Our methods creates a reliable hypoxic hepatocellular carcinoma model for noninvasive imaging of tumor hypoxia to study HCC biology in vivo, which can better recapitulate cancer development in humans. Our reliable HCC model together with a highly-producible image method serves as an important platform for translational research into the pathophysiology of human HCC. Real-time monitoring of tumor hypoxia provides insights into molecular changes leading to tumor aggressiveness, allowing determining areas of hypoxia and predicting response to treatment.
Although intraabdominal surgery can be technically challenging, adequate planning and animal care will improve the overall surgery success rate and the wellbeing of small animals. To begin, sterilize the dorsal region of an anesthetized 6 to 8-week-old mouse either at the left or right side of the lower flank with 70%ethanol. Then, use forceps to lift the skin and gently insert the injection needle underneath.
To avoid accidental leakage of the injected cell suspension during needle withdrawal, advance the needle 4 to 5 millimeters from the puncture site along the subcutaneous plane. After releasing the forceps, carefully discharge the contents of the syringe without penetrating the opposite side. After euthanizing the subcutaneous tumor-bearing mouse, make an incision on the skin of the tumor site with a scalpel blade.
Then, excise the tumor block and immediately transfer it to the culture media. Next, using sharp surgical scissors, cut the tumor block into small 1 cubic millimeter cubes, avoiding the core region of the tumor block. Keep all the cubes in culture media.
For orthotopic liver implantation, extend the limbs of an anesthetized mouse and secure them with tape to maximally expose the ventral abdomen and thorax. Then, sterilize the entire abdominal skin with 10%povidone-iodine solution followed by 70%ethanol. Next, perform a laparotomy by first making a midline incision of 10 millimeters using sharp scissors to access the peritoneum.
Then, clamp and pull the xiphoid process toward the head with forceps and open the surgical field using subcostal retractors. Using a wet gauze swab, retract the median and left liver lobes upward. Then, move the intestines outside the abdomen onto the left side of the mouse and cover them with a wet gauze swab.
Next, under a magnifying lamp for optimum visualization, perform hepatic artery ligation on the common hepatic artery, which originates from the pancreatic head to its root in the hepatic pedicle. Expose the left lobe by retracting the median and left lobe's back with a wet gauze swab. Then, using a sterile scalpel blade, make an incision of about 2 millimeters long and deep in the left lobe, and immediately insert a tumor fragment into the liver incision with sterile forceps.
Bury the tumor securely in the liver before applying a figure-eight suture, with a 6-0 nylon suture over the incision site to ensure proper tumor implantation and hemostasis. Then, close the abdominal incision with interrupted 5-0 sutures. Dispense an aliquot of 18-FMISO to a tube and dilute it with sterile saline to ensure a total activity concentration of 18 to 20 megabecquerel in 100 microliters.
Next, draw the solution with a 1 milliliter syringe and record the radioactivity and time shown on the dose calibrator. After recording the body weight of the subject mouse, carefully inject the prepared radiotracer through the tail vein. To account for decay correction, record the injection time and residual activity in the syringe.
To begin the PET acquisition, locate the mouse position in the scanner by performing a scout view. Adjust the mouse bed position to ensure that the whole mouse body with the torso region is at the center of the field of view of the MR.Next, select PET Acquisition in the study list window. For using the predetermined mouse bed positions from the scout view, choose Scan Range on Previous Acquisition.
Then, click on Prepare to automatically move the mouse bed from MR to PET, followed by Go to start the acquisition. Select the appropriate radionucleotide under the Radiopharmaceutical Editor and enter the details of injection dose and time. Additionally, under the Subject Information menu, enter the weight of the mouse.
Once the PET scan is complete, select Prepare to move the mouse from PET to MR, and click Go to perform the MR acquisition. Once all the scans are finished, move the imaging bed back to the default position by selecting Table Out. The PET imaging used in this study revealed an increase in the tumor uptake of 18-FMISO in mice with hepatic artery ligation but not in control mice.
However, tumor uptake of the glycolytic marker 18F-FDG was similar in mice with and without hepatic artery ligation. Quantification using this standardized uptake value-based approach revealed a 2.3-fold higher 18-FMISO uptake by tumors in mice with hepatic artery ligation than that in control mice. Similarly, the 18-FMISO uptake was higher in the livers of mice with hepatic artery ligation than in the livers of control mice.
Hepatic artery ligation also resulted in enhanced tumor hypoxia, as evidenced by a higher level of hypoxia-inducible factor 1-alpha expression in tumor secretions from mice with hepatic artery ligation than those from control mice. After 18-FMISO PET imaging, our sequencing can be performed to identify the roles of metabolic genes induced under hypoxia, leading to new treatment strategies. This technique can be used to monitor tumor hypoxia accurately, thereby allowing a rapid evaluation of treatment effectiveness and also the creation of new therapeutic strategy targeting the hypoxia pathway in HCC.
