Name: Drug-Induced Senescence in Liver Cells Promotes M2 Macrophage Polarization: Implications for Tyrosine Kinase Inhibitor-Associated Hepatotoxicity (Video) | JoVE Journal
Uploaded: 2025-10-17T13:10:00.000+00:00
Duration: 9 min 32 s
Description: Explore how tyrosine kinase inhibitors induce senescence in liver cells and promote M2 macrophage polarization through SASP signaling. This method reveals mechanisms underlying drug-induced hepatotoxicity.

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5 minutes

The authors thank the Shiv Nadar Institution of Eminence for infrastructure support. This study is funded by the Department of Science and Technology grant CRG/2022/003242.

1. Cell culture
<ol>
	<li>Culture HepG2 cells in Modified Eagle Medium (MEM) high glucose with 10% Fetal Bovine Serum (FBS) and RAW264.7 cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose with 10% FBS at 37 &#176;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.</li>
</ol>
2. MTT assay
<ol>
	<li>Cell counting and cell plating
	<ol>
		<li>Grow HepG2 cells in a 60 mm culture dish in a cell culture incubator in complete growth media (MEM + 10% FBS).</li>
		<li>When the cells were about 80% confluent, remove the growth media, give a quick wash with 1 mL of 1x PBS, and add 600 &#956;L of 0.5x Trypsin. Incubate for 5-6 min. 
		NOTE: All steps should be performed in a BSL 2 cell culture cabinet.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Drug treatment
	<ol>
		<li>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 &#181;M for lapatinib and 1, 2, 3, 4, 5, 6, 10 &#181;M for neratinib in growth media (MEM + 5% FBS). Keep one no-treatment control for both drugs.</li>
		<li>Discard the previous media from the 96-well plate containing cells, and add 100 &#181;L of media with the drug and incubate for 48 h inside the incubator.</li>
	</ol>
	</li>
	<li>Staining and quantification
	<ol>
		<li>After 48 h, add 10 &#181;L 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.</li>
		<li>After incubation, remove the dye, dissolve the crystals in 100 &#181;L 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.</li>
	</ol>
	</li>
</ol>
3. SA-&#946; galactosidase assay
<ol>
	<li>Drug treatment
	<ol>
		<li>Plate HepG2 cells in 35 mm dishes at a density of 40&#160;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.</li>
		<li>Add 1 &#181;L of lapatinib from 5 mM working stock and 1.43 &#181;L of neratinib from 3.5 mM main stock in 2 mL of media to make a final concentration of 2.5 &#181;M (sublethal dose) of lapatinib and neratinib. 
		NOTE: Sublethal drug dose is selected following the cell survival graph in the MTT assay.</li>
		<li>Remove the previous media from the dishes and wash with 500 &#181;L of 1x PBS once, and add drug-containing media (Inside the hood) and keep for 48 h inside the incubator.</li>
	</ol>
	</li>
	<li>Fixing and staining
	<ol>
		<li>After 48 h, remove the drug, give a quick wash with 500 &#181;L 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.</li>
		<li>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 &#176;C without CO2. 
		NOTE: Incubation period may vary according to the cell type; for HepG2, the incubation period should not exceed 8 h.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Image quantification
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. ROS detection
<ol>
	<li>Cell plating and drug treatment
	<ol>
		<li>Plate HepG2 cells in 35 mm glass-bottom dishes at a density of 40&#160;x&#160;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.</li>
		<li>Follow the same treatment strategy as described in step 3.1.</li>
	</ol>
	</li>
	<li>Staining and imaging
	<ol>
		<li>After 48 h of treatment, add 2 &#181;M cell ROX deep red reagent (5 mM stock), 1 &#181;M Mito Tracker Green (1 mM stock) for 30 min, and counterstain with 500 nM nuclear staining dye Hoechst (500 &#181;M 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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Image quantification
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Conditioned media (CM) collection
<ol>
	<li>Treat HepG2 cells with 2.5 &#181;M 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).</li>
	<li>After 48 h, remove the drug and wash with 500 &#181;L of 1x PBS once.</li>
	<li>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.</li>
	<li>Next, collect the serum-free media containing all the secretome, termed as conditioned media, and store at -80 &#176;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.</li>
</ol>
6. M1/M2 polarization in RAW264.7
<ol>
	<li>Cell plating
	<ol>
		<li>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 &#181;L 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.</li>
	</ol>
	</li>
	<li>RAW264.7 induction
	<ol>
		<li>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.</li>
		<li>After incubation, discard the CM, wash with 1 mL of 1x PBS, add 600 &#181;L 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 &#176;C for future use. 
		NOTE: Trypsinization time will vary with cell type, and centrifugation is not recommended above 180 g.</li>
	</ol>
	</li>
	<li>Protein isolation and western blotting
	<ol>
		<li>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 &#176;C, then first thaw the pellet on ice, give a short spin, and remove any residual media.</li>
		<li>After incubation, spin the cell lysate at 20,000 g for 30 min at 4 &#176;C. Collect the supernatant and proceed for western blot, or store at -20 &#176;C for future use.</li>
		<li>Prepare protein sample by taking 35-40 &#181;g of protein, add 1x loading dye, heat for 5-6 min at 95 &#176;C, run SDS-PAGE (10% and 12% acrylamide gel) at 90 V, and transfer to nitrocellulose membrane.</li>
		<li>NOTE: For more detailed information regarding the western blot, refer to Joubert et al.32.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Macrophage polarization using LPS and IFN-&#947;
	<ol>
		<li>Plate 70 x 104 RAW264.7 cells in two 60 mm dishes. 
		NOTE: Plating procedure is similar to step 6.1.</li>
		<li>After 12-16 h, mix 4 &#181;L of 1 &#181;g/mL lipopolysaccharide (LPS, stock concentration is 1 mg/mL) and 1.33 &#181;L of 33 ng/mL interferon-&#947;&#160;(IFN-&#947;, stock concentration is 100 &#181;g/mL) in 4 mL of growth media (DMEM + 10% FBS) and add to the pre-plated RAW264.7, keeping one uninduced control.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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<li>Ohtani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+roles+and+mechanisms+of+senescence-associated+secretory+phenotype+%28SASP%29%3A+can+it+be+controlled+by+senolysis%5BTitle%5D)+AND+%22Inflamm+Regen%22%5BJournal%5D)">The roles and mechanisms of senescence-associated secretory phenotype (SASP): can it be controlled by senolysis.</a> Inflamm Regen. 42 (1), 11(2022).</li>
<li>Cuollo, L., Antonangeli, F., Santoni, A., Soriani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+senescence-associated+secretory+phenotype+%28SASP%29+in+the+challenging+future+of+cancer+therapy+and+age-related+diseases%5BTitle%5D)+AND+%22Biology+%28Basel%29%22%5BJournal%5D)">The senescence-associated secretory phenotype (SASP) in the challenging future of cancer therapy and age-related diseases.</a> Biology (Basel). 9 (12), 485(2020).</li>
<li>Joubert, O., Nehmé, R., Bidet, M., Mus-Veteau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterologous+expression+of+human+membrane+receptors+in+the+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Heterologous expression of human membrane receptors in the yeast Saccharomyces cerevisiae.</a> Methods Mol Biol. 601, 87-103 (2010).</li>
</ol>

The authors declare that they have no conflict of interest.

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...

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 hepatotoxicity4,5. 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 CYP3A4/57,8. These enzymes catalyse the formation of quinone-imine intermediates, which are electrophilic and highly reactive9,10. 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 stimuli17,18. The classical M1 macrophage phenotype is typically associated with pro-inflammatory responses, including the production of cytokines such as TNF-&#945; and IL-1&#946;, and is generally considered a marker of acute inflammation19,20. In contrast, the M2 macrophage phenotype is associated with fibrosis, tissue repair21, immunosuppression, and a response to chronic inflammation22,23. 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 research24,25. 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 carcinogenesis28,29,30,31. 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 it29,30,31.
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 therapies29,30,31.

<table><tbody><tr><td>HepG2 cell line</td><td>NCCS</td><td></td><td>Adherent cell line</td></tr><tr><td>RAW264.7 cell line</td><td>NCCS</td><td></td><td>Adherent cell line</td></tr><tr><td>DMEM (Dulbacco&#039;s Modified Eagle Medium)</td><td>Himedia</td><td>AT066</td><td>High glucose, Powder</td></tr><tr><td>MEM (Modified Eagle Medium</td><td>Gibco</td><td>611100-053</td><td>Powder</td></tr><tr><td>PBS (Phosphate Buffer Saline)</td><td>Himedia</td><td>TS1101</td><td>Powder</td></tr><tr><td>Trypsin-EDTA Solution</td><td>Himedia</td><td>TCL007</td><td>Solution</td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Himedia</td><td>RM1112</td><td>Liquid</td></tr><tr><td>MTT Dye</td><td>Himedia</td><td>TC191</td><td>Powder</td></tr><tr><td>Lapatinib</td><td>Selleckchem</td><td>S2111</td><td>Powder</td></tr><tr><td>Neratinib</td><td>Cayman Chemical Company</td><td>18404</td><td>Powder</td></tr><tr><td>DMSO (Dimethyl Sulfoxide)&amp;nbsp;</td><td>Himedia</td><td>TC185</td><td>Solution</td></tr><tr><td>Formaldehyde&amp;nbsp;</td><td>Rankem</td><td>F0080</td><td>Solution</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-aldrich</td><td>340855</td><td>Solution</td></tr><tr><td>Citric Acid monohydrate, Pure</td><td>Himedia</td><td>GRM229</td><td>Powder</td></tr><tr><td>Sodium phosphate dibasic anhydrous&amp;nbsp;</td><td>Himedia</td><td>MB024</td><td>Powder</td></tr><tr><td>Potassium ferrocyanide, K&lt;sub&gt;4&lt;/sub&gt;[Fe(CN)&lt;sub&gt;6&lt;/sub&gt;]</td><td>Fisher Scientific&amp;nbsp;</td><td>P-236-500</td><td></td></tr><tr><td>Potassium ferricyanide, K&lt;sub&gt;3&lt;/sub&gt;[Fe(CN)&lt;sub&gt;6&lt;/sub&gt;]</td><td>Fisher Scientific&amp;nbsp;</td><td>P-232-500</td><td></td></tr><tr><td>NaCl</td><td>SRL</td><td>41721</td><td>Powder</td></tr><tr><td>MgCl2 hexahydrate</td><td>Himedia</td><td>GRM-1068</td><td>Powder</td></tr><tr><td>X-Gal</td><td>SRL</td><td>45904</td><td>Powder</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma-aldrich</td><td>L6529</td><td></td></tr><tr><td>Human Recombinant Interferon-&amp;gamma;</td><td>Himedia</td><td>CF040-20MCG</td><td></td></tr><tr><td>Cell ROX deep red reagent</td><td>Thermo fisher scientific</td><td>C10422</td><td></td></tr><tr><td>Hoechst</td><td>Invitrogen, Thermo Fisher Scientific</td><td>H3570</td><td>Powder</td></tr><tr><td>MitoTRACKER Green</td><td>Invitrogen, Thermo Fisher Scientific</td><td>1842298</td><td>Powder</td></tr><tr><td>Anti-ARG1 antibody</td><td>Affinity</td><td>DF6657</td><td>Solution, Calculated MW- 35kDa, Observed MW - 65kDa</td></tr><tr><td>Anti-NOS2 antibody</td><td>Cloudo Clone Corp.</td><td>PAA837Hu01</td><td>Solution, Calculated MW- 130kDa, Observed MW - 65kDa</td></tr><tr><td>Anti-iNOS antibody</td><td>Affinity</td><td></td><td>Solution, Calculated MW- 130kDa, Observed MW - 120kDa</td></tr><tr><td>Anti-GAPDH antibody</td><td>Biobharati LifeScience Pvt.Ltd</td><td>BB-AB0060</td><td>Solution</td></tr><tr><td>Anti-Rabbit Antibody</td><td>Cayman Chemical Company</td><td>10004301</td><td>Solution</td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>Merck Millipore Ltd.&amp;nbsp;</td><td>HATF00010 (Product number)</td><td></td></tr><tr><td>femtoLUCENT&lt;sup&gt;TM&lt;/sup&gt;PLUS HRP</td><td>G Bioscience&amp;nbsp;</td><td>786-003</td><td></td></tr><tr><td>60mm Dishes</td><td>NUCK</td><td>150462</td><td></td></tr><tr><td>96well plate,&amp;nbsp;</td><td>NUCK</td><td>165306</td><td></td></tr><tr><td>Cell Culture Incubator&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>371</td><td></td></tr><tr><td>Microscope&amp;nbsp;</td><td>1. LEICA Inverted Microscope&amp;nbsp;&amp;nbsp; 2. Nikon Confocal Microscope&amp;nbsp;</td><td>1. DMIL LED&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2. Ti2E, A1R</td><td>&amp;nbsp;Both are model number</td></tr><tr><td>iBright Imaging System</td><td>Invitrogen, Thermo Fisher Scientific</td><td>CL1500 (model number)</td><td></td></tr><tr><td>Plate-reader</td><td>Bio-RAD</td><td>iMark Microplate Reader</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>Sorvall ST 8R (model number)</td><td></td></tr></tbody></table>

drug-induced senescence in liver cells promotes m2 macrophage polarization: implications for tyrosine kinase inhibitor-associated hepatotoxicity

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

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.

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.
<p class="jove_content...

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.

Lapatinib and neratinib are tyrosine kinase inhibitors (TKIs) approved for treating HER2-positive metastatic breast cancer, but their clinical use is ...

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.

drug-induced-senescence-liver-cells-promotes-m2-macrophage

Research

JoVE Journal

Cancer Research

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

Techniques to Induce and Quantify Cellular Senescence

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

This protocol presents reflected light confocal microscopy for objective, single-cell detection of senescence-associated beta-galactosidase (SA-β-gal) activity in the stromal vascular fraction isolated from human adipose tissue. The method is compatible with immunocytochemistry, incorporates pH controls for quantitative assessment, and additionally provides a more sensitive and less subjective alternative to conventional SA-β-gal staining.

Senescence Detection Using Reflected Light in Adipose Stromal Vascular Fraction

This method studies how senescent skin fibroblasts affect healthy ones. Senescence is induced via UVB exposure, and the conditioned medium is applied to normal fibroblasts. It helps evaluate bioactive compounds targeting altered cell-to-cell communication in skin aging.

Novel Combination Of Antioxidants To Restore Healthy Intercellular Communication In An In vitro Model Using Conditioned Medium From Senescent Cells

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Explore how tyrosine kinase inhibitors induce senescence in liver cells and promote M2 macrophage polarization through SASP signaling. This method reveals mechanisms underlying drug-induced hepatotoxicity.

Drug-Induced Senescence in Liver Cells Promotes M2 Macrophage Polarization: Implications for Tyrosine Kinase Inhibitor-Associated Hepatotoxicity (Video) | JoVE Journal

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

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