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.

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.

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 th....

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

1. Transport of tumor tissues and microdissection
   1. Prepare the transport and culture media: complete 10% (v/v) Dulbecco's Modified Eagle Media (DMEM), 10% FBS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin (Pen Strep).
   2. Upon tissue arri.......

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.......

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 .......

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).