The authors would like to thank the Calvin Kuo Laboratory at Stanford University for providing HEK293 cells stably transfected with either HA-mouse Noggin-Fc or HA-mouse Rspo1-Fc. We would also like to thank Dr. Dean Tang for allowing us access the fluorescent dissection microscope in his laboratory. This work was supported by CA179907 to D.W.G. from the National Cancer Institute. Shared resources at Roswell Park Comprehensive Cancer Center were supported by National Institutes of Health Cancer Center Support Grant CA016056.

Animal procedures described here were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Resources, Roswell Park Comprehensive Cancer Center, Buffalo, New York.
NOTE: Male mice to be dissected to isolate prostates or prostate tumors for generation of organoids should have at least reached the age of sexual maturity &#8212; about 8-10 weeks of age. Specific ages of mice can vary amongst studies. Some fac...

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Critical steps within the protocol for prostate tumor dissection and organoid generation 
Removal of non-prostate tissue and fine dissection of the mouse prostate tumor is crucial for the optimal generation of cancer organoids since both non-prostate epithelial cells and normal prostate epithelial cells will generate organoids. For solid prostate tumors specifically, it is crucial to isolate areas of viable tumor to remove contamination with necrotic tissue that would reduce the number of viable cel...

Since the late 1980s, the ability to alter genes by homologous recombination has greatly advanced the study of biological systems1. Inducible, tissue-, or cell-specific promotor systems and site-specific recombinases, such as Cre-loxP, has advanced genetic studies by facilitating control over genetic modifications both temporally and spatially2,3,4. The combination of these genetic strategies has created a wide array of experimental model systems5,6,7.
Genetically engineered mouse models (GEMMs) are an integral tool to assess how individual genes or groups of genes affect mammalian development and disease. In pre-clinical cancer research, GEMMs are the most physiologically-relevant and rigorous method to study cancer development, progression, and treatment8. Our laboratory specializes in generating and characterizing cancer GEMMs.
The most highly diagnosed non-cutaneous cancer among men in the United States is prostate cancer (PCa). The majority of patients with PCa have low-risk disease and high likelihood of survival, but survival rates decline drastically when disease is diagnosed at advanced stages or if targeted hormonal therapy induces progression to aggressive, non-curable PCa subtypes9,10. Our laboratory has developed GEMMs that utilize floxed alleles of one or more tumor suppressor genes. Recombination and loss of tumor suppressor gene expression occurs specifically in the prostate because we have introduced a transgene with Cre recombinase downstream of the probasin promoter activated only in prostate epithelial cells11,12. We have also bred our GEMMs to contain a Cre reporter transgene called mT/mG, which induces Tomato fluorescent protein expression in cells lacking Cre and green fluorescent protein (GFP) expression in cells with Cre13. While the presentation of this method and our representative results show GEMMs we study in our laboratory, this protocol can be used to generate prostate cancer organoids from any mouse model. However, as discussed in detail in our representative results section, we have observed that certain tumor characteristics are optimal for prostate cancer organoid generation.
In the last decade, new methods of culturing cells from tissues of epithelial origin has led to significant advances in our ability to model organ systems in vitro14,15. The term &#34;3D cell culture&#34;&#160;has been attributed to the techniques involved in establishing and maintaining organoids, which can be generally defined as structures made up of cells that assemble secondary architecture driven by organ-specific cell lineage characteristics16. These new methods are distinct from classic 2D cell culture in that cells do not require transformation or immortalization for long term growth; thus, 3D cultures of normal cells can be compared to diseased cells. This is particularly valuable in cancer research where normal cell control cultures have typically not been available. In addition, organoids spontaneously form secondary tissue architectures with appropriately differentiated cell types, making them a better model system to understand cancer in vitro than 2D cell lines17. Our laboratory has created 3D organoid lines from tumor issue isolated from our PCa GEMMs to complement our in vivo data and perform experiments which would not be feasible in GEMMs.&#160;
In this article, we present written and visual protocols for the complete necropsy of PCa GEMMs, including dissection of distinct mouse prostate lobes and metastatic lesions. We describe and show a step-by-step method for generating organoids from mouse prostate tumors based on a protocol previously published by Drost et al. for deriving organoids from normal mouse prostate epithelial tissue18.

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			<td height="21" style="height:21px;width:217px;">0.25 % Trypsin+2.21 mM EDTA</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">25-053</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">1 1/4 in, 23 gauge, disposable syringe needles</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">Z192430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">10 % neutral buffered formalin</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">HT501128</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">32 % paraformaldehyde</td>
			<td style="width:177px;">Electron Microscopy Services</td>
			<td align="right" style="width:119px;">15714</td>
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			<td height="21" style="height:21px;width:217px;">A83-01</td>
			<td style="width:177px;">MedChemExpress</td>
			<td style="width:119px;">HY-10432</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:217px;">Advanced DMEM/F12+++</td>
			<td style="width:177px;">Gibco</td>
			<td align="right" style="width:119px;">12634</td>
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			<td height="21" style="height:21px;width:217px;">Analytical balance</td>
			<td style="width:177px;">Mettler Toledo</td>
			<td align="right" style="width:119px;">30216623</td>
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			<td height="21" style="height:21px;width:217px;">B27 (50X)</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">17504044</td>
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			<td height="21" style="height:21px;width:217px;">Collagenase II</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">17101015</td>
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			<td height="21" style="height:21px;width:217px;">Dissecting Board</td>
			<td style="width:177px;">Thermo-Fisher</td>
			<td style="width:119px;">36-1</td>
			<td></td>
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			<td height="147" style="height:147px;width:217px;">EHS Sarcoma matrix, Pathclear Lot#19814A10</td>
			<td style="width:177px;">Manufactured by Trevigen</td>
			<td style="width:119px;">Requistitioned from the National Cancer Institute at the Frederick National Laboratory</td>
			<td style="width:167px;">Holder of grants from the National Cancer Institute can request matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">HEPES (1M)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">25-060</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">human recombinant Epidermal growth factor (EGF)</td>
			<td style="width:177px;">PeproTech</td>
			<td>AF-100-15</td>
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			<td height="21" style="height:21px;width:217px;">L-glutamine (200 mM)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">25-005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">N-Acetyl-L-Cysteine</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">A9165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Penicillin-Streptomycin</td>
			<td style="width:177px;">Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Precision balance</td>
			<td style="width:177px;">Mettler Toledo</td>
			<td align="right" style="width:119px;">30216561</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Scalpel #23</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504176</td>
			<td></td>
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			<td height="42" style="height:42px;width:217px;">Scalpel Handle #7, 16 cm</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">500238</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Single-edge carbon razor blade</td>
			<td style="width:177px;">Fisherbrand</td>
			<td style="width:119px;">12-640</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stainless steel dissecting scissors, 10 cm, straight</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">14393</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stainless steel Iris forceps, 10 cm, curved tip, serrated</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">15915</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:217px;">Stainless steel Nugent utility forceps, straight tip, serrated</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td align="right" style="width:119px;">504489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Y-276632 (Rock Inhibitor)</td>
			<td style="width:177px;">APExBIO</td>
			<td style="width:119px;">A3008</td>
			<td></td>
		</tr>
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generation of tumor organoids from genetically engineered mouse models of prostate cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

Representative necropsy images of a mouse with a large fluid-filled primary prostate tumor in the anterior prostate region are shown in Figure 2A. In contrast, Figure 2B, shows representative necropsy images of a mouse with a large solid primary prostate tumor for which individual prostate regions are indistinguishable. Fluorescent dissection images show the same solid prostate tumor from Figure 2<s...

Watch this Scientific Journal Video about Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer at JoVE.com

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Mouse organoid cultures more closely resemble in vivo cell organization than traditional cell cultures. In cancer research, organoids model the original tumor better than 2D cell lines and can be used to test potential cancer therapies in vitro to develop new drug treatments. For en bloc extraction of the male mouse urogenital system grasp the fat pad on either side of the bladder and pull upward to expose the testicle.Carefully dissect each testicle away from the rest of the urogenital region. Gently lift the bladder so that the urogenital region is elevated together, exposing the urethra underneath. While holding the bladder, orient the scissors against the underside of the dorsal prostate to incise the urethra.The entire urogenital region will be released from the abdominal cavity. After removing the urogenital system, the pelvic lymph nodes positioned immediately behind the urogenital system and on either side of the spine will be exposed. To remove the lymph nodes orient straight forceps under the lymph tissue and pull up.Next, grasp the rectum with the straight forceps and cut, pulling up on the rectum to unravel the entire colon and small intestine to look for metastatic lesions in the mesenteric lymph nodes. When the entire ilium has been removed continue to pull on the duodenum to expose the stomach. Cut the esophagus to completely remove the stomach.If any metastatic lesions in the lymph nodes are observed, carefully dissect these nodes away from the intestine for storage in PBS. When the stomach has been removed, the spleen can be harvested from the dorsal side of the abdomen for storage in 4%paraformaldehyde. Remove the liver for storage in PBS.Remove the kidneys from either side of the spine along with any renal lymph nodes containing metastatic lesions. To expose the thoracic cavity carefully cut the diaphragm along the rib cage. The negative pressure released within the thoracic cavity will expose the heart and lungs.Pull up on the sternum to open the thoracic cavity further. Scan the ventral face of the thoracic cavity along the rib cage for metastatic lesions in the thoracic lymph nodes for dissection if present. While still holding the sternum, cut away the ventral rib cage to access the heart and lungs and pull up on the heart to cut underneath the lungs.To fully remove the heart and lungs en bloc cut all of the anterior blood vessels in the trachea. Carefully transfer the heart without damaging the lung tissue or lung metastatic lesions into a container of PBS. For dissection of the prostate tumor place the urogenital region under a dissection microscope.Use a pair of straight forceps and a pair of curved forceps to flip the urogenital region onto its dorsal surface. Locate the proximal prostate region that can be identified by the pink-red color of the urethra. Grasp the urethra firmly to allow manipulation of the urogenital tissue with the curved forceps.Locate the base of the seminal vesicles to allow their careful removal. Then use the curved side of the forceps to remove the vas deferens and as much fatty and connective tissue as possible. While still firmly holding the urethra and proximal prostate region, use a pair of fine pointed scissors to remove the bladder from the urethra.Holding the proximal prostate region and urethra firmly with the straight forceps grasp the anterior prostate region with the curved side of the forceps to firmly pull the tissue away from the bladder and the rest of the prostate. Place the anterior prostate tissue in PBS and locate the ventral prostate region. Remove the ventral prostate region in the same manner.If the lateral region can be distinguished from the dorsal region remove the lateral prostate region on both sides. Then remove the dorsal prostate region and place the proximal prostate region in 4%paraformaldehyde. After mincing, place the tumor pieces into a 15-milliliter tube of digestion buffer for a one-and-a-half to two hour digestion at 37 degrees Celsius.At the end of the digestion, sediment the digested tissue by centrifugation and discard the supernatant. Flick the tube to loosen the cell pellet. Resuspend the cells in one milliliter of pre-warmed trypsin supplemented with 10 micromolar Y-27632 ROCK inhibitor for five minutes at 37 degrees Celsius.At the end of the incubation triturate the tissue slurry five times with a standard P1000 pipette tip. Return the tube to the water bath for an additional five minute incubation and trituration. For matrix dome plating wash the digested cells in nine milliliters of cold medium, then collect the cells by centrifugation.Resuspend the cells in one milliliter of fresh medium for counting. And after counting, collect the cells by centrifugation once again. Dilute the tumor cells in the appropriate volume of matrix.Carefully drop 200 microliters of matrix cell solution into each well of a 55 degree Celsius warmed six-well plate. Allow the domes to solidify at room temperature for two minutes before placing the plate upside down in a 37 degrees Celsius incubator for 20 minutes. When the domes have fully solidified add two milliliters of prostate organoid medium supplemented with androgen R1881 and ROCK inhibitor to each well.Return the plate to the cell culture incubator. Fluorescent dissection images reveal green fluorescent protein, or GFP expression, by solid prostate tumors, indicating that the tumor cells express Cre. The liver and lungs from the same animal exhibit metastatic tumors expressing GFP demonstrating that these tumors originated from the primary prostate tumor.The pelvic lymph node from this mouse expresses GFP and not Tomato, indicating that this metastatic tumor has overtaken the lymph node and no normal tissue remains. At day one of culture small organoids generated from a solid prostate tumor begin to form with both Tomato and GFP-expressing cells present in the tumor organoid culture. By day seven and beyond when the prostate tumor organoids have fully formed however, the organoids express GFP, but not Tomato, suggesting that the organoids have originated from Cre-expressing tumor cells and not from normal epithelial cells.On day one of culture of small organoids generated from fluid-filled prostate tumor both Tomato and GFP-expressing cells are present within the organoid culture. Organoids from fluid-filled prostate tumors express either GFP or Tomato at day seven and beyond however, indicating that the organoids formed from cells that do not express Cre. To stay oriented when harvesting the prostate tissue, be aware of the bladder and urethra positions at all times.After their generation the organoids can be used in imaging applications, molecular biology analysis and drug screens.

generation-tumor-organoids-from-genetically-engineered-mouse-models

Research

JoVE Journal

Genetics

10.3K ビュー.  Roswell Park Comprehensive Cancer Center. マウスオルガノイド培養は、従来の細胞培養よりもインビボ細胞組織によく似ています。がん研究では、オルガノイドは2D細胞株よりも元の腫瘍をモデル化し、新しい薬物治療を開発するためにインビトロで潜在的な癌療法をテストするために使用することができます。男性マウス泌尿生殖器系のエンブロック抽出のために膀胱の両側の脂肪パッドをつかみ、睾丸を露出...