Quantitation of Macropinocytosis in Glioblastoma Based on High-Content Analysis

Background: Macropinocytosis serves as an internalization pathway for extracellular uid, albumin and dissolved molecules. Assessing macropinocytosis has been challenging in the past because manual acquisition in combination with visual evaluation of images is laborious, making it dicult for high-throughput applications. So, there is a need to develop sensitive and specic methods. Methods: This paper proposed a quantitative and time-saving method for macropinocytosis detection based on high-content analysis (HCA). Meanwhile, cell proliferation was tested by means of CCK8. Results: The term “macropinosome index” was dened to estimate macropinocytosis and allow comparison between different cell lines and treatments. Furthermore, we demonstrated that macropinocytosis can promote Glioblastoma (GBM) cells survival in glutamine decient conditions which resemble tumor microenvironment. Conclusions: HCA represents a novel, non-subjective and high-throughput assay for macropinocytosis assessment. Besides, Gln deprivation increased the macropinosome index in GBM cells, which points to the possible exploitation of this process in the design of GBM therapies.


Background
Macropinocytosis is a type of endocytosis that involves non-selectively uptake extracellular uid via plasma membrane ru ing into large intracellular vesicles known as macropinosomes. Macropinosomes can be visualized on the basis of the ability of cells to internalize extracellular medium containing tetramethylrhodamine labeled high-molecular-mass dextran (TMR-dextran), an established marker of macropinocytosis. Macropinocytosis functions in cancer cells as an important nutrient-scavenging pathway by which extracellular proteins move to lysosomes for degradation [1].
It was reported that intratumoral inhibition of macropinocytosis decreases amino acid levels [2].
Furthermore, macropinocytosis promotes both tumor growth and chemotherapy resistance in vivo by uptake necrotic cell debris [3,4]. The level of micropinocytosis can increase in the context of oncogenic transformation (e.g., oncogenic RAS or PI3K) or tumor suppressor mutations (e.g., loss of PTEN) [5][6]. Besides, enhanced macropinocytosis has also been found by the upregulation of growth factors [7].
Recent works have described the tumor can adapt to Glutamine (Gln) deprivation [8], implying the presence of unknown regulatory mechanisms that enable tumor progression. A full revealing of how macropinocytosis is regulated would be of great signi cance in understanding how the tumor proliferates in the microenvironment of nutrient deprivation, ultimately leading to new therapeutic approaches.
In this context, this study aimed to verify in vitro whether Gln deprivation can change the extent of macropinocytosis in GBM cells, which normally have moderate levels of macropinocytosis, in addition to proposing a new method to develop a high-throughput assay.

Methods
Preparing Gln deprivation medium Gln deprivation medium was prepared to contain inorganic salts, i.p. 4.5 g/L glucose, 2.5 g/L NaHCO 3 , 0.11 g/L C 3 H 3 NaO 3 , and Phenol Red, according to the composition of DMEM.

Seeding cells
The special 96 well plate should be irradiated by ultraviolet in biological safety cabinet for at least 12 h before seeding cells. Pipette 100 μl cells into 96 well black plates using a multichannel pipette or a single channel pipette. Incubate cells in complete fresh medium for 24 h in an incubator (37 °C, 5% CO 2 ) to ensure attachment.

Labeling macropinosomes
The visualization of macropinosomes in cells is based on the ability of cells to internalize extracellular uid containing uorescently tagged TMR-dextran (Thermo Fisher Scienti c, D1818), which is universally used to mark macropinosomes. After starvation for 24h, the former medium was replaced with complete medium including 75 μM TMR-dextran. Incubate the cells in a cell culture incubator (37 °C, 5% CO 2 ) for 30 min.

Labeling cytoplasm
After complete rinsing of the sample with pre-cooling PBS, x the sample in 4% methanol-free paraformaldehyde solution in pre-cooling PBS for 15 min at room temperature. Wash the sample three times with pre-cooling PBS, 5 min at one time. Permmeabilize the sample in 0.5% Triton TM X-100 in PBS for 15 min. Incubate the sample for 40 min in 50 μl phalloidin (Thermo Fisher Scienti c, A12379) staining solution for each well at room temperature.

Labeling nuclei
Incubate the sample in the 50 μl DAPI (Abcam, ab104139) for each well for 15 min at room temperature. Wash the sample three times with pre-cooling PBS, 5 min at one time. Add one drop of anti-uorescence quencher (BBI, E675011) for each well. Then observe at once by high content or store in the dark at 2-6℃ and observe within 12 h.

Capturing images
A High-content screening (PerkinElmer Operetta CLS) equipped for phase contrast with a standard optical iter set including DAPI, Alexa Fluor 488 and Alexa 568 was used. Select random elds of one well to test and set an appropriate exposure time to be applied to all the samples for each individual channel being analyzed. Capture images using the ×63 water-immersion objective and the Harmony software platform. The FITC and Alexa 568 exposure times range from 200 to 500 ms. Then select all the wells containing cells. To ensure the reliability of results, obtain 50-100 random elds for every well.

Data analysis
After capturing, the data will be saved automatically. Then load the data in measurement. Set script parameters including contrast, area and the detection algorithm to properly segment nuclei, cytoplasm and spots in every cell. Finally, add "De ne Result", de ne Variable a as "Number of Spots", de ne Variable b as "Cell Area", and de ne Formula as "a/b", then click Apply or Save to use next time.

Results
Macropinocytosis detection was established based on high-content analysis (HCA).
Schematic work ow of the protocol as shown in Fig. 1. For simplicity, we multiply macropinosome index values by 10. We came up with macropinosome index to measure the level of macropinocytosis. The macropinosome index values for cancer cells <0.5 and >1 are de ned as low macropinocytosis cells and highly macropinocytosis cells, respectively.
As an example of the implementation of this protocol, we assessed the extent of macropinocytosis in GBM cells. Sample images from the various steps of quanti cation are shown in Fig.2. Nuclei and cytoplasm are labeled with DAPI and phalloidin, respectively. U251 was maintained in serum-free media and treated with vehicle or 75 μM EIPA for 30 min, after which spots representing macropinosomes were labeled by uptake of TMR-dextran. After merged images capturing, the data will be saved automatically.
Then load the data in measurement. Set script parameters including contrast, area and the detection algorithm to properly segment nuclei, cytoplasm and spots in every cell. The total spots number and total cell area are then recorded and tabulated as shown in Table 1. EIPA treatment signi cantly inhibited macropinosome index campare to control group. The mean macropinosome index of EIPA treatment and control group is 1.16±0.10 and 0.43±0.19 (P<0.001), respectively.
GBM survive in the microenvironment of the Gln deprivation The apoptosis level increased and the morphology became longer for GBM cells in the microenvironment of Gln deprivation (Fig.3, A). We found that GBM cells grew slower in the absence of Gln, demonstrating that Gln starvation hindered proliferation to different extents. However, GBM cells can survive for at least ve days in such situation (Fig.3, B and C).

Gln deprivation promote macropinocytosis in GBM
To determine whether stimulated macropinocytosis is a feature of GBM exposing to nutrient stress, we compared TMR-dextran uptake of U251 and U87 cell lines in Gln deprivation medium and complete medium by the described protocol (Fig.4, A). Gln deprivation increased the macropinosome index in GBM cells (Fig.4, B and C).

Discussion
Macropinocytos is a kind of pathway for internalization involving vesicles 50-300 nm in diameter. In the analysis of micropinocytosis, the concept of micropinocytosis index was de ned by Lee SW et al [9], which is widely applied, especially in the eld of cancer research [10][11][12]. Generally, macropinocytosis assay mainly includes two discontinuous stages: (1) images capture using uorescent microscope; (2) image quanti cation by means of ImageJ software. Furthermore, the second stage also contains several steps, such as count nuclei, adjust the brightness, subtract the background, apply smooth function, adjust threshold, convert the image to a binary image and use the Analyze Particles function. Obviously, the assay is complicated and time-consuming because uorescence microscopy and analysis cannot be performed simultaneously. Besides, it is necessary to wash coverslips and seed cells on the coverslips to capture images.
In this study, we developed a quantitative image analysis protocol for macropinocytosis based on HCA. The special capability of HCA is a spot counting feature that allows the determination of the quantity and size of macropinosomes in each cell. The extent of macropinocytosis in GBM cells was quickly and accurately estimated by means of this protocol.
Glutamine (Gln) is considered a conditionally essential amino acid for tumor cells. Breakdown of Gln through glutaminolysis gives rise to glutamate (Glu), a critical precursor of most other nonessential amino acids [13]. As tumors continue to grow, increased nutrient demand and poor vascularization lead to Gln deprivation in the microenvironment. Glioma cells are able to release amounts of Glu, which potentially promotes tumorigenesis through activation of ionotropic receptors [14]. Gln addiction has been proposed as a mark of glioblastoma (GBM) [15], which means that Gln deprivation reduces proliferation and tumorigenicity. Our results are consistent with previous studies . However, We asso found that GBM cells can survive for at least ve days in such situation.
Tumor cells can survive Gln deprivation by activating cell cycle arrest genes [16], performing metabolic reprogramming [17], or sustaining purine availability by Gln synthetase [18], but other mechanisms can also contribute to cell survival. Gln deprivation-induced scavenging mechanism has received considerable attention in macropinocytosis [19]. This is an endocytic process that supports proliferation of tumor cells in the medium devoid of essential amino acids [20]. Macropinocytosis promotes survival and reduces chemosensitivity in GBM cells [21,22]. We identi ed macropinocytosis as a mechanism by which GBM cells adapt nutritionally de cient microenvironment. Thus, macropinocytosis may represent a predictive biomarker for cancer therapy.

Conclusions
In this study, we developed a novel assay for macropinocytosis based on HCA. This assay enables simultaneous analysis of spots, nuclei and cytoplasm in a wide variety of cellular models, representing a novel, non-subjective and high-throughput assay for macropinocytosis assessment. Besides, we demonstrated that Gln deprivation increased the macropinosome index in GBM cells, which points to the possible exploitation of this process in the design of GBM therapies. Ethics approval and consent to participate Not applicable.

Con ict of interest
All the authors declared that they have no con ct of interest.

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability statement
The data of macropinosome index values are available in this published article. Other data generated or analysed during the current study are available from the corresponding author on reasonable request.    The apoptosis level increased and the morphology became longer for GBM cells in the microenvironment of Gln deprivation (Fig.3, A). We found that GBM cells grew slower in the absence of Gln, demonstrating that Gln starvation hindered proliferation to different extents. However, GBM cells can survive for at least ve days in such situation (Fig.3, B and C). To determine whether stimulated macropinocytosis is a feature of GBM exposing to nutrient stress, we compared TMR-dextran uptake of U251 and U87 cell lines in Gln deprivation medium and complete medium by the described protocol (Fig.4, A). Gln deprivation increased the macropinosome index in GBM cells (Fig.4, B and C).