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.

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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.

Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models 

Venous Thrombosis Assay in a Mouse Model of Cancer

This research aims to enhance the rodents model for venous thrombosis focusing on cancer-associated thrombosis. It introduces a refined surgical technique using vascular clips in a syngeneic colon cancer xenograft mouse model to improve consistency and reproducibility, advancing cancer-associated thrombosis mechanistic understanding and drug discovery. Although the flow reduction model was introduced in the IVT stenotic models in 2012, no significant progress has been made in cancer-associated thrombosis models in the last 10 years.This was the rationale for this endeavor. In the field of cancer-associated thrombosis research, current technologies include advanced imaging techniques, like scanning, electron microscopic images, molecular biology tools, such as immunofluorescent staining, precision surgical instrument, data analysis software and data interpretation, and animal welfare. The current experimental challenges include variability in clot weights, a high mortality rate, and a prolonged learning curve in rodent models of venous thrombosis.Additionally, the venous flow direction and clot formation mechanisms differ from clinical cases. Our research protocol bridges gaps in rodent models of cancer-associated thrombosis. It introduces a novel surgical technique using vascular clips in xenograft models, yielding more consistent and reproducible thrombosis outcomes.This enhances our understanding of cancer-related thrombosis and drug discovery in translational research.

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

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.

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

Inferior Vena Cava (IVC) Ligation for Venous Thrombosis Analysis in Mouse

To begin, use a pair of fine scissors to make an incision on the abdominal skin of an anesthetized mouse, starting from the xiphoidal eminence to the bladder. With a pair of atraumatic forceps, pinch the peritoneum halfway along the skin incision, ensuring an adequate gap between the incision and the overlying intestines. Next, open the peritoneum longitudinally along the avascular linea alba.Then use wet cotton tips to pull out the intestines carefully. Then drop 200 to 300 microliters of normal saline over the intestines, and explore the route of the ureters inferior vena cava, or IVC, aorta, and renal veins. Cauterize the bilateral branches distal to the confluence of the left renal vein and infrarenal inferior vena cava.Check the ureters and vasculature for any oozing. To prevent potential complications of spinal ischemia and claudication, leave the lumbar branches intact. Check for the side branches, including the bilateral uterine horn and hypogastric vessels.Next, use a closed tip atraumatic forceps to gently pass a six-zero polypropylene suture through the avascular plane two to three times. Apply gentle pressure with a cotton tip tamponade to control any oozing. Finally, pass a five-zero polypropylene suture through the plane with gentle coddle to cranial movements to widen it.Place the vascular clamps over the suture to guarantee adequate space. Clot weights of the xenografted experimental group displayed a 1.5 fold increase relative to the control mice. Immunofluorescence assays showed that the IVC from the control group had intact CD31 and fibrin partially occupying the vessel lumen.The CD31 expression was not intact in the experimental group, with the wall showing fibrin staining. The control group had significantly lower fibrin expression compared to the experimental group. Ultrasonic graphic analysis of the mice showed the ligated infrarenal vena cava of a control group mouse had a partial clot.The experimental group, however, had a large clot nearly occluding the infrarenal vena cava.

1.2K Views.  Boston University.  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.

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

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