Name: Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue
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This study was supported by funding from the Plataforma biobancos y biomodelos - Unidades de las Plataformas ISCIII de apoyo ala I+D+i en Biomedicina y Ciencias de la Salud (PT20/00045), The European Union&#39;s Horizon 2020 Research and Innovation Programme under grant agreement No. 857381, project VISION (Strategies to strengthen scientific excellence and innovation capacity for early diagnosis of gastrointestinal cancers), Intramural call for new research projects for clinical researchers and emerging research groups IRYCIS (2021/0446), Patient Derived Organoids 2.0 Project (CIBERONC)&#160;and the TRANSCAN II project JTC 2017 call &#34;Establishing an algorithm for the early diagnosis and follow-up of patients with pancreatic neuroendocrine tumours (NExT)&#34;, grant number 1.1.1.5/ERANET/20/03. The biological samples used in this protocol were provided by the BioBank Hospital Ram&#243;n y Cajal-IRYCIS (B.0000678) and integrated into the Biobanks and Biomodels Platform of the ISCIII (PT20/00045). We would also like to thank Yvonne Kohl, Agapi Kataki Vita Rovita, and Thorsten Knoll for their invaluable support to develop this protocol as part of the NExT and VISION projects.

All procedures were performed in compliance with the institutional guidelines for the welfare of experimental animals approved by the Universidad Aut&#243;noma de Madrid Ethics Committee (CEI 103-1958-A337) and La Comunidad de Madrid (PROEX 294/19) and in accordance with the guidelines for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS). The proto...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

<ol>
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Tuveson Lab Available from: <a target="_blank" href="https://tuvesonlab.labsites.cshl.edu/protocolsreagents/">https://tuvesonlab.labsites.cshl.edu/protocolsreagents/</a> (2023)</li></ol>

Major advances in pharmacological cancer therapies are challenging, as the likelihood of the approval of drugs in phase I oncology clinical trials is 5.1%, which is the lowest of all disease types23. The main reason is that cancer is very heterogenous, and therefore, patient cohorts do not uniformly respond as expected to the given treatment, which highlights that a more personalized approach is needed. Two-dimensional (2D) cultures have been used in translational cancer research for many years bu...

Tumor organoids are ex vivo three-dimensional (3D) organized cultures that are derived from fresh tumor tissue and provide cancer models. Tumor organoids recapitulate the biological key features of the original primary tumor1,2,3,4 and can be expanded for up to several months and cryopreserved, similar to conventional immortalized cell lines. Tumor organoids provide a biobank of patient-derived tumor models for translational/personalized medicine5 and represent an important advance in cancer cell biology systems/models. Patient-derived tumor organoids can be used as ex vivo models to predict the efficacy of (neo)adjuvant oncological/pharmacological therapies, for which cultures are established from fresh tumor tissue and drug sensitivity assays or pharmacotyping are performed on a patient-specific basis to identify effective agents for subsequent lines of therapy1,4. Furthermore, tumor organoids overcome the limitation of the availability of primary tumor tissue and, more importantly, provide an excellent alternative or complementary system to in vivo mouse models, such as patient-derived xenografts (PDX)2. The complexity of tumor organoids is increased if the primary tumor cells are combined with stromal cells that are found in the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells, which mimic the functioning and complex cellularity of the primary tumor. Tumor organoids have been established for many tumor types using standardized protocols6,7,8,9,10. Organoid propagation from different solid tumors, including colorectal and breast cancer tissue, is well-established&#160;and technically affordable11,12,13,14,15.
Surgical tumor resections or tumor biopsies provide primary tumor tissue specimens. Ideally, tumor tissue specimens should come from the center of the tumor mass or the invading edge of the tumor, as well as normal-looking tissue adjacent to the tumor. Compared to conventional 2D cultures, tumor organoids require several &#34;add-ons&#34;, including a biological basement membrane (such as Matrigel, hydrogel, or a collagen-based scaffold), which mimics the extracellular TME, and a liquid growth medium that supplies specific nutrients and growth factors and supports cell proliferation and viability in culture16.
The most basic steps in primary cell culture are washing the tissue in saline solution to prevent contamination, mechanically cutting/digesting the tumor into small pieces of 1-3 mm3, and treatment with collagenase for the enzymatic digestion of the tissue. The digested mix is then filtered to remove large tissue fragments, resuspended in a biological basement membrane such as Matrigel, and plated as domes in low-attachment culture plates to enhance non-attachment growth. The basement membrane matrix domes are covered with liquid culture medium and supplemented with glutamine and antibiotics to avoid contamination, as well as with specific growth factors depending on the tissue type7,8,9,16,17. Other relevant cells present within the bulk tumor and the TME may also be isolated, such as cancer-associated fibroblasts (CAFs) and immune cells. This technique, which has recently been reviewed18, allows the establishment of co-cultures with different cell types to study the response to therapy in a more &#34;realistic&#34; tumor environment. Furthermore, cell-cell interactions and the interaction between tumor cells and components of the surrounding biological matrix can be studied.
The reported success rate of tumor organoid establishment using fresh tissue from biopsies or resected gastrointestinal tumor tissue is around 50%11, and the success rate from the latter is largely dependent on the tissue type and origin4, particularly the tumor grade and overall tumor cellularity. Three-dimensional tumor models have varying complexity, from simple unicellular aggregates to highly complex engineered models consisting of various cell types. The terminology used to describe 3D cultures in the literature is highly inconsistent19,20,21, as different terms such as spheroids, tumorspheres, and organoids are used, although the difference between them is unclear. As a clear consensus on the definition has not yet been reached, in this article, a tumor organoid is described as an organized tumor cell culture embedded into a biological basement membrane.
Herein, a validated protocol is reported for the establishment of tumor organoids from fresh tissue samples originating from fresh primary resected or PDX-derived pancreatic ductal adenocarcinoma (PDAC), and this protocol can be performed in most laboratories with basic tissue culture facilities. This protocol has been adapted from several state-of-the-art reported protocols that are currently used to establish tumor organoids or tumoroids from digestive tumor tissue from the groups of David Tuveson9, Hans Clevers8, and Aurel Perren7.
This protocol does not discuss how the fresh tissue is harvested. To obtain high-quality fresh human tumor tissue, it is important to have efficient coordination between the surgeons that harvest the tissue and the pathology department that extracts the tissue sample for organoid culture. Likewise, when using PDX as a fresh tissue source, efficient coordination with the person that harvests the tissue sample is also important. It is critical to obtain the tissue sample as quickly as possible (within 30-60 min from harvesting time) in order to maintain a high quality.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well Costar Ultra-low Attachment plates</td>
			<td>Biofil</td>
			<td>TCP011006</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m pore strainer</td>
			<td>VWR</td>
			<td>732-2758</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride Potassium (ACK) Lysis Buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator (21% O2, 5% CO2 and 37 &#186;C)</td>
			<td>Nuaire</td>
			<td>NU-4750E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell recovery solution</td>
			<td>Corning&#160;</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 (1:1)(1X) with L-Glutamine and HEPES</td>
			<td>Gibco</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-079-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing container, Nalgene</td>
			<td>Merck</td>
			<td>C1562</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator</td>
			<td>Milteny Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Placenta Growth Factor (PlGF)</td>
			<td>enQuireBio</td>
			<td>QP6485-EC-100UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunocompromised female 6-week-old NU-Foxn1nu nude mice</td>
			<td>Janvier, France&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-like growth factor-1 (IGF-1)</td>
			<td>Invitrogen</td>
			<td>RP10931</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Corning</td>
			<td>354235</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>InvivoGen</td>
			<td>ant-nr-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Deltalab</td>
			<td>200007</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution (100x)</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Basic Fibroblast Growth Factor (bFGF)</td>
			<td>Gibco</td>
			<td>PHG0026</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Epidermal Growth Factor (EGF)</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y-27632 (Dihydrochloride)</td>
			<td>STEMCELL</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blades</td>
			<td>Nahita</td>
			<td>FMB018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
	</tbody>
</table>

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  

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

The development of tumor models for translational research. We use these models to test new drugs that can be applied in the clinic for personalized medicine. Advanced in vitro and in vivo models that have been developed from many different tumor types that are more physiological relevant than the 2D models that have traditionally been used in cancer research.Design models that mimic the shape of the original organ, such as colon grips, are being used for in vitro models. Furthermore, these models and corresponding primary tissue are characterized using techniques, such as spatial transcriptomics, that allow specific areas and cell types within the tumor to be analyzed separately. The quality of the tissue is an important factor.From prior treatment of the patients prior to harvesting, time of culture establishment, from tissue harvesting, tumor cell content and contamination by microorganisms, and non-desired cell populations such as fibroblasts. We have identified molecular subgroups of patients that may respond differently to therapy in the clinic. For this, we needed to develop preclinical models to test our hypothesis before moving to clinical trials.Here, we try to address a problem of a standardized protocol that can be used for pancreatic tumor tissue, either directly from the primary tumor or from a PDX model. Many protocols have been reported, but this is the first one that has a corresponding video that is very useful for people starting to work with this type of culture. We also talk extensively about the many pitfalls and troubleshooting options that can be used.

It is important to document how the tumor organoid culture progresses over time, particularly in the first few weeks, in order to estimate how the culture will behave in downstream assays. Figure 2 shows an example of optimal tumor cell isolation and tumor organoid establishment from fresh tissue over a 15 day period. Sometimes, there is a large volume of cell debris in the sample, and it is difficult to see the developing tumor organoids, as shown in Figure 3. ...

Watch this Scientific Journal Video about Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue at JoVE.com

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. ...

establishment-pancreatic-cancer-derived-tumor-organoids-fibroblasts

Research

JoVE Journal

Cancer Research

Fresh Primary Tumor Tissue Processing to Establish Tumor Organoids and Primary Fibroblasts

Begin by placing the tissue in a sterile tissue culture plate and removing excess media. Measure the tissue with a sterile metal ruler and photograph it. Add three milliliters of advanced DMEM/F-12 media to the cut tissue and pipette the tissue up and down to wash it.Then wash the tissue with three milliliters of PBS. Aspirate the medium from the tissue culture plate and cut it into small pieces using sterile blades and two milliliters of digestion media. Then collect the tissue into a 50-milliliter tube containing five milliliters total of the digestion medium.Incubate the tube at 37 degrees Celsius for one hour. Periodically mix the suspension by pipetting up and down. Inactivate the trypsin by adding five milliliters of advanced DMEM/F-12 to the tube.Then filter the sample through a 70-micrometer pore strainer to remove any large debris. Now pipette two milliliters of DMEM containing 10%FBS to the top of the filter, and use a pipette to collect the debris and tissue remnants for fibroblast culturing. Then plate the debris for fibroblast culture.Centrifuge the filtrate at room temperature at 200 to 300 G for five minutes, and then aspirate the supernatant. Add five milliliters of advanced DMF 12 to the pellet and centrifuge the suspension again at 300 G for five minutes. For red blood cell lysis, resuspend the pellet in four milliliters of ammonium chloride potassium lysis buffer, and transfer it to a 15-milliliter tube.Incubate the cells at room temperature for two minutes. After centrifuging the mixture at the same conditions as demonstrated previously, aspirate the supernatant. To the pellet, add five milliliters of advanced DMEM/F-12, and centrifuge again at four degrees Celsius.After aspirating the supernatant, add one milliliter of commercially available cell dissociation reagent to the pellet and incubate it for two to three minutes at room temperature. After incubation, centrifuge and aspirate the supernatant once again. Then resuspend the pellet in five milliliters of advanced DMEM/F-12.Dissociate cell clumps by pipetting the suspension multiple times with a P1000 pipette. Centrifuge the suspension and remove the supernatant. Next, obtain pre-chilled P1000 and P200 pipette tips, and use the cold tips fixed onto the P1000 pipette to resuspend the cell pellet in the membrane matrix, before placing the Falcon on ice to prevent solidification.Obtain the preheated plate from the incubator. Using the P200 pipette set at 48 microliters and using cold tips, create basement membrane matrix domes in the preheated plate. Then incubate it to solidify the basement membrane mix.Once the matrix is solidified, add two to two and a half milliliters of advanced DMEM/F-12, supplemented with the growth factors and inhibitors. Tumor cells were isolated and tumor organoids were established from fresh tissue over a 15-day period. Occasionally, large cell debris volumes prevent accurate visualization of developing tumor organoids.Developing organoids were observed to phenotypically vary from isolated, rounded to spheroid or aggregate-like cultures, depending on the tumor origin. Organoid cultures may be contaminated by fibroblasts, a common PI product of primary tumor cell culture. After approximately seven days of culture, the fibroblasts migrate from the basement membrane matrix domes and adhere to the cell plates, which may compromise optimal organoid growth.Fibroblast cultures are established from the tissue debris recovered from the filter. The cells migrate out of the tissue debris and adhere to the culture plates.