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 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.
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 be used for drug screening and facilitating translational research. On the other side, PDX models faithfully recapitulate the original CRC at all relevant levels, from histology to molecular traits and drug response2,4.
In vivo PDX models are mostly grown as subcutaneous tumors in immunodeficient mice. Using this approach, PDXs have become the gold standard in cancer research, particularly for studying drug sensitivity or resistance. However, CRC related deaths are mostly associated with the presence of metastatic lesions in the liver, the lung, or the peritoneal cavity, and neither of the two approaches (PDO or PDX) can recapitulate the advanced clinical setting. In addition, the specific site of tumor growth has been shown to determine important biological characteristics that have an impact on drug efficacy and disease prognosis2. Therefore, there is an urgent need to establish preclinical models that can be used to assess the efficacy of anticancer drugs in a clinically relevant metastatic setting6.
Microcomputed tomography (µCT) scanners can function as scaled-down clinical CT scanners, providing primary tumor and metastasis imaging in mice at a scaled image resolution proportional to that of CT images of cancer patients7. To counteract the poor soft tissue contrast of the µCT technique, radiological iodinated contrast agents can be used to improve the contrast and evaluate tumor burden. Using a dual contrast approach, oral and intraperitoneal iodine is administrated at different timings. The contrast administrated orally helps to define the limits between tumor tissue and cecum content inside the bowel. On the other side, the contrast administered intraperitoneally allows for the identification of the external limits of the tumor mass, which frequently grows and invades the peritoneum8.
The manuscript describes a protocol to perform orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice, and the methodology to monitor intestinal tumor growth using µCT scanning. The present manuscript shows that the model recapitulates the clinical scenario of advanced intestinal tumors and metastatic disease in CRC patients that cannot be studied using PDO or PDXO models. Since orthotopic PDX models of CRC recapitulate the clinical scenario of CRC patients, we conclude that they are the best to date for testing the efficacy of anti-tumoral drugs in advanced intestinal tumors and metastatic disease.
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 CEEA, 47/08/10 CEEA and 12/18 CEEA).
NOTE: Female NOD-SCID (NOD. CB17-Prkdcscid/NcrCrl) mice from 8 weeks-of-age were purchased from Charles River Laboratories.
1. Derivation of patient cells
2. Orthotopic injection in cecum
NOTE: The following procedure is performed on a bench in a specific pathogen free (SPF) room at the animal facility. The equipment used is previously cleaned and sterilized. In addition, it is sterilized again in a portable sterilizer between individuals or zones in the animal facility.
3. Evaluation of orthotopic tumor growth using µCT scanning
NOTE: The following procedure is performed in the preclinical imaging platform (PIP) from the animal facility.
4. Therapeutic intervention in mice bearing orthotopic tumors
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% ethanol for at least 3 h and paraffin embedded. Hematoxylin and eosin (H&E) staining from ceca, livers, and lungs were performed using histopathology facility standard protocols to identify tumor cells (Figure 2, Figure 3, and Figure 4).
In another experiment, mice bearing orthotopic tumors were monitored weekly. When a tumor µCT scan signal was detected in most of the mice (around 2-4 weeks, depending on the PDX model), the animals were randomized in four groups and treated with either the vehicle, the testing drug (20 mg/kg), the standard of care chemotherapy irinotecan (50 mg/kg), or the testing drug with irinotecan. The drugs were administered intraperitoneally once a week until the end of the experiment. Tumor growth was monitored weekly by µCT scanning throughout the course of the experiment. The results indicated that the testing drug induced a reduction of the tumor volume, calculated by the µCT scan images, and that was enhanced in combination with irinotecan treatment (Figure 5 and Figure 6).
Previous studies in our lab have shown that the metastatic potential (carcinomatosis, lung and liver metastasis) of the orthotopic CRC-PDX models depends on the PDX model used (Table 3)2. In the present study, the therapeutic efficacy on metastasis formation was also evaluated. The results indicated that the testing drug, irinotecan, and the combination eradicated the formation of lung and liver metastasis in treated mice (Table 4)11.
Figure 1: Macroscopic images of the intestine of mice bearing orthotopic CRC-PDX tumors. Macroscopic intestine images from two mice bearing an orthotopic PDX tumor (A,B) at the end of the experiment. Cecum tumors are defined in red in the images. (C) An intestine image of a mouse without a tumor in the cecum as a control. Scale bars = 5 mm (A,B); 1 cm (C). Please click here to view a larger version of this figure.
Figure 2: Histological images of orthotopic CRC-PDX tumors. H&E staining of a PDX tumor model in the cecum at the end of the experiment at low (A) and high (a) magnification. Cecum tumors are defined in red in the images. Scale bars = 2.5 mm (A); 100 mm (a). Please click here to view a larger version of this figure.
Figure 3: Histological images of lung metastasis derived from orthotopic CRC-PDX tumor H&E staining of a lung from a mouse bearing an orthotopic PDX tumor. Lung metastasis can be observed at low (A) and high (a) magnification. Lung metastases are defined in red in the images. Scale bars = 250 mm (A); 100 mm (a). Please click here to view a larger version of this figure.
Figure 4: Histological images of liver metastasis derived from orthotopic CRC-PDX tumors. H&E staining of a liver from a mouse bearing an orthotopic PDX tumor. Liver metastasis can be observed at low (A) and high (a) magnification. Liver metastases are defined in red in the images. Scale bars are indicated in the images. Scale bars = 500 mm (A); 50 mm (a). Please click here to view a larger version of this figure.
Figure 5: Therapeutic efficacy of a testing drug in an orthotopic CRC-PDX model. Example of an experiment with four groups (vehicle, testing drug, irinotecan, and testing drug with irinotecan)11. Tumor volume obtained from the µCT scan images is represented over time (A) and at the end of the experiment (day 42) (B). Bars, ± SE (n = 15-30) and *p < 0.05, ***p < 0.001, ****p < 0.0001 versus vehicle (t-test, two-sided). Please click here to view a larger version of this figure.
Figure 6: µCT images from mice bearing orthotopic CRC-PDX tumors under treatment. Representative µCT images of mice bearing orthotopic tumors treated with a therapeutic drug. The cecum (red) and tumor mass (blue) are defined in the images. Please click here to view a larger version of this figure.
Table 1: Establishment of the subcutaneous PDX. Example of three PDX models established in the lab (P1, P2, and P3) from our biobank2 of more than 350 PDX models with the number of cells inoculated, the incidence of PDX establishment, and the passages in mice. Please click here to download this Table.
Table 2: Reagents to prepare the growth factors (GF) MIX 10X, the CoCSCM 6Ab without EGF, FGF2, and growth factors, the CoCSCM 6Ab complete medium, and the digestion medium. Please click here to download this Table.
Table 3: Metastatic potential of orthotopic CRC-PDX models. Example of three orthotopic CRC-PDX models established in the lab (P1, P2, and P3) from our biobank2. Here, the number of cells inoculated, the incidence of cecum tumor formation, and the incidence to generate carcinomatosis, lung metastasis, or liver metastasis are indicated. Please click here to download this Table.
Table 4: Therapeutic metastatic efficacy of a testing drug in an orthotopic CRC-PDX model. Example of an experiment with four groups (vehicle, testing drug, irinotecan, and testing drug with irinotecan)11. Here, the number of mice in each group and which of them developed carcinomatosis, lung metastasis, or liver metastasis at the end of the experiment are indicated. Please click here to download this Table.
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.
The manuscript describes in detail an advanced CRC orthotopic PDX model based on the implantation of patient tumor cells in the cecum wall of immunodeficient mice12.
The methodology is time consuming and concentration demanding. On average, the injection of an experiment with 30 mice may take around 11 h in total, including: 1) PDX tumor collection (1 h); tumor processing (4 h); and cecum implantation (6 h). The procedure must be performed in sterile conditions, minimizing the time for tumor processing and injection, while manipulating internal organs very carefully to avoid surgery-related mortality. It is therefore highly recommended that several pilot experiments are performed with tumor cell lines or PDX cells, to train the investigators and familiarize them with the procedure. In addition, two researchers must be involved in the procedure, one to collect and process the tumor, as well as help with the sutures of the animals, and the other to perform the actual surgery.
It is also important to consider that cecum tumors can grow into the lumen of the intestine or inside the cecum, depending on the PDX model and specific site of the injection. The outcome of the tumor growth is difficult to control and can dramatically affect the survival of the mice, resulting in smaller tumors and a severe intestinal obstruction when the tumors grow inside the lumen. The mice must therefore be monitored weekly, starting from the week after cell implantation. Once most of the mice present a tumor signal by the µCT scan, animals without signal should be excluded, and the rest randomized into experimental groups based on tumor volume. To obtain statistically significant results, each experimental group should include 12-15 mice.
Monitoring tumor bearing mice is essential to determine the efficacy of new therapeutic agents in clinically relevant orthotopic models. µCT scans enable the identification and quantification of primary tumor volume in mice. The use of a double contrast significantly increase the sensibility of the µCT technique, improving the quality of the images8. The growth of tumor cells in the cecum can lead to intraluminal tumors if they grow toward the lumen of the intestine, or extraluminal tumors if they grow out of the lumen of the intestine. Both scenarios have been observed with the previous methodology, and depend on the PDX model used and injection site.The mice recovered completely from the scanning, with no clinical evidence of renal damage or other incidences. The results show that µCT imaging can be a useful tool for monitoring the development and longitudinal growth of CRC.
Orthotopic models accurately recapitulate clinical CRC12 and are very useful to test the effect of new therapeutic drugs on primary tumor growth and liver and lung metastases2,11. However, a detailed written protocol may not suffice for a new research group to establish such complex models. In response, the present video aims to guide research groups to implement this procedure in their research. It shows the implantation procedure of cells in the cecum wall of immunodeficient mice and the methodology to monitor intestinal tumor growth using µCT scanning.
The authors have nothing to disclose.
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.
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 | |
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Phosphate-buffered saline (PBS), sterile | Labclinics S.A | L0615-500 | |
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Putrescine | MERCK LIFE SCIENCE S.L.U. | P5780-5G | |
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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 |