Name: establishment and culture of patient-derived breast organoids
Uploaded: 2023-02-17T13:20:00
Description: Watch this Scientific Journal Video about Establishment and Culture of Patient-Derived Breast Organoids at JoVE.com

We would like to thank members of the Spector lab for critical discussions throughout the course of this work. We thank Norman Sachs and Hans Clevers (Hubrecht Institute, Netherlands) for initially providing us with their organoid culturing protocol. We acknowledge the CSHL Cancer Center Histology and Microscopy Shared Resources for services and technical expertise (NCI 2P3OCA45508). We thank Dr. Qing Gao for assistance with histological sample preparation. We are grateful for the support of Dr. Karen Kostroff (Northwell Health) for providing patient tumor samples. We also appreciate the efforts of the Northwell Health Biobanking team for sample acquisition, and we thank the patients and their families for donating tissues for research. This research was supported by CSHL/Northwell Health (D.L.S.), NCI 5P01CA013106-Project 3 (D.L.S.), and Leidos Biomedical HHSN26100008 (David Tuveson and D.L.S).

Tumor resections from breast cancer patients, along with the distal and adjacent normal tissue, were obtained from Northwell Health in accordance with Institutional Review Board protocols IRB-03-012 and IRB 20-0150, and with written informed consent from the patients.
NOTE: All procedures mentioned below were performed in a mammalian tissue culture BSL2 room designated for patient samples upon approval of the biosafety committee. All procedures should be performed following safety protocols ma...

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Our lab has successfully employed the above protocols to establish organoids from na&#239;ve tumor resections or scrapings. We have also utilized this protocol to develop normal organoids from breast tissue obtained via reductive mammoplasties or from cancer patients&#39; adjacent or distal normal breast tissue. About 30%-40% of the resected primary tumors resulted in successful long-term (&#62;passage 8) tumor organoid cultures. The tumor organoid lines that tapered off after a few passages either had an outgro...

Breast cancer (BC) is the most commonly occurring malignancy in females, with 287,850 new cases estimated to be diagnosed in the United States in 20221. Despite the recent advances in early detection with annual screenings, targeted therapies, and a better understanding of genetic predisposition, it prevails to be the second leading cause of cancer deaths in females in the United States, with &#62;40,000 deaths attributed to breast cancer annually1. Breast cancer is currently classified into multiple subtypes based on histopathological and molecular evaluation of the primary tumor. Better subtype stratification has improved patient outcomes with subtype-specific treatment options2. For instance, the identification of HER2 as a proto-oncogene3 has led to the development of Trastuzumab, which has made this highly aggressive subtype manageable in most patients4. Further research into the genetics and transcriptomics of this complex disease in a patient-specific manner will aid in developing and predicting better patient-specific personalized treatment regimens2,5. Patient-derived organoids (PDOs) are a promising new model to gain insights into cancer at the molecular level, identifying novel targets or biomarkers and designing new treatment strategies6,7,8.
PDOs are multicellular, three-dimensional (3D) structures derived from freshly resected primary tissue samples8,9. They are grown three-dimensionally by being embedded in a hydrogel matrix, typically composed of a combination of extracellular matrix (ECM) proteins, and hence can be used to study tumor cell-ECM interactions. PDOs represent patient diversity and recapitulate cellular heterogeneity and genetic features of the tumor10,11,12. Being in vitro models, they allow for genetic manipulation and high-throughput drug screens13,14,15. Further, PDOs can be plausibly utilized to evaluate patient drug sensitivity and treatment strategies in parallel to the clinic and help predict patient outcomes16,17,18. Besides chemotherapy, certain organoid models have also been used to examine individual patient responses to chemoradiation19,20. Given the promising applicability of PDOs for research and clinical use, the National Cancer Institute has initiated an international consortium, The Human Cancer Models Initiative (HCMI)21, to generate and provide these tumor-derived novel cancer models. Many of the organoid models of various cancer types developed through the HCMI are available via the American Type Culture Collection (ATCC)22.
Normal breast organoids have been shown to be comprised of different epithelial cell populations present in the mammary gland11,23 and thus serve as great models to study basic biological processes, to analyze driver mutations causing tumorigenesis, and for cancer cell-of-origin lineage studies6,15. Breast tumor organoid models have been used to identify novel targets that are encouraging prospects for developing new therapies, particularly for resistant tumors24,25,26. Using patient-derived xenograft (PDX) and matched PDX-derived organoid (PDxO) models of treatment-resistant breast tumors, Guillen et al. showed that organoids are powerful models for precision medicine, which can be leveraged to evaluate drug responses and direct therapy decisions parallelly28. Furthermore, the development of new co-culture methods for culturing PDOs with various immune cells27,28,29, fibroblasts30,31 and microbes32,33 presents an opportunity to study the impact of the tumor microenvironment on cancer progression. While many such co-culture methods are actively being established for PDOs derived from pancreatic or colorectal tumors, similar established co-culture methods for breast PDOs have only been reported for natural killer cells34 and fibroblasts35.
The first biobank of &#62;100 patient-derived organoids representing different breast cancer subtypes was developed by the Hans Clevers group36,37. As part of this effort, the Clevers group also developed the first complex culture medium for breast organoid growth, which is currently widely used36. A follow-up study provided a comprehensive account of the establishment and culture of breast PDOs and patient-derived organoid xenografts (PDOXs)38. The Welm lab developed a large collection of BC PDX models and PDxOs that are cultured in a comparatively simpler growth medium containing fetal bovine serum (FBS) and fewer growth factors39,40. We have independently developed and characterized a large array of na&#239;ve patient-derived breast cancer organoid models11, and participated in developing BC PDO models as part of the HCMI initiative21. Here, we aim to provide a practical guide detailing the methodology employed by us in generating patient-derived breast organoid model systems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR</td>
			<td>525-1068</td>
			<td></td>
		</tr>
		<tr>
			<td>175 cm2 tissue culture flask</td>
			<td>VWR (Corning)</td>
			<td>29185-308</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C bead bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>VWR</td>
			<td>525-1077</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL vacuum filtration system (0.22 &#181;m Filter)</td>
			<td>Millipore Sigma</td>
			<td>SCGP00525</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>500 mL Rapid-Flow Filter Unit, 0.2 &#181;m aPES membrane, 75 mm diameter</td>
			<td>Nalgene</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates&#160;</td>
			<td>Greiner Cellstar</td>
			<td>82050-842</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask</td>
			<td>VWR (Corning)</td>
			<td>29185-304</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well opaque plates</td>
			<td>Corning</td>
			<td>353296</td>
			<td>For CTG assay</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek&#160;Synergy H4 Hybrid Microplate Reader</td>
			<td>Fisher Scientific (Agilent)</td>
			<td></td>
			<td>For dual luciferase assay and CTG assay</td>
		</tr>
		<tr>
			<td>BSA fraction V (7.5%)</td>
			<td>Thermo Fisher</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Titer-Glo (CTG) Reagent</td>
			<td>Promega</td>
			<td>G9683</td>
			<td>luminescent cell viability assay</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5804</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Millipore Sigma</td>
			<td>C5138</td>
			<td>Type IV</td>
		</tr>
		<tr>
			<td>Cryolabels</td>
			<td>Amazon</td>
			<td>DTCR-1000</td>
			<td>Direct Thermal Cryo-Tags, White, 1.05 x 0.5&#34;</td>
		</tr>
		<tr>
			<td>Cryovials&#160;</td>
			<td>Simport Scientific Inc.</td>
			<td>T311-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess 3 Automated Cell Counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAX2000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>11995040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (1X)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (hEGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-10 (human)</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-7/KGF (human)</td>
			<td>Peprotech</td>
			<td>100-19</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216&#160;</td>
			<td>For TOPFlash Assay</td>
		</tr>
		<tr>
			<td>HEK293T-HA-Rspondin1-Fc cells</td>
			<td>R&#38;D Systems</td>
			<td>3710-001-01</td>
			<td>Cultrex HA-R-Spondin1-Fc 293T Cells</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Heregulin&#946;-1 (human)</td>
			<td>Peprotech</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Phenol-red free, LDEV-free; basement membrane matrix</td>
		</tr>
		<tr>
			<td>Mr. Frosty Cell Freezing Container</td>
			<td>Thermo Fisher</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>Lonza</td>
			<td>LT07-418</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-l-cysteine</td>
			<td>Millipore Sigma</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membranes</td>
			<td>Thermo Fisher</td>
			<td>166-0045&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Millipore Sigma</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin (human)</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000, P200, P10 pipettes with tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p38 MAPK inhibitor (p38i) SB 202190</td>
			<td>Millipore Sigma</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td>transparent film</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid1: pRL-SV40P</td>
			<td>Addgene</td>
			<td>27163</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid2: M51 Super 8x FOPFlash</td>
			<td>Addgene</td>
			<td>12457</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid3: M50 Super 8x TOPFlash</td>
			<td>Addgene</td>
			<td>12456</td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 200 &#181;m</td>
			<td>pluriSelect</td>
			<td>43-50200-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ANT-PM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>12648-010</td>
			<td>cell freezing medium</td>
		</tr>
		<tr>
			<td>Red Blood Cell lysis buffer</td>
			<td>Millipore Sigma</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin conditioned media</td>
			<td>In-house or commercial from Peprotech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (No.10)</td>
			<td>Sklar Instruments</td>
			<td>Jun-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker (Incu-shaker Mini)</td>
			<td>Benchmark</td>
			<td>H1001-M</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946; receptor inhibitor A 83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), phenol red</td>
			<td>Life Technologies</td>
			<td>12605028</td>
			<td>cell dissociation reagent</td>
		</tr>
		<tr>
			<td>X-tremeGENE 9 DNA transfection reagent</td>
			<td>Millipore Sigma</td>
			<td>6365779001</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 Dihydrochloride (RhoKi)</td>
			<td>Abmole Bioscience</td>
			<td>Y-27632</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher</td>
			<td>R25001</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment and culture of patient-derived breast organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

We have established a biobank of patient-derived breast tumor organoids comprising various subtypes11. Additionally, we have established multiple normal breast organoid lines derived from reductive mammoplasty tissue samples or adjacent/distal normal breast from BC patients using the approach outlined in Figure 1.
The various patient-derived breast tumor organoid lines differ in their morphology (Figure 2) and ...

Watch this Scientific Journal Video about Establishment and Culture of Patient-Derived Breast Organoids at JoVE.com

Establishment and Culture of Patient-Derived Breast Organoids

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Human patient-derived organoids are three-dimensional in vitro model systems that represent both patient diversity and cellular heterogeneity of the tumors. This protocol and video demonstration provide a detailed, hands-on guide to establishing patient-derived breast tumor and normal organoids. Patient-derived breast tumor organoids are exciting new models, but difficult to establish.The comprehensive protocol we provide here should aid in preparing the researchers attempting to develop breast organoids and acquaint them with the expected challenges. Every PDO line derived from a different patient is unique in morphology and growth rate. Unlike two 2D cell line systems, organoids grow better when plated at a high density, allowing for better intercellular interactions.Demonstrating the procedure will be Disha Aggarwal, a graduate student in my laboratory. To begin, thaw a bottle of basement membrane matrix on ice or overnight at four degrees Celsius. Transfer the resected tissue to a 10-centimeter sterile Petri dish.Examine the tissue macroscopically and make a note if it appears morphologically fatty, vascularized, or necrotic. Additionally, record the size and shape of the tissue and take a picture of the tissue with a ruler in view. Mince the tissue into small pieces with a sterile number 10 scalpel and transfer it into a 50-milliliter conical tube.Add 10 milliliters of two milligrams per milliliter collagenase IV solution and seal the tube. Place the tube on an orbital shaker at 37 degree Celsius at 140 RPM for 30 to 90 minutes at a 30-degree angle. During the incubation, place an aliquot of complete medium to pre-warm at 37 degrees Celsius in a bead or water bath.Every 15 minutes, resuspend the tissue by mixing up and down vigorously using a five-milliliter, sterile, pre-coated serological pipette. Monitored dissociation over time by observing the tube under the microscope at a 5X or higher magnification. Once the tissue is dissociated, centrifuge at 400 G for five minutes, aspirate the supernatant, and add 10 milliliters of AdDF+Again, centrifuge and carefully aspirate the supernatant, as tissue pellets can occasionally be loose.If the tissue pellet is partially red, add two milliliters of red blood cell lysis buffer and incubate for five minutes at room temperature. After incubation, add 10 milliliters of AdDF+to the tube, centrifuge at 400 G for five minutes, and discard the supernatant. Resuspend the pellet in 50 to 300 microliters of undiluted cold basement membrane matrix and mixed by pipetting carefully to avoid forming bubbles.Using a six-well tissue culture plate that is pre-warmed in the incubator overnight, plate 300 microliters of basement membrane matrix dome containing organoids in each well. Leave the plate undisturbed in the hood for five minutes before placing it at 37 degrees Celsius for 20 to 30 minutes for the basement membrane matrix dome to fully solidify. At the end of incubation, add three milliliters of pre-warmed complete medium dropwise to each well and incubate at 37 degrees Celsius and 5%carbon dioxide.After incubation, capture images of the organoids using a 5X objective on an inverted bright-field microscope. Lift the basement membrane matrix dome into the medium in the well using a cell scraper or a one-milliliter pipette tip. Using a pre-coated pipette tip, transfer the floating dome of the organoids with medium to a 15-or 50-milliliter conical tube, depending on the number of wells being harvested.Then add DPBS to increase the volume to at least five milliliters. Spin the tubes at 400 G for five minutes. The basement membrane matrix with organoids forms a layer at the bottom.After aspirating the supernatant, add 5 to 20 milliliters of DPBS, depending on the number of wells pooled in a tube. Mix the organoid basement membrane matrix pellet in DBPS using a pre-coated sterile disposable pipette. Again, centrifuge and discard the supernatant.Using a coated pipette tip, add cell dissociation reagent at three times the volume of the basement membrane matrix and resuspend the organoids. Place the tube on an orbital shaker at 37 degrees Celsius at 140 RPM for 8 to 15 minutes in an angled position. Monitor the tube by observing it under the microscope every five minutes to ensure the organoids are broken into smaller clusters.Add AdDF+at a volume equal to or greater than the cell dissociation reagent and pipette to mix the organoids. Spin at 400 G for five minutes to obtain an organoid pellet. Once a white organoid pellet with no undissolved basement membrane matrix is obtained, discard the supernatant and resuspend the pellet in one milliliter of AdDF+Make up the volume to 10 milliliters with AdDF+Spin the tube for the wash step and discard the supernatant.Add the required amount of basement membrane matrix to the digested organoids based on the appropriate split ratio. Mix by gently pipetting up and down to avoid creating bubbles and immediately place on ice. Plate 300-microliter domes of organoids resuspended in a basement membrane matrix in a pre-warmed six-well plate.Leave the plate undisturbed in the hood for five minutes before placing it at 37 degrees Celsius with 5%carbon dioxide for 20 to 30 minutes for the domes to solidify. At the end of incubation, and three milliliters of pre-warmed complete medium to each well and place the plate back in the incubator. Add fresh complete medium every five to seven days.The various patient-derived breast tumor organoid lines differ in morphology and growth rate. The normal breast organoids and the few early ductal carcinomas in C2-derived organoids resembled the normal breast structure, with a central lumen surrounded by ductal cells. Organoids derived from invasive lobular carcinoma tend to form loosely-attached grape bunch-like structures.Meanwhile, organoids derived from invasive ductal carcinomas tend to form dense, large, and round organoids. The growth of organoids was measured using a luminescent cell viability assay on days three, six, nine, and 12, with a baseline reading on day one after plating. The bright-field images of the same organoids expanded over time are shown here.Some patient-derived organoid lines have a doubling time of two days, while some take five days. It is important to be patient with particular organoid lines that are slow to establish. In our experience, increasing plating density or filtering debris out promotes the growth of PDOs.Patient-derived organoids, or PDOs, are excellent models for drug screening. They model cell-cell as well as cell-extracellular matrix interactions that are key to studying cancer pathophysiology. Additionally, PDOs can undergo genetic manipulation and can be used to develop xenografts in co-culture systems, making them great models for mechanistic studies.

establishment-and-culture-of-patient-derived-breast-organoids

Research

JoVE Journal

Cancer Research

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...