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.

<ol><li>Siegel, R., Desantis, C., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Colorectal+cancer+statistics%2C+2014%5BTitle%5D)+AND+%22CA+Cancer+J+Clin%22%5BJournal%5D)">Colorectal cancer statistics, 2014.</a> CA Cancer J Clin. 64 (2), 104-117 (2014).</li>
<li>Hidalgo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-derived+xenograft+models%3A+an+emerging+platform+for+translational+cancer+research%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discov. 4 (9), 998-1013 (2014).</li>
<li>Aparicio, S., Hidalgo, M., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Examining+the+utility+of+patient-derived+xenograft+mouse+models%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Examining the utility of patient-derived xenograft mouse models.</a> Nat Rev Cancer. 15 (5), 311-316 (2015).</li>
<li>Puig, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+personalized+preclinical+model+to+evaluate+the+metastatic+potential+of+patient-derived+colon+cancer+initiating+cells%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.</a> Clin Cancer Res. 19 (24), 6787-6801 (2013).</li>
<li>van de Wetering, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prospective+derivation+of+a+living+organoid+biobank+of+colorectal+cancer+patients%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Prospective derivation of a living organoid biobank of colorectal cancer patients.</a> Cell. 161 (4), 933-945 (2015).</li>
<li>Fujii, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Colorectal+tumor+organoid+library+demonstrates+progressive+loss+of+niche+factor+requirements+during+tumorigenesis%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">A Colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis.</a> Cell Stem Cell. 18 (6), 827-838 (2016).</li>
<li>Fumagalli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+dissection+of+colorectal+cancer+progression+by+orthotopic+transplantation+of+engineered+cancer+organoids%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.</a> Proc Natl Acad Sci U S A. 114 (12), E2357-E2364 (2017).</li>
<li>O'Rourke, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+engineered+organoids+enables+rapid+generation+of+metastatic+mouse+models+of+colorectal+cancer%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer.</a> Nat Biotechnol. 35 (6), 577-582 (2017).</li>
<li>Roper, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+genome+editing+and+organoid+transplantation+models+of+colorectal+cancer+and+metastasis%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.</a> Nat Biotechnol. 35 (6), 569-576 (2017).</li>
<li>Kuo, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+colonization+competence+governs+colon+cancer+metastasis%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Liver colonization competence governs colon cancer metastasis.</a> Proc Natl Acad Sci U S A. 92 (26), 12085-12089 (1995).</li>
<li>Onuma, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+reconstitution+of+tumorigenesis+in+primary+intestinal+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Genetic reconstitution of tumorigenesis in primary intestinal cells.</a> Proc Natl Acad Sci U S A. 110 (27), 11127-11132 (2013).</li>
<li>Onder, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+E-cadherin+promotes+metastasis+via+multiple+downstream+transcriptional+pathways%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways.</a> Cancer Res. 68 (10), 3645-3654 (2008).</li>
<li>Cespedes, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> Am J Pathol. 170 (3), 1077-1085 (2007).</li>
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</ol>

The authors have no potential conflicts of interest to disclose.

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&amp;gamma; 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&lt;br/&gt; 2&amp;times;10mm</td><td>Natsume manufacturing, Japan</td><td>#C-21-S</td><td>Autoclave before use</td></tr><tr><td>Hamilton syringe&lt;br/&gt; 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&amp;mu;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&amp;deg;C.&lt;br/&gt; 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&amp;times;PBS. Store aliqupts at -20&amp;deg;C for up to 1 year</td></tr><tr><td>Accutase</td><td>Innovate Cell Technologies</td><td>#5V2623A</td><td>Store at 4&amp;deg;C.</td></tr><tr><td>DMEM/F-12 with GlutaMAX&amp;trade;</td><td>Gibco</td><td>#10565018</td><td>Store at 4&amp;deg;C. Warm at 37&amp;deg;C before use</td></tr><tr><td>Cell banker 1plus</td><td>ZENOAQ</td><td>#628</td><td>Store at 4&amp;deg;C. Use within 1 month</td></tr><tr><td>Penicillin</td><td>Gibco</td><td>#15140122</td><td>Store at 4&amp;deg;C. Use within 1 month</td></tr><tr><td>Streptomycin</td><td>Gibco</td><td>#15140122</td><td>Store at 4&amp;deg;C. Use within 1 month</td></tr><tr><td>hEGF</td><td>PEPROTECH</td><td>#AF-100-15</td><td>Store at -20&amp;deg;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&amp;deg;C. Add to medium on&lt;br/&gt; same day as use</td></tr><tr><td>Culture medium</td><td>Gibco</td><td></td><td>DMEM/F-12 with GlutaMAX&amp;trade; supplement supplemented with 5% FBS, 100 U/ml penicillin and 100 &amp;micro;g/ml streptomycin.&lt;br/&gt; Store at 4&amp;deg;C. Use within 1 month.</td></tr><tr><td>CRC organoid culture medium&lt;br/&gt; with 1% or 5% FCS</td><td></td><td></td><td>DMEM/F-12 with GlutaMAX&amp;trade; supplement (Gibco #10565018) supplemented with 1% or 5% FCS, 100 U/ml penicillin, 100 &amp;micro;g/ml streptomycin, 2 ng/ml hEGF and 10 &amp;micro;M Y27632, a ROCK inhibitor.&lt;br/&gt; Store at 4&amp;deg;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 &amp;micro;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 ...

high-sensitivity-detection-micrometastases-generated-gfp-lentivirus

Research

JoVE Journal

Cancer Research

10.0K Views.  Juntendo University Faculty of Medicine. Om zeer gevoelige detectie van de verspreiden menselijke colorectaal carcinoom (CRC) cellen koloniseren van weefsels, tonen we hierin een protocol voor efficiënte transductie van groen fluorescent proteïne (GFP) lentivirale deeltjes in PDX-afgeleide CRC organoid cellen voorafgaand aan hun injectie in ontvangende muizen, met stereo-fluorescentie microscopische observatie.

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

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 ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Medicine

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

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 ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

Establishing Experimental Metastases Mouse Model: Implanting GFP Expressing CRC Organoid Cells in PDX Model to Detect Micrometastases

Source: Okazawa, Y. et al.&#160; <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a> J. Vis. Exp. (2018).
This video describes the protocol for generating metastases by the PDX derived CRC organoids labeled with GFP lentivirus into the spleen of an immunodeficient mouse. Cancer micrometastases colonizing other organs can be detected using a fluorescence microscope to assess the GFP expression.

Source: Okazawa, Y. et al.&#160;  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a ...

JoVE Encyclopedia of Experiments

Colorectal Cancer

Animal model studies

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

Source: Okazawa, Y., et al. <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.&#160;

Source: Okazawa, Y., et al.  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a ...

CRC Organoid Culture: A Method to Obtain 3D Organoids from Colorectal Cancer Cells

Source: Okazawa, Y. et al. <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a> J. Vis. Exp., (2018).
This video describes the detailed protocol for generating 3D organoids from murine-model derived colorectal cancer cells by propagating them on artificial extracellular matrix coated plates. These CRC organoids can be used as a model to study the primary growth of human tumor cells and metastasis.

Source: Okazawa, Y. et al.  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived ...

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

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