Name: ex vivo organoid model of adenovirus-cre mediated gene deletions in mouse urothelial cells
Uploaded: 2022-05-05T15:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells at JoVE.com

This research work was supported in part by NIH Grants, K08CA252161(Q.L.), R01CA234162, and R01 CA207757 (D.W.G.), P30CA016056 (NCI Cancer Center Core Support Grant), the Roswell Park Alliance Foundation, and the Friends of Urology Foundation. We thank Marisa Blask and Mila Pakhomova for proofreading the manuscript.

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Roswell Park Comprehensive Cancer Center, Buffalo, NY (1395M, Biosafety 180501 and 180502). 
NOTE: Perform Steps 1-3 on the same day.
1. Dissection of mouse bladder
<ol>
	<li>Preparation for dissection
	<ol>
		<li>Prepare all the sterile instruments including scissors, forceps, sterile DPBS in 35 mm culture dishes, 70% ethanol, sterile gauze, and clean paper towels. Clean a...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+organoids+originating+from+Cd49f(high)+mouse+stem+cells+display+Notch-dependent+differentiation+capacity.">Urothelial organoids originating from Cd49f(high) mouse stem cells display Notch-dependent differentiation capacity.</a> Nature Communication. 10 (1), 4407 (2019).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+defined+disease+model+reveals+that+urothelial+cells+can+initiate+divergent+bladder+cancer+phenotypes.">A genetically defined disease model reveals that urothelial cells can initiate divergent bladder cancer phenotypes.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (1), 563-572 (2020).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intertumoral+heterogeneity+in+SCLC+is+influenced+by+the+cell+type+of+origin.">Intertumoral heterogeneity in SCLC is influenced by the cell type of origin.</a> Cancer Discovery. 8 (10), 1316-1331 (2018).</li><li>Sutherland, K. 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GEMMs have been the gold standards for cancer modeling initiated from normal cells, allowing the consequences of potential oncogenic perturbations (oncogene activation and/or loss of tumor suppressors) to be tested rigorously. Here, a rapid and efficient protocol is provided to generate bladder cancer organoids via ex vivo gene editing of normal mouse urothelial cells carrying floxed alleles in genes of interest. H&#38;E and IHC staining demonstrate that TKO organoids exhibit histology consistent with high-grade...

Bladder cancer is the fourth most common cancer in men and affects more than 80,000 people annually in the United States1. Platinum-based chemotherapy has been the standard of care for patients with advanced bladder cancer for more than three decades. The landscape of bladder cancer treatment has been revolutionized by the recent Food and Drug Administration (FDA) approval of immunotherapy (anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors), erdafitinib (a fibroblast growth factor receptor inhibitor) and enfortumab vedotin (an antibody-drug conjugate)2,3,4. However, no clinically approved biomarkers are available for predicting the responses to chemotherapy or immunotherapy. There is a critical need to generate informative preclinical models that can improve the understanding of the mechanisms driving bladder cancer progression and develop predictive biomarkers for different treatment modalities.
A major obstacle in bladder translational research is the lack of preclinical models that recapitulate human bladder cancer pathogenesis and treatment responses5,6. Multiple preclinical models have been developed, including in vitro 2D models (cell lines or conditionally reprogrammed cells), in vitro 3D models (organoids, 3D printing), and in vivo models (xenograft, carcinogen-induced, genetically engineered models, and patient-derived xenograft)2,6. Genetically engineered mouse models (GEMMs) are useful for many applications in bladder cancer biology, including analyses of tumor phenotypes, mechanistic investigations of candidate genes and/or signaling pathways, and the preclinical evaluation of therapeutic responses6,7. GEMMs can utilize site-specific recombinases (Cre-loxP) to control genetic deletions in one or more tumor suppressor genes. The process of generating desired GEMMs with multiple gene deletions is time-consuming, laborious, and expensive5. The overall goal of this method is to develop a rapid and efficient method of ex vivo Cre delivery for establishing bladder triple knockout (TKO) models from normal mouse urothelial cells carrying triple floxed alleles (Trp53, Pten, and Rb1)8. The major advantage of the ex vivo method is the fast workflow (1-2 weeks instead of years of mouse breeding). This article describes the protocol for harvesting normal urothelial cells with floxed alleles, ex vivo adenovirus transduction, organoid cultures, and in vitro and in vivo characterization in immunocompetent C57 BL/6J mice. This method can be further used to generate clinically relevant bladder cancer organoids in immunocompetent mice harboring any combination of floxed alleles.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#956;m sterile cell strainer</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>25G 1.5 inches needle</td>
			<td>EXELINT International</td>
			<td>26406</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenovirus (Ad5CMVCre High Titer, 1E11 pfu/ml)</td>
			<td>UI Viral Vector Core</td>
			<td>VVC-U of Iowa-5-HT</td>
			<td></td>
		</tr>
		<tr>
			<td>C57 BL/6J</td>
			<td>Jackson Lab</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Charcoal-stripped FBS</td>
			<td>Gibco</td>
			<td>A3382101</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase/hyaluronidase</td>
			<td>Stemcell Technologies</td>
			<td>07912</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>07913</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine substitute (GlutaMAX)</td>
			<td>Gibco</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Mammary Epithelial Cell Growth medium</td>
			<td>Lonza</td>
			<td>CC-3150</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix extracts from Engelbreth&#8211;Holm&#8211;Swarm mouse sarcomas (Matrigel)</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-CK20</td>
			<td>DAKO</td>
			<td>M7019</td>
			<td>IF 1:100</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-CK7</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-23876</td>
			<td>IHC 1:50</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-p63</td>
			<td>Abcam</td>
			<td>ab735</td>
			<td>IHC 1:100, IF 1:50</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-Upk3</td>
			<td>Fitzgerald</td>
			<td>10R-U103A</td>
			<td>IHC 1:50</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-Vimentin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-6260</td>
			<td>IF 1:100</td>
		</tr>
		<tr>
			<td>Monoclonal rat anti-CK8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I-s</td>
			<td>IF 1:100</td>
		</tr>
		<tr>
			<td>HERAcell vios 160i CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>51033557</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal chicken anti-CK5</td>
			<td>Biolegend</td>
			<td>905901</td>
			<td>IF 1:500</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant enzyme of trypsin substitute (TrypLE Express Enzyme)</td>
			<td>Thermo Fisher</td>
			<td>12605036</td>
			<td></td>
		</tr>
		<tr>
			<td>Signature benchtop shaking incubator Model 1575</td>
			<td>VWR</td>
			<td>35962-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Processing Gel (HistoGel)</td>
			<td>Thermo Fisher</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade size 10</td>
			<td>Integra Miltex</td>
			<td>4-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall T1 centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75002383</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo organoid model of adenovirus-cre mediated gene deletions in mouse urothelial cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

The workflow of adenovirus-Cre mediated gene deletions in mouse urothelial cells is shown in Figure 1A. The accompanying video demonstrates how the urothelial cells are disassociated from the fundus of the bladder and how the triple floxed cells are transduced ex vivo with Ad5CMVCre. Figure 1A showed that minimal submucosa and muscle cells were disassociated after enzyme digestion. To confirm the efficiency of adenovirus-Cre delivery, disassociated urot...

Watch this Scientific Journal Video about Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells at JoVE.com

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

We developed a rapid and efficient protocol to generate bladder cancer organoids via ex vivo gene editing of normal mouse urothelial cells carrying floxed alleles in genes of interest. This ex vivo method has a one-to-two-week workflow instead of years of mouse breeding, and it is highly efficient in adenovirus transduction on the Cre-mediated gene deletions. To begin, prepare all sterile instruments and reagents, including scissors, forceps, DPBS in 35-milliliter culture dishes, 70%ethanol, gauze, and clean paper towels.Next, clean all surfaces of the dissection area and cover the dissection area with a clean paper towel. Place the euthanized mouse on the gauze in the dissection area and spray 70%ethanol on the mouse's abdomen, then using sterile dissection scissors, make a lower midline abdominal incision to expose the bladder. Now, use the forceps to grasp and gently pull up the bladder to identify the bilateral ureterovesical junction.Using scissors, remove the bladder fundus, following the boundary line between the two ureter openings and transfer the bladder into a sterile dish containing 2 milliliters of DPBS, then remove the fatty and connective tissue and put the bladder into a new dish containing 2 milliliters of DPBS. Also remove the adenovirus from the minus 80-degree Celsius storage and keep it on ice. For cell dissociation, prepare all sterile instruments and reagents as described in the manuscript, then in a biosafety cabinet, transfer the bladder to a 35-millimeter dish and wash with 2 milliliters of sterile DPBS.Collect bladder tissues from four mice into a dish with one milliliter of complete culture medium without charcoal-stripped FBS. Next, using a surgical blade, mince each bladder into four equal pieces, then using forceps, unfold the bladder to expose the urothelium surface to the medium. Transfer the bladder pieces and medium into a 15-milliliter centrifuge tube.Wash the dish twice with 2 milliliters of complete culture medium without charcoal-stripped FBS and add the wash to the centrifuge tube, bringing the total volume to 5 milliliters. Next, add a collagenase and hyaluronidase mixture and place the tube horizontally in a 37-degree Celsius orbital shaker incubator for 30 minutes at 200 RPM. After tissue digestion, add an equal volume of 10%charcoal-stripped FBS and DPBS into the tube and centrifuge at 200 times g for 5 minutes.After discarding the supernatant, add 2 milliliters of trypsin substitute into the cell pellet and mix well, then incubate the tube at 37 degrees Celsius and 5%carbon dioxide for 4 minutes. After incubation, pipette the cells up and down 10 times with a standard P1000 tip and add 5 milliliters of 10%charcoal-stripped FBS and DPBS. Strain the dissociated cells through a 100-micrometer sterile cell strainer and collect the cell suspension in a 50-milliliter tube.Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 times g for 5 minutes. After removing the supernatant, resuspend the pellet in 1 milliliter of complete culture medium. Count the number of cells using a hemocytometer and plate 0.5 times 10 to the 6th cells into each well of a 24-well plate with 0.5 milliliters of fresh complete culture medium.Next, take out the matrix extracts of Engelbreth-Holm-Swarm mouse sarcoma cells from minus 20-degree Celsius storage and thaw them on ice. Also, prewarm a 6-well plate in a 37-degree Celsius cell incubator. For adenoviral transduction, add 2 microliters of adenovirus into the 24-well plate and mix well with the dissociated urothelial cells.To enhance transduction efficacy, perform spinoculation by centrifuging the plate at 300 times g for 30 minutes, then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 1 hour. Transfer cells and medium from the well into a 15-milliliter tube and centrifuge at 200 times g for 5 minutes. After discarding the supernatant, add 50 microliters of complete culture medium and mix well with the cells at the bottom.Next, add 140 microliters of matrix extract and gently mix well to avoid air bubbles, then create domes for organoids by quickly adding 50 microliters of the solution per dome into the prewarmed 6-well plate. Incubate the plate and allow the domes to solidify for 5 minutes. After 5 minutes, flip the 6-well plate upside down and continue solidification for an additional 25 minutes.After incubation, add 2.5 milliliters of complete culture medium to each well and place the plate in an incubator for organoid culture. The green fluorescent protein was detected in nearly 100%of cells, suggesting high efficiency of adenovirus transduction. Hematoxylin and eosin staining and immunohistochemistry of triple knockout organoids tumors showed the morphology of high-grade urothelial carcinoma with positive protein expression of basal subtype markers of CK5 and p63.The un-recombined floxed alleles were detected in urothelial cells, untreated with adenovirus, whereas the recombined alleles were observed in the triple knockout organoids. In general, 2 to 3 weeks were needed to form a 2-centimeter subcutaneous tumor. The immunohistochemistry of triple knockout organoids, in vivo xenografts, displayed positive expression of CK7, CK5, and p63 proteins.Negative expression was detected for CK8 and uroplakin proteins. The mesenchyme marker, vimentin, was detected only in the tumor capsule or stroma. The mouse bladder dissociation must not exceed 30 minutes.A longer dissociation time may increase in the percentage of the stroma cells, such as endothelial cells and the fibroblasts This approach can be combined with cell sorting or cell-type a specific adenovirus to generate the cell type of specific organoids, such as lumen versus basal organoids.

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Research

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Cancer Research

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 ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...