Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Summary

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Abstract

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

Introduction

The ability of tumor cells to evade and disseminate through the surrounding tissue is a hallmark of cancer¹. In particular, tumor cell motility is a characteristic feature of malignant tumors, whether it is a metastatic tumor type such as breast² or colorectal cancer³ or a locally invasive type such as diffuse glioma^4,5.

Imaging has a central role in the investigation of many aspects of tumor cell phenotypes; however, live-cell imaging is definitely to be preferred when studying dynamic cellular processes such as migration and invasion, when changes in morphology and cell-cell interaction^6,7 occur and can be more easily examined over time. For live-cell imaging, different optical microscopy systems can be used, from phase contrast to confocal fluorescent microscopes, and image acquisition performed over a short or long period of time on an inverted microscope equipped with a chamber for temperature and CO₂ control, or in high-content image analysis systems which have built-in chambers, or alternatively in image systems that can sit in the incubator without the need to disturb the cells during the whole duration of the experiment. The choice of the system used is often dictated by a number of factors such as resolution needed, length of the overall acquisition time and time intervals, vessel type used and throughput of the assay (single chamber or multi-well plate), the sensitivity of the cells used (precious and/or rare cells) and phototoxicity of the cells if fluorophores are present.

With regard to fluorescent imaging in live mode, this can be achieved by transducing cells for the expression of fluorescent proteins either for stable expression or as an inducible system⁸, by transient cell transfection, or by using cell dyes which are now available for live-cell labeling⁷, for live-cell tracking as well as for labeling subcellular organelles⁹.

A useful approach has been recently developed for live-cell immunocytochemistry, where an antibody recognizing a surface marker of choice can be bound to a labeling reagent, and upon addition to the culture media, cells expressing the specific marker can be readily imaged in real-time by live-cell imaging. The visualization and quantification of marker expression using such a system can be easily achieved when cells are grown in two-dimensional (2D) culture conditions¹⁰.

In this study, we optimized protocols for live-3D-cell immunocytochemistry invasion and migration of pediatric diffuse midline glioma (DMG) patient-derived cells^11,12. DMG are highly aggressive brain tumors affecting children, for the vast majority associated with the driver mutation K27M in histone H3 variants. DMG arise in the brain stem and the midline regions of the central nervous system (CNS) and are characterized by a highly infiltrative nature. This invasive capacity has been shown to be at least in part mediated by the intratumor heterogeneity and the cancer-stem-like features of DMG cells⁷.

To exemplify our assays, an antibody labeling reagent (ALR) was used in combination with an antibody for CD44. CD44 is a transmembrane glycoprotein and adhesion molecule expressed on stem-cell and other cell types, associated with cancer stem cell phenotype and tumor cell migration and invasion¹³. The protocols include the sample preparation, the image acquisition in brightfield and fluorescent mode, and the analysis on a live-cell analysis instrument that allowed to quantitatively measure in real-time the overall CD44 expression on the DMG cell membrane during 3D invasion and migration. The assays also allowed the possibility to visualize the intermittent fluorescent signal of CD44 on individual cells while migrating and invading. Interestingly an effect of the anti-CD44 antibody was also observed, which potentially acting as a blocking antibody, also seemed to reduce cell migration and invasion as well as to induce a switch of the invasion pattern from a collective mesenchymal-like to a more single-cell amoeboid-like phenotype.

Protocol

This protocol follows the guidelines of the institutions' human research ethics committees.

NOTE: This study was performed using Incucyte S3 and/or SX5 Live-Cell Analysis Instrument (referenced as live cell analysis instrument).

1. Generation of reproducibly sized tumor spheroids

NOTE: The protocol (section 1) described by Vinci et al. 2015^7,12, was used as reported below, with some modifications:

1. Collect the DMG H3K27M-mutant neurospheres (NS) and centrifuge at 170 x g for 10 minutes (min) at room temperature (RT).
2. Incubate the NS with 500 µL of the accutase solution for 3 min at 37 °C to break them up.
3. Neutralize the accutase solution with tumor stem cell (TSM) medium⁷ and centrifuge the cell suspension at 355 x g for 5 min at RT.
4. Resuspend the cell pellet in 1 mL of TSM medium and then count the cells using a cell counting chamber.
5. Dilute the cell suspension to obtain 2.5-5 x 10³cells/mL and seed 100 µL/well into ultra-low attachment (ULA) 96-well round-bottom plates (see Table of Materials). Use a proper cell density to obtain individual NS of ~300 µm diameter, 4 days after cell seeding (250-500 cells/well for highly aggressive glioma cells).
6. Visually confirm the NS formation by using an inverted microscope 4 days after cell seeding.

2. Preparation of the ALR/antibody complex and setup for the invasion assay

NOTE: For the antibody labeling procedure, the antibody labeling dyes protocol¹⁰ for live-cell Immunocytochemistry is used with some modifications, as reported below. For the invasion assay, the protocol previously described by Vinci et al. 2015¹² is followed.

1. Consider the number of wells (e.g., 60 wells) to analyze and calculate the volume needed for each reagent. Also include the wells for the negative control (samples with ALR but without antibody).
2. Add 100 µL of sterile water to the ALR to rehydrate the reagent (final concentration = 0.5 mg/mL). Pipette to mix the solution.
   NOTE: The reagent is light-sensitive; therefore, keep in the dark. Aliquot the leftover reagent and store at -80 °C (avoid freezing and thawing).
3. Mix the antibody with the ALR in the TSM medium (or appropriate cell growth media for the cell line of choice) in a round bottom multi-well plate or in an amber tube and protect from light.
4. Prepare enough quantity of the medium to dispense 25 µL/well at 3x final assay concentration. Incubate at RT for 15 min.
   NOTE: A 1:3 molar ratio of antibody to ALR is recommended, with a final (1x) concentration of the test antibody <1.5 µg/mL. For the experiments in this protocol an anti-human CD44 mouse antibody is used (starting concentration 86 µg/mL) at a final concentration of 0.1 µg/mL (3x concentration = 0.3 µg/mL).Add the reagents in the following order: i) antibody; ii) ALR; iii) TSM medium. Mix by pipetting.
5. Dilute the background suppressor reagent (BSR) in TSM medium (or appropriate cell growth media for cell line of choice) at 1.5 mM (3x) to obtain at the end of the assay a final concentration of 0.5 mM.
6. Perform the invasion assay directly in the ULA 96-well round-bottom plate where cells were initially seeded. Check the NS visually using an inverted microscope before starting.
7. Gently and slowly remove 75 µL/well of the medium, avoiding touching the bottom of the well where the NS sits. Check the presence of the NS visually.
8. Gently add 25 µL of the BSR to each well.
9. Gently add 25 µL of the ALR/antibody complex to each well. Wait 2 or 3 min to let the ALR/antibody complex mix with the medium.
10. Check visually using an inverted microscope to ensure that each NS is centrally located at the bottom of the well. Avoid the formation of bubbles. If any bubble is present, remove it by using a needle.
11. Place the plate on ice and wait 5 min to let the bottom of the plate become cold.
12. With a pre-cooled p200 tip, dispense 75 µL/well of basal membrane matrix (BMM), placing the pipette tip on the internal wall of the well and avoiding touching the bottom of the well. Avoid the formation of bubbles and remove with a sterile needle the existing ones.
    NOTE: Make sure to have thawed the BMM at 4 °C from the night before.
13. Leave the plate on ice for 5 min to let the BMM mix with the medium. Check visually using an inverted microscope the presence of the NS and that they are centrally located in the well. If not, centrifuge the plate at 4 °C at 180 x g for 5 min.
14. Transfer the plate in the live-cell analysis instrument (Table of Materials) placed within the incubator at 37 °C, 5% CO₂, 95% humidity.

3. Preparation of the ALR/antibody complex and setup for the migration assay

NOTE: For the antibody labeling procedure, the Labeling Dyes protocol¹⁰ for Live-Cell Immunocytochemistry is used.

1. Consider the number of wells to analyze and calculate the volume needed for each reagent. Include also the wells needed for the negative controls. Check the NS visually using an inverted microscope before starting.
2. Use flat-bottom 96-well plates. Perform the coating procedure as described by Vinci et al., 2013¹⁴. For this study, the BMM is used as a thin coating.
3. Once the coating is ready, remove the excess of BMM coating with a p200 tip placing the tip in the edge of the well and avoiding touching the bottom. If working with multiple wells, use a multichannel pipette.
4. Cut a p200 tip, take 50 µL of the cell medium + NS from each selected well, and transfer it to a coated flat bottom well. Check the presence and the position of the NS in each well visually.
   NOTE: Each NS must be centrally located in the well. Avoid leaning on the tip on the edge of the well during the transfer but drop the medium centrally in the well without touching the bottom. For highly migratory cells, consider a higher number of replicates than the standard three replicates. This is because when NS sits too close to the edge of the well, the migrating cells may cover a smaller area of the well.
5. Rehydrate the ALR as described above (steps 2.2-2.3).
   NOTE: Reagent is light sensitive. See above for good handling procedures.
6. Mix the antibody with ALR in the appropriate complete cell growth media in a round bottom multi-well plate or in an amber tube and protect from light. Prepare enough quantity to dispense 50 µL/well at 3x final assay concentration. Incubate at RT for 15 min.
   NOTE: Add the reagents in the order as indicated above (step 2.4).
7. Follow the same procedure as reported in step 2.5.
8. Gently add 50 µL of the BSR to each well.
9. Gently add 50 µL of the ALR/antibody to each well. Wait 2 or 3 min to let the reagents mix and check visually using an inverted microscope to ensure that most of the replicate NS are centrally located in the well.
10. Avoid the formation of bubbles and remove any existing ones by using a needle. Gently transfer the plate in the live-cell analysis instrument placed within the incubator at 37 °C, 5% CO₂, 95% humidity.

4. Live-cell analysis instrument setting for image acquisition

1. Scan the plates using the live-cell analysis instrument (for specifications, see Table of Materials) with scanning intervals starting from time point zero (t0) of the invasion and migration assays set up, respectively, after step 2.14. and 3.10. up to 96 h.
   NOTE: Ensure to be able to dispose of the live-cell analysis instrument immediately after the starting of the invasion and migration assay. Depending on the tumor type, cells can start to invade or migrate from the NS already within 1 h from the assay setup.
2. On the live-cell analysis instrument software, select the option Schedule to Acquire. Click on the + tab and select the option Scan on Schedule.
3. On the software window Create or Restore Vessel, click on the option New.
4. Select the specific application in the live-cell analysis instrument for the invasion and migration acquisition. Select Spheroid scan type, 4x objective, Phase+Brightfield and Green image channels for the invasion assay. Select Dilution Cloning scan type, 4x objective and Phase and Green for the migration assay.
5. Select the plate type and define the wells to be scanned by highlighting them on the plate map.
6. Set up the scanning frequency (for the experiments in this protocol scanning frequency was 15 min for invasion and 30 min for migration).
7. Click on Add to Schedule and start the scan.

5. Live-cell analysis instrument setting for image analysis

1. Select the tab Create New Analysis Definition.
2. Select Spheroid Invasion or Basic Analyzer application for invasion and migration, respectively, on the tab.
3. Select the invasion and migration appropriate channels (for Invasion: Phase+Brightfield-Green; for Migration: Phase-Green) in the image channel.
4. Select few representative images from 3-4 wells for previewing and refining the analysis setting.
5. For the invasion assay, in the Analysis Definition tab, adjust the application settings in the Brightfield and Green channels with the following setting to generate a precise segmentation between the Whole Spheroid and Invading Cells (see Figure 5; blue mask):
   Brightfield Segmentation: Whole Spheroid sensitivity = 50; Invading Cell sensitivity = 100; Clean Up = default.
   Whole Spheroid Filters: set all parameters as default.
   Invading Cells Filters: set all parameters as default.
   Green Segmentation: Radius = 900.
6. For migration assay, adjust the application settings in the Phase and Green channels to generate a precise segmentation between the Confluence and Green Cells (see Figure 5, yellow and pink masks) with the following setting:
   Phase: set all parameters as default.
   Green Segmentation: Radius = 300; Threshold = 1000
   Cleanup: Hole Fill = 400; Filters = default.
   Whole Well: set all parameters as default.
7. Check that the analysis settings are correct for the NS by clicking randomly on several wells. The segmentation must outline the spheroid. If not, adjust the setting accordingly.
8. Select the wells and time points to analyze.
9. Save the Analysis Definition and click on Finish.

Results

Live-3D-Cell Immunocytochemistry protocol for invasion and migration is summarized in a straightforward and reproducible workflow in Figure 1. By seeding the DMG cells in ULA 96-well round-bottom plates, reproducible sized NS are obtained and used in the steps displayed. When the NS have reached the ideal size of ~300 µm (approximately 4 days post-seeding) the invasion¹² and migration¹⁴ assays are initiated. The addition of the ALR/antibody com...

Discussion

The live-3D-cell immunocytochemistry we have adopted here for pediatric DMG invasion and migration could be easily adapted also for other highly invasive tumor cell types, including breast and colon cancer cell lines.

Different from previously performed live-2D-cell immunocytochemistry assays¹⁰, when working in 3D, it is suggested to pay attention to some critical steps. In particular, for the invasion assay we describe, it is advised to add the ALR/antibody mix direct...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 Well TC-Treated Microplates	Corning	3595	size 96 wells, polystyrene plate, flat bottom
Accutase	Euroclone	ECB3056D	solution for neurosphere dissociation
Burker chamber	Mv medical	FFL16034	cell counting chamber
CD-44 (156-3C11)	Cell Signaling Technology	3570	Mouse mAb IgG2a
Corning Matrigel Matrix	Corning	356237	Basement Membrane Matrix (BMM), Phenol Red-free, LDEV-free
Fabfluor-488 Antibody Labeling Dye	Incucyte	4743	Antibody labelling reagent (ALR): Mouse IgG2a 488 antibody for Live-Cell Immunocytochemistry
Incucyte S3 and/or SX5 Live-Cell Analysis Instrument	Sartorius	-	The Incucyte S3 and/or SX5 Instrument is used for real-time cell monitoring and surveillance, cell health and viability, migration and invasion, plus a wide range of phenotypic cell-based assays.
Inverted Microscope		-	any inverted microscope
Opti-Green Background Suppressor Reagent	Incucyte	6500-0045	Backgroung suppressor reagent (BSR)
Tumor stem cell (TSM) medium	-	-	growth cell medium (see reference in the text for details)
Ultra-Low Attachment Multiple Well Plate	Corning Costar	7007	size 96 well, round bottom clear