The authors would like to thank Kersi Pestonjamasp from the Moores Cancer Center imaging core facility for help with the microscopes UCSD Specialized Cancer Support Center P30 grant 2P30CA023100. This work was additionally supported by a JoVE publication grant (JRW), as well as generous gifts from the estate of Elisabeth and Ad Creemers, the Euske Family Foundation, the Gastrointestinal Cancer Research Fund, and the Peritoneal Metastasis Research Fund (AML).

The deidentification and acquisition of all tissues were performed under an IRB-approved protocol at the University of California, San Diego.
1. Preparation of human PMP tissues for tissue processing and culture
<ol>
	<li>Transport of tumor tissues and microdissection
	<ol>
		<li>Prepare the transport and culture media: complete 10% (v/v) Dulbecco&#39;s Modified Eagle Media (DMEM), 10% FBS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin (Pen Strep).</li>
		<li>Upon tissue arrival and according to an institutional IRB-approved protocol, transfer PMP tumor tissues into 35 dishes with a 6 cm diameter containing complete DMEM.</li>
		<li>Micro-dissect solid regions of the tumor from any liquefied mucin using a scalpel. While mucin nodules embedded within solid portions of the tissue can be cut, remove all liquified regions of the tumor. In addition to removing any liquified mucin, or highly mucinous tissue regions, remove any tissue that is difficult to cut with a scalpel. Cut tissue nodules into smaller pieces, roughly 1 cm3 in size. 
		NOTE: Obtaining tumor nodules cleaned of mucin and dense ECM will be essential for successful cutting using a vibratome. As quality control, tumor tissue that arrives within the research laboratory is first cut by a pathologist, which is subsequently distributed by the UCSD biorepository. Tissue donors who undergo palliative debulking of the omental cake are optimal cases for this protocol given a high disease burden, which enables more slice production from the increased mass of tissue availability for research. 
		CAUTION: Working with human tissues requires Biosafety Level 2 (BSL2) certification. Check with the institution for training on BSL2 procedures.</li>
	</ol>
	</li>
	<li>Agarose preparation and tissue embedding
	<ol>
		<li>Prepare a 5% solution of low melt agarose in PBS without FBS on the same day of tissue arrival. Pay close attention to the agarose solution as it tends to boil quickly. 
		NOTE: Low melt agarose is essential for embedding tissue pieces. High melt agarose will solidify at higher temperatures, reducing the viability of tissue slices if embedded. Additionally, FBS in the dissolving solution will cause precipitates to form.</li>
		<li>Place the tissue pieces obtained in 6 cm dishes without liquid. Place no more than 2-3 pieces per 6 cm dish so that they can be removed as agarose cubes without disturbing or touching other tissues.</li>
		<li>Add the liquid 5% agarose solution to 6 cm dishes containing the 1 cm3 tumor specimens. Add enough agarose to just cover the specimen. Once added, place the embedded tissues in a 4 &#176;C refrigerator for 30 min.</li>
	</ol>
	</li>
	<li>Mounting tissue specimens on the vibratome
	<ol>
		<li>Remove the solidified agar from the refrigerator and cut the agarose cubes slightly larger than the width of the tissue using a scalpel.</li>
		<li>Apply super glue to the vibratome stage and gently place the agarose cube onto the super glue. Allow 1-2 min for the glue to solidify. At this point, the agarose cube with tissue should be fixed to the stage.</li>
		<li>Fix the blade to the vibratome and set the desired thickness of the tissue section. For optimal downstream imaging using confocal microscopy, sections should be roughly 150 to 250 microns. Hit the Start button and continue to cycle through cutting of the agarose tissue block until the tissue is completely cut. 
		NOTE: If the tissue is not cutting, or dislodges from the agarose cube, consider embedding the tissue in a higher concentration agarose and trim off any additional tissue pieces that may be too dense to cut or sticks to the vibratome blade.</li>
	</ol>
	</li>
	<li>Culturing tissue slices on permeable inserts
	<ol>
		<li>Prepare a permeable insert plate for culture by adding 2 mL of complete DMEM media above and 3 mL below the permeable membrane.</li>
		<li>Gently lift the tissue slices out of the cutting chamber of the vibratome using a thin paintbrush and place two to four slices into permeable culture dishes containing complete DMEM media. After 24 h, aspirate the media using a pipette and replace the media with fresh culture media.</li>
		<li>Culture the slices for up to 7 days at 37 &#176;C/5% CO2, replacing the media every 24 h. At this time point, add controls for cell killing (bortezomib) and testable chemotherapies 5-fluoruracil (5-FU) to the culture media to perform pharmacological intervention.</li>
		<li>After 7 days of culturing, expect about a 15%-25% loss of viability compared to the day 0 time point (immediately after cutting) under non-drug treated conditions (complete DMEM). Measure the viability using live-dead viability dyes such as calcein AM (live) and propidium Iodide (dead).</li>
	</ol>
	</li>
</ol>
2. Confocal imaging of living human tissue slices
NOTE: Once the organotypic tumor slices have been prepared, it is essential to determine the tissue viability to perform an efficient and optimized downstream analysis. Given that tumor specimens are 150-250 &#181;m thick, confocal, or two-photon microscopy is highly recommended over wide-field microscopes for determining studies in situ. Flow cytometry can also be used for determining the viability and cellular populations (methods are below). However, the spatial resolution is lost during flow cytometry analysis, and viability is likely underestimated given the need to disassociate tumor slices by mechanical and chemical methods.
<ol>
	<li>Reagent preparation and experimental setup
	<ol>
		<li>Place tumor slices for confocal imaging analysis in a single well of a 12-well dish containing PBS with 1% FBS. For calcium imaging experiments, instead of PBS, incubate the slices in extracellular calcium solution prepared as follows: 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, pH 7.4, 3 mM glucose, sterile filtered. Prepare this solution ahead of time and store it for up to 1 month at 4 &#176;C (see section 2.1.8).</li>
		<li>Following the concentrations and protocols recommended by the manufacturer, stain samples with imaging dyes (see Table of Materials) to determine the viability of tissue slices. Image for 10 min-1 h after adding the viability dye.</li>
		<li>After incubation, transfer the slices to an optically clear, glass bottom Petri dish containing PBS with 1% FBS to be used for imaging on a confocal imaging device. 
		NOTE: For confocal imaging, the Nikon A1R Confocal platform was used.</li>
		<li>Open the confocal imaging software. Begin imaging at 10x and select the XYZ imaging mode. Then, configure the acquisition settings as follows.
		<ol>
			<li>Turn on the following lasers: 488 nm and 561 nm. Adjust the gain to 100 with laser power at 1%. Adjust the laser power and gain as needed during image acquisition. Depending on the image quality desired, select 512 x 512 pixels or 1024 x 1024 pixels for higher resolution.</li>
		</ol>
		</li>
		<li>Select Remove Interlock, and then select Scan to initiate imaging.</li>
		<li>For calcium imaging, incubate tissue slices with Fluo-4-AM for 1 h protected from light. Follow the manufacture&#39;s protocol for dissolving Fluo-4-AM in DMSO. After 1 h, wash slices 3x with extracellular calcium solution and follow the imaging protocol in steps 2.1.4-2.1.5.</li>
		<li>For calcium imaging experiments using Fluo-4-AM, select XYZT and unselect the 561 nm laser to increase acquisition speed. Additionally, use the resonance scanner to further increase the speed of imaging.</li>
	</ol>
	</li>
</ol>
3. Disassociation of living slices for flow cytometry
NOTE: Disassociation of living tissue slices can be used for several downstream applications, including immunotyping, assessment of the viability, and interrogation of the changes to cell populations after pharmacological intervention. Steps should be taken to ensure that tissue quality and cell viability are maintained during the disassociation process.
<ol>
	<li>Cut a small piece from the end of a 1 mL pipette tip to widen the opening to allow vigorous mechanical dissociation by pipetting for 1 min.</li>
	<li>Incubate slices at 37 &#176;C with rotation for 5-15 min in 1 mL of digestion buffer consisting of high glucose DMEM, 10% gentle collagenase/hyaluronidase (GCH), 10% FBS, and 10% DNaseI (1 mg/mL stock). 
	NOTE: Batch-dependent changes of collagenase/hyaluronidase are often found.</li>
	<li>Perform vigorous disruption of the tissue using mechanical dissociation 2-3 times during incubation. Be sure to check digestion, by eye, every 5 min for evidence of complete digestion. If needed, stop the enzymatic digestion by proceeding to step 3.1.4.</li>
	<li>Once digested, pipette the slices and the digestion media on top of a 70 &#181;m filter placed on top of a 50 mL conical tube. Pick larger pieces using sterile forceps or a pipette.</li>
	<li>Using the blunt back end of a plastic 5 mL syringe, mash any larger undissociated slices and further wash with 4 mL of PBS containing 2% FBS.</li>
	<li>Take the disassociated cell supernatant in a 50 mL conical tube and spin at 300 x g for 5-10 min. Remove the supernatant and wash the pellet with 1 mL of PBS containing 2% FBS.</li>
	<li>To prepare the sample for flow cytometry, add the blocking buffer containing 50 &#181;L of human Fc-block and leave at room temperature for 15 min. Perform extracellular staining using the respective antibodies in 50 &#181;L of PBS containing 2% FBS. 
	&#8203;NOTE: There are many flow cytometry systems. Once the sample is prepared for flow cytometry, refer to the manufacturer&#39;s protocol for operation of the device.</li>
</ol>
4. Pharmacological intervention using living tissue slices for viability and proliferation analysis
NOTE: Once the organotypic tumor slices have been prepared, interventional approaches can be performed by using several methods, including drug testing, siRNA, as well as viral infection of living tissue slices. Here we will discuss drug testing, as well as downstream functional readouts, which include viability analysis using local proliferation.
<ol>
	<li>Prepare 10 mL of 10% v/v complete DMEM with 10% FBS, 2 mM L-Glutamine, 1% Pen Strep.</li>
	<li>Aliquot 5 mL of complete media for control and treatment conditions. To the remaining 5 mL of media, add bortezomib (2 &#956;M) to induce cell death in tumor slices as a positive control. 
	NOTE: Bortezomib, at high concentrations, is a cytotoxic drug that induces cell death. The other cytotoxic drugs may be used as positive control for cell killing.</li>
	<li>Using two to four tissue slices, which have been plated onto the permeable dishes, add the media prepared in step 4.2 to the dish, as well as any additional combination therapies to be used for downstream viability LIVE/DEAD analysis using confocal microscopy as described in step 2.1.2, or use a commercial luminescent viability analysis using sequentially matched slices. 
	NOTE: Sequential slice matching is essential for luminescent viability analysis, given that luminescent viability assay relies on cellular ATP content. As internal controls are lost during cell lysis, similar-sized slices are required since results are dependent on the starting number of viable cells (i.e., unbalanced slices will result in unbalanced results). If the user does not obtain sequential slices, luminescent viability analysis is not possible, and as such another viability assessment method will be required (flow cytometry or confocal imaging based live cell analysis).</li>
	<li>Leave tissue slices in culture containing complete DMEM for 2-5 days, changing media as well as bortezomib and 5-FU every other day.</li>
	<li>For luminescent viability analysis, add 500 &#181;L of PBS into a 12-well dish and transfer the sequentially matched tissue slices to the PBS solution using a paintbrush or use the suction of a 1 mL pipette gently to transfer these.</li>
	<li>Remove the PBS and add the luminescent viability analysis solution that was prepared. See the instruction manual for preparation of the reagent.</li>
	<li>Add 500 &#181;L of the luminescent viability solution per condition and incubate with a slow rotation on the shaker at room temperature for 30 min. Read luminescence using a luminescence plate reader.</li>
</ol>

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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=The+practicalities+of+using+tissue+slices+as+preclinical+organotypic+breast+cancer+models.">The practicalities of using tissue slices as preclinical organotypic breast cancer models.</a> Journal of Clinical Pathology. 66 (3), 253-255 (2013).</li><li>Koerfer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+human+gastric+and+esophagogastric+junction+cancer.">Organotypic slice cultures of human gastric and esophagogastric junction cancer.</a> Cancer Medicine. 5 (7), 1444-1453 (2016).</li><li>Misra, 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=Ex+vivo+organotypic+culture+system+of+precision-cut+slices+of+human+pancreatic+ductal+adenocarcinoma.">Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.</a> Scientific Reports. 9 (1), 2133 (2019).</li><li>Ohnishi, T., Matsumura, H., Izumoto, S., Hiraga, S., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+glioma+cell+invasion+using+organotypic+brain+slice+culture.">A novel model of glioma cell invasion using organotypic brain slice culture.</a> Cancer Research. 58 (14), 2935-2940 (1998).</li><li>Seidman, J. 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L., Futch, H. S., Moore, B. D., Golde, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+to+model+neurodegenerative+proteinopathies.">Organotypic brain slice cultures to model neurodegenerative proteinopathies.</a> Molecular Neurodegeneration. 14 (1), 45 (2019).</li><li>Carr, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+in+the+pathology+of+peritoneal+surface+malignancy.">New insights in the pathology of peritoneal surface malignancy.</a> Journal of Gastrointestinal Oncology. 12, 216-229 (2021).</li><li>Votanopoulos, K. I., 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=Outcomes+of+repeat+cytoreductive+surgery+with+hyperthermic+intraperitoneal+chemotherapy+for+the+treatment+of+peritoneal+surface+malignancy.">Outcomes of repeat cytoreductive surgery with hyperthermic intraperitoneal chemotherapy for the treatment of peritoneal surface malignancy.</a> Journal of the American College of Surgeons. 215 (3), 412-417 (2012).</li><li>Weitz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-vivo+organotypic+culture+platform+for+functional+interrogation+of+human+appendiceal+cancer+reveals+a+prominent+and+heterogenous+immunological+landscape.">An ex-vivo organotypic culture platform for functional interrogation of human appendiceal cancer reveals a prominent and heterogenous immunological landscape.</a> Clinical Cancer Research. 28 (21), 4793-4806 (2022).</li><li>Pitoulis, F. 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The authors declare that they have no competing financial interests.

This manuscript describes a technique that can be used to culture, interrogate, and analyze human pseudomyxoma peritonei (PMP) tumor specimens. We have utilized numerous downstream functional assays to interrogate the tumor immune microenvironment and a platform for bench-to-bedside testing.
While the method is highly efficient in our hands, it will require some practice to cut tumor specimens using a vibratome. Namely, we encountered problems that were due to highly mucinous samples, as well ...

Pseudomyxoma peritonei (PMP) is rare syndrome with an incidence rate of 1 per million people per year1. Most PMP cases are caused by metastases from appendiceal neoplasms. Given that mice do not have a human-like appendix, modeling this type of cancer remains extremely challenging. While the primary disease is often curable by surgical resection, treatment options for metastatic disease are limited. Therefore, the rationale for developing this novel organotypic slice model is to study the pathobiology of PMP. To date, there are no appendiceal organoid models that can be perpetually cultured; however, a recent model was shown to be useful for the pharmacological testing of therapeutic agents and immunotherapy2. As such, we have adapted an organotypic slice culture system, which has been used in other types of human cancers, such as brain, breast, pancreas, lung, ovarian, and others3,4,5,6.
In addition to appendiceal neoplasms, PMP occasionally results from other tumor types, including ovarian cancers7, and in rare circumstances, intraductal papillary mucinous neoplasms8 and colon cancer9. Additionally, these tumors tend to grow slowly, with poor engraft rates in patient-derived xenograft (PDX) models10,11. Given these challenges, there is an unmet need to develop models to study this disease to begin to understand the pathobiology of PMP, and how these cancer cells: are recruited to the peritoneal surfaces, proliferate, and escape immune surveillance.
While cut from the systemic vascular circulation, tumor slices do contain cellular and acellular components, including the extracellular matrix, stromal cells, immune cells, cancer cells, endothelial cells, and nerves. This semi-intact microenvironment allows for the functional investigation of these cell types, which is uniquely advantageous compared to 3D organoid cultures, which consist only of cancer cells12. While organotypic slice cultures are advantageous in some respects, they are also inherently a low-throughput-based approach, compared to 3D organoids, which can be expanded, and are suitable for multiplexed investigational therapeutic drug screening13,14,15. In the case of PMP, there have been no reports documenting reliable establishment and perpetual passaging of PMP-derived organoids16. This is likely due to the slow growing nature of PMP-derived tumor cells, as well as the low number of malignant epithelial cells found within these mucinous tumors. Given the need to develop models to study PMP, organotypic slices are uniquely suited to study this disease. We present a protocol for preparing, imaging, and analyzing PMP from human specimens.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M CaCl2 solution</td>
			<td>Sigma</td>
			<td>21115</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES solution</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2 solution&#160;</td>
			<td>Sigma</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>100 micron filter</td>
			<td>ThermoFisher</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 40 glass coverslips</td>
			<td>Daiggerbrand</td>
			<td>G15972H</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M KCl solution</td>
			<td>Sigma</td>
			<td>60135</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl solution</td>
			<td>Sigma</td>
			<td>S5150</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP&#947;S&#160;</td>
			<td>Tocris&#160;</td>
			<td>4080</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM&#160;</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>CD206&#160;</td>
			<td>Biolegend</td>
			<td>321140</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Biolegend</td>
			<td>555333</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4&#160;</td>
			<td>Biolegend</td>
			<td>357410</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45&#160;</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Biolegend</td>
			<td>344721</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo&#160;</td>
			<td>Promega</td>
			<td>G9681</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Thermo Fisher</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, heat inactivated</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block&#160;</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4</td>
			<td>Thermo Fisher</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Collagenase/Hyaluronidase&#160;</td>
			<td>Stem Cell</td>
			<td>7912</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Chamber Platform</td>
			<td>Warner Instruments</td>
			<td>PH-1</td>
			<td></td>
		</tr>
		<tr>
			<td>LD-Blue&#160;</td>
			<td>Biolegend</td>
			<td>L23105</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD imaging dyes</td>
			<td>Thermofisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti microscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Includes: A1R hybrid confocal scanner including a high-resolution (4096x4096) scanner, LU4 four-laser AOTF unit with 405, 488, 561, and 647 lasers, Plan Apo 10 (NA 0.8), 20X (NA 0.9) dry objectives.&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump&#160;</td>
			<td>Isamtec</td>
			<td>ISM832C</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum silicone grease</td>
			<td>Sigma</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture and imaging of ex vivo organotypic pseudomyxoma peritonei tumor slices from resected human tumor specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

In short, human tumor specimens from PMP are obtained under an IRB-approved protocol. The tissue is prepared, micro-dissected, and solidified in an agarose mold to be cut using a vibratome (Figure 1A; Video 1). Once cut, tissue slices are placed and cultured on permeable insert membranes (Figure 1B), which can be utilized for imaging assays in situ, as well as for cellular and functional interrogation using flow cytometry, confocal imag...

Watch this Scientific Journal Video about Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens at JoVE.com

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

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Research

JoVE Journal

Cancer Research

1.6K Views.  University of California. We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Video: Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

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.

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

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...