Name: culture and imaging of ex vivo organotypic pseudomyxoma peritonei tumor slices from resected human tumor specimens
Uploaded: 2022-12-09T13:55:00
Description: 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

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 arri...

<ol>
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W., Govindarajan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+mouse+models+of+pseudomyxoma+peritonei+recapitulate+the+human+inflammatory+tumor+microenvironment.">Patient-derived xenograft mouse models of pseudomyxoma peritonei recapitulate the human inflammatory tumor microenvironment.</a> Cancer Medicine. 5 (4), 711-719 (2016).</li><li>Jiang, X., 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=Long-lived+pancreatic+ductal+adenocarcinoma+slice+cultures+enable+precise+study+of+the+immune+microenvironment.">Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment.</a> Oncoimmunology. 6 (7), 1333210 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Liu, L., Yu, L., Li, Z., Li, W., Huang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+organoid+(PDO)+platforms+to+facilitate+clinical+decision+making.">Patient-derived organoid (PDO) platforms to facilitate clinical decision making.</a> Journal of Translational Medicine. 19 (1), 40 (2021).</li><li>Croft, C. 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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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.

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			<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 ...

This research demonstrates a novel approach to the study of appendiceal cancer therapeutics and pathobiology in a preclinical setting. These methods may be more broadly applicable to the study of other malignancies. Appendiceal cancer is difficult to study given its low incidence in the absence of an appendix in mice for disease modeling.This technique allows the study of cell interactions within the tumor microenvironment. This method can be applied to study the biology of multiple types of cancers that have the propensity to metastasize to the omentum, such as colorectal and ovarian cancers. Begin by preparing the transport and culture media.Upon tissue arrival, transfer the pseudomyxoma peritonei tumor tissues into 35 complete DMEM containing six centimeter diameter dishes. In addition to removing any liquified mucin, or highly mucinous tissue regions, remove any tissue that is difficult to cut with a scalpel. Cut tumor tissue nodules roughly into one cube centimeter smaller pieces.for agarose preparation and tissue embedding, prepare a 5%solution of low-melt agarose in PBS without fetal bovine serum or FBS on the same day if tissues arrival. Add the liquid 5%agarose solution to the six centimeter dishes containing two to three smaller tumor specimens until it covers the specimen. Place the embedded tissues in a four degree Celsius refrigerator for 30 minutes.Demount the tissue specimens on the vibratome, remove the solidified auger from the refrigerator, and cut the agarose cubes slightly larger than the width of the tissue using a scalpel. Apply super glue to the vibratome stage and gently place the agarose cube onto the super glue for one to two minutes for fixing. Fix the blade to the vibratome, and set the thickness of the tissue section to roughly 150 to 250 microns for optimal downstream imaging.To culture the tissue slices, prepare a permeable insert plate by adding two milliliters of complete DMEM media above the permeable membrane and three milliliters below the permeable membrane. Next, 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. Culture the slices for up to seven days at 37 degrees Celsius in 5%carbon dioxide.During media replacement, every 24 hours, add bortezomib as controls for cell killing, and testable chemotherapies 5-Fluorouracil, or 5-FU, to the culture media to perform pharmacological intervention. 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 an extra cellular calcium solution.Stain samples with imaging dyes to determine the viability of tissue slices. Incubate for 10 minutes to one hour after adding the viability dye. 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.Cut a small piece from the end of a one milliliter pipette tip to widen the opening, allowing vigorous mechanical dissociation by pipetting. Incubate slices at 37 degrees Celsius with rotation for five to 15 minutes in one milliliter of digestion buffer, with vigorous tissue disruption two to three times during incubation using mechanical dissociation. Once the tissue is digested, transfer the disrupted tissue with digestion media on a 70 micrometer filter placed on top of a 50 milliliter conical tube.Pick larger pieces using sterile forceps or a pipette. Using the blunt backend of a plastic five milliliter syringe, mash any larger undissociated slices and wash them with four milliliters of PBS containing 2%FBS. Take the dissociated cell supernatant and centrifuge at 300G for five to 10 minutes.Remove the supernatant and wash the pellet with one milliliter of PBS containing 2%FBS. To prepare the sample for flow cytometry, add the blocking buffer containing 50 microliters of human FC block, and incubate at room temperature for 15 minutes. Perform extra cellular staining using the respective antibodies in 50 microliters of PBS containing 2%FBS.Aliquot five milliliters of complete media for control and treatment conditions. On the two to four slices plated onto the permeable dishes, add the media from the control treatment aliquots and the additional combination therapies to be used for downstream viability, live dead analysis. Leave tissue slices in a culture containing complete DMEM for two to five days, changing media as well bortezomib and 5-FU every other day.For luminescent viability analysis, add 500 microliters of PBS into a 12-well dish, and transfer the sequentially matched tissue slices to the PBS solution using a paintbrush or a one milliliter pipette. Remove the PBS from the slices. Add 500 microliters of the luminescent viability solution per condition and incubate with a slow rotation on the shaker at room temperature for 30 minutes before reading the luminescence using a luminescence plate reader.The mucin staining in tissue slices was visualized using periodic acid shift staining and mucin-specific antibodies. Incubation in calcein AM and propidium iodide determined the health and viability. The nuclei overlapped the cells proliferated during culture.These results were quantified to determine the number of proliferating cells within the tumor slice. The slices incubated in CD11b PE conjugated antibody and Fluo-4 AM, labeled the local immune cells in CTU. Pseudocolor scaled images showing intracellular calcium levels allowed visualizing differential levels of intracellular calcium.Spontaneous activity tracked and time lapsed for before, during and after intercellular calcium response within the highlighted CD11b positive immune cell is shown here. The raw traces of calcium responses in the CD11b immune cell, along with other responsive and non-responsive cells were quantified. Representative results of immunotyping of a patient tumor sample with pseudomyxoma peritonei are shown here.Quantification of the tumor immuno profile showed that the donor specimen 337 has high levels of M2 macrophages, indicating that utilizing pseudomyxoma peritonei-derived tissue slices allowed the interrogation of the unique donor cellular landscape of the patient tumor samples. Pharmacotyping and downstream analysis of tissue slices from pseudomyxoma peritonei human tissues showed the killing of cells in tumor slices as confirmed by cytotoxicity analysis and tunnel confocal imaging. In response to the cytotoxic drug, bortezomib and 5-fluorouracil.Luminescent viability analysis was performed using sequentially matched slices. Successful production of slices from mucinous tumors requires meticulous dissection and removal of cellular and acellular tissue components like fat and dense abdominal wall tissue. This technique can allow for modeling interactions between cancer cells and multiple cell types in the tumor microenvironment, such as adipose, vascular and immune cell interactions.

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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

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 ...

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

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 ...

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. ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

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 ...