Protocol for region-specific tumor dissection and protein extraction from a pancreatic neuroendocrine tumor mouse model

Summary

Isolation of high-quality protein from transgenic mouse tumors is essential for elucidating the mechanisms underlying tumor development. We describe steps for harvesting pancreatic neuroendocrine tumors from human thymidylate synthase (hTS)/Men1^−/− mice, where hTS/TYMS is overexpressed in a Men1^−/− background. We then detail procedures for protein extraction from hTS/Men1^−/− and control Men1^−/− tumors for immunoblotting. The protocol preserves protein integrity while reducing non-specific background commonly seen in pancreatic tissues. It also minimizes non-tumor tissue, enhancing the specificity of the protein extract.

For complete details on the use and execution of this protocol, please refer to Vijayakurup et al.¹

Graphical abstract

Highlights

• •Steps for controlled euthanasia and tumor dissection in PanNET mouse models
• •Guidance on PanNET tissue handling to preserve protein integrity
• •Procedure for PanNET protein extraction for immunoblotting analysis

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

Isolation of high-quality protein from transgenic mouse tumors is essential for elucidating the mechanisms underlying tumor development. We describe steps for harvesting pancreatic neuroendocrine tumors from human thymidylate synthase (hTS)/Men1^−/− mice, where hTS/TYMS is overexpressed in a Men1^−/− background. We then detail procedures for protein extraction from hTS/Men1^−/− and control Men1^−/− tumors for immunoblotting. The protocol preserves protein integrity while reducing non-specific background commonly seen in pancreatic tissues. It also minimizes non-tumor tissue, enhancing the specificity of the protein extract.

Before you begin

This protocol is optimized for the isolation of high-quality total protein from pancreatic neuroendocrine tumors generated from GEMMs which develop pancreatic neuroendocrine tumors due to the deletion of Men1, a tumor suppressor gene in islet tissues.

We had previously generated a mouse model with elevated ectopic human TYMS (hTS) expression and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1^−/−).² This GEMM develops carcinomas at a younger age with accelerated PanNET progression compared with Men1^−/− GEMMs, thereby providing a novel mouse model to study the role of TYMS in PanNET acceleration and to elucidate molecular mechanisms underlying PanNET pathogenesis.

The protocol focuses on strict anatomical attention during tumor dissection. To reduce biological variability inherent to PanNETs, tumor tissues were consistently isolated from the same region of the pancreas (splenic attachment region at the tail of the pancreas) (Figure 1). The protocol also describes carefully controlled euthanasia conditions to minimize biological variability across mouse tumor samples. Because protein expression and signaling pathways are sensitive to euthanasia conditions (including CO₂ flow rate and exposure time), these parameters must be kept constant across all experimental animals.^3,4

Figure 1.

Stepwise dissection and isolation of pancreatic tumor lobes adjacent to spleen and gastric region from hTS/Men1^-/- mouse

(A) Supine positioning and abdominal sterilization.

(B) Midline skin and peritoneal incision.

(C) Exposure of the abdominal cavity.

(D) Identification of the spleen as a landmark for isolation of pancreatic tumor lobe.

(E) Elevation of spleen revealing splenic lobe (tail) and gastric region (body) of pancreas. Dissection is shown near the splenic flexure.

(F) Isolated pancreas attached to spleen showing tumor-enriched tissue for immunoblotting.

Institutional permissions

All experiments were conducted in accordance with Institutional Animal Care Committee (IACUC)–approved protocols at the University of Florida Animal Care Service facility.

Innovation

This protocol provides a methodology to isolate proteins from mouse pancreatic neuroendocrine tumors, addressing potential sources of variability that may limit reproducibility in PanNET molecular studies. Unlike conventional protein extraction methods that treat pancreatic tissue as homogeneous, this protocol incorporates region-specific tumor dissection, ensuring that tumor samples are consistently harvested from defined pancreatic locations in all animals.

Overall, this protocol enables highly reproducible, biologically consistent protein isolation from PanNET tissues, facilitating comparative studies across cohorts and supporting mechanistic investigations into pancreatic neuroendocrine tumor development and progression.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Ethanol	Deacan	Cat# 2701
Buffered formalin (10%)	Epredia	Cat# 5701
Bio-Rad Protein Assay Dye Reagent	Bio-Rad	Cat# 5000006
RIPA	Santa Cruz	Cat# sc-24948
Protease Inhibitor	Thermo Fischer	Cat# A32953
Phosphatase Inhibitor (PhosSTOP)	Millipore Sigma	Cat# 4906837001
PBS	Corning	Cat# 21-031CV
Experimental models: Organisms/strains
Mouse: B6; FVB;129/Sv (hTS/Men1-/-) (male and female; aged 8 – 12 months)	Vijayakurup et al.2	N/A
Mouse: B6; FVB;129/Sv (Men1-/-) (male and female; aged 8 – 12 months)	Vijayakurup et al.2	N/A
Other
GLH-01 General Laboratory Homogenizer	Omni International	Cat# GLH-01
Hard Tissue Omni Tip Plastic Homogenizing Probes (7 x 110 mm)	Omni International	Cat# 30750H
1.5 ml Microcentrifuge tubes	Fischer Scientific	Cat# 02-682-002
Tissue Forceps	McKesson	Cat# 169390
Straight operating Scissors	Integra Life Sciences (Miltex Vantage)	Cat# V95-6
Splinter Forceps	McKesson	Cat# 487594
Centrifuge	Eppendorf	Model 5417 R
Mini Vortex Mixture	VWR	Cat# 58816-121

Alternatives: The ethanol and PBS listed in the key resources table may be substituted with equivalent products from other suppliers. The protease and phosphatase inhibitor cocktail can also be replaced with a comparable combined formulation from alternative vendors (e.g., Pierce; Cat# A32961). However, substitution of RIPA buffer may introduce compatibility issues with the standardized protocol.

Materials and equipment

RIPA protein extraction buffer

Reagent	Final concentration	Amount
RIPA	N/A	10 mL
Protease Inhibitor	1 tablet	N/A
Phosphatase Inhibitor	1 tablet	N/A
Total	N/A	10 mL

Should be freshly prepared; Storage is not recommended.

CRITICAL: Add Protease and Phosphatase inhibitor tablets immediately before use.

Step-by-step method details

PanNET tissue collection from hTS/Men1^−/− and Men1^−/− mice

Timing: 2 h

Here we describe the step-by-step procedure for PanNET tissue collection from hTS/Men^-/- and Men1^-/- mice, including dissection, snap freezing for protein analysis, and fixation for histopathological evaluation.

• 1.Place the mouse in a CO₂ chamber and euthanize with gradual filling of CO₂ (50% volume/min) into the chamber to prevent distress and maintain exposure for 1 min to 1.5 min after breathing stops.

Note: Make sure that the euthanasia conditions are kept constant. Alternative methods such as cervical dislocation can be used when performed by trained personnel. However, CO₂ euthanasia with gradual filling was selected based on institutional IACUC approval to ensure standardized, reproducible, and humane conditions across all animals.

• 2.Confirm euthanasia by the absence of respiration and reflexes.
• 3.Place the mouse in a supine position (Figure 1A) and disinfect the abdominal surface with 70% ethanol.
• 4.A midline longitudinal incision is made through the skin and peritoneal wall to expose the abdominal cavity (Figure 1B).
• 5.Open the abdominal cavity using sterile scissors, taking care not to damage internal organs (Figure 1C).
• 6.Locate the spleen using the forceps; it is situated in the right abdominal quadrant, adjacent to the stomach (Figure 1D).
• 7.
  Dissect the pancreatic tail and body region.

  • a.
    Gently raise the spleen using the forceps, thus elevating the attached pancreatic tail and body region of the pancreas.
  • b.
    Carefully dissect the pancreas near the splenic flexure, where the distal pancreas approaches the colon (Figure 1E).

Note: This step releases the pancreatic tail and body while preserving their attachment to the spleen for easier handling. (Figure 1F). The excised tumor should be transferred immediately to cold PBS in a petri dish, as described in step 8. In some mice, the tumor-bearing pancreatic region may become adherent to or partially fused with the colon, making clean separation challenging. Dissecting the pancreas just proximal to the region helps maintain uniformity of the pancreatic specimen while minimizing the risk of colon contamination during dissection. Moreover, pancreatic islets in mice are enriched in the pancreatic tail region, which is adjacent to the spleen. Standard islet isolation protocols commonly prioritize this region due to its higher islet density and ease of access.⁵ Because the PanNET model originates from islet tissue, the pancreatic tail regions are expected to contain a higher tumor burden and to allow more consistent and reproducible isolation.

• 8.Rinse the Isolated specimen consisting of the pancreatic region along with the spleen in 20 ml ice-cold PBS placed in a 100-mm Petri dish for 15 to 20 s to remove blood. Remove and discard the spleen.
• 9.
  Keep tissues in ice-cold PBS and carefully cut a small piece of the pancreas, (approximately 35–40 mg; about 3–4 mm in dimension) that was previously attached to the spleen.

  • a.
    Transfer the piece into a 1.5 ml microcentrifuge tube.
  • b.
    Immediately snap freeze the tissue by immersing the microcentrifuge tube in liquid nitrogen for 1 to 2 min.

Note: Representative examples of pancreatic tissues isolated from several mice (8 to 12 months old) are shown in Figure 2A. These specimens may display variable external morphology, including differences in size, shape, and the extent of external nodulation. Figure 2B provides a guide for the recommended dissection region for isolating the tumor region mentioned in step 9, indicated by dotted lines.

• 10.Fix the remaining pancreas tissue in biopsy cassette and fully immerse it in buffered formalin for 48 h.
• 11.After 48 h, remove the formalin and store the tissue in 70 % ethanol until further processing for histological evaluation. Histological evaluation is performed according to the standard protocol.⁶

Note: Formalin is used initially to fix and preserve tissue morphology, while subsequent storage in 70% ethanol prevents over-fixation and maintains tissue quality before histological processing.

Figure 2.

Representative pancreatic tumor morphology from hTS/Men1^-/- mouse

(A) Representative pancreatic tumor specimens from 8–12-months-old hTS/Men1^-/- mice showing variability in gross morphology, including size, shape, and nodulation.

(B) Schematic representation of the recommended dissection region (dotted lines) of pancreatic neuroendocrine tumor (PanNET) from a 9.5-months-old hTS/Men1^-/- mouse.

Dotted lines indicate the pancreatic region that was originally attached to the spleen. This tumor region was excised for protein isolation. Arrows mark the nodular tumor regions.

Pancreatic tissues in hTS/Men1^-/- and Men1^-/- mice exhibit distinct stages ranging from hyperplasia to adenoma and carcinoma.² Therefore, histological evaluation is essential to selecting suitability for tissues used for immunoblotting. In our workflow, tissues are initially harvested without pre-selection based on stage of disease, as external morphology does not reliably reflect tumor progression in this model. For each specimen the pancreatic tail region attached to spleen is cut and snap-frozen and stored at −80 °C for protein analysis. This region is selected primarily for consistency in dissection, though it is also known to contain a higher density of islet tissue, and not due to any pathological differences between different pancreatic regions. The remaining portion is fixed for histological evaluation.

After fixation and histological processing, immunohistochemistry (IHC) is performed to classify the tumor as hyperplasia, adenoma, or carcinoma. Based on this pathological assessment, only samples confirmed to contain carcinoma (Figure 3) are selected for subsequent immunoblotting analysis. This workflow ensures that immunoblotting are performed using well-defined and histologically validated tumor stages.

Figure 3.

H&E-stained pancreatic carcinoma in hTS/Men1^-/- mice

Representative hematoxylin and eosin (H&E)-stained sections showing carcinoma developed in hTS/Men1^-/- mice.

Scale bars, 200 μm.

Protein isolation from PanNET tissues

Timing: ∼4 h

Here we describe total protein extraction from snap-frozen PanNET tissue for immunoblotting, including lysis, homogenization, and protein quantification.

• 12.Retrieve snap-frozen PanNET tissue from −80 °C storage, thaw on ice for 4 – 5 min and then transfer it to a pre-chilled 5-mL Falcon tube.
• 13.Add ice-cold RIPA lysis buffer supplemented with freshly prepared protease and phosphatase inhibitors at a ratio of 100 μL per 10 mg of tissue (e.g., 400 μL for ∼40 mg tissue), ensuring a minimum total volume of 300 μL for proper sonication.

Note: Protease and phosphatase inhibitor tablets (Sigma) were used. Half of a tablet was dissolved in 5 mL of RIPA buffer immediately before use (key resources table) to prepare a small volume as needed and reduce reagent waste. The tablet can be cut in half using a disposable scalpel, and the remaining half can be stored in a microcentrifuge tube for future use. Alternatively, one full tablet each can be dissolved in 10 ml of RIPA buffer.

• 14.Homogenize the tissue using a probe sonicator with short pulses while keeping the tube immersed in ice. Sonication was performed using alternating pulses at level 3 (3–5 s) followed by level 2 (3–5 s).

Note: Complete homogenization is indicated by a uniformly turbid lysate (Figure 4). An OMNI International GLH-01 sonicator with a disposable probe was used (Item 60CE76; Mfr. Model 30750H). This instrument does not report amplitude as a numerical setting but instead operates using instrument-specific power levels (levels 2–3 in this protocol). Because sonicator performance and amplitude settings may vary across manufacturers and models, users employing alternative sonicators should optimize settings to achieve complete tissue homogenization while avoiding overheating. If visible tissue fragments remain, repeat the sonication cycle once more.

• 15.Transfer the homogenized lysate to a pre-chilled microcentrifuge tube and incubate on ice for 45 min, vortexing briefly every 15 min.
• 16.Centrifuge the lysate at 13,000 rpm for 15 min at 4 °C.

Note: Ensure that the centrifuge has reached 4 °C before starting centrifugation. Maintaining low temperature is critical to minimize nonspecific background and preserve protein integrity.

• 17.Carefully transfer the clarified supernatant (total protein lysate) to a new pre-chilled microcentrifuge tube. Discard the pellet. Aliquot a separate volume for protein quantification.
• 18.Determine protein concentration using a Bio Rad protein assay (key resources table) according to the manufacturer’s instructions.

Note: When 30–40 mg of PanNET tissue is lysed in ∼300–400 μL of RIPA buffer, using 100 μL of RIPA per 10 mg of tissue, the typical protein concentration ranges from 2 to 5 μg/μL. 100 μL of RIPA is needed per 10 mg of tissues. However, note that the minimum RIPA volume required for proper sonication is 300 μL.

Figure 4.

Tumor tissue before and after sonication in RIPA buffer

Representative image of tumor tissue before and after sonication in RIPA buffer, showing intact tissue prior to lysis and homogenized lysate following sonication.

Expected outcomes

This protocol yields highly reproducible and high-quality total protein lysates from pancreatic neuroendocrine tumors isolated from hTS/Men1^-/- and Men1^-/- mice (2 to 5 μg/μL). Key elements are the harvesting of tumor tissue from a specific location (such as the splenic lobe of the pancreas) and the controlled euthanasia conditions. Using the spleen attachment lobe of the pancreas ensures that the extracted samples are enriched for carcinoma-derived proteins with minimal contamination from exocrine pancreatic tissue.

Protein lysates generated using this method are expected to exhibit excellent protein integrity and reduced nonspecific background during immunoblotting² (Figure 5), making them well-suited for protein analysis from PanNET tissues developed in genetically engineered mice model. Overall, this approach enables reliable investigation of signaling mechanisms associated with PanNET progression using Immunoblotting.

Figure 5.

Representative immunoblot analysis of TYMS and p-AMPK expression in Men1^-/- and hTS/Men1^-/- tumor lysates

Representative whole western blot showing expression of thymidylate synthase (TYMS) and phosphorylated AMPK (p-AMPK) in tumor lysates from Pan NET tissues.

The same membrane was cut according to the molecular weight marker (MW) and probed separately with the indicated antibodies.

Lanes 1–4 represent protein extracts from Men1^-/- mice, whereas lanes 5–7 represent extracts from hTS/Men1^-/- mice.

Limitations

First, this protocol was optimized and validated primarily for the isolation of total non–membrane-bound and cytosolic proteins from pancreatic neuroendocrine tumor tissues. Although RIPA buffer can solubilize some membrane-associated proteins, the efficiency of isolating integral membrane proteins using this method has not been systematically evaluated. Therefore, this protocol may not be suitable for studies focusing on membrane proteins or membrane-enriched fractions without additional optimization.

Second, a small amount of non-tumorous pancreatic tissue contamination cannot be completely excluded due to the heterogeneous nature of pancreatic tumors and their close anatomical association with surrounding pancreatic tissue.

Troubleshooting

Problem 1

Incomplete tissue homogenization or visible tissue fragments after lysis (step 14).

Potential solution

Increase the volume of ice-cold RIPA buffer by an additional 100–150 μL and repeat the total homogenization cycle using the probe sonicator. Ensure the sample remains on ice throughout the process. If tissue fragments are still visible, perform one additional short sonication cycle. Avoid excessive sonication to prevent protein degradation.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maria Zajac-Kaye (mzajackaye@ufl.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Vinod Vijayakurup (vijayakurupvinod@ufl.edu).

Materials availability

This study did not generate any new unique reagents.

Data and code availability

Detailed western blotting protocol information is available from the lead contact upon request. This study did not generate any new code.

Acknowledgments

We thank Liliana Katz for genotyping. This work has been funded in part by Public Health Service grant R01 CA-188132 from the National Cancer Institute (to M.Z.-K.); the Gatorade Trust through funds distributed by the University of Florida, the Department of Medicine (to M.Z.-K.); and UF Health Cancer Center Pilot grant P0167056 (to M.Z.-K.).

Author contributions

This study was designed by M.Z.-K. and V.V. The experiments were performed by V.V. Data were analyzed by V.V. and M.Z.-K. Histology interpretation was provided by M.F. The article was written by M.Z.-K. and V.V. Supervision was provided by M.Z.-K. All authors reviewed the article.

Declaration of interests

The authors declare no competing interests.