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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 others^3,4,5,6.

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) models^10,11. 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 screening^13,14,15. 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.

Protokół

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 arrival and according to an institutional IRB-approved protocol, transfer PMP tumor tissues into 35 dishes with a 6 cm diameter containing complete DMEM.
   3. 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.
2. Agarose preparation and tissue embedding
   1. 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.
   2. 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.
   3. 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 °C refrigerator for 30 min.
3. Mounting tissue specimens on the vibratome
   1. Remove the solidified agar from the refrigerator and cut the agarose cubes slightly larger than the width of the tissue using a scalpel.
   2. 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.
   3. 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.
4. Culturing tissue slices on permeable inserts
   1. Prepare a permeable insert plate for culture by adding 2 mL of complete DMEM media above and 3 mL below the permeable membrane.
   2. 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.
   3. Culture the slices for up to 7 days at 37 °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.
   4. 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).

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 µm 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.

1. Reagent preparation and experimental setup
   1. 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 °C (see section 2.1.8).
   2. 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.
   3. 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.
   4. Open the confocal imaging software. Begin imaging at 10x and select the XYZ imaging mode. Then, configure the acquisition settings as follows.
      1. 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.
   5. Select Remove Interlock, and then select Scan to initiate imaging.
   6. For calcium imaging, incubate tissue slices with Fluo-4-AM for 1 h protected from light. Follow the manufacture'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.
   7. 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.

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.

1. 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.
2. Incubate slices at 37 °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.
3. 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.
4. Once digested, pipette the slices and the digestion media on top of a 70 µm filter placed on top of a 50 mL conical tube. Pick larger pieces using sterile forceps or a pipette.
5. 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.
6. 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.
7. To prepare the sample for flow cytometry, add the blocking buffer containing 50 µL of human Fc-block and leave at room temperature for 15 min. Perform extracellular staining using the respective antibodies in 50 µL of PBS containing 2% FBS.
   ​NOTE: There are many flow cytometry systems. Once the sample is prepared for flow cytometry, refer to the manufacturer's protocol for operation of the device.

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.

1. Prepare 10 mL of 10% v/v complete DMEM with 10% FBS, 2 mM L-Glutamine, 1% Pen Strep.
2. Aliquot 5 mL of complete media for control and treatment conditions. To the remaining 5 mL of media, add bortezomib (2 μM) 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.
3. 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).
4. Leave tissue slices in culture containing complete DMEM for 2-5 days, changing media as well as bortezomib and 5-FU every other day.
5. For luminescent viability analysis, add 500 µL 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.
6. Remove the PBS and add the luminescent viability analysis solution that was prepared. See the instruction manual for preparation of the reagent.
7. Add 500 µL 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.

Wyniki

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...

Dyskusje

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 ...

Materiały

Name	Company	Catalog Number	Comments
1 M CaCl2 solution	Sigma	21115
1 M HEPES solution	Sigma	H0887
1 M MgCl2 solution	Sigma	M1028
100 micron filter	ThermoFisher	22-363-549
22 x 40 glass coverslips	Daiggerbrand	G15972H
3 M KCl solution	Sigma	60135
5 M NaCl solution	Sigma	S5150
ATPγS	Tocris	4080
Bovine Serum Albumin	Sigma	A2153
Calcein-AM	Invitrogen	L3224
CD11b	Biolegend	101228
CD206	Biolegend	321140
CD3	Biolegend	555333
CD4	Biolegend	357410
CD45	Biolegend	304006
CD8	Biolegend	344721
CellTiter-Glo	Promega	G9681
DMEM	Thermo Fisher	11965084
DPBS	Sigma Aldrich	D8537
FBS, heat inactivated	ThermoFisher	16140071
Fc-block	BD Biosciences	564220
Fluo-4	Thermo Fisher	F14201
Gentle Collagenase/Hyaluronidase	Stem Cell	7912
Imaging Chamber	Warner Instruments	RC-26
Imaging Chamber Platform	Warner Instruments	PH-1
LD-Blue	Biolegend	L23105
L-Glutamine 200 mM	ThermoFisher	25030081
LIVE/DEAD imaging dyes	Thermofisher	R37601
Nikon Ti microscope	Nikon		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.
Peristaltic pump	Isamtec	ISM832C
Propidium Iodide	Invitrogen	L3224
Vacuum silicone grease	Sigma	Z273554-1EA