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A Hepatocellular Cancer Patient-Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth
* These authors contributed equally
In This Article
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 resection1,2,3. 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 gen....
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 therefo.......
Representative 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 .......
Acknowledgements
We thank everyone assisting in this project, especially Karolina Guja and Dr. Daniela Liberati, for maintaining the organoids and guaranteeing reproducible quality over time. We thank Eva Breuer and Anurag Gupta from the Hepatobiliary and Transplant Surgery Lab of the University Hospital of Zürich for their support concerning anesthesia. This work was supported by the Surgical Department program "Personenförderung" to G.F.H and F.H.; the St. Clara Forschung AG and the "Stiftung zur Krebsbekämpfung" to G.F.H. and University of Basel, Research Fund Junior Researcher (3MS1087) to M.C-LL.
....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 |
References
- Huang, J., et al. A randomized trial comparing radiofrequency ablation and surgical resection for HCC conforming to the Milan criteria. Annals of surgery. 252 (6), 903-912 (2010).
- Lim, K. -. C., et al.
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