Drug-Induced Senescence in Liver Cells Promotes M2 Macrophage Polarization: Implications for Tyrosine Kinase Inhibitor-Associated Hepatotoxicity

The research examines how TKI lapatinib and neratinib induce senescence in liver cells, triggering secretin production to influence macrophage polarization. Such phenomenon may be key to the TKI-induced hepatotoxicity. The current technologies include culturing of liver and immune cells in cellular acids with the conditioned media, which is from the senescent liver cells, confocal microscopy, western blotting, and cytokine profiling, proteomics, and examining the ROS production and senescence biomarkers.

The current challenges basically include variability in senescence induction. It could be residual drug contamination. Limited relevance of immortalized cell lines is one of the challenges, differences between urine and human macrophages, neglecting cell-cell interactions, and optimizing conditioned media collection.

Through our study, we link SASP production via senescent liver cell to macrophage polarization and provide insight into TKI-induced immune modulation. Traditional hepatotoxicity assay or animal model often miss such paracrine interaction. Our lab aims to identify key SASP components and senotherapeutics to mitigate TKI-induced hepatotoxicity, investigate human macrophage responses, and explore the long-term effect of TKI-induced senescence on liver damage and tumorigenesis.

To begin, grow Hep G2 cells in a 60-millimeter culture dish containing complete growth media, composed of minimum essential medium, and 10%FBS, and place the dish in a cell culture incubator. Once the cells reach approximately 80%confluency, remove the growth media using a pipette, rinse the dish quickly with one milliliter of PBS, then add 600 microliters of 0.5 times trypsin to the dish. Place the dish back into the incubator for five to six minutes.

Inside a biosafety level two cabinet, neutralize the trypsin by adding 1.2 milliliters of complete growth media to the dish. Using a pipette, transfer the cell suspension into a five-milliliter microcentrifuge tube. Centrifuge the tube at 180 G for three minutes at room temperature.

Discard the supernatant using a pipette. Resuspend the cell pellet in one milliliter of complete growth media to create a single cell suspension, and count the cells using a hemocytometer under a microscope. Plate Hep G2 cells in a 96-well plate at 5, 000 to 7, 000 cells per well in triplicate for each drug dose using complete growth media.

Place the plate in the incubator for 12 to 16 hours to allow the cells to adhere. Inside the biosafety cabinet, prepare serially increasing working concentrations of lapatinib and neratinib in DMSO from their stock concentrations of 30 millimolar and 3.5 millimolar respectively. Dilute the drugs in growth media containing minimum essential medium and 5%FBS.

Prepare one untreated control for each drug. Aspirate the old media from the 96-well plate containing the cells, and add 100 microliters of drug-containing media to each well. Incubate the plate for 48 hours inside the incubator.

After 48 hours, add 10 microliters of MTT dye at five milligrams per milliliter, dissolved in PBS directly into the media in each well. Incubate the plate for at least two hours until purple crystals appear. Remove the media containing the dye from each well, then add 100 microliters of DMSO to dissolve the purple crystals.

After 15 minutes, record the optical density at 595 nanometers. Plate Hep G2 cells in 35-millimeter dishes at a density of 400, 000 cells per dish, creating three sets, one for lapatinib, one for neratinib, and one for untreated control. Add one microliter of lapatinib from a five-millimolar working stock and 1.43 microliters of neratinib from a 3.5 millimolar main stock to two milliliters of media to prepare drug-containing media.

Inside the biosafety cabinet, remove the old media from each dish and wash once with 500 microliters of PBS. After adding the drug-containing media, place the dishes into the incubator for 48 hours. Next, remove the drug-containing media and rinse the dishes once with 500 microliters of PBS.

Incubate the cells with 1.5 milliliters of a fixative solution containing 2%formaldehyde and 0.2%glutaraldehyde in PBS at room temperature for 10 minutes. Wash each dish twice with one milliliter of PBS to remove the fixative solution, then incubate the dishes with the staining solution at 37 degrees Celsius for eight hours without carbon dioxide. After staining, wash each plate twice with one milliliter of PBS.

Capture bright-field images at 20 and 40 X magnification using an epifluorescence microscope covering a minimum of four to five fields per treatment group. Now select one captured field to count the total number of cells along with the number of cells exhibiting a blue signal. Manually calculate the percentage of blue stained cells for at least four images, average the results, and plot them in a spreadsheet graph.

Plate Hep G2 cells in 35-millimeter glass bottom dishes at a density of 400, 000 cells per dish, setting up three experimental sets, one for lapatinib, one for neratinib, and one for untreated control. Treat the plated cells using the same procedure as previously described for lapatinib and neratinib, including drug preparation, media replacement, and 48-hour incubation. After 48 hours of treatment, add two-micromolar CellROX Deep Red Reagent, one-micromolar MitoTracker Green, and 500-nanomolar hooks dye to each dish.

Incubate the cells for 30 minutes. After 30 minutes, aspirate the staining solution and wash the cells with PBS to remove excess dye. Acquire images using a confocal microscope at 100 X oil immersion magnification with appropriate excitation wavelengths.

Use Fiji ImageJ software to quantify the red signal from CellROX Deep Red and compare it with the signal from the untreated control. Use the green MitoTracker signal to confirm mitochondrial localization. Plot the average signal intensities as bar graphs and perform a T-test for statistical comparison.

Hep G2 cell viability decreased in a dose-dependent manner after treatment with lapatinib and neratinib. Treatment with 2.5-micromolar lapatinib or neratinib caused a strong increase in mitochondrial reactive oxygen species as shown in CellROX Deep Red staining, and colocalization with MitoTracker Green confirmed mitochondrial origin. SA beta-galactosidase staining revealed a marked increase in senescent cells after treatment with lapatinib and neratinib compared to the control.

RAW 264.7 macrophages exposed to conditioned media from drug-treated Hep G2 cells exhibited a larger, elongated morphology consistent with polarization. Western blotting showed increased expression of arginase 1 in RAW 264.7 macrophages treated with conditioned media from the lapatinib and neratinib groups while inducible nitric oxide synthase levels were decreased.