This work was supported by AHA Cardio-oncology SFRN CAT-HD Center grant 857078 (KR, VCC, XY, and SL) and R01HL166608 (KR and VCC).

For this study, 16 female C57Bl6/J mice, 8-12 weeks in age, and a body weight of 20 to 25 g were used. The mice were housed under standard conditions and were fed with chow and water ad libitum. This study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at Boston University. The open procedures described here were undertaken in a sterile condition.
1. Xenograft model
<ol>
	<li>Cell culture
	<ol>
		<li>Prior to heterotopic subcutaneous implantation, grow MC-38 cells as a monolayer to 80% confluency.</li>
		<li>Following trypsinization and resuspension in 10% FBS-containing media, centrifuge cells at 277 x g, 4 &#176;C for 5 min. Then, resuspend the cells in serum-free media to a concentration of 1 x 104 MC-38 cells/&#181;L serum-free media.
		<ol>
			<li>For trypsinization, culture the MC-38 cells until they reach a desired confluence (usually around 70%-80%) and then aspirate the culture medium from the Petri dish.</li>
			<li>Add 1 mL of trypsin-EDTA solution and incubate the cells with trypsin for 1 min at 37 &#176;C to allow detachment. Gently tap or shake the Petri dish to ensure even detachment.</li>
			<li>Add a culture medium containing serum or a trypsin inhibitor to neutralize the activity and prevent further cell dissociation. Collect the detached cells along with the neutralized trypsin solution.</li>
			<li>For resuspension, once the cells are detached through trypsinization, prepare them for transplantation or further experiments. Centrifuge the collected cell suspension at 277 x g to pellet the cells.</li>
			<li>Carefully remove the supernatant (liquid above the cell pellet). Add 9 mL of culture medium, buffer, or solution to the cell pellet to achieve the desired cell concentration for transplantation or experimentation.</li>
			<li>Gently resuspend the cells by pipetting up and down or&#160;using a culture flask shaker. Avoid vigorous pipetting that could damage the cells. 
			NOTE: Depending on the variant or cell line used, cells can be resuspended in a 1:1 ratio of solubilized basement membrane matrix and serum-free media to slow highly aggressive mouse cell growth and dissemination after implantation. All cell culture steps should be undertaken in a complete aseptic hood in a cell culture specifically allocated space and were tested for mycoplasma regularly.</li>
		</ol>
		</li>
		<li>Count the cells with an automated cell counter as per the provided manual protocol.</li>
	</ol>
	</li>
	<li>Cell implantation
	<ol>
		<li>Randomly assign eight mice to the experimental group, and eight mice to the control group as follows. In the control group, use mice with MC38 xenograft and perform IVC ligation with a polypropylene 5-0 suture. In the experimental group, use mice with MC38 xenograft and perform IVC ligation with a customized vascular clip.</li>
		<li>Place the mouse in left lateral recumbency and shave the right side between the forelimbs and hindlimbs. Apply alcohol and iodine along the dorsal lumbar area to aseptically prepare the area for injection.</li>
		<li>Inject 2 x 106 MC-38 cells prepared in 200 &#181;L of medium subcutaneously in the flank, halfway between the animal&#39;s most distal rib and pelvic eminence using a 27G needle while holding the animal gently in the non-dominant hand.</li>
		<li>Following injection, inspect the animals carefully and put them back into the cage. The animals should be inspected for the followings; 1. Changes in behavior, activity levels, and overall mobility of the mice. Any significant changes could indicate discomfort or health issues. 2. The overall physical condition of the mice, including fur quality, grooming habits, and any visible signs of distress or abnormalities 3. Return to food consumption and water intake 4. Any changes in their activity level, posture, or interactions with cage mates. Lethargy or increased aggression could be signs of distress.</li>
	</ol>
	</li>
</ol>
2. Follow-up of tumor growth
<ol>
	<li>Monitor the animals on a bi-weekly basis for tumor growth trends for 3-4 weeks. Measure the tumors with a caliper and record the tumor dimensions.
	<ol>
		<li>Measure the tumors along their longest axis (L) and the axis perpendicular to the long axis (W). Calculate the tumor volume (V) using the equation L x (W2) = V. If the tumor size surpasses 20 mm in each dimension and or the tumor has evidence of ulceration, euthanize the animals before the termination of the experiment.</li>
	</ol>
	</li>
	<li>Once the tumor volume reaches 400 mm3, plan to use the mice for IVC ligation.</li>
</ol>
3. Anesthesia and preparation
<ol>
	<li>Anesthetize the mouse in an isoflurane chamber and inject the analgesic subcutaneously: Hold the animal from the base of the tail. Keep the animal on the dorsum surface of the non-dominant hand.</li>
	<li>Transfer the animal to the continuous anesthetic induction chamber filled with 3%-4% isoflurane. Confirm adequate general anesthesia with the absence of a toe-pinch reflex. Use a maintenance dose of 1%-3% isoflurane. Apply vet ointment on both eyes.</li>
	<li>Subcutaneous injection with buprenorphine (opioid for mitigating pain): Dissolve the stock of buprenorphine at a concentration of 0.3 mg/mL in 0.9% sodium chloride (NaCl) to achieve the concentration of 0.03 mg/mL.</li>
	<li>Inject a dosage of 0.05-0.1 mg/kg of 0.03 mg/mL buprenorphine together with 500 &#181;L of sterile 0.9% NaCl subcutaneously before surgery. In a 20 g mouse amount of 2 &#181;g or 66 &#181;L of 0.03 mg/mL buprenorphine is required.</li>
	<li>Skin preparation: Place the fully anesthetized mouse in a supine position over the heating blanket. Disinfect the anterior abdominal area with 0.5% chlorhexidine tincture, 2x. Frequently check the temperature of the heating blanket during the procedure and make sure that the temperature does not fall.</li>
</ol>
4. IVC ligation
<ol>
	<li>Midline laparotomy
	<ol>
		<li>Incise the abdominal skin with fine scissors starting from xiphoidal eminence to the bladder. Pinch the peritoneum halfway along the skin incision with atraumatic forceps. Ensure adequate space between the incision and the overlying intestines (Figure 2A).</li>
		<li>Open the peritoneum in a longitudinal manner along with avascular linea alba.</li>
	</ol>
	</li>
	<li>Exteriorizing the intestines to the right abdominal side
	<ol>
		<li>Pull the intestines with wet cotton tips. Cover the intestines in a completely moist sterile gauze. Ensure that the covered intestines are in a traction-free direction without any excessive traction on the mesenteric vessels.</li>
	</ol>
	</li>
	<li>Full exposure of the IVC and branches, including the confluence of the left renal vein to the IVC (Figure 2B).
	<ol>
		<li>Drop 200-300 &#181;L of normal saline. Explore the ureters&#39;, IVC, Aorta, and renal veins course (Figure 2).</li>
	</ol>
	</li>
	<li>Avascular plane at the confluence point of the infra-renal IVC and the left renal vein confluence.
	<ol>
		<li>Pass the polypropylene 6-0 suture through the avascular plane gently 2x-3x with caudal to cephalad movements with the closed tip atraumatic forceps.</li>
		<li>Ensure that any minimal oozing is controlled with gentle pressure provided with a cotton tip tamponade. Widen the provided plane by passing the 6-0 polypropylene suture with gentle caudal to cranial movements.</li>
	</ol>
	</li>
	<li>Cauterizing the gonadal and lumbar branches (Figure 2D)
	<ol>
		<li>Up to 4 posterior or lumbar branches might be present. To avoid potential complications of spinal ischemia and claudication, leave the lumbar branches intact in the current protocol.</li>
		<li>Check that 2 to 3 side branches, including the bilateral uterine horn arterial supply and hypogastric vessels are present. Ensure consistent disruption of the first bilaterally present branches. Cauterize the bilateral branches just distal to the confluence of the left renal vein and IVC (infra-renal IVC). Ensure that the venous draining vessels of the uterine horn are not disrupted. 
		NOTE: Side-branch cauterization is opted for because this method provides a smooth and uninterrupted surface, reducing any potential for uncontrolled bleeding. Moreover, cauterization is faster and more feasible than suture ligation with 10-0 sutures. Therefore, the animals will be exposed to a shorter and safer procedure.</li>
	</ol>
	</li>
	<li>Double-check the ureters&#39; course and the vasculature to ensure the absence of any oozing or inadvertent cauterizing (Figure 2C).</li>
	<li>IVC ligation with vascular clips in the experimental group
	<ol>
		<li>Customize the vascular clips with a width of 2-0 nylon suture by atraumatic forceps. Apply the customized vascular clips in the experimental group circumferentially around the infrarenal IVC.</li>
		<li>Apply the vascular clips over the suture. The primary suture passage provides a sufficient avascular plane with two to three meticulous caudal to cranial movements (Figure 2E-F).</li>
		<li>Further pass the vascular clips through the prepared plane. Prepare the plane at the confluence of the left renal vein and IVC wide enough to allow an uneventful clip passage (Figure 2E).</li>
		<li>Place the customized vascular clip horizontally, perpendicular to the IVC course, through the prepared plane. Place the vascular clips over the 5-0 polypropylene suture to ensure adequate remaining space (Figure 2G).</li>
		<li>Ensure that the above steps are performed gently to prevent any vessel damage or bleeding at the site of vascular clips. Observe the surgical field for potential oozing. Apply gentle pressure with the cotton tip to the surgical field in case of oozing.</li>
	</ol>
	</li>
	<li>IVC Ligation with suture in the control group
	<ol>
		<li>Suture ligation with 6-0 polypropylene suture. Make a surgical square knot suture around the infra-renal IVC in the prepared plane over a 5-0 silk suture.</li>
		<li>Once the suture is complete, gently remove the silk suture to ensure adequate remnant space.</li>
	</ol>
	</li>
	<li>Perform subcutaneous injection of 200 &#181;L of normal saline. 
	NOTE: To provide a comparable technique for partial IVC occlusion with clamp application, the vascular clip is customized in the current study to provide space in between lips. The vascular clip is then placed in the prepared avascular plane in the confluence of the left renal vein and IVC.</li>
	<li>Close the laparotomy with continuous running 4-0 vicryl suture.</li>
</ol>
5. Follow-up after the index surgery
<ol>
	<li>Monitor the animal continuously post-operation for 1 h or until balance and righting capability are recovered, whatever comes first in an isolated clean cage until fully recovered.</li>
	<li>Monitor the animal daily for 2 days. If the animal looks distressed, monitor it twice a day for 2 days. Administer postoperative analgesia and fluids per protocol.
	<ol>
		<li>Dissolve the stock of buprenorphine with a concentration of 0.3 mg/mL in 0.9% sodium chloride (NaCl) to achieve the concentration of 0.03 mg/mL.</li>
		<li>Inject a dosage of 0.05-0.1 mg/kg of 0.03 mg/mL buprenorphine subcutaneously together with 500 &#181;L of sterile 0.9% NaCl, every 8 to 12 h during the first 48 h following the index surgery. In a 20 g mouse, 2 &#181;g or 66 &#181;L of 0.03 mg/mL buprenorphine would be required.</li>
	</ol>
	</li>
</ol>
6. Euthanasia and harvesting the IVC containing the clot
<ol>
	<li>Anesthetize the mouse in an isoflurane chamber, hold the animal from the base of the tail and keep the animal on the dorsum surface of your hand.</li>
	<li>Transfer the animal to the continuous anesthetic induction chamber filled with 3%-4% isoflurane. Place the animal in the supine position.</li>
	<li>Perform a long midline laparotomy starting from the xiphoid to the bladder as described.</li>
	<li>Exteriorize the intestine to the right side of the abdomen and harvest the clamped IVC distal to the iliac bifurcation to the extent of infra-renal IVC (Figure 2G).</li>
	<li>Following the clot and tumor harvest, euthanize the animal with cervical dislocation.</li>
</ol>
7. Statistical analysis
<ol>
	<li>Present statistical analysis as the mean, median, and standard deviation (SD) or standard error of the mean (SEM), 25th or 75th percentile, or the entire range of values, including minimum and maximum values, as appropriate. Perform a Student t-test, and the F-test as appropriate. Assess statistical significance at the p &#60;0.05 level.</li>
</ol>

Optimized Venous Thrombosis Assay in a Mouse Cancer Model Using Vascular Clips

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The authors have nothing to disclose.

In a syngeneic xenograft colon cancer model, we observe higher thrombogenicity and expressions of coagulation markers in the experimental group compared to the control group. Importantly, the variance in all these parameters was lower in the experimental group compared to the control group. The modification involved introducing a vascular clip with a specific pressure profile at the confluence point of the IVC and the left renal vein. The clip was placed over a spacer, which was a 5-0 polypropylene suture. This modificat...

Cancer-associated venous thromboembolism (VTE) 
Venous thromboembolism (VTE) risk is 4 to 7 times higher in cancer survivors compared to the general population1,2,3. This condition proves fatal in one out of seven patients with cancer. The incidence of VTE varies depending on the type of cancer and the tumor burden and is highest among patients with pancreatic and gastric cancers4.
Cancer-associated VTE in cancer patients has prognostic significance. It is associated with unfavorable overall survival in the first year after a cancer diagnosis, even after adjusting for age, race, and stage of underlying cancer5. These findings highlight the importance of examining cancer associated VTE and the need to probe its mechanism using an animal model. The translational relevance of this area is further emphasized by the fact that VTE in cancer patients is preventable and treatable with thromboprophylaxis and antithrombotic therapy6.
Animal models of cancer and venous thrombosis 
Cancer models are conventionally termed xenografts, which entail the injection of cancer cells in mice. The injection of cancer cells at a site like its origin is referred to as an orthotopic model, while at a different site (subcutaneous plane over the flank) is known as a heterotopic model. The species of origin of cancer cells determines them as an allogeneic model, such as the HT-29 cell line (human colon cancer)7,8,9. On the contrary, syngeneic models use the murine cancer cell lines, including RenCa and MC-38 cell lines3,10.
The literature has described arterial, venous, and capillary thrombosis models in rodents. Venous thrombosis is induced in the inferior vena cava (IVC) by mechanical injury (guide wire) or complete IVC ligation, chemical (Ferric chloride), or electrolytic injury. Ferric chloride-induced thrombosis or IVC ligation represents complete occlusion models. The latter results in the stasis of blood and inflammatory infiltrates in veins11,12,13. The complete ligation model results in a high rate of thrombosis formation in 95% to 100% of mice. The partial IVC ligation model might include interruption of lateral iliolumbar branches, and the venous return is abrogated by applying suture ligations in the distal target points of IVC12. Sometimes, a space holder is used to interrupt the venous return partially. However, the thrombus weight is inconsistent in the current partial occlusion model, resulting in high variability in clot weights and heights12,14.
Both these large vein mechanical models (partial and complete) have limitations. First, IVC ligation (stasis model) often results in hypotension. The blood gets shunted through vertebral veins. Though in experienced hands, the mortality with this model ranges from 5%-30%, with the higher rate expected during the learning curve. Importantly, the complete occlusion model does not reproduce deep vein thrombosis (DVT) in humans, where a thrombus typically is nonocclusive. Complete occlusion is likely to alter hemorheological factors and pharmacodynamic parameters, altering the bioavailability of compounds at the local site. Due to these limitations, complete occlusion models may not be optimal for testing novel chemical compounds for therapeutic purposes and drug discoveries12.
It should be noted that to provide a more clinically relevant murine model of venous thrombosis with decreased flow with endothelial damage, a venous thrombosis model has been introduced, where DVT is triggered by the restriction of blood flow in the absence of endothelial disruption. The model was validated by scanning electron microscopy15. A preferred clinically relevant thrombosis model is one with near complete thrombosis that enables drug discoveries. The clot formation in the current partial occlusion models is inconsistent, resulting in high variability in the clot weight and heights12,16. Furthermore, the clot weight is variable with the conventional methods, requiring&#160;more&#160;mice per studies12.
Previous cancer-associated thrombosis models focused on colon, pancreatic, and lung cancer and were all complete occlusion models17,18,19. This manuscript modifies the partial occlusion thrombosis model to provide clots with lower variability and mouse mortality (Figure 1). Former studies used allogeneic cancer cell lines on immunocompromised athymic mice background19,20,21. This manuscript uses an MC-38 cell syngeneic xenograft in C57Bl6/J mice, which allows the use of immunocompetent mice and examination of immune components to thrombogenesis.

<table><tbody><tr><td>Buprenorphine 0.3 mg/mL</td><td>PAR Pharmaceutical</td><td>&amp;nbsp;NDC 42023-179-05</td><td></td></tr><tr><td>C57BL/6J mice</td><td>The Jackson Lab</td><td>IMSR_JAX:000664</td><td></td></tr><tr><td>Caliper</td><td>VWR International, Radnor, PA</td><td>12777-830</td><td></td></tr><tr><td>CD31</td><td>Abcam</td><td>Ab9498</td><td></td></tr><tr><td>Cell Counter</td><td>MOXIE</td><td>MXZ000</td><td></td></tr><tr><td>Clamp</td><td>&amp;nbsp;Fine Science Tools</td><td>&amp;nbsp;&amp;nbsp; 13002-10</td><td></td></tr><tr><td>Clips ASSI.B2V Single Clamp, General Purpose,</td><td>Accurate Surgical &amp;amp; Scientific Instruments</td><td>PR 2 144.50 289.00</td><td></td></tr><tr><td>Dumont #5SF Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Fibrin</td><td>Millipore</td><td>MABS2155-100UG</td><td></td></tr><tr><td>Fine Scissors - Large Loops</td><td>Fine Science Tools</td><td>14040-10</td><td></td></tr><tr><td>Forceps</td><td>&amp;nbsp;Fine Science Tools</td><td>11002-12</td><td></td></tr><tr><td>Hill Hemostat</td><td>Fine Science Tools</td><td>13111-12</td><td></td></tr><tr><td>Isoflurane, USP</td><td>&amp;nbsp;Covetrus</td><td>NDC 11695-6777-2</td><td></td></tr><tr><td>MC-38 cell</td><td>Sigma Aldrich</td><td>SCC172</td><td></td></tr><tr><td>Microscope</td><td>Nikon Eclipse Inverted Microscope</td><td>TE2000</td><td></td></tr><tr><td>Scissors</td><td>&amp;nbsp;Fine Science Tools</td><td>&amp;nbsp; 14079-10</td><td></td></tr><tr><td>Suture- Vicryl</td><td>AD-Surgical</td><td>#L-G330R24</td><td></td></tr><tr><td>Suture-Nylon 2-0</td><td>Ethilon</td><td>664H</td><td></td></tr><tr><td>Suture-Prolene 5-0</td><td>Ethicon</td><td>8661G</td><td></td></tr><tr><td>Suture-Prolene 6-0</td><td>Ethicon</td><td>PDP127</td><td></td></tr><tr><td>VEV03100</td><td>VisualSonics</td><td>FujiFilm</td><td></td></tr><tr><td>Vitrogel Matrigel Matrix</td><td>The Well Bioscience</td><td>VHM01&amp;nbsp;</td><td></td></tr></tbody></table>

venous thrombosis assay in a mouse model of cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

A group of female C57Bl6/J mice, 8-12 weeks of age, were injected with MC-38 cells at the logarithmic phase of the cell growth. The xenografts grew rapidly between the third- and fourth -weeks post-injection18. Once the tumors reached an average volume of 400 mm3, mice were randomized to the control and experimental groups. The control group underwent IVC ligation with suture, while the experimental mice were subjected to IVC ligation with vascular clip application. The tumor volumes in...

Watch this Scientific Journal Video about Advancing Cancer Associated Thrombosis Research in Rodent Models at JoVE.com

Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...

venous-thrombosis-assay-in-a-mouse-model-of-cancer

Research

JoVE Journal

Cancer Research

2.1K Views.  Boston University. Dit onderzoek heeft tot doel het knaagdiermodel voor veneuze trombose te verbeteren, met de nadruk op kanker-geassocieerde trombose. Het introduceert een verfijnde chirurgische techniek met behulp van vasculaire clips in een syngeen xenotransplantaatmuismodel voor darmkanker om de consistentie en reproduceerbaarheid te verbeteren, waardoor het mechanistische begrip van kankergeassocieerde trombose en de ontdekking van geneesmiddelen worden bevorderd. Hoewel het flowreductiemodel in 2012 werd ...

Video: Auteur in de schijnwerpers: Bevorderen van kankergeassocieerde trombose-onderzoek in knaagdiermodellen 

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Medicine

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

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

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

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

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

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

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

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

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

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

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Immunology and Infection

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...

Venous Thrombosis Assay in a Mouse Model of Cancer

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