Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

Summary

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Abstract

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

Introduction

The Radial Monolayer Cell Migration assay was originally developed to measure the infiltrative properties of adherent tumor cells^1-4 on slides coated with extracellular matrix (ECM) proteins^5-7 or with individual ECM components, such as fibronectin or laminin^1,2. The technique involved seeding a single cell suspension of tumor cells in the center of wells using a stainless steel cell sedimentation manifold (CSM). After sedimentation, the tumor cells would adhere to the bottom of the well and the change in the diameter of the initial cell population over time was used to establish a rate of horizontal motility. The Radial Monolayer Cell Migration assay provided a visual advantage over other existing methods that employed transwell plates to assay the in vitro migratory capabilities of cells; these assays are non-conducive to imaging⁸. As well, it also provided a great amount of freedom in choosing the timepoints when migration is assessed, with no limit on the number of timepoints a researcher could choose to image after sedimentation.

Because the ability to migrate is an important functionality for non-adherent cells, especially in the area of immunotherapy or where they may be used as delivery vehicles for viral vectors, we adapted the use of the CSM to evaluate the migration of non-adherent cell types on tumor cell monolayers, in addition to ECM proteins. The added benefit of microscopically visualizing the migration of non-adherent cells on viable tumor cell monolayers, on complex ECM isolated from the tumor, or on individual ECM components makes this assay versatile. Assays that employ wells coated with a single extracellular protein do not reflect accurately the ECM tissue substrate or tumor the cells would migrate through in vivo.

Here, we used alloreactive cytotoxic T lymphocytes (alloCTL), sensitized to major histocompatibility complex (MHC) proteins using one-way mixed lymphocyte tumor cell reactions (MLTR) or mixed lymphocyte reactions (MLR)⁹, as our representative non-adherent cell type. We tested cells of both human and murine origin. When migration was measured on tumor monolayers, the tumor cells employed were either partially relevant targets, displaying some of the same MHC proteins found on the cell population used to sensitize the effectors, or fully relevant targets, with a full set of MHC molecules that the effectors had been sensitized towards. In some experiments, we used fluorescent CellTracker Red CMPTX or cell proliferation dye eFluor 670 to differentiate between effector and target cells. We also used transduction with viral vectors encoding for fluorescent proteins as an additional way to visualize the cells. For certain assays, we transduced the alloCTL with retroviral replicating vectors (RRV) coding for Emerald Green (EMD) fluorescent protein^10,11; for others, tumor cells were transduced with lentiviral vectors coding for mStrawberry.

The alloCTL were seeded through a channel of the manifold into the center of either tumor cell monolayers or ECM harvested from tumor cell monolayers. Adherent and non-adherent cell interactions were visualized by light and/or by fluorescence microscopy over time. Disruption in the tumor cell monolayer at low power, or tumor cells with fragmented nuclei at high power were indicators of cell injury by lysis and apoptosis, respectively. We digitally created surface intensity fluorescent maps showing the migration of non-adherent fluorescing T cells over the monolayer cultures. We also noted the cytotoxicity engendered to the adherent glioma cell monolayer after cluster formation of the overlaid non-adherent alloCTL. As well, horizontal transduction of RRV-EMD from the alloCTL to the glioma monolayer was observed.

Protocol

1. Slide Preparation

1. Insert Cell Sedimentation Manifold slides into sterilization pouches and seal with autoclave tape. Face the Teflon-coated side of the slide the paper-side of the pouch to avoid plastic deposits on the wells.
2. Autoclave pouches for 15 min at 121 °C.
3. Remove slide from sterilization pouch inside a biosafety cabinet and place in a sterile 150 x 15 mm sterile Petri dish. Up to four slides can fit per dish. Place a 35 x 10 mm Petri dish next to the slides. Add 2-3 ml of sterile H₂O to provide humidification.
4. Pre-coat slides with poly-D-lysine at 100 µg/ml by using enough volume to cover the entire well. After one hr at RT, aspirate the solution and rinse the surface of the well twice by pipetting 1x PBS over the surface of the wells.
5. Optionally, add fibronectin or other ECM components to ensure stronger tumor cell adherence. Add fibronectin at 5 µg/ml in enough volume that the wells will not dry within a short period of time at RT.
   1. After 1 hr, aspirate the leftover solution and wash twice by pipetting up and down with 1x PBS.
6. Keep PBS on the wells until tumor is ready for plating. Use slides the same day.
7. Harvest a confluent flask of the desired adherent tumor cell type. Count viable cells using Trypan Blue dye exclusion by light microscopy and resuspend at 5 x 10⁶ cells/ml in adequately-buffered growth medium—for example, Dulbecco’s minimal essential medium (DMEM) with 10% fetal bovine serum (FBS), L-glutamine and sodium pyruvate.
8. If fluorescent imaging of the adherent cell population is desired, label the adherent cell population with a vital fluorescent dye or a cell proliferation dye by following the protocols provided by the manufacturer.
9. Smoothly pipette 10 μl of complete medium into each well of the slide taking care to avoid creating bubbles in the wells, as these can prevent uniform tumor cell adherence.
10. Add 2.5-5.0 x 10⁴ cells (5-10 μl of cell suspension) to the medium in each well (Figure 1A). Adjust the optimal number depending on tumor cell type and the number of days of growth desired. Larger tumor cells or fast growing cells may require fewer cells initially. Bring well volume up to 40 μl by adding medium and pipette up and down to ensure an even distribution of cells.
11. After all wells are seeded, allow the tumor cells to adhere at RT, then place the lid on the 150 x 15 mm Petri dish and carefully move the dish to a 37 °C/5% CO₂ humidified incubator for at least 24 hr.
12. Change the culture medium daily. Carefully pipette a portion of the spent medium from the monolayer and replace with fresh complete medium.
    NOTE: Because of evaporation, more volume will need to be added than was taken out. Wells will be ready for assay when an even, confluent layer of cells is present. This is typically 1-3 days after initial seeding.
13. Wash monolayer gently once with PBS as described in step 1.4 and either proceed to step 2 if monolayers are desired, or to step 1.13 below to extract ECM proteins from the monolayer.
14. Make a 0.5% Triton-X (v/v) solution in complete medium and add 30 μl to each well. Let monolayer digest in hood for 2 min, then wash twice with complete medium by pipetting as described above.
    NOTE: The ECM layer may be visible by light microscopy (Figure 2). Use slides right away, or keep in complete medium in the humidified CO₂ incubator for 1-2 days. Do not allow wells to dry out.

2. Sedimentation of Non-adherent T Cells onto Slide

1. If fluorescent imaging of the non-adherent cell population is desired, label the non-adherent cell populations with a vital fluorescent dye, such as CFSE, Cell Tracker Red CMPTX, or eFluor 670 by following the protocols provided by the manufacturer at least 1 hr in advance of the assay. Alternatively, manipulate cells to express fluorescent proteins encoded by viral vectors.
2. Autoclave the cell sedimentation manifold in an autoclave pouch as described in 1.2 above.
3. Remove the humidified chamber containing Teflon coated slides from the incubator and place in biological safety cabinet.
4. Wash wells with 1x PBS by pipetting up and down, then add 45 μl of complete medium containing at least 10% serum.
5. Remove the manifold from the sterilization envelope and carefully slide in over the wells until the ‘hook’ at the end of the manifold touches the bottom of the slide (Figure 1B).
6. Ensure that the culture medium is visible in each of the channels. If none is seen, take off the manifold and add more medium to the corresponding well, then replace and check again.
7. Repeat step 2.5 for each channel of the manifold, or for as many wells as desired.
8. Count non-adherent cells and resuspend at no less than 1.0-2.0 x 10⁵/μl. Draw up 1 μl of cells and slowly pipette them into the channel (Figure 1C). Do this for each well desired.
9. Leave the entire cell loaded apparatus, i.e., manifold and slide, inside the bio-hood for 20-30 min to allow the cells in the channel to settle.
10. Remove the manifold by touching both ends and gently lifting it straight up from the slide so as to not disturb the cells focally seeded in the center of the wells (Figure 1D).
11. Inspect each well to ensure that the non-adherent cells have been successfully seeded at the center of the well staying within the circumference of the manifold channel. The non-adherent cells will ideally slightly adhere to the monolayer or ECM and to each other, therefore gently moving the slide around will not cause the cell pellet to disperse. Do not jostle the slide.

3. Fluorescence Microscopy

1. With appropriate experimental equipment, perform the imaging after sedimentation is confirmed. Ideally, use a microscope that is equipped with a CO₂ and humidity chamber. Image at low power, i.e., 4X or 10X, to allow for multiple images to be taken of a larger area so that the entirety of the well can be seen.
2. Acquire digital images using image capture software. Perform brightfield and/or multi-channel fluorescence imaging.
3. If desired, set up a time-lapse to record images in different channels over a given area, or keep the slide humidified and in a 37 °C/5% CO₂ incubator and manually take out to image at the time points desired (Figure 1E & F, recommended for longer time points).

4. Analysis and Display

1. Using image editing software, merge light and fluorescent images into one layer so multiple cell types and markers can be seen in the same image. Drag and drop the image files for each individual color into the program and, when prompted, select the ‘Add Layer’ option to create an image file with multiple layers.
   1. At higher magnification, take, align, and stitch together images across the well digitally. Align files by looking at conserved regions between two images and overlaying one image on top of the other until a complete picture is generated.
2. Add micron bars to images during acquisition using the capture software to determine distance individual cells have migrated.
3. To determine the cell migration at various times, make surface intensity fluorescence maps with image software, i.e., ImageJ, to quantify fluorescent T cell aggregation or spread over time.
   1. To generate surface plots, take a layered image generated in step 4.1 and under the Layers window, uncheck all layers except the ones corresponding to the channel desired. For instance, if a surface plot is desired to visualize EMD expression, only the ‘green’ layers corresponding to the filter used for this marker should be visible.
   2. Flatten the image and save it in a file format recognized (e.g., .png) and load the image into the program. To generate the surface plot, change the image type to 8-bit, change the brightness/contrast as needed to reduce background, and select the ‘Analyze/Surface Plot’ option. The images in Figure 5 were generated by checking the ‘Shade’, ‘Draw Axis’, ‘One Polygon Per Line’, and ‘Smooth’ checkboxes.
   3. Alternatively, take measurements using the radius or diameter from the focal cell deposit at time zero and compare to the cells at the outermost point at different times. The diameter of the Teflon wells is 6.0 mm.

Results

Viral vectors encoding for fluorescent proteins can be used in addition to, or instead of, fluorescent dye. Viral transduction should be performed in advance of the motility assay. Both adherent and non-adherent cell types can be differentially-labeled. The protocol for transduction will depend on the type of vector employed. Here, we transduced the alloCTL in Figures 3, 5 and 6 with RRV-EMD at least two days in advance of the assay using the protocol described¹²

Discussion

Tumor cells in a single cell suspension were pipetted into the wells of a Teflon-masked slide. The cells were allowed to adhere and then formed monolayers in a humidified 5% CO₂, 37°C incubator (Figure 1A). Established monolayers or ECM proteins derived from the monolayer could be harvested for these assays (Figure 1B). Effector T lymphocytes labeled with vital fluorescent dyes or transduced with vectors coding for EMD were seeded into the center of the well using a CSM. ...

Materials

Name	Company	Catalog Number	Comments
Teflon-masked microscope slides	Creative Scientific Methods	CSM002
Cell Sedimentation Manifold	Creative Scientific Methods	CSM001
Petri Dish, 150mm	Corning	430597
Petri Dish, 35mm	Corning	430588
Phosphate-Buffered Saline (PBS)	Mediatech	21-040
20 μl Pipetman	Gilson	F123600
200 μl Pipetman	Gilson	F123601
200 μl pipette tips	VWR	89003-060
Distilled, deionized water (sterile)	Mediatech	25-055
Trypsin	Mediatech	25-054-CL
Celltracker Red CMPTX	Invitrogen	C34552
TrypLE Express (optional)	Gibco	12604
Tumor cell culture media (e.g. DMEM)	Mediatech	10-013
AIM-V serum-free media	Invitrogen	12055-083
Fetal Bovine Serum	Omega Scientific	FB-02
Inverted microscope
SPOT Advanced	Diagnostic Instruments
Poly-D-Lysine	Millipore	A-003-E
Fibronectin	BD	354008
31/2 x 51/4 Autoclave Pouches	Crosstex	SCXS2
Trypan Blue	Mediatech	25-900-CI
Cell Proliferation eFluor 670	eBioscience	65-0840-85
ImageJ	NIH