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

Summary

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.

Abstract

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 IL2Rγ^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.

Introduction

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....

Protocol

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.

1. Prepare the primary CRC tissue immediately after surgical resection of the tumor from the patient.
2. Wash the primary CRC tissue by gentle agitation several ....

Results

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.......

Discussion

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 models^4,10. To detect micrometastases with high sensitivity, we developed a protocol for transducing GFP lentiviral particles into PDX-derived CRC organoids prior to their o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NOD/Shi-scid IL2Rγ null (NOG) mice	The Central Institute for Experimental Animals,Kanagawa, Japan		Breed 6-week-old male mice under germ-free and specific pathogen-free conditions
wound clips 2×10mm	Natsume manufacturing, Japan	#C-21-S	Autoclave before use
Hamilton syringe needle size:22 gauge	Tokyo Science, Japan		Disinfect with 70% alcohol and sterile PBS.
6-well plate	BMBio	#92006
12-well plate	BMBio	#92412
15ml conical tube	Sumitono Bakelite	MS-57150
50ml conical tube	Sumitomo Bakelite	MS-57500
microtube	Eppendorf	#0030120086	Autoclave before use
Hemocytometer	Erma	#03-202-1
40μm cell strainer	Corning	#352340
Matrigel basement membrane matrix	Corning	#354234	Store aliqupts at -20°C. Place on ice until use
Collagenase type 1	Sigma	#C1030	150 mg/ml collagenase type1 in 1×PBS. Store aliqupts at -20°C for up to 1 year
Accutase	Innovate Cell Technologies	#5V2623A	Store at 4°C.
DMEM/F-12 with GlutaMAX™	Gibco	#10565018	Store at 4°C. Warm at 37°C before use
Cell banker 1plus	ZENOAQ	#628	Store at 4°C. Use within 1 month
Penicillin	Gibco	#15140122	Store at 4°C. Use within 1 month
Streptomycin	Gibco	#15140122	Store at 4°C. Use within 1 month
hEGF	PEPROTECH	#AF-100-15	Store at -20°C. Add to medium on same day as use
Y27632, a ROCK inhibitor	Wako	#253-00591	Store at -20°C. Add to medium on same day as use
Culture medium	Gibco		DMEM/F-12 with GlutaMAX™ supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Store at 4°C. Use within 1 month.
CRC organoid culture medium with 1% or 5% FCS			DMEM/F-12 with GlutaMAX™ supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 ng/ml hEGF and 10 µM Y27632, a ROCK inhibitor. Store at 4°C. Use within 1 month.
the FuGENE 6 transfection regent	Roche	11814 443001
Minisart 0.45 µm filter	Sartorius stedim	17598-K
5 ml polypropylene centrifuge tubes	Beckman Coulter	326819
PRRL-GFP vector	Gift from Dr. Robert A. Weinberg
pCMV-VSV-G	Gift from Dr. Robert A. Weinberg
pCMV-dR8.2 dvpr	Gift from Dr. Robert A. Weinberg
the SW55Ti swinging bucket rotor	Beckman Coulter
a Zeiss Axioplan 2 stereo-fluorescence microscope	Zeiss