Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.
Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.
The non-specific endocytic pathway of macropinocytosis allows cells to internalize a variety of extracellular components, including nutrients, proteins, antigens, and pathogens, through bulk uptake of extracellular fluid and its constituents1. Though important for the biology of numerous cell types, increasingly, the macropinocytosis pathway is described to play an essential role in tumor biology, where, through macropinocytic uptake, tumor cells are able to survive and proliferate in the presence of a nutrient-depleted microenvironment2,3. The uptake of extracellular macromolecules, including albumin and extracellular matrix, and necrotic cell debris, provides an alternative nutrient source for biomass production by creating amino acids, sugars, lipids and nucleotides through macropinosome and lysosome fusion-mediated cargo catabolism4,5,6,7,8.
The induction and regulation of macropinocytosis are complex and can vary depending on cellular context. Thus far, several inducers of macropinocytosis have been identified and include ligands, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), galectin-3, and Wnt3A9,10,11,12,13. In addition, culturing conditions that mimic the tumor microenvironment can trigger activation of the pathway. Pancreatic ductal adenocarcinoma (PDAC) tumors are nutrient-deprived, especially for the amino acid glutamine, which causes both cancer cells and cancer-associated fibroblasts (CAFs) to rely on macropinocytosis for survival7,13,14,15. Moreover, tumor stresses, such as hypoxia and oxidative stress, can activate this scavenging pathway16. In addition to the numerous extrinsic influencers that can induce macropinocytosis, a variety of intracellular pathways control macropinosome formation. Oncogenic Ras-mediated transformation is sufficient to initiate the macropinocytic machinery, and multiple cancer types exhibit oncogenic Ras-driven constitutive macropinocytosis4,5,9,17. Alternatively, wild-type Ras activation and Ras-independent pathways have been identified to activate macropinocytosis in cancer cells and CAFs10,11,15,18. The use of various in vitro models in combination with inhibitor treatments has resulted in the identification of several macropinocytosis modulators, which include sodium-hydrogen exchangers, the small GTPase Rac1, phosphoinositide 3-kinase (PI3K), p21-activated kinase (Pak), and AMP-activated protein kinase (AMPK)4,13,15. However, given the multitude of described factors and conditions that regulate macropinocytosis, it is conceivable that many more modulators and stimuli remain undiscovered. The identification of novel modulators and stimuli can be facilitated by automated assessment of a multitude of conditions in a single experiment. This methodology can shed light on the factors involved in macropinosome formation and may allow for the discovery of novel small molecules or biologics that target this pathway.
Here, we have adapted our previously established protocol for determining the extent of macropinocytosis in cancer cells in vitro to a 96-well microplate format and automated imaging and quantification19,20. This protocol is based on fluorescent microscopy, which has become a standard in the field to determine macropinocytosis in vitro and in vivo4,5,6,7,9,10,11,12,13,15,16,17,18,19,20,21,22. Macropinosomes can be distinguished from other endocytic pathways through their ability to internalize large macromolecules, such as high molecular weight dextran (i.e., 70 kDa)2,3,4,20,21,22,23. Thus, macropinosomes can be defined through uptake of extracellularly administered fluorophore-labeled 70 kDa dextran. As a result, macropinocytic vesicles manifest as intracellular clusters of fluorescent puncta with sizes ranging from 0.2-5 µm. These puncta can be microscopically imaged and subsequently quantified to determine the extent of macropinocytosis in the cell – 'the macropinocytic index'.
In this protocol, the essential steps to visualize macropinosomes in adherent cells in vitro on a 96-well microplate and coverslips using standard laboratory equipment are described (Figure 1). In addition, the directions to automate the image acquisition and quantification of the macropinocytic index using a cell imaging multi-mode plate reader are provided. This automation reduces time, cost, and effort compared to our previously described protocols19,20. In addition, it avoids unintentionally biased imaging acquisition and analysis and thereby enhances reproducibility and reliability. This method can easily be adapted to different cell types or plate readers or be utilized to determine alternative macropinosome features, such as size, number, and location. The herein described method is especially suitable for the screening of cell culture conditions that induce macropinocytosis, the identification of novel modulators, or optimization of drug concentrations of known inhibitors.
Figure 1: Schematic of the automated assay to determine the 'macropinocytic index' in adherent cells. Created using BioRender. Please click here to view a larger version of this figure.
1. Preparation of materials
2. Preparation of cells
3. Macropinosome labeling
Figure 2: Placing coverslips on a microscope slide with silicone isolators. (A) Silicone isolators are pressed and adhered to a microscope slide. (B) The entire microscope slide can be populated with a total of 3 isolators, resulting in even spacing and reproducible localization of the coverslips. (C) For each coverslip, add a drop of fluorescence mounting media on the microscope slide within the open space of the isolator. (D) Using forceps, pick up a coverslip from the 24-well plate and place it upside down on the drop of mounting media. (E) When bubbles are present between the coverslip and microscope slide, gently tap the coverslip using closed forceps to remove bubbles. Created using BioRender. Please click here to view a larger version of this figure.
Figure 3: Rinsing the 96-well microplate to prepare for fixation. (A) Empty the microplate of media into a 5 L beaker by manually flicking. (B) Vertically and at a slight angle, slowly submerge the microplate in a 2 L beaker filled with ice-cold PBS. (C) Empty the microplate of PBS into the 5 L beaker by manually flicking. Repeat the wash steps as described in B two times. (D) After emptying the PBS in the microplate for the last time, add 100 µL 3.7% formaldehyde to the wells, using a multichannel pipette. Created using BioRender. Please click here to view a larger version of this figure.
4. Automated macropinosome imaging
Images of macropinosomes can be captured using a standard fluorescent microscope, as previously described19,20. However, such a procedure can be improved upon in terms of efficiency through automation, especially when assessing numerous different cell culture conditions. Automation of image acquisition can be accomplished via a cell imaging multi-mode plate reader, which decreases effort by reducing handling procedures and, importantly, increases data reproducibility and reliability by acquiring images in an unbiased fashion. Multiple imaging systems are commercially available, and directions will differ between instruments. Here, acquiring images using a Cytation 5 is described. However, the protocol below can be tailored to each individual instrument by adhering to the following guidelines:
Figure 4: Optimization of conditions for image acquisition. (A) Increasing the glycerol concentration increases TMR-dextran fluorescence, as determined in AsPC-1 cells treated with EGF. (B) Example coordinates of imaging beacons for automatic image acquisition when using the 24-well plate with coverslips format. The bar graph shows the average relative fluorescence with SEM of 5 experiments. Statistical significance was determined by two-way ANOVA, relative to PBS. ** p < 0.01; *** p < 0.001. Please click here to view a larger version of this figure.
Figure 5: Control conditions for assessing macropinocytosis in PDAC cells. (A) AsPC-1 cells display macropinocytosis in response to 100 ng/mL EGF stimulation for 5 min or glutamine deprivation for 24 h. For image acquisition, picture frames of 4 x 4, 3 x 3, 2 x 3, or 2 x 2 were taken to determine the influence of the number of photos on data quality. (B) MIA PaCa-2 cells show constitutive macropinocytosis that is inhibited by 30-min treatment with 75 µM EIPA or 2-h treatment with 10 µM EHop-016. Picture frames were taken as in A. Scale bar = 25 µm. Bar graphs show the average relative macropinocytic index with SD of 1 experiment with 4 replicates. Statistical significance was determined by two-way ANOVA relative to the +Q or vehicle condition. *** p < 0.001 Please click here to view a larger version of this figure.
5. Determining the macropinocytic index
The 'macropinocytic index' is the extent of cellular macropinocytosis that is determined by quantifying fluorescent dextran uptake per cell using microscopic imaging19. To this end, the acquired images are used to determine the amount of internalized dextran by measuring the total fluorescence intensity or fluorescence-positive area and the total number of cells as determined by DAPI staining. This analysis can be performed with open-source image processing and analysis software, such as Cell Profiler or FIJI/ImageJ, as previously described19,20. However, when working with a multi-mode plate reader the software provided with the instrument may include built-in analysis applications that can be used for the purposes of computing the macropinocytic index. In some cases, the built-in software analysis pipeline may not be completely apparent to the user. It is therefore recommended to validate the software at an early stage by comparison with a non-automated procedure, such as Cell Profiler or FIJI/ImageJ. This protocol can be adapted to other image processing and analysis software tools by adhering to the following general instructions:
6. Addition of treatments
Cell treatments (small molecules, biologics, growth factors, metabolites etc.) can be incorporated at any stage of the protocol, and the precise timing will depend on the goals and aims of the study.
Figure 6: Performing a dose-response curve for macropinocytosis inhibitors. Example data obtained when testing known macropinocytosis inhibitors in a new cell line. PATU8998T cells were used for the 96-well microplate format and treated for 2 h and 30 min with the indicated concentrations of (A) EHop-16 and (B) EIPA, respectively. Comparison of results obtained through image analysis by the Gen5 software or ImageJ shows no significant differences between the two approaches as indicated by ns in (A). Scale bar = 25 µm. Bar graphs show the average and SD of a single experiment with 4 replicates. Statistical significance was determined by one- or two-way ANOVA, compared to untreated conditions. * p < 0.05; *** p < 0.001. Please click here to view a larger version of this figure.
When the steps and adjustment of the above-described protocol are followed accordingly, the final experimental results should provide information about whether the studied cell culture conditions or inhibitors induce or reduce macropinocytosis in the cell line of interest. To strengthen the validity of these findings, the inclusion of control conditions will allow for the scrutinization of the results to determine whether the experiment has been completed successfully. Macropinocytosis induction controls will provide information about the relative level of macropinocytosis. For this purpose, the ligand EGF is most commonly used13. In AsPC-1 PDAC cells, adding EGF at 100 ng/mL for 5 min before adding the dextran activates macropinocytosis (Figure 5A). Moreover, autocrine EGF activation of macropinocytosis can be induced by depriving the cells of glutamine for 16-24 h (Figure 5A). Alternatively, other inducers may be incorporated depending on the cell type and available literature; these may include PDGF or Wnt3A10,11,12. Not all cells behave similarly, and KRAS mutant cells may exhibit constitutive macropinocytosis13. One example is the MIA PaCa-2 PDAC cell line, which does not respond to EGF treatment. Here, the addition of known macropinocytosis inhibitors will validate that the observed and quantified fluorescent puncta are indeed macropinosomes. The inhibitory controls most commonly used include the sodium-hydrogen exchanger inhibitor EIPA, which is a specific macropinocytosis inhibitor (Figure 5B)4. Other controls may also be included, such as the Rac1 inhibitor EHop-016 (Figure 5B), the Pak inhibitor FRAX597, or the PI3K inhibitor LY294002; however, their effect on macropinocytosis may be model specific13.
For cell lines that have previously not been assessed for macropinocytosis and inhibitors, dose-response curves are an excellent approach to confirm macropinocytosis and to determine optimal drug concentration for future use. For these purposes, the 96-well microplate format protocol was used to determine constitutive macropinocytosis in a cell line previously not assessed in the lab, the PATU8998T PDAC cells. To determine whether these cells exhibit macropinocytosis, the effect of two known inhibitors of dextran uptake (EHop-016 and EIPA) were studied. As illustrated, the dose-response experiment gradually decreased the macropinocytic index at higher drug concentrations (Figure 6A,B), thereby confirming the existence of constitutive Rac1-dependent macropinocytosis in these cells. In addition, the results provide information for optimal drug concentrations to be used for future experiments when studying macropinocytosis in this cell line.
Figure 7: Trouble-shooting conditions that may be encountered. (A) The image is overexposed, as indicated in pink. (B) The image is out-of-focus. (C) The image contains dextran blotches or smudges. (D) The image contains residue. (E) The image contains a bubble. (F) The cells show drug-induced autofluorescence. Scale bar = 25 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Screenshot of image preprocessing. (A) Process button. (B) Image roll-out. (C) Analyze button. Please click here to download this File.
Supplementary Figure 2: Screenshot of image quantification settings. (A) ANALYSIS SETTINGS. (B) View Line Profile tool. (C) Line over dextran positive area. (D) View Line Profile results Please click here to download this File.
Supplementary File 1. Please click here to download this File.
Supplementary File 2. Please click here to download this File.
The quality of the experiments and data acquisition highly depends on the quality of the reagents, the optimization of the settings, and the cleanliness of the coverslips and microplate. The final results should give minimal variation between replicates; however, biological variations do naturally occur or may otherwise be caused by a number of factors. Cell density may cause cells to respond more or less to macropinocytosis inducers or inhibitors. It is, therefore, crucial to adhere to the 80% confluency as proposed here in the protocol. Alternatively, it is well documented by microplate manufacturers that media evaporation occurs on a 96-well microplate. Here, the outer wells are subject to more evaporation relative to the inner wells and thereby may affect macropinocytosis. Thus, the choice can be made to not include the outer wells in the analysis and instead fill these wells with PBS to build a 'buffer wall' to protect the inner wells from extensive evaporation.
It is highly recommended to visually inspect images for each condition to determine if the acquisition was completed successfully. This may be done during the imaging of the first set of conditions to ascertain that the applied settings are indeed correct and allow for intervention if required. Specific issues that can occur are as follows; the images are overexposed (Figure 7A), the images are out-of-focus (Figure 7B), the images contain blotches of fluorescent dextran (Figure 7C) or residue (Figure 7D), the images contain bubbles (Figure 7E), or autofluorescence is visible in the dextran or DAPI channel (Figure 7F). For the first two issues, overexposure and out-of-focus (Figure 7A,B), the image acquisition settings have to be adjusted, and the acquisition has to be repeated. In addition, the plate and coverslips should be cleaned for the autofocus to work properly. If only a few images are out-of-focus, the specific images or the entire well may also be excluded from the analysis using the 'Mask' image function. This same function can be applied to exclude images that contain blotches in the dextran channel (Figure 7C), impurities (Figure 7D), or bubbles (Figure 7E). In addition, make sure that the samples have been washed well, the dextran has completely been dissolved, which can be improved by heating the dextran solution at 37 °C and vortexing, and that the experiment is performed with freshly prepared and clean reagents. Finally, drug-induced autofluorescence can occur and is quite common for the macropinocytosis inhibitor EIPA which fluoresces in the FITC and DAPI channel, especially when previously excited in the DAPI channel. A workaround is the acquisition of the FITC channel before DAPI. Alternatively, TMR-dextran can be utilized instead of FITC-dextran, or for the exposure settings, the light intensity and exposure time can be lowered while increasing the gain.
To compute the macropinocytic index, nuclear DAPI staining is utilized to determine the cell number in each image. This staining procedure is easy to apply and is a reliable readout for the number of cells and thus for the calculation of the relative dextran uptake per cell. One caveat to this approach is that it does not consider differences in cell size, which may occur when comparing different cell lines or in response to pharmacological treatments. In these cases, where cell size is suspected to affect the macropinocytic index, the cell area can be used for normalization of dextran uptake, as previously described4. This can be achieved by slightly modifying the protocol to include a cell mask stain after fixation or incorporating phase contrast or bright field imaging. It is also highly recommended to follow up any findings using the described assay to evaluate whether macropinocytosis is a nutrient supply route that contributes to cellular fitness in the particular cell-based system being employed. This may be achieved by assessing proliferative capacity, survival, or viability in nutrient-depleted conditions with and without the addition of extracellular albumin4,7. Additionally, a DQ-BSA pulse-chase assay can provide evidence for whether the changes in macropinocytosis translate to changes in albumin degradation in lysosomes. Like high molecular weight dextran, DQ-BSA is internalized through macropinocytosis and is delivered to the lysosomes, where it fluoresces after proteolytic digestion4. Given the similarities to fluorescent dextran uptake, the described method can be adapted to assess DQ-BSA uptake. Likewise, this protocol may be used to evaluate the uptake of other fluorescently tagged cargo, such as albumin, lipids, or necrotic cell debris known to enter cells through macropinocytosis. On all occasions, these adaptations should be in line with the manufacturer's protocols, and experimental controls should be used to validate the assay.
Microscopic image analysis and quantification is a favored approach to assess macropinocytosis and has become the standard in the field4,5,6,7,9,10,11,12,13,15,16,17,18,19,20,21,22. It allows for the visual inspection of the samples and can provide additional information about macropinosome number, size, and location. Moreover, the possibility to visually inspect images permits the assessment of cell fitness and viability and identification of artifacts and/or autofluorescence caused by drug administration. Compared to other proposed quantification methods, such as flow cytometry, these data could otherwise be lost or overlooked and may cause false positives or negatives23. However, imaging is more labor-intensive and more prone to biased data acquisition as flow cytometry. Here, we have overcome these obstacles through automation of the protocol, thereby reducing bias, labor, cost, and time.
Many modulators of macropinocytosis most likely remain undiscovered and, given the importance of the macropinocytic pathway in certain pathologies, such as cancer, their discovery could potentially be of great importance for the development of novel therapeutic approaches2,3. The identification of these modulators can be achieved through high-content screening of compounds, for which the herein proposed method may function as a cornerstone. The outlined protocol is aimed at performing the experiments with standard laboratory equipment. However, the 96-well plate format may be optimized and adapted for high-content screening. Increasing the well format to 384 or 1536-well microplates would allow the user to increase the number of compounds that can be tested on one plate. Moreover, robotization of cell seeding, plate washing, compound and dextran administration would reduce manual handling and well-to-well variability, and thereby improve scalability. Ultimately, an adaption of this protocol to high-content screening would greatly facilitate identifying new factors that regulate macropinocytosis.
Altogether, the herein proposed method is an excellent approach to determine the level of macropinocytosis in cells of interest, the identification of regulators of the process, and the optimization of drug concentrations for known inhibitors. Also, the protocol can serve as the starting point for assessing macropinocytic uptake of other cargo besides dextran, and high content screening aimed at identifying lead compounds that could potentially result in the development of novel therapeutic strategies.
The authors have nothing to disclose.
This work was supported by NIH/NCI grants (R01CA207189, R21CA243701) to C.C. KMO.G. is a recipient of a TRDRP Postdoctoral Fellowship Award (T30FT0952). The BioTek Cytation 5 is a part of the Sanford Burnham Prebys Cell Imaging Core, which receives financial support from the NCI Cancer Center Support Grant (P30 CA030199). Figures 1-3 were created using BioRender.
0.25% Trypsin | Corning | 25053CI | 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate |
1.5 mL Microcentrifuge tube | Fisherbrand | 05-408-129 | |
10-cm Tissue culture dish | Greiner Bio-One | 664160 | CELLSTAR |
15 mL Centrifuge tube | Fisherbrand | 07-200-886 | |
2 L Beaker | Fisherbrand | 02-591-33 | |
24-well Tissue culture plate | Greiner Bio-One | 662160 | CELLSTAR |
25 mL Reagent reservoir | Genesee Scientific Corporation | 28-121 | |
500 mL Beaker | Fisherbrand | 02-591-30 | |
6-cm Tissue culture dish | Greiner Bio-One | 628160 | CELLSTAR |
8-Channel aspiration adapter | Integra Biosciences | 155503 | |
8-Channel aspiration adapter for standard tips | Integra Biosciences | 159024 | |
95% Ethanol | Decon Laboratories Inc | 4355226 | |
Ammonia-free glass cleaner | Sparkle | FUN20500CT | |
Black 96-well high-content screening microplate | PerkinElmer | 6055300 | CellCarrier-96 Ultra |
Cotton-tipped applicator | Fisherbrand | 23-400-101 | |
Coverslips | Fisherbrand | 12-545-80 | 12 mm diameter |
Cytation 5 Cell Imaging Multi-Mode Reader | Biotek | CYT5FW | |
DAPI | Millipore Sigma | 5.08741 | |
Dextran 70 kDa – FITC | Life Technologies | D1822 | Lysine-fixable |
Dextran 70 kDa – TMR | Life Technologies | D1819 | |
DMSO | Millipore Sigma | D1435 | |
DPBS | Corning | 21031CV | Without Calcium and Magnesium |
Forceps | Fine Science Tools | 11251-20 | Dumont #5 |
Formaldehyde, 37% | Ricca Chemical | RSOF0010-250A | ACS Reagent Grade |
Glycerol | Fisher BioReagents | BP229-1 | |
Hardening fluorescence mounting media | Agilent Tech | S302380-2 | DAKO |
Hoechst 33342 | Millipore Sigma | B2261 | |
Hydrochloric acid (HCl) | Fisher Chemical | A144-212 | Certified ACS Plus, 36.5%–38.0% |
Lint-free wipes | Kimberly-Clark | 34155 | Kimwipes |
Miscroscope slides | Fisherbrand | 12-544-1 | Premium plain glass |
Multichannel pipette | Gilson | FA10013 | 8 channels, 0.5–10 µL |
Multichannel pipette | Gilson | FA10012 | 12 channels, 20–200 µL |
Multichannel pipette | Gilson | FA10011 | 8 channels, 20–200 µL |
Parafilm M | Pechiney | PM996 | |
Plastic wrap | Kirkland Signature | 208733 | Stretch-Tite |
Silicone isolators | Grace Bio Labs Inc | 664107 | 13 mm Diameter X 0.8 mm Depth ID, 25 mm X 25 mm |
Slide adapter | Biotek | 1220548 | |
Wash bottle | Fisherbrand | FB0340922C |