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

Summary

Here, we study hepatotoxicity caused by lapatinib and neratinib through SASP-driven macrophage polarization, which offers a deeper mechanistic understanding beyond traditional toxicity tests. This approach demonstrates how senescent hepatocytes influence immune responses. This is particularly valuable for assessing long-term drug effects and discovering new therapeutic targets for managing drug-induced hepatotoxicity.

Abstract

Lapatinib and neratinib are tyrosine kinase inhibitors (TKIs) approved for treating HER2-positive metastatic breast cancer, but their clinical use is limited by hepatotoxicity. Within hepatocytes, cytochrome P450 enzymes produce reactive metabolites of these drugs, causing DNA damage, mitochondrial dysfunction, and oxidative stress, which lead to hepatocyte injury. This cellular stress triggers hepatocyte senescence, a state of growth arrest accompanied by the secretion of pro-inflammatory and tissue-remodeling factors called the senescence-associated secretory phenotype (SASP). SASP significantly modifies the liver microenvironment, influences immune cell behavior, and worsens tissue damage. We show that lapatinib and neratinib induce senescence and SASP in the HepG2 liver cell line. Additionally, SASP from these senescent cells promotes M2-like polarization of RAW264.7 macrophages. Although M2 macrophages are associated with anti-inflammatory responses and tissue repair, their excessive or prolonged activity can lead to tissue damage and fibrosis. Our findings highlight a mechanism by which TKI-induced hepatocyte senescence and SASP modulate the immune response and may contribute to liver toxicity in breast cancer patients. This underscores the importance of targeting senescent cells or SASP factors to reduce liver toxicity and improve treatment outcomes, making the identification of effective senotherapeutics a vital research focus. There hasn't been any literature report demonstrating the effect of TKI-induced liver cell secretome on macrophage polarization.

Introduction

Lapatinib and neratinib, both small-molecule tyrosine kinase inhibitors (TKI), received FDA approval in 2007 and 2017, for the treatment of metastatic breast cancer¹. These drugs are known to inhibit the human epidermal growth factor receptor 2, HER2², a receptor tyrosine kinase that is overexpressed in certain breast cancers³. Despite their clinical success, lapatinib and neratinib are also associated with adverse effects, particularly hepatotoxicity^4,5. Acute liver injury, manifested by elevated aminotransferase levels, occurs in approximately 14% of patients undergoing treatment with either lapatinib or neratinib⁶. The hepatotoxicity is largely attributed to the hepatic metabolism of these drugs, which involves cytochrome P450 (CYP) enzymes, predominantly CYP3A4/5^7,8. These enzymes catalyse the formation of quinone-imine intermediates, which are electrophilic and highly reactive^9,10. These reactive metabolites, including O-dealkylated lapatinib, N-dealkylated lapatinib, and N-hydroxy lapatinib, O-dealkylated neratinib, and N-demethylated neratinib, can form covalent adducts with cellular macromolecules, thereby disrupting normal cellular functions and ultimately resulting in liver injury¹¹.

Reactive metabolites of lapatinib and neratinib can also induce oxidative stress, a key trigger of cellular senescence¹². Cellular senescence is a growth-arrested but metabolically active state, characterized by the secretion of various pro-inflammatory cytokines, growth factors, and proteases. This secretory phenotype is known as the senescence-associated secretory phenotype (SASP)^13,14,15. The SASP can profoundly affect the surrounding microenvironment, often promoting inflammation and tissue remodelling. In the context of cancer therapy, the SASP can enrich the cancer stem cell population and modify the immune response to favour tumor survival¹⁶. Additionally, the inflammatory cytokines can exacerbate tissue damage. We speculated that in patients treated with lapatinib and neratinib, the liver injury is partly contributed to by TKI-induced senescence and pro-inflammatory SASP production.

Our experiments demonstrated that lapatinib and neratinib induce the production of reactive oxygen species (ROS) in hepatic cells, leading to DNA damage and mitochondrial dysfunction, which in turn triggers cellular senescence. This senescence may contribute to lapatinib/neratinib-induced hepatotoxicity by releasing SASP factors, which can influence macrophage polarization.

Macrophages play a critical role in the immune response, and their functional phenotype can shift in response to various stimuli^17,18. The classical M1 macrophage phenotype is typically associated with pro-inflammatory responses, including the production of cytokines such as TNF-α and IL-1β, and is generally considered a marker of acute inflammation^19,20. In contrast, the M2 macrophage phenotype is associated with fibrosis, tissue repair²¹, immunosuppression, and a response to chronic inflammation^22,23. The inflammatory cytokines in the SASP can influence macrophage polarization, promoting the conversion of M1 macrophages to an M2-like phenotype²³. This shift in polarization can have significant implications for the immune microenvironment, potentially leading to immunosuppression, tumor progression, and remodelling of tissue architecture.

To investigate the macrophage polarizartion induced by lapatinib and neratinib, we used the RAW264.7 cell line, a murine macrophage-like cell line commonly used in immunological research^24,25. RAW264.7 cells are derived from BALB/c mice and are widely used to study inflammation, phagocytosis, and immune responses²⁵. They respond to various stimuli, including lipopolysaccharides (LPS), and are particularly valuable for studying macrophage function, cytokine production, and cancer biology²⁶. In this study, HepG2, a human hepatoma cell line, was treated with lapatinib and neratinib to mimic the hepatotoxic effects observed in patients. The HepG2 cell line is often used as a drug metabolism and hepatotoxicity study model due to its human origin and hepatocellular carcinoma lineage²⁷. After treating the HepG2 cells with the TKIs, the conditioned media were collected and added to RAW264.7 cells. The subsequent macrophage phenotypic changes were monitored, focusing on whether the treatment induces a shift from the pro-inflammatory M1 phenotype to the immunosuppressive M2 phenotype.

Drug-induced hepatotoxicity is generally studied through cytotoxicity assays, assessment of inflammatory markers, and the use of animal models. Compared to these, our method of examining TKI lapatinib and neratinib-induced hepatotoxicity through senescence-associated secretory phenotype (SASP) factors and their influence on macrophage polarisation has several advantages. First, our method establishes a connection between SASP factors produced by drug-induced senescent liver cells and innate immunity, which can be further linked to tissue remodelling, chronic inflammation, and carcinogenesis^28,29,30,31. Conventional toxicity screens cannot help us capture the short-term interactions between the liver and immune cells, nor can they reveal the long-term impact of TKIs on liver inflammation and fibrosis, particularly in the context of chronic drug exposure. When combined with a suitable proteomics approach to identify SASP constituents, this technique can help identify predictive biomarkers of TKI-induced hepatotoxicity and inform the design of therapeutic interventions to mitigate it^29,30,31.

For implementation, researchers should utilize co-culture or conditioned media systems with hepatic and immune cell lines, as described, and evaluate biomarkers of senescence as well as M1 and M2 macrophages. This method is most effective for assessment of drug-induced acute or chronic liver injury, or for exploring strategies to minimize off-target effects of cancer therapies^29,30,31.

Protocol

1. Cell culture

1. Culture HepG2 cells in Modified Eagle Medium (MEM) high glucose with 10% Fetal Bovine Serum (FBS) and RAW264.7 cells in Dulbecco's Modified Eagle Medium (DMEM) high glucose with 10% FBS at 37 °C with 5% CO ₂ and refresh every 3 days.
   NOTE: Antibiotic usage is not recommended unless there is any fungal or bacterial contamination. RAW264.7 cells are very fast-growing and should be passaged after 2 days.

2. MTT assay

1. Cell counting and cell plating
   1. Grow HepG2 cells in a 60 mm culture dish in a cell culture incubator in complete growth media (MEM + 10% FBS).
   2. When the cells were about 80% confluent, remove the growth media, give a quick wash with 1 mL of 1x PBS, and add 600 μL of 0.5x Trypsin. Incubate for 5-6 min.
      NOTE: All steps should be performed in a BSL 2 cell culture cabinet.
   3. Neutralize trypsin by adding 1.2 mL of complete growth media inside the BSL 2.
      NOTE: To prevent over-trypsinization, complete media, twice the volume of added trypsin, should be added.
   4. Take the cell suspension in a 5 mL microcentrifuge tube (MCT) and pellet it down by centrifuging at 180 g (1000 rpm) for 3-4 min at room temperature (RT).
   5. Discard the supernatant, resuspend the cell pellet in 1 mL of media, pipette to mix as a single cell suspension, and count using a hemocytometer under a microscope.
   6. Next, plate HepG2 cells in a 96-well plate at 5000-7000 cells per well, in complete growth media, in triplicate for each drug dose, and keep in the incubator for 12-16 h.
      NOTE: Cell confluency should be 50-60% for drug treatment, and this step should be performed inside the hood.
2. Drug treatment
   1. Prepare gradually increasing working concentrations of lapatinib and neratinib in dimethyl sulfoxide (DMSO) from the stock concentration (30 mM and 3.5 mM, respectively) inside the hood. The final concentrations are 1, 2.5, 5, 10, 15, 25, 50 µM for lapatinib and 1, 2, 3, 4, 5, 6, 10 µM for neratinib in growth media (MEM + 5% FBS). Keep one no-treatment control for both drugs.
   2. Discard the previous media from the 96-well plate containing cells, and add 100 µL of media with the drug and incubate for 48 h inside the incubator.
3. Staining and quantification
   1. After 48 h, add 10 µL of 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT; 5 mg/mL), dissolve in 1x phosphate buffer saline (PBS) in each well, and incubate for at least 2 h inside the incubator until purple colored crystals appear.
      NOTE: Add the dye directly into drug-containing media and do not incubate cells with dye for more than 4 h.
   2. After incubation, remove the dye, dissolve the crystals in 100 µL of DMSO in each well, and incubate for 15 min, followed by taking the optical density (OD) at 595 nm.
      NOTE: This step can be conducted outside the hood.

3. SA-β galactosidase assay

1. Drug treatment
   1. Plate HepG2 cells in 35 mm dishes at a density of 40 x 10⁴ cells per plate in three sets, one for lapatinib, one for neratinib, and one untreated control.
      NOTE: Cell counting and plating steps are as described in step 2.1.
   2. Add 1 µL of lapatinib from 5 mM working stock and 1.43 µL of neratinib from 3.5 mM main stock in 2 mL of media to make a final concentration of 2.5 µM (sublethal dose) of lapatinib and neratinib.
      NOTE: Sublethal drug dose is selected following the cell survival graph in the MTT assay.
   3. Remove the previous media from the dishes and wash with 500 µL of 1x PBS once, and add drug-containing media (Inside the hood) and keep for 48 h inside the incubator.
2. Fixing and staining
   1. After 48 h, remove the drug, give a quick wash with 500 µL of 1x PBS once, and fix with 1.5 mL of 2% formaldehyde and 0.2% glutaraldehyde in 1x PBS for 10 min at RT.
   2. Following fixation, wash the plates with 1 mL of 1x PBS twice and ensure the fixative solution is removed correctly. Add 1.5 mL of staining solution (40 mM citric acid/ Na-phosphate buffer, 5 mM K4[Fe(CN)6], 5 mM K3[Fe(CN)6], 150 mM NaCl, 2 mM MgCl ₂, 1 mg/mL X-gal in autoclaved water) and incubate for 8 h at 37 °C without CO₂.
      NOTE: Incubation period may vary according to the cell type; for HepG2, the incubation period should not exceed 8 h.
   3. Following staining, wash the plates quickly in 1 mL of 1x PBS twice and capture brightfield images at 20x and 40x magnification with an epi-fluorescence microscope. Capture a minimum of four to five fields for each treatment group.
      NOTE: The volume of fixative and staining solution will vary with the plate size, and all the steps may be performed outside the hood.
3. Image quantification
   1. Take a picture from one field, count the total number of cells as well as the cells showing a blue signal. Manually calculate the percentage of cells with a blue signal. Calculate at least four images, take the average, plot in a spreadsheet graph, and conduct a t-test.

4. ROS detection

1. Cell plating and drug treatment
   1. Plate HepG2 cells in 35 mm glass-bottom dishes at a density of 40 x 10⁴ cells per plate in three sets: one for lapatinib, one for neratinib, and one untreated control.
      NOTE: Cell counting and plating steps are as described in step 2.1.
   2. Follow the same treatment strategy as described in step 3.1.
2. Staining and imaging
   1. After 48 h of treatment, add 2 µM cell ROX deep red reagent (5 mM stock), 1 µM Mito Tracker Green (1 mM stock) for 30 min, and counterstain with 500 nM nuclear staining dye Hoechst (500 µM stock).
      NOTE: Dye should be added inside the hood, and direct exposure to light should be avoided. Counterstaining is done by adding nuclear staining dye Hoechst during the administration of other dyes.
   2. After 30 min, remove the dye, and wash with 1x PBS to remove excess dye and image with a confocal microscope at 100x magnification (oil emersion, 640 nm, 488 nm, and 405 nm excitation wavelength, respectively).
3. Image quantification
   1. Quantify the signal of cell ROX red using Fiji ImageJ software and compare it to the signal of the untreated control. Use MitoTracker green to indicate that the signal originates from mitochondria. Following plotting the bar graphs with average values, conduct a t-test.

5. Conditioned media (CM) collection

1. Treat HepG2 cells with 2.5 µM of lapatinib or neratinib for 48 h (treatment strategy similar to step 3.1, except for this experiment, plate 70 x10⁴ cells in a 60 mm dish).
2. After 48 h, remove the drug and wash with 500 µL of 1x PBS once.
3. On the maximum day of senescence (after 48 h of drug treatment), add 4 mL of serum-free MEM media (without FBS or any other supplements) to the drug-treated cells as well as the untreated control for 48 h.
   NOTE: The volume of serum-free media will depend upon the plate size.
4. Next, collect the serum-free media containing all the secretome, termed as conditioned media, and store at -80 °C for future use.
   NOTE: After collecting the CM, it should be centrifuged for 5 min at 2800 g (4000 rpm) to remove dead cells and other cellular debris. RAW264.7 cells are typically cultured in DMEM, but CM is collected in MEM as per HepG2 culture conditions. To maintain compatibility, conditioned media should be mixed with serum-free DMEM in 1:1 ratio prior to RAW264.7 incubation.

6. M1/M2 polarization in RAW264.7

1. Cell plating
   1. Follow the cell counting and cell plating as described in step 2.1, except the trypsinization step. While trypsinizing RAW264.7 cells, add 600-700 µL of 0.5x trypsin, incubate for 15-20 min in the incubator, and for neutralizing trypsin, add 2 mL of growth media (DMEM + 10% FBS) and plate 70 x10⁴ cells in a 60 mm dish in three sets: lapatinib, neratinib and one untreated control.
2. RAW264.7 induction
   1. Remove the regular growth media, wash with 1 mL of 1x PBS once, and add the corresponding CM for 48 h.
      NOTE: Before adding the CM, make sure to condition it with serum-free DMEM in a 1:1 ratio, as RAW264.7 cells grow in DMEM, so keeping the cells in MEM will cause stress, which may hamper the result. Culturing RAW264.7 in CM (serum-free media) for 48 h may also cause stress; in that case, 24-30 h incubation can be done before collecting the data.
   2. After incubation, discard the CM, wash with 1 mL of 1x PBS, add 600 µL of trypsin in each plate, incubate for 10 min, and then pellet down at 180 g (1000 rpm) for 3 min at RT. Use the pellet for protein purification or store it at -80 °C for future use.
      NOTE: Trypsinization time will vary with cell type, and centrifugation is not recommended above 180 g.
3. Protein isolation and western blotting
   1. Prepare the lysis buffer by mixing radioimmunoprecipitation assay (RIPA) lysis buffer and protease inhibitor cocktail at a 100:1 ratio. Add the lysis buffer to the cell pellet, resuspend, and incubate on ice for 30 min.
      NOTE: The volume of lysis buffer will vary with the size of the cell palette, and lysis can be done outside the hood. If the pellet is stored at -80 °C, then first thaw the pellet on ice, give a short spin, and remove any residual media.
   2. After incubation, spin the cell lysate at 20,000 g for 30 min at 4 °C. Collect the supernatant and proceed for western blot, or store at -20 °C for future use.
   3. Prepare protein sample by taking 35-40 µg of protein, add 1x loading dye, heat for 5-6 min at 95 °C, run SDS-PAGE (10% and 12% acrylamide gel) at 90 V, and transfer to nitrocellulose membrane.
   4. NOTE: For more detailed information regarding the western blot, refer to Joubert et al.³².
   5. Check the expression of M1 and M2 polarization markers, via developing the blot using a horseradish peroxidase (HRP) developer, iNOS (M1) and arginase1 (M2), respectively, by using anti-NOS2 and anti-ARG1 antibodies.
      NOTE: Concentration for both primary antibodies, anti-NOS2 and anti-ARG1 is 1:1000, and secondary anti-rabbit antibody is 1:10,000.
4. Macrophage polarization using LPS and IFN-γ
   1. Plate 70 x 10⁴ RAW264.7 cells in two 60 mm dishes.
      NOTE: Plating procedure is similar to step 6.1.
   2. After 12-16 h, mix 4 µL of 1 µg/mL lipopolysaccharide (LPS, stock concentration is 1 mg/mL) and 1.33 µL of 33 ng/mL interferon-γ (IFN-γ, stock concentration is 100 µg/mL) in 4 mL of growth media (DMEM + 10% FBS) and add to the pre-plated RAW264.7, keeping one uninduced control.
   3. After 48 h, collect the cell pellet via trypsinization, isolate protein, run a western blot, and check the expression of iNOS (M1) and ARG1 (M2), respectively, by using anti-iNOS and anti-ARG1 antibodies.
      NOTE: All cell culture waste and biochemical waste should be discarded in a biohazard bin, properly autoclaved, and then disposed of.

Results

A series of assays was conducted to evaluate the impact of TKI lapatinib and neratinib on hepatocellular carcinoma (HepG2) cells and their downstream paracrine effects on macrophages. HepG2 cells were treated with varying concentrations of lapatinib or neratinib, and cell viability was measured at 48 h using the MTT assay (Figures 1A,B). Based on the survival curves, a sublethal dose of 2.5 µM was chosen for further experiments.

Discussion

The current study explores the indirect effects of lapatinib or neratinib treatment on macrophage polarization through conditioned media collected from TKI-treated HepG2 liver cells. Utilizing RAW264.7 macrophages, we observe an M2 phenotype upon exposure to the conditioned media, as indicated by the upregulation of ARG1. Morphologically, the treated macrophages displayed a characteristic enlarged and elongated shape, further supporting the induction of an M2-like state. Below, we discuss the protocol's critical steps, p...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HepG2 cell line	NCCS		Adherent cell line
RAW264.7 cell line	NCCS		Adherent cell line
DMEM (Dulbacco's Modified Eagle Medium)	Himedia	AT066	High glucose, Powder
MEM (Modified Eagle Medium	Gibco	611100-053	Powder
PBS (Phosphate Buffer Saline)	Himedia	TS1101	Powder
Trypsin-EDTA Solution	Himedia	TCL007	Solution
FBS (Fetal Bovine Serum)	Himedia	RM1112	Liquid
MTT Dye	Himedia	TC191	Powder
Lapatinib	Selleckchem	S2111	Powder
Neratinib	Cayman Chemical Company	18404	Powder
DMSO (Dimethyl Sulfoxide)	Himedia	TC185	Solution
Formaldehyde	Rankem	F0080	Solution
Glutaraldehyde	Sigma-aldrich	340855	Solution
Citric Acid monohydrate, Pure	Himedia	GRM229	Powder
Sodium phosphate dibasic anhydrous	Himedia	MB024	Powder
Potassium ferrocyanide, K4[Fe(CN)6]	Fisher Scientific	P-236-500
Potassium ferricyanide, K3[Fe(CN)6]	Fisher Scientific	P-232-500
NaCl	SRL	41721	Powder
MgCl2 hexahydrate	Himedia	GRM-1068	Powder
X-Gal	SRL	45904	Powder
Lipopolysaccharide	Sigma-aldrich	L6529
Human Recombinant Interferon-γ	Himedia	CF040-20MCG
Cell ROX deep red reagent	Thermo fisher scientific	C10422
Hoechst	Invitrogen, Thermo Fisher Scientific	H3570	Powder
MitoTRACKER Green	Invitrogen, Thermo Fisher Scientific	1842298	Powder
Anti-ARG1 antibody	Affinity	DF6657	Solution, Calculated MW- 35kDa, Observed MW - 65kDa
Anti-NOS2 antibody	Cloudo Clone Corp.	PAA837Hu01	Solution, Calculated MW- 130kDa, Observed MW - 65kDa
Anti-iNOS antibody	Affinity		Solution, Calculated MW- 130kDa, Observed MW - 120kDa
Anti-GAPDH antibody	Biobharati LifeScience Pvt.Ltd	BB-AB0060	Solution
Anti-Rabbit Antibody	Cayman Chemical Company	10004301	Solution
Nitrocellulose membrane	Merck Millipore Ltd.	HATF00010 (Product number)
femtoLUCENTTMPLUS HRP	G Bioscience	786-003
60mm Dishes	NUCK	150462
96well plate,	NUCK	165306
Cell Culture Incubator	Thermo Fisher Scientific	371
Microscope	1. LEICA Inverted Microscope   2. Nikon Confocal Microscope	1. DMIL LED     2. Ti2E, A1R	Both are model number
iBright Imaging System	Invitrogen, Thermo Fisher Scientific	CL1500 (model number)
Plate-reader	Bio-RAD	iMark Microplate Reader
Centrifuge	Thermo Fisher Scientific	Sorvall ST 8R (model number)