		<li>Emerge the slide in EA65 for 1 - 3 min.</li>
		<li>Dip the slide into H2O, 10 times, with each dip taking about 1 s.</li>
		<li>Dip the slide into 95% ethanol, 10 times, with each dip taking about 1 s.</li>
		<li>Dip the slide in xylene until the slide becomes clear. 
		NOTE: This is a modified Papanicolaou staining procedure. This method is used as a more rapid stain method for cases with limited time or space like on-site fine needle aspiration and STAT specimens. This procedure provides comparable quality staining as the automatized Non-Gyn stain procedure. The procedure is illustrated in Table 2.</li>
	</ol>
	</li>
</ol>
5. Spin Cells onto Slides
NOTE: Three cytospin slides are made for each specimen. One slide is H&#38;E stained for the morphological comparison with the cytology slide. Two unstained slides were made for the future IHC staining study. The detailed steps of this section are not the focus of this paper; therefore, we will not discuss them in extended details.
<ol>
	<li>Prepare a cytocentrifuge with a labeled slide, specimen funnel and filter for each sample to be examined.</li>
	<li>Concentrate the cells by centrifuging for 5 min at 200 x g.</li>
	<li>Remove about half of the supernatant and then re-suspend the cells by gentle pipetting.</li>
	<li>Add 200 &#181;L of each cell suspension to a specimen funnel.</li>
	<li>Spin at 250 x g for 5 min. Carefully remove the slide(s) from the cytocentrifuge and transfer them to 95% Ethanol for fixation.</li>
	<li>Air dry prior to staining and for long term storage. 
	NOTE: Depending on the cell density on the final slides, step 5.2 and step 5.3 can be skipped or adjusted. In this study, we require at least 2 cell clusters present per high power view for adequate cellular density.</li>
</ol>
6. Sectioning and Extensively Examining the Fimbriated End (SEE-FIM) Protocol
<ol>
	<li>Fix the entire specimen (including uterus, bilateral fallopian tubes, and ovaries) in 10% formalin prior to grossing to minimize exfoliation.</li>
	<li>Gently and carefully detach the fallopian tubes from the uterus at the isthmus using small forceps and a scissors. If adhesion is present, carefully dissect the fimbriated end from the ovary surface using small forceps and a scissors; these steps are crucial to minimize tumor cross contamination between ovary and fallopian tube due to their intimate proximity.</li>
	<li>Amputate the infundibulum and fimbriae segment (the distal 1.0 - 2.0 cm of fallopian tube) from the rest of the specimen.</li>
	<li>Section the infundibulum and fimbriae longitudinally at 2 mm intervals. Submit all sections in toto for histologic examination.</li>
	<li>Section the isthmus and ampulla at 2 - 3 mm intervals and submit entirely.</li>
</ol>

<ol>
	<li>Carlson, J. W., 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=Serous+tubal+intraepithelial+carcinoma:+its+potential+role+in+primary+peritoneal+serous+carcinoma+and+serous+cancer+prevention.">Serous tubal intraepithelial carcinoma: its potential role in primary peritoneal serous carcinoma and serous cancer prevention.</a> J Clin Oncol. 26 (25), 4160-4165 (2008).</li><li>Kurman, R. J., Shih Ie, 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+origin+and+pathogenesis+of+epithelial+ovarian+cancer:+a+proposed+unifying+theory.">The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.</a> Am J Surg Pathol. 34 (3), 433-443 (2010).</li><li>Lee, 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=A+candidate+precursor+to+serous+carcinoma+that+originates+in+the+distal+fallopian+tube.">A candidate precursor to serous carcinoma that originates in the distal fallopian tube.</a> J Pathol. 211 (1), 26-35 (2007).</li><li>Li, J., Fadare, O., Xiang, L., Kong, B., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+serous+carcinoma:+recent+concepts+on+its+origin+and+carcinogenesis.">Ovarian serous carcinoma: recent concepts on its origin and carcinogenesis.</a> J Hematol Oncol. 5, 8 (2012).</li><li>Przybycin, C. G., Kurman, R. J., Ronnett, B. M., Shih Ie, M., Vang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+all+pelvic+(nonuterine)+serous+carcinomas+of+tubal+origin?.">Are all pelvic (nonuterine) serous carcinomas of tubal origin?.</a> Am J Surg Pathol. 34 (10), 1407-1416 (2010).</li><li>Zheng, W., Fadare, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fallopian+tube+as+main+source+for+ovarian+and+pelvic+(non-endometrial)+serous+carcinomas.">Fallopian tube as main source for ovarian and pelvic (non-endometrial) serous carcinomas.</a> Int J Clin Exp Pathol. 5 (3), 182-186 (2012).</li><li>Carcangiu, M. L., 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=Atypical+epithelial+proliferation+in+fallopian+tubes+in+prophylactic+salpingo-oophorectomy+specimens+from+BRCA1+and+BRCA2+germline+mutation+carriers.">Atypical epithelial proliferation in fallopian tubes in prophylactic salpingo-oophorectomy specimens from BRCA1 and BRCA2 germline mutation carriers.</a> Int J Gynecol Pathol. 23 (1), 35-40 (2004).</li><li>Leunen, 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=Prophylactic+salpingo-oophorectomy+in+51+women+with+familial+breast-ovarian+cancer:+importance+of+fallopian+tube+dysplasia.">Prophylactic salpingo-oophorectomy in 51 women with familial breast-ovarian cancer: importance of fallopian tube dysplasia.</a> Int J Gynecol Cancer. 16 (1), 183-188 (2006).</li><li>Piek, J. 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=Dysplastic+changes+in+prophylactically+removed+Fallopian+tubes+of+women+predisposed+to+developing+ovarian+cancer.">Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer.</a> J Pathol. 195 (4), 451-456 (2001).</li><li>Crum, C. 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=The+distal+fallopian+tube:+a+new+model+for+pelvic+serous+carcinogenesis.">The distal fallopian tube: a new model for pelvic serous carcinogenesis.</a> Curr Opin Obstet Gynecol. 19 (1), 3-9 (2007).</li><li>Jarboe, E. 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=Tubal+and+ovarian+pathways+to+pelvic+epithelial+cancer:+a+pathological+perspective.">Tubal and ovarian pathways to pelvic epithelial cancer: a pathological perspective.</a> Histopathology. 53 (2), 127-138 (2008).</li><li>Menon, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+screening+has+no+effect+on+disease-specific+mortality.">Ovarian cancer screening has no effect on disease-specific mortality.</a> Evid Based Med. 17 (2), 47-48 (2012).</li><li>Buys, S. 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=Effect+of+screening+on+ovarian+cancer+mortality:+the+Prostate,+Lung,+Colorectal+and+Ovarian+(PLCO)+Cancer+Screening+Randomized+Controlled+Trial.">Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial.</a> JAMA. 305 (22), 2295-2303 (2011).</li><li>Otsuka, I., Kameda, S., Hoshi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+ovarian+and+fallopian+tube+cancer+by+examination+of+cytological+samples+from+the+endometrial+cavity.">Early detection of ovarian and fallopian tube cancer by examination of cytological samples from the endometrial cavity.</a> Br J Cancer. 109 (3), 603-609 (2013).</li><li>Dhanani, M., Nassar, A., Dinh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+Fallopian+Tube+Cytology+Sampling+to+Develop+a+Model+for+Future+Peritoneal+and+Ovarian+Cancer+Screening.">Classification of Fallopian Tube Cytology Sampling to Develop a Model for Future Peritoneal and Ovarian Cancer Screening.</a> J Am Soc Cytopathol. 3 (5), S35-S36 (2014).</li><li>Rodriguez, E. F., Lum, D., Guido, R., Austin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytologic+findings+in+experimental+in+vivo+fallopian+tube+brush+specimens.">Cytologic findings in experimental in vivo fallopian tube brush specimens.</a> Acta Cytol. 57 (6), 611-618 (2013).</li><li>Chen, H., Klein, R., Arnold, S., Chambers, S., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytologic+studies+of+the+fallopian+tube+in+patients+undergoing+salpingo-oophorectomy.">Cytologic studies of the fallopian tube in patients undergoing salpingo-oophorectomy.</a> Cancer Cell Int. 16, 78 (2016).</li></ol>

The authors declare that they have no competing financial interests.

Prophylactic bilateral salpingectomy or salpingo-oophorectomy has been shown to decrease the risk of developing HGSC in high risk populations, such as women with BRCA gene mutations and strong familial cancer history. However, the sterility and surgical menopause caused by the surgery are serious consequences and make for a difficult decision, especially for women of childbearing age. The previous study has shown excellent correlation between tubal cytology and histology17. We believe that tubal c...

Ovarian high-grade serous carcinoma (HGSC) is the most common and lethal type of ovarian cancer, and remains a major threat to the public health. In the past decade, researchers have suggested that the vast majority of HGSC cases arise from the fallopian tube instead of the ovary itself 1,2,3,4,5,6. Serous tubal intraepithelial carcinoma (STIC) is widely accepted as the precursor of HGSC 1,7,8,9,10,11. So far, early detection of ovarian cancer remains difficult. The current screening protocol with combined cancer antigen 125 (CA-125) and transvaginal ultrasound has shown little effect on patient mortality12,13. Endometrial sampling for ovarian cancer detection has shown an extremely low sensitivity14. Two pioneer studies15,16 explored the usage of laparoscopic direct sampling of benign fallopian tubes and characterized the cytological features of benign tubal epithelium. We believe that direct sampling from the fallopian tube can also be a practical and straightforward way to detecting STIC or HGSC at an early stage when the tumor is not visualized by imaging.
Although the ultimate goal is the utility of minimally invasive laparoscopic screening in patients with high-risk factors, the immediate aims of current study are: 1) To establish the baseline cytological features of benign tubal epithelia; and 2) To test the sensitivity and specificity of tubal cytology in detecting ovarian cancers and cancer precursors. We therefore developed the following baseline tubal cytology method to test the effectiveness of this protocol in detection of HGSC and its precursor STIC17. In this study, we took advantage of the large volume of regularly received surgical specimens, and performed direct sampling of the surgically excised fallopian tube in addition to the routine grossing procedures.
Our recent study17 showed that the cytological diagnosis of malignant or suspicion for malignancy is highly correlated with the histological diagnosis of HGSC (100%). In addition, the tubal cytological evaluation identified intratubal neoplasia in the only two histologically confirmed STIC cases involved in this study. We believe that tubal cytology has great potential in early detection and will provide valuable information for patient management.

<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

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

Our previous study showed that sampling directly from the excised fallopian tube using a gentle touch brush could yield quantitatively and qualitatively adequate specimen for further cytological evaluation. In addition, the combination of automated slide preparation and modified Papanicolaou staining enable the pathologist to better visualize the cytological details to make an accurate diagnosis. An example of a manual quick pap stain of a specimen from a benign fallopian tube is illustra...

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

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

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

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12.2K Views.  University of Arizona College of Medicine. We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Video: 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.

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 ...

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.

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.

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.

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.

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.

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.

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.

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.

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