Name: portal vein injection of colorectal cancer organoids to study the liver metastasis stroma
Uploaded: 2021-09-03T13:45:00
Description: Watch this Scientific Journal Video about Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma at JoVE.com

This study was supported by grants from the National Health and Medical Research Council (APP1156391 to D.L.W., S.L.W.) (APP1081852 to D.L.W., APP1140236 to S.L.W., APP1099283 to D.L.W.,); Cancer Council SA Beat Cancer Project on behalf of its donors and the State Government of South Australia through the Department of Health (MCF0418 to S.L.W., D.L.W.); a Grant-in-Aid for Scientific Research (B) (20H03467 to M.T.) commissioned by the Ministry of Education, Culture, Sports, Science and Technology of Japan; AMED-CREST (Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (19gm0810007h0104 and 19gm1210008s0101 to A.E.); the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from AMED (19cm0106332h0002 to A.E.); Japan Society for the Promotion of Science Overseas Challenge Program for Young Researchers (to H.K.), Takeda Science Foundation Fellowship (to H.K.), Greaton International Ph.D. Scholarship (to H.K.), Lions Medical Research Foundation Scholarship (to K.G.).
We thank Dr. Leszek Lisowski at Vector and Genome Engineering Facility (VGEF), Children&#39;s Medical Research Institute (CMRI) (NSW, AUSTRALIA) for producing recombinant AAV vectors.

All animal procedures in this article were reviewed and approved by the South Australian Health and Medical Research Institute Animal Ethics Committee (Approval number, SAM322).
1. Tail vein injection of adeno-associated virus
NOTE: Adeno-associated virus (AAV) should be handled as a biohazard under Biosafety Level 1 guidelines. Please refer to the published protocol for AAV preparation, purification, and titration33. Hepatocyte-tropic AAV, AAV...

In this study, we have shown that portal vein injection of mouse CRC organoids reproducibly generates fibroblast-rich liver metastases that mimic histological features of human CRC hepatic metastases. Furthermore, when combined with stroma-directed therapeutics such as AAV8-mediated gene therapy, this preclinical model serves as a useful tool to assess therapeutic effects on mouse survival and tumor growth.
There are, at least, two critical steps in the protocol. Firstly, it is important to pr...

Colorectal cancer (CRC) is a major cause of cancer mortality worldwide1. More than half of the CRC patients develop hepatic metastasis that occurs through the portal vein dissemination1. Currently, there are no effective therapeutics that can cure advanced liver metastasis, and most patients succumb to metastatic disease.
The metastatic niche or tumor microenvironment plays a key role in engraftment and growth of disseminated CRC cells2. Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, promote or restrain cancer progression through secreting growth factors, remodeling the extracellular matrix (ECM), and modulating immune landscapes and angiogenesis3,4,5. CAFs also confer resistance to chemotherapies and immunotherapies3. Moreover, CAFs regulate initiation and progression of CRC liver metastasis and predict prognosis in patients with CRC3,6,7,8. Thus, CAF-related factors could be exploited for the development of therapeutic strategies to inhibit CRC liver metastasis. However, the lack of satisfactory mouse models to study the metastatic tumor stroma has been a major obstacle to developing stroma-targeted therapies.
Currently, animal models to study CRC liver metastasis include primary CRC models that spontaneously develop hepatic metastasis and cancer cell transplantation models into the liver. Primary CRC mouse models, such as genetically engineered mouse models and colonic injection of cancer cells, rarely show metastasis to the liver9,10,11,12. Moreover, even if a liver metastasis is observed, these models show long latency from the primary tumor induction to metastasis, and potentially die of primary tumor burden12. To efficiently generate CRC liver metastases, cultured CRC cells are transplanted into the liver&#160;using three injection approaches: intra-splenic injection, direct intra-parenchymal injection into the liver, and portal vein injection. Intra-splenically injected cancer cells spread into the splenic vein, the portal vein, and ultimately to the liver13,14. However, the intra-splenic injection yields a lower tumor take ratio compared with other transplantation models15,16. With intra-splenic injection, surgical removal of the spleen is performed to avoid cancer growth in the spleen, which can potentially compromise immune cell maturation17. Furthermore, intra-splenic injection can also result in unintended tumor growth in the spleen and abdominal cavity18, complicating liver metastasis analyses. Direct intra-parenchymal injection into the liver efficiently induces hepatic metastasis16,19,20. Nonetheless, this approach does not fully recapitulate a biological step of liver metastasis that naturally occurs through portal vein dissemination. Using direct injection into the liver, entry of cancer cells into a non-portal, but systemic circulation can also result in multiple large lung metastases16. Although a majority of patients with CRC liver metastasis show multiple tumor nodules in the liver21, direct injection into a specific liver lobe generates a single tumor mass19,20. Portal vein injection or mesenteric vein injection, though technically challenging, allows efficient delivery of tumor cells into the liver in a manner that recapitulates the growth patterns seen in patients17. This strategy can minimize the possibility of secondary-site metastases and enables rapid growth of cancer cells in the liver, simplifying mouse survival analyses.
Historically, colorectal cancer cell lines such as mouse MC-38, human HT-29, and SW-620 were used to generate mouse models of hepatic metastasis22,23. However, these colorectal cancer cell lines do not induce a desmoplastic stromal reaction. Low stromal content in the tumors makes it difficult to investigate the biological roles of cancer-associated fibroblasts. Recent advances in CRC organoids and their transplantation have offered useful platforms to assess vital roles of the stroma in cancer progression24. Liver transplantation of CRC organoids generates a fibroblast-rich tumor microenvironment and has provided novel insights into stromal research6,25. Currently, portal or mesenteric vein injection of organoids has become a gold standard approach to generate CRC liver metastasis6,25,26,27,28. Nonetheless, to our knowledge, no previous papers have described detailed methods for the portal vein injection of colorectal tumoroids. Here, we present a methodology for using portal vein injection of CRC organoids to develop novel adeno-associated virus (AAV)-mediated stroma-directed therapy.
Hepatocytes are an important constituent of the metastatic tumor microenvironment in the liver and play a critical role in metastatic cancer progression29. Inspired by the success of AAV gene therapy approaches to induce protein expression in hepatocytes in non-neoplastic patients30,31, we investigated a similar approach but aimed at modifying the liver tumor microenvironment in CRC25. As such, we also describe herein the tail vein injection of AAV8 to induce expression of anti-tumorigenic proteins to modify the liver tumor microenvironment. The AAV8 serotype, designated by the choice of viral capsid protein during virus production, leads to high transduction efficiency specifically of hepatocytes (i.e., targeted gene expression in the liver tumor microenvironment)32. We have previously shown that Islr (immunoglobulin superfamily containing leucine-rich repeat) is a CAF-specific gene that induces bone morphogenetic protein (BMP) signaling, reduces CRC tumoroid growth, and promotes Lgr5+ intestinal stem cell differentiation25. We tested whether AAV8-mediated overexpression of the cancer-restraining stromal gene, Islr, in hepatocytes could attenuate hepatic metastasis progression by performing portal vein injection of CRC tumoroids in AAV8-Islr-treated mice.
In this paper, we first describe the tail vein injection procedure of liver tropic AAV. Then, we describe a method for tumoroid cell preparation and portal vein injection into the AAV-treated mice. Finally, we present approaches to monitor metastatic tumor progression to assess the efficacy of stroma-directed therapeutics.

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			<td>10% Formalin</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
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			<td>15 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
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			<td>33-gauge needle</td>
			<td>TSK</td>
			<td>LDS-33013</td>
			<td>For portal vein injection</td>
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			<td>4-0 vicryl suture</td>
			<td>ETHICON</td>
			<td>J494G</td>
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			<td>5 mL Syringe</td>
			<td>BD</td>
			<td>302130</td>
			<td>Used to apply saline to the intestine after portal vein injection</td>
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			<td>50 mL centrifuge tube</td>
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			<td>50 mL syringe</td>
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			<td>SS*50LE</td>
			<td>Luer lock syringe for perfusion fixation</td>
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			<td>70% Isopropyl alcohol wipe</td>
			<td>Briemar</td>
			<td>5730</td>
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			<td>Anaesthesia machine</td>
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			<td>&#945;SMA antibody</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td>Clone 1A4. 1/500 dilution for immunohistochemistry</td>
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			<td>Buprenorphine</td>
			<td>TROY</td>
			<td>N/A</td>
			<td>ilium Temvet Injection, 300 &#181;g/ml Buprenorphine</td>
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			<td>Cotton buds</td>
			<td>Johnson &#38; Johnson</td>
			<td>N/A</td>
			<td>Johnson&#39;s pure cotton bud applicators. Need to be autoclaved before use.</td>
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			<td>D-luciferin</td>
			<td>Biosynth</td>
			<td>L-8220</td>
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			<td>Electric shaver</td>
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			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Hamilton syringe</td>
			<td>HAMILTON</td>
			<td>81020</td>
			<td>For portal vein injection</td>
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			<td>Heat box (animal warming chamber)</td>
			<td>Datesand</td>
			<td>MK3</td>
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			<td>Heat lamp</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Hemostatic sponge</td>
			<td>Pfizer</td>
			<td>09-0891-04-015</td>
			<td>Gelfoam absorbable gelatin sponge, USP, 12-7 mm</td>
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			<td>India ink</td>
			<td>Talens</td>
			<td>44727000</td>
			<td></td>
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			<td>Injection syringe and needle</td>
			<td>BD</td>
			<td>326769</td>
			<td>For tail vein injection</td>
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			<td>Islr probe (RNAscope)</td>
			<td>ACD</td>
			<td>450041</td>
			<td></td>
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		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>988-3244</td>
			<td></td>
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			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
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			<td>Living Image Software</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td></td>
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			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
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			<td>MRI fibrosis tool</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/MRI_Fibrosis_Tool</td>
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			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
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			<td>RNAscope kit</td>
			<td>ACD</td>
			<td>322300</td>
			<td></td>
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			<td>Rodent restrainer</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Rosa26-Cas9 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>024858</td>
			<td></td>
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			<td>Saline</td>
			<td>Pfizer</td>
			<td>PHA19042010</td>
			<td></td>
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			<td>Scissors</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Skin staplers</td>
			<td>Able Scientific</td>
			<td>AS59028</td>
			<td>9 mm wound clips</td>
		</tr>
		<tr>
			<td>Stapler applicator</td>
			<td>Able Scientific</td>
			<td>AS59026</td>
			<td>9 mm wound clip applicator</td>
		</tr>
		<tr>
			<td>Stapler remover</td>
			<td>Able Scientific</td>
			<td>AS59037</td>
			<td>Wound clip remover</td>
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			<td>Surgical drape</td>
			<td>Multigate</td>
			<td>29-220</td>
			<td></td>
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			<td>Surgical gauze</td>
			<td>Sentry Medical</td>
			<td>GS001</td>
			<td></td>
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			<td>Topical anesthesia cream</td>
			<td>EMLA</td>
			<td>N/A</td>
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			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td>Recombinant cell-dissociation enzyme mix</td>
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			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
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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.

To induce AAV-mediated overexpression of a tumor-restraining stromal gene, Islr4,25,43,44, in hepatocytes, we intravenously injected Islr-encoding AAV8. 1.0 x 1011 viral genomes (vg) of AAV8-Islr, or as a control, AAV8-mRuby2, was injected into the adult mouse tail vein (Figure 1A). Two weeks after the tail vein inject...

Watch this Scientific Journal Video about Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma at JoVE.com

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

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 ...

More than 50%of colorectal cancer patients develop liver metastasis through portal vein dissemination. And current animal models to study colorectal cancer liver metastasis remain inadequate to recapitulate the biological process. Portal vein injection of colorectal cancer tumoroids provides a fibroblast-rich tumor environment that allows investigation of the crosstalk between metastatic cancer cells and CAS in colorectal cancer liver metastasis.This technique provides a platform to assess the efficacy of therapeutic strategies targeting the tumor microenvironment of colorectal cancer liver metastasis. This method is particularly useful for investigating interactions between CAS, tumor cells, and the tumor microenvironment. If the cotton tip isn't placed correctly onto the injection site, excessive bleeding may occur after needle removal.Soaking of the cotton tip stops once the injection site is located. Identifying the portal vein and visualizing the injection site can be difficult without visual demonstration of this procedure. To begin, prepare an aseptic surgical area using sterile drapes on a heating pad.Additionally, prepare all instruments required for surgery as described in the text manuscript. Then illuminate the surgical area by adjusting the position of the light source. Next, subcutaneously inject 0.1 milligrams per kilogram of body weight of buprenorphine into a mouse for surgical pain management.Then anesthetize the mouse and using an electric shaver, shave the middle to the upper abdomen in an area distant from the aseptic surgical area to avoid hair contaminating the site. Clean the incision site alternating with Betadine and 80%ethanol to sterilize the surgical area. Repeat three times.Place the mouse in the surgical area on a heating pad in a supine position with isoflurane anesthesia maintenance. Then place a surgical drape with a hole over its abdomen. Lift the abdominal skin with forceps and make a two to three centimeter skin incision at the midline with scissors, cutting only the skin, making sure that the incision ranges from the mid-abdomen to the xiphoid process of the sternum and not above the lower end of the xiphoid process.Then fully lift the peritoneal wall with forceps and make a similar two to three centimeter incision to the peritoneum with scissors. Avoid cutting the intestine and the diaphragm. Next, soak a surgical gauze with warm saline and place it next to the incision on the surgeon's right.Gently pull the small and large intestines out using a cotton bud soaked with saline and place them on the saline soaked gauze. Cover the intestines with additional wet gauze to keep them moist. Next, gently pull the intestine to the surgeon's right with the wet gauze.Then apply gentle tension to visualize the portal vein. If visualization of the portal vein is difficult, gently adjust the position of the stomach with a wet cotton bud. After gently piping the tumoroid suspension several times to obtain a homogenous cell suspension, draw up 100 microliters of the cell suspension into a Hamilton or one milliliter syringe attached to a 33 gauge needle, avoiding air bubbles.Slowly insert the needle bevel up into the portal vein with an insertion depth of three to four millimeters and the needle angle almost parallel to the portal vein. Inject the tumor cells slowly over 30 seconds to prevent occlusion of the portal vein. The color of the liver temporarily changes from red to white.After the injection, remove the needle slowly and immediately apply gentle pressure to the injection site with a dry cotton bud for five minutes. Remove the cotton bud and immediately apply a hemostatic sponge to the injection site. Hold the sponge with a cotton bud or forceps and apply gentle pressure for five more minutes.After five minutes, remove the pressure to the hemostatic sponge and confirm that there is no bleeding from the injection site. If bleeding occurs, immediately perform pressure hemostasis with a cotton bud for about 10 minutes. Then apply an additional hemostatic sponge for a further five minutes.After confirming that there is no bleeding, remove the surgical gauzes on the intestines. Then using a syringe filled with five milliliters of saline, squirt the saline to the intestines to prevent organ adhesion. Gently place the intestines back inside the abdominal cavity and suture the peritoneum using 4-0 polyglactin sutures.Then lifting both sides of the skin with forceps, apply skin staplers to close the skin incision, being careful not to staple the intestine. Turn off the isoflurane, but keep the oxygen flow running. Carefully monitor the mouse.And when the mouse awakens, place it in an empty cage on a heating pad. Monitor the mouse until it is conscious and ambulating normally. Four hours post-surgery, subcutaneously inject 0.1 milligrams per kilogram of buprenorphine.Carefully monitor the mouse daily for a week, checking the sutures and wound healing. Seven to 10 days after surgery, remove the skin staplers using a stapler remover. RNA in situ hybridization two weeks after tail vein AAV-8 Islr injection showed overexpression of Islr in the liver.Two weeks after the AAV mRuby2 injection, single cell suspensions of colon cancer organoids were injected into the mouse portal vein. Macroscopically, this portal vein injection resulted in multiple white tumor nodules in the liver. Hematoxylin and eosin staining of the colorectal cancer hepatic metastases induced by the portal vein injection demonstrated histopathology of moderately differentiated tubular adenocarcinoma accompanied with a desmoplastic stromal reaction and necrosis.This stroma-rich histology faithfully recapitulated that of human colorectal cancer liver metastases, making this model suitable for translational research investigating the metastatic tumor stroma. Immunohistochemistry for alpha smooth muscle actin confirmed the presence of cancer-associated fibroblasts, whereas Picro-Sirius red staining demonstrated abundant extracellular matrix in the tumor mesenchyme. Immunochemistry for EPCAM revealed that the metastatic colorectal cancer showed tumor budding, which is a characteristic of poor prognosis colorectal cancer.And the Ki67 labeling index was approximately 80%indicating that most tumor cells are mitotically active. The mice showed a median survival of 57 days after tumoroid injection into the portal vein. AAV-8 Islr-treated mice showed improved mouse survival, decreased in vivo imaging signals from tumors, augmented bone morphogenetic protein signaling, and decreased Ki67 proliferating cells.Make sure that the cotton tip is applied to the right injection site and the hemostatic sponge is applied only after the bleeding has stopped. Tumor establishment can be monitored using the IVIS imaging to make sure that tumor cells are growing within the liver.

portal-vein-injection-colorectal-cancer-organoids-to-study-liver

Research

JoVE Journal

Cancer Research

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

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

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

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

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 ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

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 ...

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

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 ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...