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 Z&#252;rich for their support concerning anesthesia. This work was supported by the Surgical Department program &#34;Personenf&#246;rderung&#34; to G.F.H and F.H.; the St. Clara Forschung AG and the &#34;Stiftung zur Krebsbek&#228;mpfung&#34; to G.F.H. and University of Basel, Research Fund Junior Researcher (3MS1087) to M.C-LL.

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
<ol>
	<li>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.</li>
</ol>
2.&#160;Patient and clinical samples
<ol>
	<li>Place 12-, 24-, and 6-well plates in the incubator at 37 &#176;C before starting. If possible, do this 1 day in advance.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Liver cancer tissue dissociation for the generation of organoids
	<ol>
		<li>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.</li>
		<li>Place the tissue pieces in a 15 mL tube with 5 mL of dissociation buffer (physiological pH). Place at 37 &#176;C (warming cabinet/incubator) in a rotation device.</li>
		<li>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.</li>
		<li>Collect 10 &#181;L of cell-dissociation buffer mixture from step 2.2.3 in a 0.5 mL plastic tube, add 10 &#181;L 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.</li>
		<li>Take a 50 mL tube, place a 100 &#181;m 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.</li>
		<li>Transfer the filtrate to a 15 mL conical tube. Centrifuge for 15 min at 300 &#215; g.</li>
		<li>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.</li>
		<li>Centrifuge for 5 min at 300 &#215; g. Remove the supernatant and resuspend in 1 mL of advanced medium.</li>
	</ol>
	</li>
	<li>Counting cells and assessing cell viability
	<ol>
		<li>Add 10 &#181;L of Trypan blue to a 1.5 mL tube and mix with 10 &#181;L of the cell suspension. Place 10 &#181;L of the mix on the counting slides and count the cells.</li>
		<li>Separate the necessary number of cells for the organoid generation and centrifuge for 5 min at 300 &#215; g. Generally, seed 80,000 cells per dome and 20 &#181;L of basement membrane matrix per dome (10 drops per well per 6-well plate).</li>
		<li>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 &#176;C freezer overnight. The next day, transfer into liquid nitrogen until needed.</li>
	</ol>
	</li>
	<li>Plating for organoid generation
	<ol>
		<li>Remove the supernatant. Place the tube on ice and resuspend the pellet with a basement membrane matrix.</li>
		<li>Take the prewarmed plate out of the incubator (prewarmed overnight). Make small drops in the plate (20 &#181;L of basement membrane matrix per dome [10 drops per well]). Invert the plate and leave the plate for 5 min in the flow cabinet.</li>
		<li>After 5 min, transfer the plate to the 37 &#176;C incubator and leave the plate for ~20 min, allowing the matrix to solidify. Add 2 mL of organoid culture medium6,7.</li>
	</ol>
	</li>
	<li>Maintenance of organoid culture
	<ol>
		<li>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.</li>
		<li>Remove the medium from the corner of the well using a P1000 tip, taking care not to touch the matrix domes.</li>
		<li>Add 100 &#181;L 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.</li>
		<li>Incubate for 3 min in the 37 &#176;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.</li>
		<li>Add at least the same volume of Dulbecco&#39;s modified Eagle&#39;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.</li>
		<li>Centrifuge at 300 &#215; 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.</li>
		<li>Take the prewarmed plate out of the incubator (prewarmed overnight). Make small drops in the plate (20 &#181;L of basement membrane matrix per dome [10 drops per well]). Invert the plate and leave the plate for 5 min in the flow cabinet.</li>
		<li>After 5 min, transfer the plate to the 37 &#176;C incubator. Leave the plate for ~20 min, allowing the matrix to solidify. Add 2 mL of organoid culture medium.</li>
	</ol>
	</li>
	<li>Preparation of organoids for making a formalin-fixed paraffin-embedded (FFPE) block
	<ol>
		<li>Remove the medium from the corner of the well using a P1000 tip, taking care not to touch the matrix domes. Add 200 &#181;L of Dispase (2 mg/mL) and disrupt the drops with a P1000 pipette tip. Leave in the incubator for 80 min.</li>
		<li>Add 2 mL of Dulbecco&#39;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 &#215; g for 5 min, aspirate the supernatant, and resuspend the pellet in 1 mL of 4% paraformaldehyde. Refrigerate for at least 1 h.</li>
		<li>Centrifuge at 300 &#215; 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 staining8,9.</li>
	</ol>
	</li>
	<li>Preparation of organoids for injection
	<ol>
		<li>Take the plate containing the growing organoid out of the incubator and place it in the flow cabinet.</li>
		<li>Follow steps 2.5.2 to 2.5.4.</li>
		<li>Add 2 mL of DMEM and mix.</li>
		<li>Add 10 &#181;L of Trypan blue to a 1.5 mL tube and mix with 10 &#181;L of cell suspension.</li>
		<li>Place 10 &#181;L 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.</li>
		<li>Calculate the volume of cell suspension required for 106 cells and dispense this volume to 1.5 mL tubes. Centrifuge at 300 &#215; g for 5 min and place the tubes on ice.</li>
		<li>Carefully remove the supernatant and add 5 &#181;L of DMEM and 5 &#181;L of basement membrane matrix. Carefully mix the suspension by pipetting while keeping the suspension cold during the process.</li>
		<li>Place the 1.5 mL tubes containing the organoids on ice during the transport to the mice and proceed with injection.</li>
	</ol>
	</li>
</ol>
3. Injection of organoid into mouse
<ol>
	<li>Perioperative steps
	<ol>
		<li>Remove the experimental animal from the main cage and place it into an isolated, fresh cage with water.</li>
		<li>Anesthetize the mouse with 1.5% to 2.5% Isoflurane.</li>
		<li>Perform a subcutaneous injection into the neck with 10 &#181;L of Buprenorphine (0.3 mg/mL) 30 min prior to the start of the surgery.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Laparotomy 
	NOTE: Before the surgical procedure started, the depth of the anesthesia was checked by the toe pinch test. During the procedure, adequate anesthesia was provided through routine checks and monitoring of the respiration rate and adjusted if there was a relevant increase. The surgical equipment was cleaned and disinfected according to the in-house guidelines of our animal facility.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Make an incision along the linea alba with forceps and microsurgical scissors from the lower abdomen to the xiphoid cartilage.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Injection of organoids
	<ol>
		<li>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.</li>
		<li>Expose the right superior lobe (RSL) until an injection is possible.</li>
		<li>Prepare the Hamilton syringe, rinse it with water, and place a mark 2-3 mm from the tip as a reference for the injection.</li>
		<li>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.</li>
		<li>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. 
		NOTE: The surgical site was covered by a sterile drape. The surgical instruments used during the procedure were primarily cleaned with water and cleaning agents to remove macroscopic remnants. The instruments were then processed in a sterilization solution. Consumables like threads, gloves or cotton swabs were only used once for hygienic reasons and were sterile.</li>
	</ol>
	</li>
</ol>
4. Minor resection (30% of liver tissue)
<ol>
	<li>Perform the laparotomy as stated above in step 3.2.</li>
	<li>Use moist cotton swabs and push the ML cranially to create more space in the surgical field of the LLL.</li>
	<li>Flip the LLL cranially and expose the caudate lobe (CL) and ligament, which is habitually present between the CL and LLL (Figure 2C).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Resect the liver lobe. Cut close to the ligature without cutting it.</li>
</ol>
5. Major resection (65% of liver tissue)
<ol>
	<li>Perform the minor resection of the LLL as stated above (steps 3.4.1-3.4.8).</li>
	<li>Transect the falciform ligament to mobilize the ML. Flip the lobe cranially. Insert the ligature below the lobe and flip the lobe caudally.</li>
	<li>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.</li>
	<li>Tie a second half knot in the opposite direction and tighten it completely. Add a third half knot in the opposite direction.</li>
	<li>Resect the lobe.</li>
	<li>Flush the abdomen with sterile saline solution and place the intestines in their anatomical place.</li>
</ol>
6. Closure of the abdomen
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Continue the running suture and finish with a knot at the bottom end of the incision.</li>
	<li>Clean the abdomen of the mouse of any remaining blood.</li>
</ol>
7. Postoperative care
<ol>
	<li>After the closure of the abdomen, inject another 10 &#181;L 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.</li>
	<li>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 &#181;L of Bupivacaine if needed.</li>
	<li>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.</li>
	<li>After the final postoperative control 1 day after the surgery, proceed with the regular weighing of the animals. 
	NOTE: Analgesia was administered 4 and 8 hours postoperatively. Further monitoring was performed at 12, 24 and 48 hours postoperatively, and mice were checked for any kind of pain or distress. If any signs of pain were observed, another dose of Buprenorphine was administered.</li>
</ol>
8. Monitoring tumor growth
<ol>
	<li>Immobilize the mice using isoflurane anesthesia and use sonography to visualize tumor implantation beginning 2 weeks after injection10.</li>
	<li>Euthanize the mice using CO2.</li>
</ol>

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

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All authors have no financial or personal relationships with other people or organizations that could inappropriately influence their work. This includes any potential conflicts of interest, such as financial interests, affiliations, or relationships that might affect their ability to present or interpret data objectively.

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 ...

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 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&#39;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.

<table><tbody><tr><td>Advanced DMEM/F12 (adDMEM/F12)</td><td>Life Technologies</td><td>12634-034</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3733-50G</td><td></td></tr><tr><td>Bupaq Buprenorphinum 0.3 mg pro 1 mL</td><td>Streuli Tiergesundheit AG</td><td>1121915AB</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Collagenase IV</td><td>Worthington</td><td>LS004189</td><td></td></tr><tr><td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (10 mL)</td><td>Corning (Merk)</td><td>CLS356231-1EA</td><td></td></tr><tr><td>Countess II Automated Cell Counter</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Deoxyribonuclease I Type IV from Bovine (DNAse)</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td></td></tr><tr><td>DMEM (1x)</td><td>Gibco</td><td>41965-039</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium (500 mL)</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) (South America). Heat Inactivated &amp;ndash; (500 mL)</td><td>Lubio Science</td><td>S181H-500</td><td></td></tr><tr><td>Glutamax (100x)</td><td>Gibco</td><td>9149793</td><td></td></tr><tr><td>Grant SUB Aqua Pro Water Bath</td><td>Grant Instruments</td><td></td><td></td></tr><tr><td>HEPES (1 M)</td><td>Thermo Fisher</td><td>15630056</td><td></td></tr><tr><td>Histogel</td><td>Thermo Fisher</td><td>R904012</td><td>specimen-processing&amp;nbsp; gel</td></tr><tr><td>Hyaluronidase Type IV from sheep (Tested)</td><td>Sigma-Aldrich</td><td>H6254-500MG</td><td></td></tr><tr><td>Inverted Microscope Olympus CKX53</td><td>Olympus</td><td></td><td></td></tr><tr><td>MacsMix Tube Rotator</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin-Glutamine (100x)</td><td>Gibco</td><td>10378-016</td><td></td></tr><tr><td>Red Blood Cell Lysis</td><td>Roche</td><td>11814389001</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>72400-021</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red (100 mL)</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Vitaris CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Vitaris AG</td><td></td><td></td></tr><tr><td>Y-27632 dihydrochloride (Rock Inhibitor)</td><td>Abmole Bioscience</td><td>M1817</td><td></td></tr></tbody></table>

a hepatocellular cancer patient&#45;derived organoid xenograft model to investigate impact of liver regeneration on tumor growth

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.

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...

Read this Scientific Article Protocol about Author Spotlight: Hepatocellular Carcinoma Research Through Patient-Derived Organoid Engraftment in Liver Regeneration Models at JoVE.com

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

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.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

681 Views.  University Center for Gastrointestinal and Liver Diseases, St. Clara Hospital and University Hospital Basel. Tumorrecidief is een zeer prominent probleem bij resectie van de bevalling. In deze studie proberen we meer te begrijpen over de tumorgroei in een regeneratieve omgeving. Van tumororganoïden is aangetoond dat ze de tumor van de patiënt histologisch en genetisch recapituleren, hoewel in vivo modellen schaars zijn en niet stabiel zijn.We hebben met succes de van de patiënt afgeleide organoïde geënt in de lever van de muis. We hebben dit aan de hand ...

Video: Een van patiënten afgeleide organoïde xenotransplantaatmodel voor hepatocellulaire kanker om de impact van leverregeneratie op tumorgroei te onderzoeken

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials (&#8220;Phase II-like mouse clinical trials&#8221;); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 &#181;m cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 &#181;M Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...

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

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