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

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.