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&#39;Hebron Research Institute (VHIR), where the experiments were performed.

Written informed consent was obtained from all patients. The project was approved by the Research Ethics Committee of the Vall d&#39;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&#39;s animal care directive (86/609/EEC) and were approved by the Ethical Committee of Animal Experimentation of the VHIR-the Vall d&#39;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
<ol>
	<li>Tumor extraction 
	NOTE: The following procedure is performed in a biological cabinet at room temperature (RT) in the animal facility.
	<ol>
		<li>Obtain tumor samples from the patients&#39; surgeries or biopsies and from PDXs growing subcutaneously in mice.</li>
		<li>For the orthotopic injection, prepare the tumor cells from established subcutaneous PDX tumor models instead of tissue obtained directly from patients9.</li>
		<li>Generate PDX tumors by inoculating a suspension of 1 x 105 tumor cells in phosphate buffered saline (PBS) (50 &#181;L) mixed with Matrigel matrix (50 &#181;L) subcutaneously in the flank of NOD-SCID mice2.
		<ol>
			<li>Measure tumor growth using a caliper every other day. 
			NOTE: Importantly, our laboratory has generated a biobank of more than 350 PDX models. The CRC-PDX tumor models used in the protocol are established PDX models in the lab that have been amplified more than three passages in mice, and passed the inclusion/exclusion criteria with a positive result (Table 1).</li>
		</ol>
		</li>
		<li>Euthanize the mice by cervical dislocation when subcutaneous tumors reach the maximum size established by the CEEA (1 cm of diameter), or when the animals reach endpoint criteria.</li>
		<li>Extract the tumor and carefully remove it from the skin and surrounding non-tumor tissue using scissors and forceps.</li>
		<li>Store the harvested tumors in PBS at 4 &#176;C until the next step. 
		NOTE: Dissociate the tumors in cell suspension as soon as possible after removal from their original location in the patients&#39; lesions or subcutaneous xenografts in mice. Cell viability is significantly reduced 24 h after tissue removal, resulting in an inefficient implantation in recipient mice.</li>
	</ol>
	</li>
	<li>Cell preparation 
	NOTE: The following procedure is performed in a biological cabinet at room temperature (RT) in the tissue culture room.
	<ol>
		<li>Dissociate the tumors using a blade in a 10 cm culture plate with 1 mL of complete CoCSCM 6Ab medium (Table 2) (to make mincing easier). Place the homogeneous dissociated sample into a 15 mL conical tube.</li>
		<li>Add complete CoCSCM 6Ab medium to a final volume of 5 mL (use no more than 3 mL of dissociated sample in the same tube). 
		NOTE: Primary CRC resected from patients are naturally contaminated with bacteria and fungus. It is essential to remove pathogens present in the original patient sample using a cocktail of six antibiotics (penicillin, streptomycin, fungizone, kanamycin, gentamycin, and nystatin). Injection of tumor cells contaminated with bacteria in immunodeficient mice may result in animal death.</li>
		<li>Incubate with 50 &#181;L of DNase I (0.08 kU/mL) and 50 &#181;L of collagenase (1.5 mg/mL) (digestion medium; Table 2) for 1 h at 37 &#176;C in a cell culture incubator, at a 45&#176; position. Mix the solution well every 15 min with a 5 mL pipette before the incubation. 
		NOTE: Dissociate the tumor tissue and digest it by pipetting several times to obtain a single cell solution. This is essential for counting the cells before injecting in recipient mice and therefore achieving a homogeneous implantation of tumor cells.</li>
		<li>Add 5 mL of complete CoCSCM 6Ab medium and mix well with a 5 mL pipette.</li>
		<li>Sort the solution with a 100 &#181;m cell strainer using a new sterile 50 mL tube.</li>
		<li>Spin the sorted cells at 500 x g for 8 min at RT.</li>
		<li>Aspirate the supernatant.</li>
		<li>Resuspend the pellet in 3 mL of 1x RBC lysis buffer solution.</li>
		<li>Incubate for 10 min at RT.</li>
		<li>Add 3 mL of complete CoCSCM 6Ab medium, pipette the sample, and spin at 500 x g for 10 min at RT. Aspirate the supernatant.</li>
		<li>Resuspend the pellet with 5-10 mL of complete CoCSCM 6Ab medium, and use a cell counter to calculate the total number of cells.</li>
		<li>Spin the cells at 500 x g for 10 min at RT, and resuspend in 10 mL of PBS.</li>
		<li>Resuspend the pellet to obtain a concentration of 20 x 106 cells/mL, and mix well to obtain a homogeneous cell suspension.</li>
		<li>Prepare 29 G syringes (0.5 mL U 100 needle, 0.33 mm [29 G] x 12.7 mm) for cecum injection in the tissue culture (one syringe/mouse). Load 50 &#181;L of the tumor cell suspension (1 x 106 cells/injection) into the syringe and keep it on ice. Ensure that air bubbles are removed from the cell suspension. 
		NOTE: Eliminating air bubbles when the tumor cells are loaded in the syringe is essential to avoid injection of an excessive volume in the cecum wall, which could result in tissue rupture and sample loss. It is imperative to mix the cell suspension well when loading the syringe to avoid uneven tumor size among mice in the same experiment.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Clean the surgical site by spraying with disinfecting detergent and wiping.</li>
	<li>Depilate the mice abdomen using a mouse hair removal machine.</li>
	<li>Place the mouse in a supine position. Use 2% isoflurane to anesthetize the animal. Confirm the effect of anesthesia by gently pinching the extremity and observing the absence of stimulation.</li>
	<li>Place a 50-100 &#181;L drop of veterinary ointment (3 mg/g Lacryvisc) in the eyes to prevent dryness during anesthesia.</li>
	<li>Disinfect the mouse abdomen by scrubbing with chlorhexidine or povidone-iodine several times in a circular motion.</li>
	<li>Make a 1 cm longitudinal incision over the lower abdomen using surgery scissors. Carefully separate the skin to each site to present the peritoneum that is under the skin.</li>
	<li>Make a 0.5-1 cm incision in the peritoneum membrane, big enough to exteriorize the cecum. 
	NOTE: Exteriorize the cecum without excessively manipulating the internal organs, which could dramatically increase the lethality of the procedure.</li>
	<li>Carefully isolate the cecum from the mouse using a pre-cut, sterile gauze.</li>
	<li>Moisten the cecum with saline solution throughout the whole procedure.</li>
	<li>Immobilize the cecum by carefully taking hold of it with forceps, and introduce the needle superficially into the cecum wall. Avoid capillaries and vessels in the injection site. Remove bubbles from the cell suspension.</li>
	<li>Inject the whole 50&#181;L of tumor cell suspension slowly. It usually takes around 10 s to administer. Avoid perforating the cecum lumen with the needle, as this results in the elimination of the tumor cell suspension from the body through intestinal peristalsis. 
	NOTE: Injecting the tumor cell suspension in the cecum of the mice is the most challenging step of the entire procedure. A bright light focused on the injection site and a magnifying loupe should be used in this part of the protocol. Introduce the needle parallel to the cecum surface. The cecum is a very fragile tissue; therefore, immobilization of the cecum should be performed using surgical forceps and by applying gentle pressure to avoid tissue rupture resulting in hemorrhage. Successful implantation results in a white bubble (pellet of cells) in the cecum wall. If the bubble cannot be visualized, it may indicate that the cecum has been perforated and the cells have ended in the cecum lumen, resulting in their clearance by the intestinal tract.</li>
	<li>After injection, slowly remove the needle from the cecum and apply gentle pressure on the injection site with a cotton-tipped applicator, to avoid tumor cells escaping and reduce slight bleeding.</li>
	<li>Clean the cecum with saline solution to remove any debris.</li>
	<li>Return the cecum back into the abdomen of the animal.</li>
	<li>Close the peritoneum using 5/0 sutures.</li>
	<li>Close the skin of the abdomen using 5/0 sutures.</li>
	<li>Administer post-operative antibiotics (100 mg/kg amoxicillin or 20 mg/kg enrofloxacin) and analgesics (5 mg/mL metacam/meloxicam) by subcutaneous injection. Place the mice on a heating pad and keep them there until they have fully recovered. Then, return them to the cage with other animals.</li>
</ol>
3. Evaluation of orthotopic tumor growth using &#181;CT scanning
NOTE: The following procedure is performed in the preclinical imaging platform (PIP) from the animal facility.
<ol>
	<li>Perform all animal procedures following institutional ethical committee regulations.</li>
	<li>Start monitoring the tumor volume by &#181;CT 2 weeks after cell injection, and every week thereafter.</li>
	<li>Freshly dilute the contrast agent iopamiro (300 mg/mL) in saline solution, in a ratio of 3:1 for both doses. Administer 300 mL of iopamiro by oral gavage. 
	NOTE: Due to the poor soft tissue contrast of the &#181;CT, a contrast agent is recommended to improve the sensitivity of the technique. The protocol includes an oral administration of an iodine-based agent (iopamiro) to delimitate the intraluminal tumor burden, and a secondary intraperitoneal administration of the same agent to define the tumor burden in the visceral face of the intestine. Pilot experiments have been previously performed to determine the exact time that the contrast agent needs to arrive to the cecum of mice before its elimination. In the case of iopamiro, it is around 2 h.</li>
	<li>After 2 h, administer an intraperitoneal injection of 300 mL of previously diluted iopamiro. The administration helps to define the tumor limits in the parietal face.</li>
	<li>Anesthetize the animals using 2% isoflurane.</li>
	<li>After confirming that the animal is correctly anesthetized by pinching the foot of the mouse, place the animal in the scanning bed of the &#181;CT. The best position is supine (face up).</li>
	<li>In the control software, start the live mode (fluoroscopy mode) to place the abdominal area into the field of view (FOV) of the scanner. To do that, move the bed forward and backward and laterally until the desired position is achieved. Rotate the X-ray tube and the detector 90&#176;, and move the scanning bed in the y-axis to center the animal completely.</li>
	<li>Use the following parameters for the &#181;CT scan images: 30 mm FOV, 26 s of acquisition time, 90 kV of current voltage, and 200 &#181;A of current amperage, using an FX &#181;CT imaging system.</li>
	<li>Return the animals to their cages for recovery when the scan is finished. Provide thermal support and monitor the animals until they recover from anesthesia before returning to housing.</li>
	<li>The &#181;CT acquisition yields a file 250 Mb in size for each scan. The created data files have a VOX format. In order to make them accessible for any imaging analysis software, convert the files to DICOM format using the database managing software of the &#181;CT. Store the batch of created files in a portable hard disk in order to analyze them using any computer with available imaging software. 
	NOTE: During image analysis, the cecum is localized as a dilated bowel with a radiodense content (iopamiro), frequently in the left side of the caudal abdomen. In the visceral flexure of the cecum, a wall thickening is observed, compared to the adjacent intestinal regions. The thickening corresponds to tumor growth.</li>
	<li>Once the tumor is localized, find the highest diameter in the different views (axial, coronal, and sagittal). Measure these three axes and calculate the tumor volume following the ellipsoid formula: volume = 4/3&#960; x (x-semiaxis x y-semiaxis x z-semiaxis)10.</li>
</ol>
4. Therapeutic intervention in mice bearing orthotopic tumors
<ol>
	<li>Monitor the mice bearing orthotopic tumors weekly.</li>
	<li>When a tumor &#181;CT scan signal is detected in most of the mice, perform another &#181;CT scan the following week to confirm the presence of the tumor. 
	NOTE: The time to start the treatment depends on the PDX model used, and varies from 3-12 weeks after tumor cell inoculation in the cecum.</li>
	<li>Randomize the mice in four groups: a vehicle group (n = 10-15 mice), a testing drug group (n = 10-15 mice), a standard of care chemotherapy group (n = 10-15 mice), and a combination treatment group (n = 10-15 mice).</li>
	<li>Treat the mice intraperitoneally with saline (vehicle group), the testing drug (20 mg/kg) (testing drug group), irinotecan (50 mg/kg) (standard of care group), or the testing drug (20 mg/kg) with irinotecan (50 mg/kg) (combination treatment group). Perform the administration once a week until the end of the experiment.</li>
	<li>Monitor the tumor growth weekly by &#181;CT scanning throughout the course of the experiment.</li>
	<li>At the end of the experiment, euthanize the mice by cervical dislocation and collect livers, lungs, and any other possible lesions in other organs.</li>
	<li>Include the tissue samples in cassettes and incubate them in 4% formalin overnight. Use intestine tissue from a mouse without a tumor in the cecum as a control.</li>
	<li>Remove the cassettes from formalin and incubate with 70% ethanol for at least 3 h.</li>
	<li>Embed the cassettes with paraffin, using histopathology facility standard protocols.</li>
	<li>Perform hematoxylin and eosin (H&#38;E) staining from the cecum, liver, and lung, using histopathology facility standard protocols.</li>
</ol>

<ol>
	<li>Sung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Cancer+Statistics+2020:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: a Cancer Journal for Clinicians. 71 (3), 209-249 (2021).</li><li>Puig, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+personalized+preclinical+model+to+evaluate+the+metastatic+potential+of+patient-derived+colon+cancer+initiating+cells.">A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.</a> Clinical Cancer Research. 19 (24), 6787-6801 (2013).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Byrne, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+open+issues+in+cancer+precision+medicine+with+patient-derived+xenografts.">Interrogating open issues in cancer precision medicine with patient-derived xenografts.</a> Nature Reviews. Cancer. 17 (4), 254-268 (2017).</li><li>Vatandoust, S., Price, T. J., Karapetis, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer:+Metastases+to+a+single+organ.">Colorectal cancer: Metastases to a single organ.</a> World Journal of Gastroenterology. 21 (41), 11767-11776 (2015).</li><li>Cespedes, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites.">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> The American Journal of Pathology. 170 (3), 1077-1085 (2007).</li><li>Durkee, B. Y., Weichert, J. P., Halberg, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+micro-CT+colonography.">Small animal micro-CT colonography.</a> Methods. 50 (1), 36-41 (2010).</li><li>Boll, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-contrast+micro-CT+colonoscopy+in+live+mice.">Double-contrast micro-CT colonoscopy in live mice.</a> International Journal of Colorectal Disease. 26 (6), 721-727 (2011).</li><li>O'Brien, C. A., Pollett, A., Gallinger, S., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+colon+cancer+cell+capable+of+initiating+tumour+growth+in+immunodeficient+mice.">A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.</a> Nature. 445 (7123), 106-110 (2007).</li><li>Jensen, M. M., Jorgensen, J. T., Binderup, T., Kjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+volume+in+subcutaneous+mouse+xenografts+measured+by+microCT+is+more+accurate+and+reproducible+than+determined+by+18F-FDG-microPET+or+external+caliper.">Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper.</a> BMC Medical Imaging. 8, 16 (2008).</li><li>Herpers, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+patient-derived+organoid+screenings+identify+MCLA-158+as+a+therapeutic+EGFR+x+LGR5+bispecific+antibody+with+efficacy+in+epithelial+tumors.">Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR x LGR5 bispecific antibody with efficacy in epithelial tumors.</a> Nature Cancer. 3 (4), 418-436 (2022).</li><li>Chicote, I., Camara, J. 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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.
<p class="jove_...

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 (&#181;CT) 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 &#181;CT 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. &#160;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 &#181;CT 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.

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			<td>11360039</td>
			<td></td>
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			<td>Sodium Selenite</td>
			<td>MERCK LIFE SCIENCE S.L.U.</td>
			<td>S5261-25G</td>
			<td></td>
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		<tr>
			<td>ESSENTIAL SUPPLIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 weeks-old NOD.CB17-Prkdcscid/NcrCrl mice</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro-Fine 0.5 ml U 100 needle 0.33 mm (29G) x 12.7 mm&#160;</td>
			<td>BECTON DICKINSON, S.A.U.</td>
			<td>320926</td>
			<td></td>
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		<tr>
			<td>Blade #24</td>
			<td>-</td>
			<td>-</td>
			<td></td>
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		<tr>
			<td>Cell Strainer 100 &#181;m</td>
			<td>Cultek, SLU</td>
			<td>45352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps and Surgical scissors</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Lacryvisc, 3 mg/g, ophthalmic gel</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfasafe</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture PROLENE 5-0&#160;</td>
			<td>JOHNSON&#38;JOHNSON S, A.</td>
			<td>8720H</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT/SOFTWARE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum FX &#181;CT Imaging system</td>
			<td>Perkin Elmer</td>
			<td>Perkin Elmer</td>
			<td>http://www.perkinelmer.com/es/product/quantum-gx-instrument-120-240-cls140083</td>
		</tr>
	</tbody>
</table>

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.

Mice orthotopically implanted with patient-derived cancer cells were monitored weekly by &#181;CT scanning. At the end of the experiment, the animals were euthanized. Intestines, ceca (Figure 1A,B), livers, lungs,&#160;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 chan...

Watch this Scientific Journal Video about Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer at JoVE.com

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

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 ...

orthotopic-implantation-patient-derived-cancer-cells-mice

Research

JoVE Journal

Cancer Research

1.5K Views.  Vall d’Hebron Institute of Oncology (VHIO). 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.

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...