Name: Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer (Video) | JoVE Journal
Uploaded: 2017-07-18T13:00:00.000+00:00
Duration: 6 min 19 s
Description: Learn a microsurgical technique for isolating circulating tumor cells using orthotopic colorectal cancer models in mice. This method enables reproducible tumor formation and reliable CTC analysis for metastasis research.

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5 minutes

This work was supported by the German Research foundation (WE 3548/4-1) and Roland-Ernst-Stiftung f&#252;r Gesundheitswesen (1/14).

The animal experiments presented here were independently reviewed and permitted by an institutional and a governmental Animal Care and Use Committee and were conducted according to Federation of Laboratory Animal Science Associations (FELASA) guidelines. All possible measures were taken to minimize suffering including anesthesia and analgesia or, if necessary, premature euthanasia.
1. Preparation of Cells and Organoids
NOTE: Use a volume of 20 &#181;L with 100,000 cells for each injection. Use basement membrane matrix (BMM) in order to prevent leakage and ensure standardized injection. In order to ensure reproducible results, conduct cell line authentication assays (e.g., via STR profiling) at regular intervals.
<ol>
	<li>Preparation of primary cell suspensions from fresh tissue 
	NOTE: Always work under sterile conditions. Immediate transfer of the freshly resected tissue from the operating room to the laboratory on ice is required to ensure high viability of the cells. 
	The patient&#39;s well-being and optimal treatment must always be the first priority. Therefore, tissue samples for research must be obtained in a manner that does not interfere with the subsequent pathological work-up and staging of the resected tumor. In most cases, it is therefore reasonable to have the samples for research obtained by a trained pathologist in order to ensure non-interference with the pathological diagnosis.
	<ol>
		<li>Immediately after sterile removal from the resected specimen (ideally ~1 cm3), place the tissue sample in a 50 mL tube pre-filled with 20-30 mL of phosphate-buffered saline (PBS). Store the tube on ice and transfer to the laboratory immediately.</li>
		<li>Put the tissue in a Petri dish and wash it twice with plenty of PBS to remove the remaining blood.</li>
		<li>Cut the tissue into small pieces (~ 2-4 mm) with a scalpel (e.g., with a #20 or #36 blade).</li>
		<li>Use the dissociator according to the manufacturer&#39;s instructions in order to further dissociate the tumor tissue to a single-cell suspension. Alternatively, use other protocols of enzymatic tumor digestion.</li>
		<li>Count the cells of the resulting single-cell suspension (e.g., in a coulter counter or a hemocytometer).</li>
		<li>Centrifuge (5 min at 1,500 x g) and wash the cell suspension twice with PBS.</li>
		<li>Resuspend the cells in BMM at a concentration of 5 x 106 cells/mL and keep them on ice.</li>
	</ol>
	</li>
	<li>Preparation of cell lines for injection
	<ol>
		<li>Grow all colorectal cancer cell lines under standard culturing conditions (37 &#176;C, 5% CO2) and prepare them at the day of surgery.</li>
		<li>Harvest the cells according to standard cell culture protocols, count the cells (e.g., in a coulter counter or a hemocytometer) and calculate the required amount for all injections depending on the numbers of animals to be injected.</li>
		<li>Prepare 3-5x of the actually needed volume to account for pipetting losses and the dead volume of the injection syringe.</li>
		<li>Centrifuge (5 min at 1,500 g) and wash the cell suspension twice with PBS.</li>
		<li>Resuspend the cells in BMM at a concentration of 5 x 106 cells/mL and keep them on ice.</li>
	</ol>
	</li>
	<li>Organoid preparation 
	NOTE: All 3D organoid cultures are grown under standard culturing conditions (37 &#176;C, 5% CO2). Detailed protocols for organoid culture have been published before.18,19,20 Similar to conventional cell lines, we recommend a volume of 20 &#181;L BMM with 100,000 cells per injection. The organoids are prepared at the day of surgery as follows:
	<ol>
		<li>Prepare the following medium (in the following referred to as DMEM/F12+++): Advanced Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)/F12 + 1% HEPES (1M) + 1% Glutamine (200 mM) + 1% Penicillin/Streptomycin.</li>
		<li>Pre-fill 15 mL tubes with 1 mL DMEM/F12+++.</li>
		<li>Carefully aspirate the organoids from the surface of the culture plate and transfer the contents of 3 to 5 wells into one 15 mL tube.</li>
		<li>Carefully break the organoids up into smaller pieces by pipetting them up and down with an extended glass pipette.</li>
		<li>Add 5 mL DMEM/F12+++ to the tube, followed by centrifugation at 1,000 x g for 5 min.</li>
		<li>After centrifugation, remove the supernatant, resuspend the pellet in 600 &#181;L enzymatic dissociation buffer and transfer the suspension to one well of a 6-well plate.</li>
		<li>Incubate at 37 &#176;C for 1-5 min and then carefully pipet the suspension up and down in order to dissolve the organoids. 
		NOTE: Verify the digestion via microscope; it is complete when all cell clusters have been dissociated to single cells.</li>
		<li>Upon successful digestion, add 1.4 mL of DMEM/F12+++ to stop the digestion. Then transfer all wells of digested organoids to a 50 mL tube.</li>
		<li>Count the cells and calculate the required amount for all injections.</li>
		<li>Prepare 3 - 5x of the actually needed volume to account for pipetting losses and the dead volume of the injection syringe</li>
		<li>Centrifuge (5 min at 1,500 x g) and wash the cell suspension twice with PBS.</li>
		<li>Resuspended the cells in BMM to a concentration of 5 x 106 cells/mL and keep them on ice.</li>
	</ol>
	</li>
</ol>
2. Orthotopic Mouse Model
<ol>
	<li>Preparation of recipient animals for surgery 
	NOTE: Use 6-8 week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD scid gamma, NSG) mice as recipients.21 NSG are among the most immunocompromised mice, lacking mature B, T and NK cells along with multiple other immune defects.21 They reliably grow tumors even if low cell numbers are injected and are highly prone to distant metastases. NSG mice are excellent breeders and can be kept in conventional specific pathogen free (SPF) units.
	<ol>
		<li>Prior to the first incision, inject 0.05 mg/kg of buprenorphine subcutaneously.</li>
		<li>Use sevoflurane at 3-3.5 vol% for general anesthesia. A loss of the toe pinch reflex indicates sufficient anesthesia.</li>
		<li>Cover the eyes of the anesthetized mice with ophthalmic ointment to avoid desiccation of the cornea.</li>
		<li>Restrain the mice in a supine position on a small table.</li>
		<li>Shave the abdomen with an electric shaver (depilatory cream can be used alternatively) and disinfect with at least 3 times clorhexidine/iodine and 70% alcohol alternatively.</li>
		<li>Cover the surgical field with sterile drapes. 
		NOTE: The use of perioperative antibiotics is optional and subject to institutional guidelines.</li>
	</ol>
	</li>
	<li>Midline laparotomy and exposure of the cecum
	<ol>
		<li>Use scissors (scalpels can be used alternatively) to make a small midline incision (3 - 5 mm) of the skin on the lower abdomen. Pick up the abdominal wall musculature with forceps and carefully incise it with scissors, thus opening the abdominal cavity.</li>
		<li>Identify and carefully exteriorize the cecum with atraumatic forceps. Position the blind ending pouch of the cecum on the abdomen pointing cranially.</li>
		<li>Once exteriorized, keep the cecum moist using warm saline swabs at all times.</li>
	</ol>
	</li>
	<li>Orthotopic injection, closure of the abdomen and postoperative recovery
	<ol>
		<li>For intracecal injection, use a standard 1 mL syringe with a 30 G cannula. Mount this syringe on a microinjection pump, which is in turn mounted on a micromanipulator.</li>
		<li>Carefully grasp the tip of the cecum with atraumatic forceps and gently smoothen it by stroking it downward with a second set of forceps moistened with warm saline.</li>
		<li>Position the cannula directly above the cecum.</li>
		<li>Perform the following steps under visual control with a binocular surgical microscope.</li>
		<li>Carefully grasp the cecum with two atraumatic forceps at both ends of the exteriorized part of the cecum, slightly stretch it and then slowly pull it over the cannula which is positioned parallel and directly above. It is crucial not to perforate the entire bowel wall (thus injecting the cells into the lumen) as well as not to perforate the serosa beyond the initial point of penetration, as this would lead to leakage and peritoneal dissemination.
		<ol>
			<li>Move the bowel towards the cannula, not the cannula towards the bowel. Hold and stretch the bowel between two forceps. The surgeon&#39;s hands must be resting on a surface in order to reduce tremor.</li>
		</ol>
		</li>
		<li>Inject the cells between the serosa (seen as very thin, translucent lining above the intramural blood vessels) and the muscularis. The cannula must therefore be visually placed above the blood vessels and underneath the thin translucent membrane.</li>
		<li>Use a foot switch to start the injection in order to reduce tremor while the cannula is inside the bowel wall.</li>
		<li>Once the cannula is in position, start the injection. Use a duration of 20 s, resulting in 1 &#181;L/s setting on the control unit of the pump. 
		NOTE: The injection into or near a damaged blood vessel leads to direct intravascular dissemination and distant metastasis and should therefore be avoided.</li>
		<li>After completion of the injection, carefully remove the cannula by pulling it backward.</li>
		<li>Place a dry swab under the cecum, and then thoroughly rinse the cecum with distilled water in order to lyse leaked cells and thus prevent artificial peritoneal dissemination.</li>
	</ol>
	</li>
	<li>Closure of the abdomen and postoperative recovery
	<ol>
		<li>After rinsing, gently return the cecum to the abdominal cavity.</li>
		<li>Close the abdominal wall with 6-0 rapidly absorbable running sutures.</li>
		<li>Close the skin with surgical wound clips.</li>
		<li>Place the mouse on a heating map set to 38 &#176;C until it has fully recovered from the anesthesia.</li>
		<li>Closely observe the postoperative condition of the mice for the following 48 h. In case of distress, treat with 0.05 mg/kg buprenorphine every 12 h.</li>
		<li>Monitor the mice at least once daily for signs of distress due to tumor growth.</li>
	</ol>
	</li>
</ol>
3. Isolation of Circulating Tumor Cells from Whole Blood Samples
NOTE: Obtain blood by transcutaneous cardiac puncture on anesthetized mice, followed by euthanasia. Ideally, 1,000 &#181;L of blood are drawn into a syringe prefilled with 100 &#181;L of an anticoagulant (Ethylenediaminetetraacetic acid (EDTA) or heparin). Use an anti-human-EpCAM (epithelial cell adhesion molecule) antibody to identify the CTCs. This works very well if human epithelial cell lines are used in the mouse model. For other appliances, different antibodies may be required.
<ol>
	<li>CTC enrichment:
	<ol>
		<li>Pre-fill 15 mL tubes with 5 mL density gradient medium and carefully transfer the blood into the 15 mL tube.</li>
		<li>Centrifuge (30 min at 300 x g without brake) and carefully remove the upper supernatant.</li>
		<li>Pour the rest into a new 15 mL tube and centrifuge (15 min, 300 x g with brake).</li>
		<li>Recover the interphase containing the mononuclear cells (discard the rest) and wash the cells twice with PBS.</li>
		<li>Resuspend the pellet in 200 &#181;L PBS/1% EDTA and add 4 &#181;L of EpCAM antibody. Incubate 20 min on ice in the dark.</li>
	</ol>
	</li>
	<li>Screening and picking
	<ol>
		<li>Prepare the following buffer (in the following referred to as picking buffer): PBS + 10% fetal calf serum (FCS) + 1% Penicillin/Streptomycin (1%) + 0.8% EDTA.</li>
		<li>Use a PAP pen to draw a ~1 cm circle in a 6 cm sterile Petri dish (prevents the fluid from dispersing in the dish), and pipet 700 &#181;L of picking buffer into this circle.</li>
		<li>Add 50 &#181;L of cell suspension to the 700 &#181;L picking buffer within the circle. Check the density of cells with the microscope. Split the sample into different dishes if it is too dense.</li>
		<li>Wait for the cells to settle down (~5 min).</li>
		<li>Screen the drop of cell suspension for stained (= EpCAM-positive) cells.</li>
		<li>Once a cell is found, pick the cell with the micromanipulator and put it in 50 &#181;L buffer depending on the intended downstream analysis (e.g., RNA extraction buffer or culturing medium).</li>
		<li>Depending on the intended downstream analyses, isolate EpCAM-negative cells and/or medium as negative controls.</li>
		<li>Proceed with downstream analyses (e.g., cell culture, PCR).</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

Despite their preclinically proven activity in subcutaneous mouse models, the great majority of novel compounds fail in clinical trials and never reach the clinic.11 This obvious insufficiency of subcutaneous mouse models to accurately simulate the biology and growth patterns of tumors has led to the development of orthotopic mouse models based on the injection of tumor cells directly into the original organ.
Orthotopic mouse models are able to simulate the biology and ...

Colorectal cancer (CRC) is one of the most common causes of cancer death in western countries.1 While the primary tumor can often be resected, the occurrence of distant metastases dramatically worsens the prognosis and often leads to death.2,3 The biological correlate of metastasis is circulating tumor cells (CTCs), which detach from the tumor, survive in circulation, attach to the epithelium in the target organ, invade the organ and ultimately outgrow to new lesions.4 Although CTCs are known to be of prognostic relevance,5,6,7,8,9 their biology is only partly understood as a result of their extreme rarity in CRC.10
Mouse models are a powerful tool to study various aspects of cancer biology. Classical, subcutaneous tumor models are produced by subcutaneous injection of tumor cells into recipient mice, which can be either immunocompetent (if syngeneic murine tumor cells are used) or immunodeficient. Subcutaneous tumor models are inexpensive and produce data fast; their end-point tumor growth can be easily and non-invasively measured. However, 88% of new compounds that have demonstrated antitumor activity in such models fail in clinical trials.11 This is in part due to interspecies differences between humans and mice; however, a large part of this failure is due to the low predictive value of subcutaneous mouse models.
Orthotopic mouse models, in which the tumor cells are injected into the organ of origin and thus grow in their original microenvironment, are therefore increasingly used in cancer research.11,12,13,14 Orthotopic models do not only simulate local tumor growth conditions; as a result of the anatomically correct site of tumor growth, orthotopic mouse models also allow realistic simulation of metastasis and are therefore used to study CTC biology8,15,16 or their response to different treatments in CRC.13,17
A major disadvantage of orthotopic mouse models is their technical complexity. Depending on the organ in which the cells are to be injected, the learning curve until the experimenter is able to induce reproducible tumors is rather long. This especially applies to colorectal cancer models, as the tumor cells need to be injected into the bowel wall, which often results in perforation, tumor cell leakage or endoluminal tumor cell loss. This article is intended to describe the method of cell preparation from primary tissue samples, 2D cell lines and 3D organoid culture and their injection into the cecum of mice. The technique described here leads to highly uniform tumors and, depending on the tumor biology of the cell line used for injection, reproducible formation of distant metastases and CTCs in the recipient mice.15

<table><tbody><tr><td>&lt;strong&gt;Cell culture Media and Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced DMEM F12</td><td>Invitrogen</td><td>12634010</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>HEPES (1 M)</td><td>Life Technologies GmbH</td><td>15630056</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>Glutamax-I Supplement (200 mM)</td><td>Life Technologies GmbH</td><td>35050038</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>Penicillin/Streptomycin (PenStrep)</td><td>Life Technologies GmbH</td><td>15140122</td><td>DMEM/ F12 +++ medium</td></tr><tr><td>DMEM</td><td>Life Technologies GmbH</td><td>61965026</td><td>basic medium of 2D cell lines (DMEM/10% FCS)</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>BIOCHROM AG</td><td>S 0115</td><td>basic medium of 2D cell lines (DMEM/10% FCS)</td></tr><tr><td>TrypLE Express enzymatic dissociation buffer</td><td>Life Technologies GmbH</td><td>12604021</td><td></td></tr><tr><td>Matrigel basement membrane matrix (BMM, phenol red free)</td><td>CORNING B.V. Life Sciences</td><td>356231</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Life Technologies GmbH</td><td>14190169</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%, Phenol-Red)</td><td>Life Technologies GmbH</td><td>25200072</td><td></td></tr><tr><td>6-/48-well plates with lid</td><td>CORNING</td><td>3516/3548</td><td></td></tr><tr><td>cell culture flask 75 cm&amp;sup2;, 250 mL</td><td>VWR International GmbH</td><td>734-2066</td><td></td></tr><tr><td>cell culture flask 150 cm&amp;sup2;, 600 mL</td><td>Corning B.V. Life Sciences</td><td>355001</td><td></td></tr><tr><td>Eppendorf tubes 1,5 mL / 2 mL</td><td>Sarstedt AG &amp;amp; Co.</td><td>72.706.400/ 72.695.400</td><td></td></tr><tr><td>15 mL, 50 mL centrifuge tubes</td><td>Greiner-Bio-One GmbH</td><td>188271/227270</td><td></td></tr><tr><td>TC10 Counting Slides (for TC20 Counting Machine)</td><td>Bio-Rad Laboratories GmbH</td><td>1450016</td><td></td></tr><tr><td>Pasteur pipettes (glass, 150 mm)</td><td>Fisher Scientific GmbH</td><td>11546963/ FB50251</td><td>thinly pulled by using a bunsen burner</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>for primary tumor tissue preparation</td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td>for primary tumor tissue preparation</td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td>for primary tumor tissue preparation</td></tr><tr><td>Human Tumor Dissociation Kit</td><td>Miltenyi Biotec</td><td>130-095-929</td><td>for primary tumor tissue preparation</td></tr><tr><td>Falcon 70 &amp;micro;m Cell Strainer</td><td>Corning B.V. Life Sciences</td><td>352350</td><td>for primary tumor tissue preparation</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sevoflurane</td><td>AbbVie Germany GmbH &amp;amp; Co. KG</td><td>-</td><td></td></tr><tr><td>Medical oxygen</td><td>Air Liquide Medical GmbH</td><td>-</td><td></td></tr><tr><td>Buprenorphine</td><td>Temgesic</td><td>-</td><td></td></tr><tr><td>Bepanthen - opthalmic ointment</td><td>Bayer Vital GmbH</td><td>10047757</td><td></td></tr><tr><td>Normal saline 0.9% (E154)</td><td>Serumwerk Bernburg AG</td><td>10013</td><td></td></tr><tr><td>Aqua ad injectabilia</td><td>Braun</td><td>235144</td><td></td></tr><tr><td>1 mL Syringe (without dead volume) - Injekt-F SOLO</td><td>Braun/neoLab</td><td>194291661</td><td></td></tr><tr><td>30G injection needle</td><td>BECTON DICKINSON</td><td>304000</td><td></td></tr><tr><td>cellulose swabs</td><td>Lohmann &amp;amp; Rauscher Deutschland</td><td>13356</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>FST - Fine Science Tools</td><td>11018-12</td><td></td></tr><tr><td>Iris Scissor - ToughCut</td><td>FST - Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Olsen-Hegar Needle Holder</td><td>FST - Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>AutoClip Kit</td><td>FST - Fine Science Tools</td><td>12020-00</td><td></td></tr><tr><td>PDS Z1012H 6/0 C1 (surgical suture)</td><td>Johnson &amp;amp; Johnson Medical GmbH</td><td>Z1012H</td><td></td></tr><tr><td>Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer</td><td>Parkland Scientific</td><td>V3000PS/PK</td><td></td></tr><tr><td>UltraMicro Pump with Micro4 Controller</td><td>World Precision Instruments</td><td>UMP3-4</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Footswitch for SYS-Micro4 Controller</td><td>World Precision Instruments</td><td>15867</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Three-axis Manual Micromanipulator</td><td>World Precision Instruments</td><td>M325</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Magnetic Stand for Micromanipulator</td><td>World Precision Instruments</td><td>M10</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Steel Base Plate for M10 Magnetic Stand</td><td>World Precision Instruments</td><td>5479</td><td>equipment for highly controlled orthotopic injection</td></tr><tr><td>Hot Plate 062</td><td>Labotect</td><td>13854</td><td></td></tr><tr><td>Isis - Hair shaver</td><td>AESCULAP - Braun</td><td>-</td><td></td></tr><tr><td>Binocular Surgical Microscope</td><td>Parkland Scientific</td><td>VS-2Z</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;CTC isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EDTA</td><td>Roth</td><td>8040.1</td><td></td></tr><tr><td>Density gradient medium &amp;ndash; Ficoll</td><td>StemCell - Lymphoprep</td><td>7801</td><td></td></tr><tr><td>Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4</td><td>BioLegend</td><td>324210</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8</td><td>BioLegend</td><td>118210</td><td></td></tr><tr><td>Petri Dish, &amp;oslash; 60 x 15 mm, 21 cm&amp;sup2;, Vent</td><td>Greiner bio-one</td><td>628102</td><td></td></tr><tr><td>Fluorescence Cell Culture Microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Transferman 4r Micromanipulator</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>CellTram Air</td><td>Eppendorf</td><td></td><td>aspiration pump connected to the micromanipulator</td></tr><tr><td>Dmz Universal Microelectrode Puller</td><td>Dagan Corporation</td><td></td><td>required for the manufacturing of micro capillaries for single cell aspiration</td></tr><tr><td>Prism Glass Capillaries</td><td>Dagan Corporation</td><td></td><td></td></tr><tr><td>PAP pen</td><td>Abcam</td><td>ab2601</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Life Technologies GmbH</td><td>14190169</td><td>picking buffer</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>BIOCHROM AG</td><td>S 0115</td><td>picking buffer</td></tr><tr><td>Penicillin/Streptomycin (PenStrep)</td><td>Life Technologies GmbH</td><td>15140122</td><td>picking buffer</td></tr><tr><td>EDTA</td><td>Roth</td><td>8040.1</td><td>picking buffer</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunohistochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Purified anti-human CD326 (EpCAM) antibody clone 9C4</td><td>BioLegend</td><td>324201</td><td>EpCAM immunohistochemistry (cf, fig 2C)</td></tr><tr><td>HRP rabbit anti-mouse IgG</td><td>Abcam</td><td>ab97046</td><td>EpCAM immunohistochemistry (cf, fig 2C)</td></tr></tbody></table>

isolation of circulating tumor cells in an orthotopic mouse model of colorectal cancer

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.

The successful and reproducible generation of colorectal tumors in this model critically depends on accurate injection of the cells without spillage or leakage. If this is achieved, this model is extremely reliable and very rarely results in artificial peritoneal dissemination. The growth kinetics of the tumors as well as their dissemination patterns depend on the biology of the used organoids and cells.15 While HCT116 cells reliably metastasize to the liver in thi...

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 ...

The overall goal of this microsurgical technique is to inject colorectal tumor cells into the cecal wall to induce reproducible and metastatic colorectal tumors in mice. This method can answer key questions in the field of circulating tumor cell biology, its main advantages are the highly reproducible and uniform tumors, allowing reliable stage prediction and circulating tumor cell prediction. Begin by using atraumatic forceps to carefully exteriorize the cecum of a six to eight week old immunocompromised mouse.Placing the blind end of the cecum on the abdomen so that the pouch points cranially. For the intra-cecal injection, mount a standard one milliliter syringe equipped with a 30 gauge canella and load it with the tumor cells onto a micro injection pump that is mounted on a micro manipulator. Next, use the atraumatic forceps to carefully grasp the tip of the cecum and use a second forceps, moistened with warm saline to gently smooth the cecum with downward strokes.Now move the animal under a binocular surgical microscope and with the canella directly parallel to and above the cecum, use two atraumatic forceps to carefully stretch the tissue at both ends of the exteriorized end of the cecum. Slowly pull the cecum over the canella, taking care not to perforate the entire belt of wall or the serosa beyond the initial point of penetration and place the canella above the blood vessels and under the thin translucent membrane inside belt of wall. When the canella is in place, use a footswitch to inject 20 microliters of cells over a period of 20 seconds.Between the thin translucent serosa lining, above the intramural blood vessels and the muscularis. When all of the cells have been injected, carefully remove the canella and place a dry swap under the cecum. To prevent artificial peritoneal dissemination, lyse any leaked cells with a thorough distilled water rinse and gently return the cecum to the abdominal cavity.Close the abdominal wall with rapidly absorbable running sutures, in the skin with surgical wound clips. Then place the mouse on a heating mat set to 38 degrees celsius with monitoring until full recumbency. To isolate the circulating tumor cells, at the appropriate experimental end point, fill 15 milliliter conical tubes with five milliliters of density gradient medium per tube and carefully transfer whole blood samples collected from tumor bearing animals onto the density gradient layers.Separate the cells by centrifugation, and carefully recover the interface containing the mononuclear cells. Pipette the mononuclear cells into a new 15 milliliter tube for a second centrifugation. Followed by tube washes in PBS.After the second wash, re-suspend the pellets in 200 microliters of PBS, supplemented with EDTA and add four microliters of anti amp camp antibody to each sample, for 20 minute incubation on ice in the dark. Next, use a hydrophobic barrier pen to draw an approximately one centimeters circle in one sterile six centimeter petri dish per sample and add 700 microliters of picking buffer to each circle, followed by 50 microliters of cell suspension. Use a microscope to check the density of the cells, and allow the samples to settle for about five minutes.Then screen the drop of cells for amp camp positivity and use the micromanipulator to transfer the cells of interest into 15 microliters of the appropriate buffer for the intended subsequent downstream analysis. The use of colorectal cells in this model, reliably results in more abund mice within 35 days of tumor cell injection, with the primary tumors measuring about 10 millimeters in size, and with liver, and lung metastasis, and circulating tumor cells, almost invariably present. Further, the circulating tumor cells can be easily isolated for downstream analysis.Once mastered and if performed properly, this technique can be performed within 10 minutes, facilitating the formation of uniform and metastatic tumors that can both be used for therapeutic and molecular studies. Limitations of the model include, its dependency on cell lyse, which have their own limitations, as well as the immunodeficiency of the mice, both of which, can be overcome by the use of mutant colorectal cancer cells. Due to its simplicity, this model maybe a valuable addendum to the currently available models of colorectal cancer.

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Research

JoVE Journal

Cancer Research

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Orthotopic Mouse Model of Colorectal Cancer

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.

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

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.

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

The video describes the technique for intracecal injection of cancerous cells to develop an orthotopic mouse model with highly uniform tumors. Therefore, the orthotopic mouse model allows realistic simulation of metastasis, providing a valuable tool to study the phenotype changes during tumor growth and dissemination.

Intracecal Tumor Cell Injection: A Technique to Deliver Cancer Cells for Establishing Orthotopic Colorectal Cancer Mouse Model

This video describes a detailed protocol for isolating circulating tumor cells from the whole blood sample of a euthanized colorectal tumor mouse model. These isolated CTCs can be cultured and used for intended downstream analysis.

CTC Isolation from a Whole Blood Sample: A Method to Obtain CTCs from Murine Colorectal Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

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.

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

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.

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

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.

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

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.

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

This study focuses on the in vivo subcutaneous induction of luminescent colorectal cancer cells in a murine model and the subsequent luminescence measurement using an imaging system. The procedure aims to establish a reproducible tumor growth model for real-time monitoring and therapeutic evaluation.

Subcutaneous Injection of Human Colorectal Cancer Cells in Athymic Nude Mice to Evaluate Antitumor Efficacy

Learn a microsurgical technique for isolating circulating tumor cells using orthotopic colorectal cancer models in mice. This method enables reproducible tumor formation and reliable CTC analysis for metastasis research.

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

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