Processing and Analysis of Ascites

Abstract

The accumulation of peritoneal fluid, referred to as ascites, is common in ovarian cancer. This fluid is a complex mixture that may include cells as well as a diverse array of cytokines and growth factors. Here we describe a comprehensive method to process ascites to maximize data collection. The cellular fraction and fluid are first separated by centrifugation. The fluid can be frozen for later analysis of soluble factors or for use in in vitro experiments. The cellular fraction can be processed to analyze its composition or stored for future use.

1. Introduction

Abnormal peritoneal fluid accumulation, called ascites, occurs in more than one third of patients with ovarian cancer [1]. Ascites results from leaky vasculature and increased lymphatic drainage associated with cancer-induced inflammation. The presence of ascites has been linked to a poor prognosis in patients, with 89% of advanced-stage patients (stages III and IV) displaying ascites [1]. Additionally, larger ascites volumes have been correlated with shorter overall survival and suboptimal cytoreductive surgery [2]. Ascites is a complex mixture and frequently includes soluble growth factors and cytokines, as well as several cell types (e.g., tumor, mesothelial, and immune). As such, ascites likely contributes to the continued expansion of the tumor and conditioning of the tumor microenvironment. Ascites can be removed during cytoreductive surgery or by paracentesis, providing an opportunity to study its composition and effects on tumor progression.

The use of ascites in in vitro models has shown that this fluid contains soluble factors that facilitate crosstalk between diverse cell types in the tumor microenvironment, with the potential to promote migration, invasion, and adhesion. For example, when ascites was added to wound healing migration and transwell invasion assays of ovarian cancer cells, both migration and invasion increased [3]. Through the incorporation of ascites into in vitro models of ovarian cancer cells and mesothelial cells, we recently determined that MIP-1β in ascites led to changes in mesothelial cell expression of P-selectin, resulting in increased adhesion of ovarian cancer cells [4]. In addition to soluble factors, tumor cells can be isolated from ascites for study. Epithelial tumor cells in ascites can be found both as single cells and in multicellular aggregates; tumor cells in aggregates may have advantages over single cells due to increased chemoresistance and anoikis resistance [5–7]. While most studies model these aggregates through formation of cellular spheroids by in vitro methods (e.g., hanging drop, microwells [8,9]), we describe here a process to isolate aggregates from patient ascites. In addition to tumor cells, the ascites often contains immune and stromal cells; the ratio of epithelial to stromal cells has been shown to be higher in chemo-naïve patients [10].

Thus, collection and analysis of ascites is a useful practice to garner valuable insights into the progression of ovarian cancer. This chapter will detail approaches to collect, store, and utilize this fluid, as well as how to isolate and prepare the cellular fraction of the ascites for downstream analysis.

2. Materials

Prepare all solutions with ultrapure deionized water (18.2 MΩ-cm) at 25°C. All pipettes, pipette tips, tubes, and other disposable waste should be bagged in a biohazard waste bag. This waste should be disposed of properly as human biohazard waste in accordance with institutional policies.

2.1. Ascites Processing and Analysis

1. Heat-inactivated fetal bovine serum (HI FBS)

2. 50 mL conical tubes, sterile

3. 1.5 mL low binding microcentrifuge tubes, sterile

4. Benchtop centrifuge with temperature control

2.2. Cellular Processing, Storage, and Analysis

1. HI FBS

2. Trypan Blue

3. DMSO

4. Cryovials

5. Cell Freezing Container

6. Isopropanol

7. Liquid nitrogen storage for cell stocks

8. Cell Strainers, 40 μm

9. 24-well plates

10. PBS, sterile

11. 4% paraformaldehyde (PFA) in PBS (v/v)

12. 1.5% agarose in water (w/v), made fresh during procedure

13. Microwave (approximately 1000 W)

14. Microscope with phase optics and camera

15. Computer with ImageJ (FIJI) software installed [11]

3. Methods

Ascites can be obtained from debulking surgery for ovarian cancer or from paracentesis. Institutional Review Board (IRB) approval and patient consent is needed, but the specifics will vary depending on your institution. At many research universities a biospecimen collection facility can collect and de-identify ascites to simplify administrative burden. Clear records should be kept regarding each samples identity that comply with local IRB regulations.

Ascites should be kept on ice for the duration of processing. Processing should be done in a sterile laminar flow biosafety cabinet.

3.1. Ascites Collection and Storage

1. Pipette ascites up and down several times to resuspend any settled cells.

2. Aliquot the ascites into 50 mL conical tubes.

3. Using a temperature-controlled centrifuge, spin down the ascites at 300 g for 5 min at 4°C. After spinning, there should be a visible cell pellet at the bottom of the tube (Figure 1A, see Note 1).

4. Collect the supernatant, taking care to avoid tissue pieces that may be in the fluid and the cell pellet (Figure 1B).

5. Aliquot the supernatant into 1.5 mL microcentrifuge tubes to collect the desired number of 1 mL samples (see Notes 2 – 3).

6. Aliquot the remaining supernatant into 50 mL conical tubes by pipetting ~45 mL of supernatant per tube (see Note 4).

7. Label all tubes with sample code (see Note 5). Freeze all fluid samples at −80°C.

8. Record the number of tubes and where they are stored in your lab’s sample management system. These samples can be analyzed later for cytokines and growth factors (see Note 6) or used for in vitro culture experiments (see Note 7).

Figure 1.

Representative images taken during the fluid collection and aggregate separation process. A) Ascites after spinning at 300 g for 5 minutes at 4°C. Two different samples are shown demonstrating the variability in bloodiness (see Note 1). B) Large piece of tissue floating in ascites following centrifugation; these are discarded before proceeding. C) Separate tubes of ascites cell suspension in FBS for different applications of ascites cells (see Note 8). D) Aggregates become trapped in the cell strainer (top), where single cells will pass through into the 50 mL conical tube (bottom). E) After inverting the cell strainer on top of a new 50 mL conical tube and passing FBS through the cell strainer, the aggregates are collected into the 50 mL conical tube.

3.2. Initial Cell Suspension

1. After the fluid has been removed, remove any large tissue pieces (not the cell pellet) from the 50 mL conical tubes with a pipette. This tissue can be discarded (Figure 1B).

2. Resuspend each cell pellet in 12 mL HI FBS.

3. Combine all cell pellets and bring to an appropriate total volume with additional HI FBS based on the planned number of applications (Figure 1C, see Note 8). Split the combined cellular fraction, with 12 mL of solution transferred to a 50 mL conical tube for each application. These tubes are referred to as ‘initial cell suspension’ in later steps.

3.3. Cryopreservation of Ascites Cells

1. Mix one tube of the initial cell suspension using a 1 mL pipette.

2. Dilute cell suspension 1:1 with Trypan Blue and load Trypan Blue-cell solution to a hemocytometer and count cells (see Note 9).

3. Spin down 50 mL conical tube of cell suspension at 300 g for 5 minutes at 4°C.

4. While cells are spinning down, prepare freezing medium (10% v/v DMSO in HI FBS) and label cryovials with sample code (see Note 5).

5. Aspirate the supernatant (this is HI FBS, and not ascites) and resuspend to a concentration of 1 × 10⁶ cells/mL in freezing medium.

6. Pipette 1 mL of cell solution to each cryovial.

7. Add cryovials to cell freezing container with isopropanol and place overnight in −80°C freezer.

8. The next day, move cells from freezing container to liquid nitrogen storage and inventory their location.

3.4. Separation of Aggregates and Single Cells

1. Mix one tube of the initial cell suspension using a 5 mL pipette to homogenize cell solution.

2. Collect cell suspension with a pipette and pass through 40 μm cell strainer fitted into a new 50 mL conical tube. The solution that passes through the filter will be the single cell solution. These cells can be frozen as in Section 3.3 or analyzed by flow cytometry (see Note 10).

3. Carefully flip the cell strainer upside down on top of a new 50 mL conical tube. Pass 12 mL of HI FBS through the upside-down cell strainer to collect any aggregates. There will be a drop of FBS that does not pass through the strainer – tilt the cell strainer on the edge of the 50 mL conical tube to release this drop into the tube. The solution that been released from the filter is the aggregate solution (Figure 1D).

3.5. Analysis of Aggregate Size Distribution

1. Separate aggregates from single cells as described in Section 3.4.

2. In a 24-well plate, pipette 0.5 mL of aggregate solution (what has passed through the filter when inverted) into each well.

3. Using a phase microscope, take images of aggregate or single cells per well in the phase channel (Figure 2A, see Note 11).

4. Open cell aggregate images in ImageJ/FIJI [11].

5. Make the image binary by selecting Process: Binary: Make Binary (Figure 2B).

6. Fill holes in the aggregates by selecting Process: Binary: Fill Holes (Figure 2C). The aggregates will now appear as single objects.

7. Analyze particles with size exclusion 1257 μm² – infinity by selecting Analyze: Analyze Particles (Figure 2D, see Notes 12 – 13).

Figure 2.

Representative images in the aggregate image analysis process. A) Unprocessed phase image of ascites aggregates. B) Image after conversion to binary. C) Image after steps to fill holes, note that aggregates now appear as single objects. D) Outlines of analyzed particles, comparison of C and D demonstrates the particles are indeed aggregates.

3.6. Embedding Aggregates for Histological Analysis

1. Separate aggregates from single cells as described in Section 3.4.

2. Spin down the aggregate tube at 300 g for 5 min at 4°C.

3. Aspirate supernatant from aggregate tube, taking care not to aspirate cell pellet.

4. Add 5 mL of PBS to aggregate tube and transfer aggregates to 15 mL conical tube.

5. Resuspend aggregates by gently pipetting up and down until solution is well mixed.

6. Spin down the aggregate tube once more at 300 g for 5 min at 4°C. The remainder of the protocol can be performed in non-sterile conditions.

7. Aspirate supernatant.

8. Add 1 mL of 4% PFA and pipette to mix.

9. Incubate for 15 minutes at room temperature to fix the cells.

10. During incubation, prepare agarose embedding medium composed of 1.5% agarose in ultrapure water. To melt the agarose, microwave on high at 1000 W for 3 minutes until in solution, swirling every 30 seconds.

11. After cells are fixed, spin down the spheroid tube at 300 g for 5 min.

12. Aspirate PFA and add 0.5 mL of the liquid agarose to the spheroid tube.

13. Pipette up and down to distribute spheroids. Using a 1 mL pipette, take up the entire volume into the pipette tip and transfer spheroid-agarose solution to a 1.5 mL microcentrifuge tube. Pipette up and down once more to distribute (Figure 3, see Note 14).

14. Spheroids will become embedded into the agarose as the agarose solidifies. Store at room temperature.

15. This agarose plug can be subsequently paraffinized and sectioned for histological staining [12].

Figure 3.

Representative images of ascites aggregates embedded in agarose.

4. Notes

1. The volume of blood that is incorporated into ascites can vary widely from patient to patient. While we have not observed obvious trends in our measurements that correlate to the amount of blood, cell counting is considerably more challenging due to the large number of red blood cells that remain.

2. This is done as most assays only require a small volume of ascites for analysis. 1 mL aliquots are more convenient and prevents freeze-thaw cycles compared to larger aliquots. As the total volume of ascites is often large (>1 L is common), it may be impractical to save all of the fluid. Fluid volumes from patients treated with neoadjuvant chemotherapy may be much smaller.

3. Thaw aliquots of ascites on ice. When thawing a large aliquot, aliquot any remaining ascites into 1 mL aliquots prior to refreezing, so that at most samples are frozen/thawed twice.

4. Do not aliquot more than 45 mL of ascites into the tube as it will expand once frozen and may crack the tube, resulting in a leak.

5. The coding system will be set by the IRB regulations of each institution and may not allow the use of identifying information, including date of collection. Careful record keeping is essential to utilize the sample across multiple experiments for comparative analysis or to obtain patient outcome data, where allowed.

6. Quantification of the cytokines and growth factors in the ascites is necessary to fully characterize the ovarian cancer tumor microenvironment. Samples collected by this protocol are appropriate for such analysis. Enzyme-linked immunosorbent assays (ELISAs) provide quantitative and precise measurement of soluble factors present in ascites [4]. Additionally, multiplex immunoassays have been utilized to measure cytokine levels in ascites and present a higher-throughput option for characterization [13]. There are commercially-available multiplex immunoassays and ELISAs specific to a myriad of relevant cytokines, chemokines, and growth factors that may be used to profile the composition of ascites. As it has been shown that there is significant variability in the levels of soluble factors between patients, it is recommended that multiple patients are profiled to obtain a representative picture [13]. Furthermore, it is important to consider patient disease stage in analyses, as increased levels of factors such as IL-8 and IL-6 have been linked to increased tumorigenicity [14,15].

7. Incorporation of ascites fluid into cell culture models is a facile approach to recapitulate the soluble tumor microenvironment in vitro. Ascites can be added to tumor cell cultures or co-cultures of tumor cells and other relevant cell types such as macrophages or mesothelial cells to study the impact of soluble factors in the ascites on outcomes such as adhesion [4]. A 10% (v/v) dilution of ascites in the culture medium is recommended for in vitro experiments to maintain proper cell media nutrients for viability.

8. For example, if you plan to freeze ascites cells, perform aggregate size distribution analysis, embed aggregates for histological analysis, and analyze cell populations with flow cytometry, there are four applications and the cell pellets should be combined and reconstituted in 48 mL of HI FBS. Once the cells are resuspended and well mixed, transfer 12 mL of this cell suspension to 4 different conical tubes. Each conical tube will be used for one of the downstream cellular applications (Figure 1C).

9. When counting cells, avoid counting platelets that may be present. These cell fragments are non-nucleated and much smaller than the other cell types in the ascites.

10. The cellular fraction in ascites is complex and can include multiple cell types (e.g. tumor cells, immune cells, mesothelial cells) as well as both single cells and aggregates of cells. Characterization of the composition of the cellular fraction may provide a better understanding of the metastatic process or provide prognostic markers. Flow cytometry of common cell types in the ascites including tumor cells (CD326+), immune cells (CD45+), and mesothelial cells (MSLN+) can be performed to profile cell types in the ascites (Figure 4).

11. Depending on aggregate size, a recommended objective to use is 5X. If you take multiple images in one well, ensure that the capture area of the image does not overlap, or some aggregates may be double counted. While images can be captured with brightfield, phase optics may improve automated image analysis.

12. With the assumption that the aggregates are spherical, this size range includes aggregates with a diameter of 40 μm and greater. 40 μm-diameter aggregates should be the smallest aggregates that will be trapped with the 40 μm cell strainer and so define the lower bound of the size analysis. If you would like to determine sizes of individual aggregates, this can be approximated from:

    $$\mathit{\text{Area}}=\pi {(\mathit{\text{radius}})}^2$$

    Note that scaling for images can be set in Analyze: Set Scale in FIJI. The specific scaling factor to convert micron to pixels is dependent on the objective and binning used during image capture.

13. A macro script can be created in ImageJ to perform all the analysis steps with one click.

14. It is useful to confirm that aggregates were embedded in the agarose using a light microscope. Hold the microcentrifuge tube on the stage and adjust focus until cells come into focus.

Figure 4.

Representative flow cytometry plots of cells isolated from ascites. A) Selection of cell population from debris based on FSC-A vs. SSC-A. B) Selection of single cells based on FSC-A vs. FSC-H. C) Selection of live cells using a fixed cell-compatible viability dye. D) CD45 (immune cells) and CD326 (tumor cells) expression of cells. E) CD326 and MSLN (mesothelial cells) expression of cells.