Name: high-sensitivity detection of micrometastases generated by gfp lentivirus-transduced organoids cultured from a patient-derived colon tumor
Uploaded: 2018-06-14T13:30:00
Description: Watch this Scientific Journal Video about High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor at JoVE.com

This work was supported by the Juntendo University Young Investigator Award (2013, 2014 and 2015) to Y.O., the Joint Project Award (2013 and 2014) to K. M., and Grants in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (16K15625 to Y. K. and 16K15598 to M.G.). We are especially thankful to all members of the Dept. of Coloproctological Surgery and Molecular Pathology for useful discussions and technical support. We also thank Dr. Hiroyuki Konno (Hamamatsu University Schoolof Medicine) and Dr. Hideki Kitajima (International University of Health and Welfare) for generous technical guidance in the surgical procedures for orthotopic implantation into mice and Dr. Yoshitaka Hippo (Chiba Cancer Center) for technical advice on performing the tumor organoid culture.

The patient provided written informed consent and the project was approved by the Research Ethics Committee of the Juntendo University Faculty of Medicine. The mouse experiments were also approved by the Animal Research Ethics Committee of the Juntendo Faculty of Medicine.
1. Establishment of CRC PDXs in Immunodeficient Mice
Experimental procedures for establishing CRC PDXs (step 1) are outlined in Figure 1A.
<ol>
	<li>Prepare the pri...

<ol>
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Although the CRC PDX model has been widely employed to study primary tumor growth, whether this model is also applicable to investigating tumor metastasis has not yet been fully elucidated. Spontaneous metastases were also barely detectable in the liver and lungs of various reported colon PDX models4,10. To detect micrometastases with high sensitivity, we developed a protocol for transducing GFP lentiviral particles into PDX-derived CRC organoids prior to their o...

Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide1. The insufficient response to conventional therapies of patients with advanced stage disease indicates the ineffectiveness of attempts to radically cure CRCs. To develop more effective therapeutic approaches, various preclinical mouse models of cancer that mimic the characteristics of CRCs have been established. Various CRC cell lines have been widely used to generate tumor xenografts due to their convenience and ease of manipulation. Long-term culture of cancer cell lines, however, often causes selection of unique cell populations that are quite proliferative under a particular culture condition, thereby resulting in unreliable outcomes and crucial limitations in preclinical drug development.
Without being cultured in vitro, patient-derived tumor xenografts (PDXs) have also been generated by implantation into animal models of human CRC tissues surgically dissected from patients2,3,4. PDXs are widely recognized as recapitulating the major histopathological features and genetic alterations originally present in tumors of the patients from which they were derived. Moreover, patient-derived tumor organoids composed of tumor cell clusters were established by culture under 3D conditions that closely mimicked the biological properties of the original tumors5,6. These tumor organoids were also applied to high-throughput drug screening, thereby allowing personalized therapies to be designed5. However, markedly heterogeneous CRC populations are assumed to be present within a tumor mass. Particular CRC populations might selectively proliferate and expand during the in vivo and in vitro series of passages of PDX and tumor organoids, respectively. This may also allow the overall gene expression profiles and epi/genetic status of the affected CRCs to change, thereby resulting in minimal resemblance to the parental CRC.
Patient-derived CRC organoids and those extracted from noncancerous human colon engineered to harbor combinations of oncogenic mutations have also been employed to investigate the hallmarks of human tumor cells exemplified by tumor invasion and metastasis6,7,8,9. However, the very low incidence of spontaneous metastasis arising from orthotopic implantation of patient-derived CRC into immunodeficient mice, has made it difficult to study the multistep process of the invasion-metastasis cascade that includes local invasion, intravasation, transport in the bloodstream, extravasation and colonization of distant organs4,10. Micrometastasis as represented by tumor cell deposition of &#8804;2 mm, formed by patient-derived CRC organoids, has often been overlooked on histopathological analysis of sections from affected distant organs in experimental mouse models. Visualization of spontaneous micrometastases has also been minimal in vivo due to the difficulty of efficiently introducing fluorescent markers into tumor organoid cells prior to their injection into recipient mice. In this study, we developed a protocol to efficiently transduce GFP lentivirus into PDX-derived CRC organoid cells in 3D culture prior to injection into recipient mice and to allow highly sensitive detection, employing stereo-fluorescence microscopy, of their colonization of different organs to form micrometastases.

<table>
	<tbody>
		<tr>
			<td>NOD/Shi-scid IL2R&#947; null (NOG) mice</td>
			<td>The Central Institute for Experimental Animals,Kanagawa, Japan</td>
			<td></td>
			<td>Breed 6-week-old male mice under germ-free and specific pathogen-free conditions</td>
		</tr>
		<tr>
			<td>wound clips 
			2&#215;10mm</td>
			<td>Natsume manufacturing, Japan</td>
			<td>#C-21-S</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Hamilton syringe 
			needle size:22 gauge</td>
			<td>Tokyo Science, Japan</td>
			<td></td>
			<td>Disinfect with 70% alcohol and sterile PBS.</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>BMBio</td>
			<td>#92006</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>BMBio</td>
			<td>#92412</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml conical tube</td>
			<td>Sumitono Bakelite</td>
			<td>MS-57150</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical tube</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-57500</td>
			<td></td>
		</tr>
		<tr>
			<td>microtube</td>
			<td>Eppendorf</td>
			<td>#0030120086</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Erma</td>
			<td>#03-202-1</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m cell strainer</td>
			<td>Corning</td>
			<td>#352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel basement membrane matrix</td>
			<td>Corning</td>
			<td>#354234</td>
			<td>Store aliqupts at -20&#176;C. 
			Place on ice until use</td>
		</tr>
		<tr>
			<td>Collagenase type 1</td>
			<td>Sigma</td>
			<td>#C1030</td>
			<td>150 mg/ml collagenase type1 in 1&#215;PBS. Store aliqupts at -20&#176;C for up to 1 year</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovate Cell Technologies</td>
			<td>#5V2623A</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>DMEM/F-12 with GlutaMAX&#8482;</td>
			<td>Gibco</td>
			<td>#10565018</td>
			<td>Store at 4&#176;C. Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Cell banker 1plus</td>
			<td>ZENOAQ</td>
			<td>#628</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>#15140122</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>#15140122</td>
			<td>Store at 4&#176;C. Use within 1 month</td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>PEPROTECH</td>
			<td>#AF-100-15</td>
			<td>Store at -20&#176;C. Add to medium on same day as use</td>
		</tr>
		<tr>
			<td>Y27632, a ROCK inhibitor</td>
			<td>Wako</td>
			<td>#253-00591</td>
			<td>Store at -20&#176;C. Add to medium on 
			same day as use</td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td>Gibco</td>
			<td></td>
			<td>DMEM/F-12 with GlutaMAX&#8482; supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. 
			Store at 4&#176;C. Use within 1 month.</td>
		</tr>
		<tr>
			<td>CRC organoid culture medium 
			with 1% or 5% FCS</td>
			<td></td>
			<td></td>
			<td>DMEM/F-12 with GlutaMAX&#8482; supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 2 ng/ml hEGF and 10 &#181;M Y27632, a ROCK inhibitor. 
			Store at 4&#176;C. Use within 1 month.</td>
		</tr>
		<tr>
			<td>the FuGENE 6 transfection regent</td>
			<td>Roche</td>
			<td>11814 443001</td>
			<td></td>
		</tr>
		<tr>
			<td>Minisart 0.45 &#181;m filter</td>
			<td>Sartorius stedim</td>
			<td>17598-K</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polypropylene centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326819</td>
			<td></td>
		</tr>
		<tr>
			<td>PRRL-GFP vector</td>
			<td>Gift from Dr. Robert A. Weinberg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Gift from Dr. Robert A. Weinberg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-dR8.2 dvpr</td>
			<td>Gift from Dr. Robert A. Weinberg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>the SW55Ti swinging bucket rotor</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>a Zeiss Axioplan 2 stereo-fluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-sensitivity detection of micrometastases generated by gfp lentivirus-transduced organoids cultured from a patient-derived colon tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

A primary colorectal adenocarcinoma diagnosed as moderately differentiated had been surgically resected from a 76 year-old female with TNM classification, stage IIIa, followed by post-operative chemotherapy. The primary CRC cells immunohistochemically stained positive for carcinoembryonic antigen, Ki-67, pan-cytokeratin and E-cadherin. Pieces of the resected tumor were also subcutaneously implanted into NOG mice to generate the colon PDX model. CRC organoid cells were then extracted for t...

Watch this Scientific Journal Video about High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor at JoVE.com

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

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