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.
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Method Article
Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer
In This Article
Summary
Abstract
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 (µCT), 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.
Introduction
Colorectal cancer (CRC) is the second leading cause of cancer death worldwide1. The ability to generate in vitro or in vivo tumor models derived from individual patient tumor cells has advanced precision medicine in oncology. Over the last decade, patient derived organoids (PDOs) or xenografts (PDXs) have been used by many research groups around the world2. PDOs are multicellular in vitro structures that resemble the features of the original tumor tissue and can self-organize and self-renew3. These promising in vitro models can successfully....
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Protocol
Written informed consent was obtained from all patients. The project was approved by the Research Ethics Committee of the Vall d'Hebron University Hospital, Barcelona, Spain (approval ID: PR(IR)79/2009 PR(AG)114/2014, PR(AG)18/2018). Human colon tissue samples consisted of biopsies from non-necrotic areas of primary adenocarcinomas or liver metastases, corresponding to patients with colon and rectal cancer who underwent tumor resection. Experiments were conducted following the European Union's animal care directive (86/609/EEC) and were approved by the Ethical Committee of Animal Experimentation of the VHIR-the Vall d'Hebron Research Institute (ID: 40/08 C....
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Results
Mice orthotopically implanted with patient-derived cancer cells were monitored weekly by µCT scanning. At the end of the experiment, the animals were euthanized. Intestines, ceca (Figure 1A,B), livers, lungs, and any other possible lesion were collected, included in a cassette, and fixed with 4% formalin overnight. Intestine tissue from a mouse without a tumor in the cecum was used as a control (Figure 1C). Finally, cassettes were changed to 70%.......
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Discussion
Over the last few decades, many new anti-cancer therapies have been developed and tested in patients with different tumor types, including colorectal cancer (CRC). Although promising results in preclinical models have been observed in many cases, the therapeutic efficacy in patients with advanced metastatic CRC has been frequently limited. Therefore, there is an urgent need for preclinical models that allow for testing the efficacy of new therapeutic drugs in a clinically relevant metastatic scenario.
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Acknowledgements
We acknowledge the Cellex Foundation, CIBERONC network, and Instituto de Salud Carlos III for their support. Moreover, we also thank the preclinical imaging platform at the Vall d'Hebron Research Institute (VHIR), where the experiments were performed.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| REAGENT | |||
| Apo-Transferrin | MERCK LIFE SCIENCE S.L.U. | T1147-500MG | |
| B27 Supplement | Life Technologies S.A (Spain) | 17504044 | |
| Chlorhexidine Aqueous Solution 2% | DH MATERIAL MÉDICO, S.L. | 1111696250 | |
| Collagenase | MERCK LIFE SCIENCE S.L.U. | C0130-500MG | |
| D-(+)-Glucose | MERCK LIFE SCIENCE S.L.U. | G6152 | |
| DMEM /F12 | LIFE TECHNOLOGIES S.A. | 21331-020 | |
| DNase I | MERCK LIFE SCIENCE S.L.U. | D4263-5VL | |
| EGF | PEPRO TECH EC LTD. | AF-100-15-500 µg | |
| FGF basic | PEPRO TECH EC LTD. | 100-18B | |
| Fungizone | Life Technologies S.A (Spain) | 15290026 | |
| Gentamycin | LIFE TECHNOLOGIES S.A. | 15750037 | |
| Heparin Sodium Salt | MERCK LIFE SCIENCE S.L.U. | H4784-250MG | |
| Insulin | MERCK LIFE SCIENCE S.L.U. | I9278-5ML | |
| Iopamiro | |||
| Isoflurane | - | - | |
| Kanamycin | LIFE TECHNOLOGIES S.A. | 15160047 | |
| L-Glutamine | LIFE TECHNOLOGIES S.A. | 25030032 | |
| Matrigel Matrix | CULTEK, S.L.U. | 356235/356234/354234 | |
| Metacam, 5 mg/mL | - | - | |
| Non-essential amino acids | LIFE TECHNOLOGIES S.A. | 11140035 | |
| Nystatin | MERCK LIFE SCIENCE S.L.U. | N4014-50MG | |
| Pen/Strep | Life Technologies S.A (Spain) | 15140122 | |
| Phosphate-buffered saline (PBS), sterile | Labclinics S.A | L0615-500 | |
| Progesterone | MERCK LIFE SCIENCE S.L.U. | P0130-25G | |
| Putrescine | MERCK LIFE SCIENCE S.L.U. | P5780-5G | |
| RBC Lysis Buffer | Labclinics S.A | 00-4333-57 | |
| Sodium Pyruvate | LIFE TECHNOLOGIES S.A. | 11360039 | |
| Sodium Selenite | MERCK LIFE SCIENCE S.L.U. | S5261-25G | |
| ESSENTIAL SUPPLIES | |||
| 8 weeks-old NOD.CB17-Prkdcscid/NcrCrl mice | - | - | |
| BD Micro-Fine 0.5 ml U 100 needle 0.33 mm (29G) x 12.7 mm | BECTON DICKINSON, S.A.U. | 320926 | |
| Blade #24 | - | - | |
| Cell Strainer 100 µm | Cultek, SLU | 45352360 | |
| Forceps and Surgical scissors | - | - | |
| Heating pad | - | - | |
| Lacryvisc, 3 mg/g, ophthalmic gel | - | - | |
| Surfasafe | - | - | |
| Suture PROLENE 5-0 | JOHNSON&JOHNSON S, A. | 8720H | |
| EQUIPMENT/SOFTWARE | |||
| Quantum FX µCT Imaging system | Perkin Elmer | Perkin Elmer | http://www.perkinelmer.com/es/product/quantum-gx-instrument-120-240-cls140083 |
References
- Sung, H., et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a Cancer Journal for Clinicians. 71 (3), 209-249 (2021).
- Puig, I., et al.
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