Name: Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models
Uploaded: 2024-01-05T13:30:00
Description: Watch this Scientific Journal Video about Advancing Cancer Associated Thrombosis Research in Rodent Models at JoVE.com

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
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	<li>Cell culture
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		<li>Prior to heterotopic s...

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

<ol>
	<li>Blom, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+venous+thrombosis+in+a+large+cohort+of+66,329+cancer+patients:+results+of+a+record+linkage+study.">Incidence of venous thrombosis in a large cohort of 66,329 cancer patients: results of a record linkage study.</a> Journal of Thrombosis and Haemostasis. 4 (3), 529-535 (2006).</li><li>Gabre, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+protein+C+accelerates+venous+thrombus+resolution+through+heme+oxygenase-1+induction.">Activated protein C accelerates venous thrombus resolution through heme oxygenase-1 induction.</a> Journal of Thrombosis and Haemostasis. 12 (1), 93-102 (2014).</li><li>Chang, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorafenib+(BAY+43-9006)+inhibits+tumor+growth+and+vascularization+and+induces+tumor+apoptosis+and+hypoxia+in+RCC+xenograft+models.">Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models.</a> Cancer Chemotherapy and Pharmacology. 59 (5), 561-574 (2007).</li><li>Khorana, A. A., Kuderer, N. M., Culakova, E., Lyman, G. H., Francis, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+predictive+model+for+chemotherapy-associated+thrombosis.">Development and validation of a predictive model for chemotherapy-associated thrombosis.</a> Blood. 111 (10), 4902-4907 (2008).</li><li>Chew, H. K., Wun, T., Harvey, D., Zhou, H., White, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+venous+thromboembolism+and+its+effect+on+survival+among+patients+with+common+cancers.">Incidence of venous thromboembolism and its effect on survival among patients with common cancers.</a> Archives of Internal Medicine. 166 (4), 458-464 (2006).</li><li>Leiva, O., Newcomb, R., Connors, J. M., Al-Samkari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+and+thrombosis:+new+insights+to+an+old+problem.">Cancer and thrombosis: new insights to an old problem.</a> Journal de Medecine Vasculaire. 45 (6S), 6S8-6S16 (2020).</li><li>Chen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bevacizumab+promotes+venous+thromboembolism+through+the+induction+of+PAI-1+in+a+mouse+xenograft+model+of+human+lung+carcinoma.">Bevacizumab promotes venous thromboembolism through the induction of PAI-1 in a mouse xenograft model of human lung carcinoma.</a> Molecular Cancer. 14, 140 (2015).</li><li>Goto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+a+new+vascular+targeting+agent,+ZD6126,+in+pulmonary+metastases+by+human+lung+adenocarcinoma+in+nude+mice.">Activity of a new vascular targeting agent, ZD6126, in pulmonary metastases by human lung adenocarcinoma in nude mice.</a> Cancer Research. 62 (13), 3711-3715 (2002).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+anchorage-independent+growth+and+lung+metastasis+of+A549+lung+carcinoma+cells+by+IkappaBbeta.">Inhibition of anchorage-independent growth and lung metastasis of A549 lung carcinoma cells by IkappaBbeta.</a> Oncogene. 20 (18), 2254-2263 (2001).</li><li>Salup, R. R., Wiltrout, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+immunotherapy+of+established+murine+renal+cancer+by+interleukin+2-stimulated+cytotoxic+lymphocytes.">Adjuvant immunotherapy of established murine renal cancer by interleukin 2-stimulated cytotoxic lymphocytes.</a> Cancer Research. 46 (7), 3358-3363 (1986).</li><li>Deatrick, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+matrix+metalloproteinase+2+and+matrix+metalloproteinase+2/9+deletion+in+experimental+post-thrombotic+vein+wall+remodeling.">The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling.</a> Journal of Vascular Surgery. 58 (5), 1375.e2-1384.e2 (2013).</li><li>Diaz, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+a+mouse+model+of+venous+thrombosis:+a+consensus+assessment+of+utility+and+application.">Choosing a mouse model of venous thrombosis: a consensus assessment of utility and application.</a> Journal of Thrombosis and Haemostasis. 17 (4), 699-707 (2019).</li><li>Henke, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+9+signaling+is+critical+for+early+experimental+deep+vein+thrombosis+resolution.">Toll-like receptor 9 signaling is critical for early experimental deep vein thrombosis resolution.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 31 (1), 43-49 (2011).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+vena+cava+stenosis-induced+deep+vein+thrombosis+is+influenced+by+multiple+factors+in+rats.">Inferior vena cava stenosis-induced deep vein thrombosis is influenced by multiple factors in rats.</a> Biomedicine &amp; Pharmacotherapy. 128, 110270 (2020).</li><li>von Br&#252;hl, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes,+neutrophils,+and+platelets+cooperate+to+initiate+and+propagate+venous+thrombosis+in+mice+in+vivo.">Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.</a> The Journal of Experimental Medicine. 209 (4), 819-835 (2012).</li><li>Brill, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor-mediated+platelet+adhesion+is+critical+for+deep+vein+thrombosis+in+mouse+models.">von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models.</a> Blood. 117 (4), 1400-1407 (2011).</li><li>Stark, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Pathogenesis+of+Pancreatic+Cancer+Microvesicle-Associated+Venous+Thrombosis+Identifies+New+Antithrombotic+Targets+In+Vivo.">Distinct Pathogenesis of Pancreatic Cancer Microvesicle-Associated Venous Thrombosis Identifies New Antithrombotic Targets In Vivo.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 38 (4), 772-786 (2018).</li><li>Belghasem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolites+in+a+mouse+cancer+model+enhance+venous+thrombogenicity+through+the+aryl+hydrocarbon+receptor-tissue+factor+axis.">Metabolites in a mouse cancer model enhance venous thrombogenicity through the aryl hydrocarbon receptor-tissue factor axis.</a> Blood. 134 (26), 2399-2413 (2019).</li><li>Tracz, A., Mastri, M., Lee, C. R., Pili, R., Ebos, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+spontaneous+metastatic+renal+cell+carcinoma+(mRCC)+in+mice+following+nephrectomy.">Modeling spontaneous metastatic renal cell carcinoma (mRCC) in mice following nephrectomy.</a> Journal of Visualized Experiments. (86), e51485 (2014).</li><li>Lertkiatmongkol, P., Liao, D., Mei, H., Hu, Y., Newman, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+functions+of+platelet/endothelial+cell+adhesion+molecule-1+(CD31).">Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31).</a> Current Opinion in Hematology. 23 (3), 253-259 (2016).</li><li>Payne, H., Brill, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stenosis+of+the+Inferior+Vena+Cava:+A+Murine+Model+of+Deep+Vein+Thrombosis.">Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis.</a> Journal of Visualized Experiments. (130), e56697 (2017).</li><li>Yabit, F., Hughes, L., Sylvester, B., Tiesenga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypersensitivity+Reaction+Post+Laparoscopic+Cholecystectomy+Due+to+Retained+Titanium+Clips.">Hypersensitivity Reaction Post Laparoscopic Cholecystectomy Due to Retained Titanium Clips.</a> Cureus. 14 (6), e26167 (2022).</li><li>Nagorni, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-laparoscopic+cholecystectomy+Mirizzi+syndrome+induced+by+polymeric+surgical+clips:+a+case+report+and+review+of+the+literature.">Post-laparoscopic cholecystectomy Mirizzi syndrome induced by polymeric surgical clips: a case report and review of the literature.</a> Journal of Medical Case Reports. 10, 135 (2016).</li><li>Zemelka-Wiacek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+Allergy:+State-of-the-Art+Mechanisms,+Biomarkers,+Hypersensitivity+to+Implants.">Metal Allergy: State-of-the-Art Mechanisms, Biomarkers, Hypersensitivity to Implants.</a> Journal of Clinical Medicine. 11 (23), 6971 (2022).</li><li>Poyyamoli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=May-Thurner+syndrome.">May-Thurner syndrome.</a> Cardiovascular Diagnosis and Therapy. 11 (5), 1104-1111 (2021).</li><li>Streiff, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NCCN+Guidelines+Insights:+Cancer-Associated+Venous+Thromboembolic+Disease,+Version+2.2018.">NCCN Guidelines Insights: Cancer-Associated Venous Thromboembolic Disease, Version 2.2018.</a> Journal of the National Comprehensive Cancer Network. 16 (11), 1289-1303 (2018).</li></ol>

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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			<td>Buprenorphine 0.3 mg/mL</td>
			<td>PAR Pharmaceutical</td>
			<td>&#160;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>&#160;Fine Science Tools</td>
			<td>&#160;&#160; 13002-10</td>
			<td></td>
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		<tr>
			<td>Clips ASSI.B2V Single Clamp, General Purpose,</td>
			<td>Accurate Surgical &#38; Scientific Instruments</td>
			<td>PR 2 144.50 289.00</td>
			<td></td>
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			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
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			<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>&#160;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>
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			<td>Isoflurane, USP</td>
			<td>&#160;Covetrus</td>
			<td>NDC 11695-6777-2</td>
			<td></td>
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		<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>&#160;Fine Science Tools</td>
			<td>&#160; 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>
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			<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&#160;</td>
			<td></td>
		</tr>
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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.