Examining MLKL phosphorylation to detect necroptosis in murine mammary tumors

Summary

Necroptosis occurs predominantly in the center of late-stage tumors and necroptotic cells are dispersed and difficult to be detected by Western blotting of key markers without enrichment by microdissection. To overcome these obstacles, this protocol provides a detailed immunohistochemistry-oriented approach including the steps of tumor isolation from mouse mammary tumor models, necrotic region identification by H&E staining, and necroptosis detection through examining mixed lineage kinase domain-like protein (MLKL) phosphorylation. This protocol could be applied to other types of solid tumors.

For complete details on the use and execution of this protocol, please refer to Baik et al. (2021).

Graphical abstract

Highlights

• •An optimized protocol for identifying tumor cell necroptosis in murine mammary tumors
• •For use on solid tumors from orthotopic and genetically engineered models
• •Identification of tumor necrosis by H&E staining is critical for necroptosis detection

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Necroptosis occurs predominantly in the center of late-stage tumors and necroptotic cells are dispersed and difficult to be detected by Western blotting of key markers without enrichment by microdissection. To overcome these obstacles, this protocol provides a detailed immunohistochemistry-oriented approach including the steps of tumor isolation from mouse mammary tumor models, necrotic region identification by H&E staining, and necroptosis detection through examining mixed lineage kinase domain-like protein (MLKL) phosphorylation. This protocol could be applied to other types of solid tumors.

Before you begin

Tumor necrosis commonly happens under hypoxic and nutrient deficient conditions in the advanced solid tumors (Jiao et al., 2018), and is closely related with poor prognosis of cancer patients. We recently reported that necroptosis, a programmed necrosis, plays a pivotal role in tumor necrosis and promotes tumor metastasis (Baik et al., 2021). Since tumor necrosis mainly happens in the core areas of solid tumors, identification of the necrotic regions before examining cell death with IHC is critical for necroptosis detection in solid tumors. This protocol guides you step-by-step from tumor model preparation and tumor isolation, identifying necrotic areas with H&E staining, to the IHC labeling of MLKL phosphorylation.

Although this protocol specifically focuses on murine mammary tumor, it can be easily adapted for necroptosis detection in other types of solid tumors.

Institutional permissions

The animal protocols of MVT-1 and MMTV-PyMT animal models are approved by NCI (IACUC no. CCBB-029).

MVT-1 orthotopic tumor model

Timing: 5 weeks

• 1.Female 6–8 weeks old FVB/J mice are purchased from The Jackson Laboratory. All mice are housed in specific pathogen free (SPF) condition.
• 2.
  Preparation of the MVT-1 cells.

  • a.
    MVT-1 cells, which are originally derived from mammary tumor in MMTV-Myc-VEGF bitransgenic mouse (Pei et al., 2004) are cultured in DMEM.
  • b.
    2 × 10⁶ of MVT-1 cells are suspended in 100 μL of Matrigel Matrix solution (diluted 1:1 with 1× DPBS).
• 3.
  Injection of the MVT-1 cells.

  • a.
    The 100 μL of Matrigel Matrix solution containing MVT-1 cells are injected into the mammary fat pad of FVB/J mice (Figure 1A).

Figure 1.

MVT-1 model and tumor isolation

(A) MVT-1 cells are injected into fat pad of no.2 mammary gland in FVB mouse.

(B) Tumor isolation. Lines indicate the incision area.

MMTV-PyMT tumor model

Timing: 15 weeks

Female 5–6 weeks old FVB/N-Tg (MMTV-PyVT) 634 Mul/J mice are purchased from The Jackson Laboratory. All mice are housed in specific pathogen free (SPF) condition.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-phospho-MLKL (mouse specific) antibody (1:1000 dilution)	Abcam	ab196436
Anti-cleaved caspase-3 (cl. casp-3) antibody (1:1000 dilution)	Cell Signal Technology	9664
Chemicals, peptides, and recombinant proteins
Matrigel Matrix	Corning	354234
30% hydrogen peroxide (H2O2) solution	Sigma	H1009
10× Antigen retrieval solution (citrate pH 6.0)	Dako	S1699
1× DPBS (pH 7.4)	KD medical	RGF-3200
Sodium bicarbonate	Mallinckrodt	7412
Permount (Mounting medium)	Fisher	SP15-100
Methanol	Fisher chemical	A411-4
Ethanol	Fisher chemical	A407P-4
Xylene	Lab Alley	XYL-1GAL
Hematoxylin	Vector Laboratories	H-3404
4% buffered formalin (Z-fix)	ANATECH LTD	170
Critical commercial assays
VECTASTIN ABC Elite kit, Peroxidase (Rabbit IgG) (containing biotinylated goat anti-rabbit IgG)	Vector Laboratories	PK-6101
ImmPACT® DAB Substrate, Peroxidase (HRP)	Vector Laboratories	SK-4105
TUNEL Assay kit	Abcam	ab206386
Experimental models: Cell lines
MVT-1 cells	Described in (Jiao et al., 2018)	MVT-1 cells
Experimental models: organisms/strains
FVB/J: female 6–8 weeks old	The Jackson Laboratory	001800
MMTV-PyMT: female 5–6 weeks old FVB/N-Tg (MMTV-PyVT) 634 Mul/J mice	The Jackson Laboratory	002374
Software and algorithms
ZEN software	ZEISS	N/A
Other
Miltex Eye Dressing Forceps, 4″ Half Curved, Serrated	Miltex	V918-782
Miltex Straight 4.5 in. Iris Scissors	Miltex	V95-304
Scintillation vial	Electron Microscopy Sciences	72632-PT
SLIP-RITE Cover Glass	Thermo Scientific	102250
IHC slide staining holder	N/A	N/A
IHC slide staining tray (black) with lid	N/A	N/A
Manual IHC staining dish with lid	N/A	N/A
Super HT PAP Pen	Research Products International Corp.	195505
Culture dish (10 cm and 6 well plate)	Corning	430167 and 353046
Cork board	N/A	N/A
50 mL conical tube	Corning	430828
100 μm nylon cell strainer	Fisher	22-363-549
Razor blade	N/A	N/A
Shake’N’Bake Hybridization Oven	Boekel	Model 136400
Black & Decker steamer	Black & Decker	N/A
PYREX Griffin beaker	Fisher scientific	N/A
37°C incubator	N/A	N/A
−20°C freezer	N/A	N/A
Swing bucket centrifuge	N/A	N/A
Shaker	Benchmark	BT300
Traceable™ Digital Carbon Fiber Calipers	Fisher Scientific	15-077-957
Thermometer	611 S-35C	Cole-Parmer

Materials and equipment

1× Antigen retrieval solution

Reagent	Final concentration	Amount
10× Antigen retrieval solution (pH 6.0)	1× (10%)	15 mL
H2O (molecular biology grade)	N/A	135 mL
Total	N/A	150 mL

Freshly prepare the 1× Antigen retrieval solution each time, do not store it.

CRITICAL: Adjust the pH to 6.0 with 0.1 M HCl. Bring up the volume to 1 liter with H₂O.

3% Hydrogen peroxide solution

Reagent	Final concentration	Amount
30% Hydrogen peroxide (H2O2) solution	3%	20 μL
Prechilled methanol	N/A	180 mL
Total	N/A	200 mL

Methanol should be prechilled in −20°C for 30 min before use.

3% Hydrogen peroxide solution can be stored in 4°C up to 1 week.

Primary antibody dilution ratio chart

Reagent	Final concentration	Amount
Anti-phospho-MLKL (mouse specific) antibody	1:1000	N/A
Anti-cleaved caspase-3 (cl. casp-3) antibody	1:1000	N/A

Freshly dilute the primary antibody using 1× blocking buffer each time, do not store it.

CRITICAL: Anti-phospho-MLKL antibody dilution ratio varies from batch to batch. It can be used up to 1:5000 dilution.

1× blocking buffer (from VECTASTIN ABC Elite kit)

Reagent	Final concentration	Amount
Normal Goat serum (1:150 dilution)	2%	3 drops
DPBS (pH 7.4)	1×	10 mL
Total	N/A	10 mL

1× blocking buffer should be stored in 4°C up to 1 week.

Secondary antibody solution (biotinylated goat anti-rabbit IgG from VECTASTAIN ABC Elite kit)

Reagent	Final concentration	Amount
Biotinylated secondary antibody (1:200 dilution)	N/A	5 μL
1× Blocking buffer	N/A	1 mL
Total	N/A	1 mL

Freshly dilute the secondary antibody using 1× blocking buffer each time, do not store it.

1× Avidin-Biotin Complex (ABC) solution (from VECTASTAIN ABC Elite kit)

Reagent	Final concentration	Amount
A	N/A	20 μL
B	N/A	20 μL
1× DPBS (pH 7.4)	N/A	1000 μL
Total	N/A	1040 μL

Freshly prepare the 1× ABC solution and should be incubated in 22°C for 1 h before use.

ImmPACT DAB Solution

Reagent	Final concentration	Amount
ImmPACT DAB Reagent 1	N/A	1 drop
ImmPACT DAB Diluent	N/A	1 mL
Total	N/A	1 mL

Freshly prepare the ImmPACT DAB solution each time, do not store it.

1× DPBST

Reagent	Final concentration	Amount
10× DPBS	1×	100 mL
Tween 20	0.05%	0.5 mL
H2O (molecular biology grade)	N/A	900 mL
Total	N/A	1 L

1× DPBST can be stored in 22°C up to 6 months.

0.1% Sodium bicarbonate solution

Reagent	Final concentration	Amount
Sodium bicarbonate	1%	1 g
H2O (molecular biology grade)	N/A	1 L
Total	N/A	1 L

0.1% Sodium bicarbonate solution can be stored in 22°C up to 6 months.

95% Ethanol solution

Reagent	Final concentration	Amount
100% Ethanol	95%	950 mL
H2O (molecular biology grade)	N/A	50 mL
Total	N/A	1 L

95% Ethanol should be stored in 22°C up to 6 months.

70% Ethanol solution

Reagent	Final concentration	Amount
100% Ethanol	70%	700 mL
H2O (molecular biology grade)	N/A	300 mL
Total	N/A	1 L

70% Ethanol should be stored in 22°C up to 6 months.

Step-by-step method details

Tumor isolation

Timing: 20 min per mouse

Timing: 20 min per mouse

Preparation before isolation:

• 1.Euthanize mice by carbon dioxide (CO₂) inhalation.
• 2.Pin down mouse on its back using 25-gauge needles (Figures 1B and 2A). Spray the mouse with 70% Ethanol.

Figure 2.

Tumor isolation from MMTV-PyMT model

(A) MMTV-PyMT mouse is pinned down on its back.

(B) Incision area is labeled with dashed lines.

(C) Localization of multiple mammary glands after cutting open the skin.

Orthotopic model: Tumors from MVT-1 tumor bearing FVB mice.

Note: Tumor size should be measured with digital caliper every week. Once a tumor volume reaches 1500 mm³, the tumor can be collected. For tumor measurements, W is the tumor width (mm), L is the tumor length, and a tumor volume (V) is calculated using formula, V $=\frac{\text{L}(\text{W }\times \text{ W})}{2}$.

This step describes how the tumors are collected from both FVB mice bearing MVT-1 tumor and MMTV-PyMT mice.

• 3.Cut open the mouse skin from the area near the bottom of rib cage to neck with scissors (as shown in Figure 1B).
• 4.Gently hold the tumor capsule with half-curved forceps and dissect out the MVT-1 tumor.

CRITICAL: All the surgical tools should be autoclaved before use.

Genetically engineered transgenic mouse (GEMM) model: Tumors from MMTV-PyMT mice.

MMTV-PyMT mice are euthanized at 12-week-old or 15-week-old for tumor collection.

• 5.Cut open the skin from the area of the urethral orifice to the neck with scissors (Figure 2B).
• 6.Pin down the skin as shown below (Figure 2C) for an easy access to the tumors.
• 7.Gently hold the tumor capsule with half-curved forceps and dissect out the tumor.

CRITICAL: Although MMTV-PyMT mice have multiple tumors, MMTV-PyMT tumors are only collected from mammary glands no.3 and no.4 of each mouse as shown in the Figure 2C. The tumors from other mammary glands are not collected (Plante et al., 2011).

Tumor sample fixation

Timing: 5 min

• 8.Trim off any visible contamination of yellowish fat tissue or brachial lymph node from tumor samples.
• 9.Immediately put the tumor into a 10 cm culture dish and rinse once with cold sterile phosphate buffered saline (DPBS, pH 7.4).
• 10.The ball-shaped mammary tumor can be bisected along the longest diameter using a sharp razor.

Note: The longest diameter needs to reach 15 mm.

• 11.Transfer one half of each tumor sample into scintillation vials containing 10 mL of 4% buffered formalin (Z-fix) for histology.

Note: The other halves of tumors will be used for isolating primary tumor cells or Western blot assay.

• 12.After fixing tumor samples with Z-fix for 72 h at 22°C, rinse them once with 1× DPBS (pH 7.4). Dehydrate the fixed tumors and embed them in paraffin.

Paraffin-embedding and H&E staining

Timing: 2 h for staining

Because necroptosis is a major cause of tumor necrosis (Jiao et al., 2018), which normally happens in the core areas of solid tumors, identification of the necrotic region before necroptosis detection is critical. Therefore, serial sectioning in tumor samples should be used for identifying the necrotic region. Then those samples with tumor necrosis can be used in evaluating necroptosis by phospho-MLKL IHC labeling.

The formalin-fixed tumor tissue is entrusted to Histoserv company for paraffin embedding and H&E staining following a general protocol (https://www.histoservinc.com/). The brief procedures of H&E staining (Fischer et al., 2008) are provided below.

• 13.Trim the top of the paraffin-embedded tumor block and cut 5 slides by 5 μm-serial-sectioning.

Note: H&E staining is performed on the 3rd slide from serial sections (Figure 3). The unstained slides will be used for IHC.

• 14.Place an unstained tumor slide in IHC slide staining holder. Deparaffinize the slide with 100% Xylene for 2 min.
• 15.Repeat step 2.
• 16.Hydrate tumor slide in 100% Ethanol for 2 min.
• 17.Repeat step 4.
• 18.Hydrate tumor slide once with 95% Ethanol for 2 min.
• 19.Wash tumor slide once with ddH₂O for 2 min.
• 20.Stain tumor slide with Carazzi’s hematoxylin for 3 min.
• 21.Wash tumor slide once with tap water and place the slide in 95% Ethanol before proceeding to the next step.
• 22.Counterstain tumor slide with eosin-phloxine for 30 s.
• 23.Dehydrate slide once with 95% Ethanol for 1 min and twice with 100% Ethanol for 1 min.
• 24.Rinse twice with 100% Xylene for 2 min.
• 25.Mount tumor slide with 200 μL of Permount solution dropwise to cover tumor tissue, followed by applying a coverslip.

Figure 3.

An example of tumor sample serial sections

The slide in the middle was stained with H&E.

Deparaffinization for IHC

Timing: ∼40 min

The necrotic areas of tumors can be identified based on the unique morphology of tumor necrosis from H&E-stained tumor samples as shown in expected outcomes. After tumor samples with necrosis regions are identified by H&E staining, the serial sectioned slides of these samples will be stained with anti-p-MLKL antibody to examine necroptosis.

This step describes how to dissolve the paraffins from tumor slides for IHC.

• 26.Place tumor slides in slide staining holder (Figure 4C) and incubate at 56°C for 30 min in Shake’N’Bake hybridization oven.
• 27.Rinse three times with 100% of Xylene for 5 min: dip tumor slide holder in staining dish (Figure 4B) containing 150 mL of 100% Xylene. Move the slide holder up and down for a couple of times.
• 28.Rinse two times with 100% of Ethanol for 2 min: dip tumor slide holder in staining dish (Figure 4B) containing 150 mL of 100% Ethanol. Move the slide holder up and down after 1 min.
• 29.Rinse two times with 95% of Ethanol for 2 min: dip tumor slide holder in staining dish (Figure 4B) containing 150 mL of 95% Ethanol. Move the slide holder up and down after 1 min.
• 30.Rinse two times with 70% of Ethanol for 2 min: dip tumor slide holder in staining dish (Figure 4B) containing 150 mL of 70% Ethanol. Move the slide holder up and down after 1 min.

CRITICAL: All the deparaffinization steps are performed in a chemical hood. All the solution should be freshly made before use.

• 31.Wash tumor slides in ddH₂O for 5 min.

Note: All wash steps are performed on a shaker at 0.005 × g (50 rpm) at 22°C.

Figure 4.

Slide staining holder and container

(A) Slide staining holder.

(B) Staining dish.

(C) Slide staining holder with slide.

Antigen retrieval

Timing: ∼60 min

This step describes the antigen retrieval method specific for p-MLKL antibody. Antigen retrieval is critical for the paraffin-embedded tissue section, which unmasks antigens to improve the binding of p-MLKL antibody.

• 32.Take out tumor slide from the slide holder and transfer into a 250 mL beaker (Figure 5B) containing 150 mL of 1× antigen retrieval solution.

Note: Steamer should be preheated to 95°C before use. Check the temperature with thermometer before proceeding to the next step.

• 33.Steam (Figures 5C and 5D) tumor slide for 10 min at 95°C.
• 34.Take out the beaker from the steamer and let slide cool down to 22°C.

Note: It will take about 45 min to cool down.

• 35.Take out tumor slide from the beaker and place it in a slide holder.
• 36.Wash tumor slides in 200 mL of ddH₂O for 5 min.

Figure 5.

Antigen retrieval

(A) Unstained tumor slide.

(B) A beaker with an unstained tumor slide inside.

(C) A beaker in the steamer.

(D) Steaming process.

Blocking non-specific binding of antibody

Timing: ∼90 min

This step describes how to reduce the endogenous peroxidase activity, which is critical for eliminating the background staining in tumor samples.

• 37.Transfer slide holder into a staining dish (Figure 4B) containing 200 mL of 3% hydrogen peroxide (H₂O₂) for 10 min at 22°C.
• 38.Transfer slide holder into a staining dish (Figure 4B) containing 200 mL of ddH₂O and wash for 5 min.
• 39.Take out one slide each time and remove the residual ddH₂O with a clean paper towel, then place it on a IHC slide staining tray (Figure 6).
• 40.Draw a circle around the tumor tissue with Super PAP Pen.
• 41.Add 100 μL of 1× blocking buffer dropwise to the tumor tissue.
• 42.Close the tray lid and incubate for 60 min at 22°C on bench.

Figure 6.

Slide staining tray

Primary antibody incubation

Timing: ∼16 h

This step describes how to incubate the primary antibody.

• 43.Remove the blocking buffer by tapping the slide edge on a clean paper towel.
• 44.Place tumor slide on a IHC slide staining tray. Add 1:1000 diluted p-MLKL antibody dropwise to tumor tissues.

Note: The primary antibody solution should be freshly prepared before use.

• 45.Close the tray lid and incubate at 4°C for 16 h.

Secondary antibody incubation

Timing: 90 min

This step describes how to incubate the secondary antibody.

• 46.Take out tumor slide from the IHC slide staining tray and place them in slide holder.
• 47.Wash tumor slide in 200 mL of 1× DPBST for 10 min, repeat the wash step for three times.
• 48.Remove the excessive DPBST by tapping the slide edge on a clean paper towel.
• 49.Place tumor slide on a IHC slide staining tray and add 100 μL of secondary antibody solution dropwise to the tumor tissues.

Note: Secondary antibody solution should be freshly prepared before use.

• 50.Close the tray lid and incubate for 60 min at 22°C on bench.

Note: During the incubation time, prepare ABC solution and equilibrate to 22°C.

ABC solution incubation

Timing: 90 min

This step describes how antigen-antibody complexes will be detected by avidin and biotinylated enzyme (ABC solution).

• 51.Take out tumor slide from the IHC slide staining tray and place them in a slide holder.
• 52.Wash tumor slide in 200 mL of 1× DPBST for 10 min, repeat the wash step for three times.
• 53.Place tumor slide on a IHC slide staining tray and add 100 μL of ABC solution dropwise to the tumor tissues.
• 54.Close the tray lid and incubate for 60 min at 22°C on bench.

Develop IHC staining signal

Timing: ∼40 min

This step describes DAB staining in tumor slides. The DAB HRP substrate will produce a dark brown reaction against avidin and biotinylated antigen-antibody complexes.

• 55.Take out tumor slide from the IHC slide staining tray and place them in slide holder.
• 56.Wash tumor slides in 200 mL of 1× DPBST for 10 min, repeat the wash step for three times.
• 57.Take out one slide each time and remove the residual 1× DPBST by tapping the slide edge on a clean paper towel.
• 58.Add 100 μL of DAB solution dropwise to the tumor tissue and incubate for up to 5 min at 22°C.

Note: DAB solution should be freshly prepared before use.

• 59.Once visible coloring is observed, immediately put the slide in fresh ddH₂O to stop the reaction.

Note: The DAB solution should be washed out immediately after coloring to inhibit background staining (Figure 7). If there is no visible color change within 5 min, then increase the incubation time for up to 10 min. See troubleshooting.

Figure 7.

Example of sample coloring with DAB solution

Counter staining with hematoxylin

Timing: ∼40 min

This step describes hematoxylin staining in tumor slides. The hematoxylin stains cell nuclei with a purplish blue.

• 60.Place tumor slide in slide holder and wash them in 200 mL of ddH₂O for 10 min, repeat the wash step twice.
• 61.Take out tumor slide from the slide holder and remove the residual ddH₂O.
• 62.Place one tumor slide each time on bench top and add two drops of hematoxylin solution directly to tumor tissue and incubate for 4 min at 22°C.
• 63.Immediately put the slide in fresh ddH₂O to stop the reaction.
• 64.Place tumor slide in slide holder and wash them in ddH₂O for 5 min.
• 65.Take out slide holder with tumor tissues and dip in 200 mL of 0.1% sodium bicarbonate solution. Move slides up and down for three times to ensure evenly bluing on slides.

Note: The bluing step should be less than 1 min to avoid over staining.

• 66.Put slide in holder and dip into 200 mL of ddH₂O to stop the bluing reaction.
• 67.Transfer slide into 200 mL of fresh ddH₂O and wash for 5 min.

Dehydration and mounting

Timing: 30 min for dehydration

This step describes dehydration and mounting of tumor cells.

• 68.Rinse two times with 70% of Ethanol: put slide holder in staining dish (Figure 4B) containing 150 mL of 70% Ethanol for 2 min.
• 69.Rinse two times with 95% of Ethanol: put slide holder in staining dish (Figure 4B) containing 150 mL of 95% Ethanol for 2 min.
• 70.Rinse two times with 100% of Ethanol: put slide holder in staining dish (Figure 4B) containing 150 mL of 95% Ethanol for 2 min.
• 71.Rinse three times with 100% of Xylene: put slide holder in staining dish (Figure 4B) containing 150 mL of 100% Xylene for 5 min. Move the slide holder up and down for a couple of times during wash.
• 72.Take out one tumor slide each time and place it on bench top.
• 73.Remove the excessive Xylene solution and add 200 μL of Permount solution dropwise to cover the tumor tissue. Slowly apply the coverslip without any air bubbles.
• 74.Dry the tumor slide in a chemical hood for 16 h.

Note: All dehydration procedures should be performed in a chemical hood.

Expected outcomes

Representative H&E-stained tumor slides are shown in Figures 8 and 9. In the MVT-1 orthotopic tumors (1500 mm³), the necrotic areas are commonly observed in all of 8–12 slides sectioned between upper third and the lower third of a tumor block (Baik et al., 2021). Necroptotic tumor cells are characterized by organelles swelling, collapse, and deficiency of nuclear chromatin as dark-hematoxylin-stained cells (Figure 8, left panel). These features of necroptosis are quite distinct from the blebbing, nuclear fragmentation and chromatin condensation in apoptosis (Christofferson and Yuan, 2010); (Galluzzi and Kroemer, 2008); (Galluzzi et al., 2018). Typical apoptotic cell death is observed in MVT-1 ZBP1 knock out tumor by H&E staining (Figure 8, right panel). A typical necrotic area with a mixture of viable cells, cells undergoing necroptosis, and necrotic cell debris is outlined with yellow dashed lines in Figure 9.

Figure 8.

Tumor cell necroptosis in MVT-1 WT tumor and tumor cell apoptosis in MVT-1 ZBP1 KO tumor with H&E staining

Scale bar, 50 μm.

Figure 9.

Tumor necrosis area (outlined with yellow dashed lines) in H&E (left) or p-MLKL IHC (right) stained slides

Lower 4 panels (1, 2, 3, 4) show the enlarged parts of each slide. Scale bar, 50 μm.

Although tumor necroptosis features are quite distinct from the apoptosis in H&E staining, it should be confirmed by IHC labeling of p-MLKL, a hallmark of necroptosis. As shown in Figure 9, tumor cell necroptosis is located surrounding the core of necrosis (no.2 of Figure 9), with positive p-MLKL signal (no.4 of Figure 9). Viable tumor cells with the negative staining of p-MLKL were also presented in Figure 9 (no. 1 and 3). P-MLKL positive necroptotic tumor cells are also detected surrounding the core of necrosis in the MMTV-PyMT tumor (Figure 10).

Figure 10.

MMVT-PyMT tumor necrosis area shown in H&E (upper panel) and p-MLKL IHC (lower panel) staining

Tumor necrosis area is outlined with yellow dashed lines. Red arrows indicate the necroptotic cell death with positive p-MLKL staining in IHC. Scale bar, 50 μm. Reprinted by permission from CCC RightsLink: Springer Nature, Cell Research, Necroptosis of tumor cells leads to tumor necrosis and promotes tumor metastasis, Jiao, D., et al., [COPYRIGHT] (2018).

This feature of p-MLKL staining in tumor samples is unique and critical for the detection of necroptosis in tumors. These data demonstrated that identifying the necrotic areas with H&E staining in serial tumor sections is an important prerequisite for an ideal IHC staining-oriented necroptosis detection with anti-p-MLKL antibody in solid tumors.

Limitations

Quality of the p-MLKL antibody

Successful necroptosis detection is highly dependent on the quality of the p-MLKL antibody. The dilution ratio varies from batch to batch. A pilot study is recommended with titrations from 1:1000 to 1:10,000 for each lot of primary antibodies. However, a dilution of the antibody lower than 1:1000 is not recommended because of background staining.

Absence of RIPK3 or ZBP1 expression in tumor samples

Reports showed that RIPK3 or ZBP1, key molecules of necroptosis pathway, was often epigenetically silenced (e.g., DNA methylation) in tumors (Koo et al., 2015). Therefore, necroptosis could not happen in these RIPK3 or ZBP1-silenced tumor samples (Baik et al., 2021). If no necroptosis is detected by p-MLKL IHC in tumor samples, examining the expression levels of RIPK3 and ZBP1 in tumor lysates by Western blotting may be helpful in troubleshooting.

Troubleshooting

Problem 1

Fail to identify a necrotic area in tumor samples.

Potential solution

Tumor necroptosis only happens in solid tumors at their advanced stages when tumor cells are under nutrient-deficient stresses, particularly glucose deprivation (Baik et al., 2021). Early-stage solid tumors normally do not undergo necroptosis. As shown in Figure 10, in MMTV-PyMT tumors, the necrosis areas are gradually enlarged along with the tumor size and growth time (Jiao et al., 2018). The p-MLKL positive necroptotic cells are also gradually increased in these samples (Figure 10, bottom two panels). Therefore, it is important to conduct a time course (or size-dependent) collection of tumor samples, if possible, for H&E assay, and this will help to get an idea of necrosis development.

Problem 2

pH of the antigen retrieval buffer.

Potential solution

pH of the antigen retrieval buffer is a critical factor in p-MLKL IHC. Although the commercial 10× antigen retrieval buffer (Dako) supposed to be pH 6.0, an obvious pH increase was often noticed upon dilution to working solution. Therefore, it is highly recommended to routinely check the pH of the 1× antigen retrieval buffer before use. If only weak p-MLKL signal can be detected with the commercial antigen retrieval solution, it would be helpful to make fresh antigen retrieval buffer in house.

1× Antigen retrieval buffer

Reagent	Final concentration
Sodium citrate	10 mM
Tween 20	0.05%
H2O (molecular biology grade)	N/A
Total	1 L

CRITICAL: Adjust the pH to 6.0 with 0.1 M HCl. Bring up the volume to 1 liter with H₂O.

Problem 3

Lack of positive control for p-MLKL IHC staining.

Potential solution

To have a positive control of p-MLKL IHC staining, primary tumor cells can be isolated from tumors and are then cultured under glucose deprivation (GD) condition to induce necroptosis as shown in the recent study (Baik et al., 2021). If primary tumor cells are not available, it is recommended to epigenetically reprogram MVT-1 cells with 5AD before challenging them with GD. These cells can be fixed and embedded in paraffin to serve as the positive control for p-MLKL IHC.

Expected outcomes: Primary tumor cells isolated from MVT-1 or MMTV-PyMT tumors are highly responsive to GD. As presented in Figure 11, GD triggered the induction of p-MLKL in cultured MVT-1 and MMTV-PyMT primary tumor cells. Also, as shown in Figure 12, p-MLKL IHC staining is achieved in 5AD reprogrammed MVT-1 cells after GD treatment.

Figure 11.

p-MLKL is increased in MVT-1 or MMTV-PyMT primary tumor cells under GD condition

(A) Primary MVT-1 tumor cells were isolated from 5-week tumors of FVB/NJ mice implanted with MVT-1 cells. Isolated MVT-1 tumor cells were treated for the indicated time points with 0.5 mM glucose (GD) and examined by Western blotting.

(B) Primary MMTV-PyMT tumor cells from 15-week mice were isolated and treated for the indicated time points with GD then examined by Western blotting.

Material from: ‘Baik, J.Y, et al., ZBP1 not RIPK1 mediates tumor necroptosis in breast cancer, Nature Communications (2021) Nature Publishing Group [COPYRIGHT].

Figure 12.

Example of a positive control for p-MLKL IHC staining

MVT-1 cells were pretreated with 5AD (upper panel), followed by GD for 16 h (middle panel) or 24 h (lower panel). Scale bar, 50 μm.

Problem 4

Classification of necroptotic vs apoptotic cell death in tumor samples.

Potential solution

Although necroptosis may be the main cause of tumor necrosis, other forms of cell death, e.g., apoptosis could also occur in tumors, particularly when necroptosis is defective (Baik et al., 2021). It is highly recommended to examine apoptosis by TUNEL assay or cleaved caspase-3 (cl. casp-3) IHC in tumor samples. For cl. casp-3 IHC, general IHC method can be followed with the use of cl. casp-3 antibody with 1:1000 dilution.

Expected outcomes: Here we used ZBP1 knock out tumors, where cell death switched from necroptosis to apoptosis, as an example. In Figure 13, tumor cell apoptosis was identified with positive cl. casp-3 (B) and TUNEL (C) signal.

Figure 13.

Representative images of tumor cell necroptosis and apoptosis

(A–C) IHC staining with p-MLKL (A), cl. casp-3 (B), and TUNEL assay (C) in MVT-1 CRISPR control (CRISPR CT) tumor or ZBP1 depleted (sgZBP1) tumor. TN: tumor necrosis, Scale bar, 50 μm.

Problem 5

Background staining.

Potential solution

The DAB solution incubation time is important to get a nice IHC image. If the background staining is too strong in tumor samples, there are two ways to fix it. First, figure out the optimal DAB incubation time as shown in Figure 7 (5 min incubation is ideal in this example). Second, reduce the secondary antibody incubation time. Incubation between 40 min to 60 min is ideal for murine mammary tumor.

Problem 6

Is necroptosis detection by p-MLKL IHC specific for tumor cells?

Potential solution

Solid tumors are composed of tumor cells and a mixture of other types of cells, including immune cells and fibroblasts. So, there is always the question whether other types of cells in tumors undergo necroptosis. In other words, are p-MLKL IHC detected necroptosis mainly from tumor cells? As presented in our previous study (Jiao et al., 2018), when the execution protein, MLKL, was depleted from xenograft tumor cells, the p-MLKL signal almost completely disappeared in tumor samples. Neither fibroblasts nor infiltrating immune cells from wildtype hosts have any obvious p-MLKL staining (Figure 14), which indicated that tumor cells is the major type of cells that underwent necroptosis in necrotic areas of solid tumors (Jiao et al., 2018).

Figure 14.

Representative images of tumor cell necroptosis (p-MLKL IHC staining) and apoptosis (TUNEL staining) from MVT-1-CRISPR-CT or MVT-1-MLKL KO cells orthotopic models

Reprinted by permission from CCC RightsLink: Springer Nature, Cell Research, Necroptosis of tumor cells leads to tumor necrosis and promotes tumor metastasis, Jiao, D., et al., [COPYRIGHT] (2018).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Zheng-gang Liu, zgliu@helix.nih.gov.

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate or analyze datasets.