A Hepatocellular Cancer Patient-Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Summary

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Abstract

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.

Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.

In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Introduction

Recurrence following hepatocellular carcinoma (HCC) treatment is a significant challenge, impacting over 70% of patients undergoing surgical resection^1,2,3. This recurrence may result from undetected micro-metastasis (multicentric tumor) or the development of de novo cancer⁴. Both clinical and experimental investigations propose that the liver regeneration process triggered by surgical resection could activate latent micro-metastases, contributing to tumor recurrence.

Further research is necessary to compare the gene and protein expression in normal and malignant liver cells within a regenerative liver environment. In addition to elucidating the mechanisms underlying recurrence, identifying targetable signaling pathways specific to cancer cells holds the potential for substantial therapeutic advancements. This approach aims to reconcile the seemingly contradictory notions of an anti-tumor microenvironment and favorable conditions for liver regeneration.

Previous models have used orthotopic injection of HCC cell lines to investigate the impact of liver regeneration⁵. This pioneering model employs patient-derived HCC organoids, offering several advantages over traditional cell lines. Organoids maintain diverse cell populations, mirroring the model template's structure and function, unlike cell lines and spheroids. Their genetic diversity enhances representativeness. Additionally, organoids provide stability for extended culture, enabling prolonged intervention studies. We recommend RSL injection due to its superior regeneration potential compared to the right inferior lobe (RIL). The method described here provides a platform using cancer organoids with pronounced advantages compared to cancer cell lines to investigate the aforementioned research question.

Protocol

Liver samples used for organoid generation were obtained from patients operated at the University Hospital of Basel (USB) following written informed consent under approval by the Ethical Committee of Northwestern and Central Switzerland (BASEC number 2019-02118). All mouse experiments were approved by and performed in accordance with the guidelines and regulations of the Animal Care Committee of Kanton Basel-Stadt, Switzerland (3123-33896). All mice were of the non-obese diabetic (NOD) SCID gamma mouse strain and therefore genetically modified to be immunosuppressed; only male animals were used. See Table 1 and Table of Materials for details related to solutions, reagents, materials, and instruments used in the protocol. See Figure 1 for an overview of the experiment.

1. Mouse husbandry

1. Breed and maintain mice in the animal facility under specific pathogen-free conditions on a 12 h day and 12 h night schedule with ad libitum access to food and drinking water.
   NOTE: Implantation of the organoids is performed at an age of 6-8 weeks.

2. Patient and clinical samples

1. Place 12-, 24-, and 6-well plates in the incubator at 37 °C before starting. If possible, do this 1 day in advance.
   1. Collect the tissue within 1 h after resection in RPMI 1640 medium and transport it to the laboratory to start the dissociation process immediately. Place the tissue in a 10 cm Petri dish (prewarmed dish) and add a sufficient amount of sterile saline to avoid the tissue drying out.
2. Liver cancer tissue dissociation for the generation of organoids
   1. Mince the tissue into small fragments of 1-2 mm² using scalpels. Use additional tissue forceps to stabilize the tissue in case cutting is difficult.
   2. Place the tissue pieces in a 15 mL tube with 5 mL of dissociation buffer (physiological pH). Place at 37 °C (warming cabinet/incubator) in a rotation device.
   3. Mix by pipetting every 10 min and leave for approximately 1-1.5 h, repeating the mixing process every 10 min.
      NOTE: Mixing may require cutting the end of the pipette to increase the orifice diameter, thereby preventing oversize tissue from getting stuck in the pipette tip.
   4. Collect 10 µL of cell-dissociation buffer mixture from step 2.2.3 in a 0.5 mL plastic tube, add 10 µL of Trypan Blue, and use the automated cell counter to assess cell viability and cell clusters. If clusters are visible, repeat the mixing process (step 3.3.); otherwise, proceed with the next step.
   5. Take a 50 mL tube, place a 100 µm cell strainer on the top, and pass the mix through it. With the plunger of a 5 mL syringe, smash all remaining tissue fragments through the filter. Wash everything with 5 mL of Ad-DF+++ and be sure to collect the drop from the bottom of the filter.
   6. Transfer the filtrate to a 15 mL conical tube. Centrifuge for 15 min at 300 × g.
   7. Aspirate the supernatant carefully, taking care to not touch the pellet. Add 5 mL of Red Blood cell lysis buffer. Incubate for 5-10 min at room temperature.
   8. Centrifuge for 5 min at 300 × g. Remove the supernatant and resuspend in 1 mL of advanced medium.
3. Counting cells and assessing cell viability
   1. Add 10 µL of Trypan blue to a 1.5 mL tube and mix with 10 µL of the cell suspension. Place 10 µL of the mix on the counting slides and count the cells.
   2. Separate the necessary number of cells for the organoid generation and centrifuge for 5 min at 300 × g. Generally, seed 80,000 cells per dome and 20 µL of basement membrane matrix per dome (10 drops per well per 6-well plate).
   3. If any excess cells are remaining in suspension, suspend the pellet in Cell Suspension Freezing medium (10% DMSO in FBS), transfer it into a cryotube, and place it in a freezing container. Immediately transfer the tube to a -80 °C freezer overnight. The next day, transfer into liquid nitrogen until needed.
4. Plating for organoid generation
   1. Remove the supernatant. Place the tube on ice and resuspend the pellet with a basement membrane matrix.
   2. Take the prewarmed plate out of the incubator (prewarmed overnight). Make small drops in the plate (20 µL of basement membrane matrix per dome [10 drops per well]). Invert the plate and leave the plate for 5 min in the flow cabinet.
   3. After 5 min, transfer the plate to the 37 °C incubator and leave the plate for ~20 min, allowing the matrix to solidify. Add 2 mL of organoid culture medium^6,7.
5. Maintenance of organoid culture
   1. Replace the medium every 2 days. Once the organoids are confluent in the basement membrane matrix dome and ready for expansion, calculate the splitting ratio e.g., 2x or 3x accordingly.
      NOTE: Record this information; it will be needed in the next steps.
   2. Remove the medium from the corner of the well using a P1000 tip, taking care not to touch the matrix domes.
   3. Add 100 µL of 0.25% trypsin-EDTA per dome, scrape it, collect it into a 15 mL tube, and add again a small amount of trypsin into the well to collect everything. Pipette it ~10x.
   4. Incubate for 3 min in the 37 °C water bath and pipette 10x to dissociate the cells with a P1000 tip. Repeat this step every 3 min for a maximum of 10 min.
   5. Add at least the same volume of Dulbecco's modified Eagle's medium (DMEM)/10% Fetal bovine serum (FBS)/1% Penicillin-Streptomycin (Pen-Strep) medium and mix up and down slowly 2x. Fill up with DPBS.
   6. Centrifuge at 300 × g for 5 min and carefully remove the supernatant. Resuspend according to the splitting ratio decided based on the organoid confluency e.g., 2x or 3x the initial amount of basement membrane matrix.
   7. Take the prewarmed plate out of the incubator (prewarmed overnight). Make small drops in the plate (20 µL of basement membrane matrix per dome [10 drops per well]). Invert the plate and leave the plate for 5 min in the flow cabinet.
   8. After 5 min, transfer the plate to the 37 °C incubator. Leave the plate for ~20 min, allowing the matrix to solidify. Add 2 mL of organoid culture medium.
6. Preparation of organoids for making a formalin-fixed paraffin-embedded (FFPE) block
   1. Remove the medium from the corner of the well using a P1000 tip, taking care not to touch the matrix domes. Add 200 µL of Dispase (2 mg/mL) and disrupt the drops with a P1000 pipette tip. Leave in the incubator for 80 min.
   2. Add 2 mL of Dulbecco's phosphate-buffered saline (DPBS) to the well, transfer it to a 15 mL conical tube, and fill it up with DPBS. Centrifuge at 300 × g for 5 min, aspirate the supernatant, and resuspend the pellet in 1 mL of 4% paraformaldehyde. Refrigerate for at least 1 h.
   3. Centrifuge at 300 × g for 5 min, aspirate the paraformaldehyde, and resuspend the pellet with the specimen-processing gel to create a drop. Wait for 40 min for the drops to solidify. Place the gel drop containing the cells in a histology cassette. Move the cassette to dehydrate and afterward embed it in paraffin for cutting and H/E staining^8,9.
7. Preparation of organoids for injection
   1. Take the plate containing the growing organoid out of the incubator and place it in the flow cabinet.
   2. Follow steps 2.5.2 to 2.5.4.
   3. Add 2 mL of DMEM and mix.
   4. Add 10 µL of Trypan blue to a 1.5 mL tube and mix with 10 µL of cell suspension.
   5. Place 10 µL of the mix on the counting slides and count the cells. Calculate the average cell count of both chambers. If the difference is too big between the two chambers, consider redoing the count.
   6. Calculate the volume of cell suspension required for 10⁶ cells and dispense this volume to 1.5 mL tubes. Centrifuge at 300 × g for 5 min and place the tubes on ice.
   7. Carefully remove the supernatant and add 5 µL of DMEM and 5 µL of basement membrane matrix. Carefully mix the suspension by pipetting while keeping the suspension cold during the process.
   8. Place the 1.5 mL tubes containing the organoids on ice during the transport to the mice and proceed with injection.

3. Injection of organoid into mouse

1. Perioperative steps
   1. Remove the experimental animal from the main cage and place it into an isolated, fresh cage with water.
   2. Anesthetize the mouse with 1.5% to 2.5% Isoflurane.
   3. Perform a subcutaneous injection into the neck with 10 µL of Buprenorphine (0.3 mg/mL) 30 min prior to the start of the surgery.
   4. Immediately prior to the start of the surgery, carefully paint the eyes of animals with Carbomerum (e.g., Lacrinorm) to avoid any dryness of the eyes during surgery. Do not touch the cornea during the application to avoid any erosion of the cornea.
   5. Place the animals onto a clean and sterile surface and handle them with fresh plastic gloves, hairnet, and a special laboratory working coat used only in the mouse facility.
   6. Use two heat-emitting headlights close to the animal to provide a sufficient view. If there is any doubt or concern about hypothermia during the surgery, use another heating lamp or heating mat.
2. Laparotomy
   1. Place the anesthetized mouse in the supine position, extend the four extremities, and fix them with tape. Remove the fur by applying a depilatory cream and use a wet tissue to remove the excess cream and fur.
      NOTE: Depilation works best if a cotton swab is used to massage the cream into the fur against the grain.
   2. Localize the linea alba, which is the target for the laparotomy. Identify the superior and inferior epigastric arteries as well. Take care not to damage the vessels to avoid injuries.
   3. Make an incision along the linea alba with forceps and microsurgical scissors from the lower abdomen to the xiphoid cartilage.
   4. Insert the retractors and adjust the direction of traction for optimal exposure of the upper abdomen (Figure 2A,B).
      NOTE: We use bent paper clips as retractors with attached rubber bands for fixation.
3. Injection of organoids
   1. Place a clean napkin right next to the incision and put some drops of saline onto it. Exteriorize the small intestine and the cecum and place it on the wet napkin.
   2. Expose the right superior lobe (RSL) until an injection is possible.
   3. Prepare the Hamilton syringe, rinse it with water, and place a mark 2-3 mm from the tip as a reference for the injection.
   4. Draw up the syringe with the prepared organoid mixture and inject the needle until the mark. Slowly inject the organoids in a steady position; do not move the needle during this time.
   5. Wait for 15-20 s to avoid backflow of the mixture. Remove the needle and put some pressure with a cotton swab on the injection site. Check the abdomen for a rupture of the liver lobe and backflow. Rinse the abdomen with 0.5 mL of sterile saline and close the abdomen with a running suture using a resorbable suture.

4. Minor resection (30% of liver tissue)

1. Perform the laparotomy as stated above in step 3.2.
2. Use moist cotton swabs and push the ML cranially to create more space in the surgical field of the LLL.
3. Flip the LLL cranially and expose the caudate lobe (CL) and ligament, which is habitually present between the CL and LLL (Figure 2C).
4. Cut the ligament completely and insert the ligature (non-resorbable thin thread, e.g. Prolene 5-0) below the LLL. To ensure that it stays in this position, moisten the ligature and move it towards the right upper quadrant.
5. Flip the LLL caudally. Guide the left free end of the ligature around the left tip of the LLL. Guide the right free end of the ligature around the right tip of the LLL. Apply caudal traction on the LLL with a cotton swab and ensure the correct and central positioning of the ligature.
6. Tie a double half knot. Secure the knot with a second half knot tied in the other direction without fully tightening it. Tighten the knots a little bit, check the correct placement of the ligature, and reposition it to guarantee good positioning.
7. Tighten the knot completely by holding the cranial free end in position with blunt-tipped forceps and pull the caudal free end with another pair of forceps (Figure 2D). Secure the knot with a second half knot tied in the opposite direction.
8. Resect the liver lobe. Cut close to the ligature without cutting it.

5. Major resection (65% of liver tissue)

1. Perform the minor resection of the LLL as stated above (steps 3.4.1-3.4.8).
2. Transect the falciform ligament to mobilize the ML. Flip the lobe cranially. Insert the ligature below the lobe and flip the lobe caudally.
3. Loop the free ends around the lobule. Repeat placement of the ligature in case of misplacement during manipulation. Once the ligature is positioned correctly, place a double half knot, tie it slowly, and reposition the ligature if needed.
4. Tie a second half knot in the opposite direction and tighten it completely. Add a third half knot in the opposite direction.
5. Resect the lobe.
6. Flush the abdomen with sterile saline solution and place the intestines in their anatomical place.

6. Closure of the abdomen

1. Grab the skin and peritoneum with forceps and make a stitch with a resorbable suture (e.g., Monocryl 5-0) on the top end of the incision line. Be sure to exit on the peritoneal side.
2. Perform the second stitch on the counterpart of the incision, exiting on the skin side. Tie the knot tightly while leaving the free end without the needle short and the end with the needle long. Cut the short end to a length of 2 mm.
3. Continue the running suture and finish with a knot at the bottom end of the incision.
4. Clean the abdomen of the mouse of any remaining blood.

7. Postoperative care

1. After the closure of the abdomen, inject another 10 µL of Buprenorphine (0.3 mg/mL) subcutaneously into the neck. Place the animals in the previously used, isolated cage, partially placed on a heating mat. Provide some easily accessible food on the floor of the cage.
   NOTE: Do not leave the animal unattended until it has regained consciousness.
2. Carry out postoperative controls at 2 h and 6 h after the surgery. Closely observe the behavior with special attention to any sign of pain; administer another 10 µL of Bupivacaine if needed.
3. Once the mouse is fully recovered, but not prior to 6 h after surgery and not later than 24 h after surgery, re-introduce it to the main cage. Closely monitor the behavior of all the involved animals regarding any aggression or other suspicious behavior. When in doubt of any postoperative weakness of the animal, leave it isolated overnight to recover until final re-introduction.
4. After the final postoperative control 1 day after the surgery, proceed with the regular weighing of the animals.

8. Monitoring tumor growth

1. Immobilize the mice using isoflurane anesthesia and use sonography to visualize tumor implantation beginning 2 weeks after injection¹⁰.
2. Euthanize the mice using CO₂.

Results

We looked at the contribution of the single liver lobes to the total liver volume and found that the LLL represents 33% of the total liver volume. The ML represents 32% of the total liver volume, the RSL 13%, the RIL 10%, and the CL 10% (Table 2). The boxplots show that the relative contribution of the liver lobes is comparable between the different mice of the same strain (Figure 3B). Hence, a resection of the LLL results in a resection of 35% of the total liver volume, and...

Discussion

Critical steps in the protocol
Mouse anesthesia
The goal of the anesthesia is to convert the mouse safely into a state in which it will tolerate surgical manipulation. At the same time, the effect of the anesthesia should not be too strong, to prevent cardiopulmonary arrest and ensuing death. Inhalant anesthesia is easier to dose, allowing the researcher to adjust the dose by manipulating the flow rate of the vaporizer. The effect of the change is quick. This helps prevent a ...

Materials

Name	Company	Catalog Number	Comments
Advanced DMEM/F12 (adDMEM/F12)	Life Technologies	12634-034
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A3733-50G
Bupaq Buprenorphinum 0.3 mg pro 1 mL	Streuli Tiergesundheit AG	1121915AB
Centrifuge 5810R	Eppendorf
Collagenase IV	Worthington	LS004189
Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (10 mL)	Corning (Merk)	CLS356231-1EA
Countess II Automated Cell Counter	Thermo Fisher
Deoxyribonuclease I Type IV from Bovine (DNAse)	Sigma-Aldrich	D5025-150KU
DMEM (1x)	Gibco	41965-039
DPBS, no calcium, no magnesium (500 mL)	Gibco	14190-094
Fetal Bovine Serum (FBS) (South America). Heat Inactivated – (500 mL)	Lubio Science	S181H-500
Glutamax (100x)	Gibco	9149793
Grant SUB Aqua Pro Water Bath	Grant Instruments
HEPES (1 M)	Thermo Fisher	15630056
Histogel	Thermo Fisher	R904012	specimen-processing  gel
Hyaluronidase Type IV from sheep (Tested)	Sigma-Aldrich	H6254-500MG
Inverted Microscope Olympus CKX53	Olympus
MacsMix Tube Rotator	Miltenyi Biotec
Penicillin-Streptomycin-Glutamine (100x)	Gibco	10378-016
Red Blood Cell Lysis	Roche	11814389001
RPMI 1640 Medium	Gibco	72400-021
Trypsin-EDTA (0.25%), phenol red (100 mL)	Gibco	25200-056
Vitaris CO2 Incubator	Vitaris AG
Y-27632 dihydrochloride (Rock Inhibitor)	Abmole Bioscience	M1817