Transfemoral approach TACE technique in orthotopic PDX hepatocellular carcinoma models of nude rats

Introduction

Transcatheter arterial chemoembolization (TACE) is a minimally invasive treatment modality for hepatocellular carcinoma (HCC) and plays a key role in HCC management (1-4). Immunodeficient mouse tumor models are widely used in HCC research, including studies of tumor biology (5), antitumor drug development and screening (6), gene therapy (7), tumor angiogenesis (8), and as platforms for non-invasive imaging biomarkers to examine hypoxia and prognosis in liver cancer (9,10). The patient-derived tumor xenograft (PDX) immunodeficient mouse orthotopic HCC model, which preserves the biological characteristics of the original tumor and accurately simulates the complexity of human HCC, is a commonly used animal model for studying human HCC (11). Previous studies have largely employed rat-derived HCC models for hepatic arteriography and treatment (12,13); however, reports on TACE in nude rat orthotopic PDX HCC models are lacking.

As part of a systematic investigation, this study successfully performed TACE using the transfemoral approach in six Foxn1^rnu nude rat orthotopic PDX HCC models, and summarized the technical experience and key points. The findings aim to provide technical support and a reference for transcatheter arterial approaches in nude rat orthotopic PDX HCC research. We present this article in accordance with the ARRIVE reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1248/rc).

Surgical techniques

Experimental animals and ethical considerations

The experiments were performed under a project license (Nos. II2025-036-01 and SYSU-IACUC-2024-002867) granted by the Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University and the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University, in accordance with institutional guidelines for the care and use of animals.

Six male Foxn1^rnu nude rats (average body weight: 307±28.3 g; range, 273–350 g) were housed in a specific pathogen-free (SPF) barrier environment at the Sun Yat-sen University Laboratory Animal Center. Magnetic resonance imaging (MRI) confirmed successful tumor formation (average tumor diameter: 6.2±0.8 mm; range, 4.9–7.2 mm) in the orthotopic PDX HCC models (Figure 1).

Figure 1 MRI scans demonstrating intrahepatic tumor nodules (highlighted by blue circles) in orthotopic PDX HCC models in six nude rats (A-F). Imaging parameters (GE scanner, T2 fat suppression sequence: repetition time =6,613 ms, echo time =74.5 ms). A, anterior; HCC, hepatocellular carcinoma; MRI, magnetic resonance imaging; PDX, patient-derived tumor xenograft.

Transfemoral approach TACE technique: step-by-step details

Two experienced interventional radiologists jointly performed the procedures. All the surgical procedures adhered to aseptic principles and animal welfare guidelines. The detailed steps for establishing femoral artery access are illustrated in Figure 2, and the TACE procedure is shown in Figure 3.

Figure 2 Technical steps for FA access establishment. (A) Exposure of the femoral sheath revealing the dark-red, thicker FV (green arrow) and adjacent slender FA (red arrow). (B) Isolation of the FA. (C) Proximal and distal suspension of the FA with distal ligation to achieve arterial engorgement. (D) Anterior arteriotomy followed by micro-guidewire and microcatheter insertion. FA, femoral artery; FV, femoral vein.

Figure 3 TACE procedure. (A) Abdominal aortography delineating the origin and morphology of the CT. (B) CT angiography. (C) Superselective catheterization with chemotherapeutic emulsion injection (black arrow). (D) Post-procedural angiography. CHA, common hepatic artery; CT, celiac trunk; GDA, gastroduodenal artery; LGA, left gastric artery; PHA, proper hepatic artery; SA, splenic artery; SMA, superior mesenteric artery; TACE, transcatheter arterial chemoembolization.

The procedure was performed in the nude rats as follows:

• Step 1. Tribromoethanol (2.5%) was administered via intraperitoneal injection at a dose of 6 mL/kg for anesthesia. If available, isoflurane gas was used as an alternative anesthetic.
• Step 2. Once the anesthesia took effect, the hair in the right groin area was shaved using a hair clipper.
• Step 3. The nude rats were positioned supine on a digital subtraction angiography (DSA) examination bed, and a heating pad was used to maintain body temperature at 37 ℃. Note: Maintaining a stable body temperature after anesthesia was essential, as anesthesia can easily induce hypothermia, which, if not properly managed, can be fatal.
• Step 4. The skin of the right groin area was disinfected with povidone-iodine and draped with a sterile sheet.
• Step 5. Local anesthesia was administered to the groin area using an appropriate amount of 1% lidocaine.
• Step 6. A longitudinal incision of about 3 cm was made in the skin of the right groin area using ophthalmic scissors. Gauze was applied for hemostasis as needed, and two retractors were crossed under the incision to widen the field of view.
  Note: Transverse skin incisions were avoided, as they can easily sever subcutaneous veins and cause bleeding.
• Step 7. The subcutaneous fascial tissue was bluntly dissected using a hemostatic clamp, exposing the femoral artery sheath, including the dark-red femoral vein medially, the white femoral nerve laterally, and the bright red, slender femoral artery centrally.
• Step 8. The fascial tissue around the femoral artery sheath was carefully separated using toothed forceps to prevent damage to blood vessels and bleeding.
  Note: The assistant gently pulled the fascial tissue around the femoral artery sheath in both directions perpendicular to the sheath, expanding the gap between the femoral artery and vein to facilitate the passage of a curved vascular clamp. If the fascial gap could not be opened after the curved clamp passed through the posterior femoral artery to the gap between the femoral nerve and artery, a sharp instrument was used to assist in breaking the fascia, with care taken to avoid damaging nerves and blood vessels.
• Step 9. A curved hemostatic clamp was passed through the gap between the femoral artery and vein from posterior to anterior, and a 6-0 suture (approx. 5 cm) was clamped back and pulled through. This step was repeated once.
• Step 10. The sutures at both ends of the femoral artery were suspended at intervals of about 1 cm.
• Step 11. The distal suture was tied to fill the femoral artery, and a few drops of lidocaine were dripped into the femoral artery sheath to prevent vasospasm.
• Step 12. A small oblique incision was made in the anterior wall of the femoral artery using micro-ophthalmic scissors. After observing blood flow out, the proximal suture was gently suspended.
  Note: This incision was critical to avoid severing the femoral artery due to improper use of instruments.
• Step 13. With the help of an assistant, the assembled curved micro-guidewire (KangFly^R, 0.008 inch, RICOTON) and microcatheter (FloatSky^TM, 1.2 Fr, RICOTON) set was inserted through the femoral artery incision. A surgical magnifying glass was used to improve visualization as necessary. Micro-forceps were used to gently pull the anterior wall of the femoral artery incision to allow the microcatheter to pass smoothly during its insertion.
  Note: Cutting the micro-guidewire and microcatheter was avoided, as it can damage product characteristics and decrease controllability. The micro-guidewire and microcatheter were used as an assembled unit, with the guidewire tip pre-shaped and extending 1cm beyond the catheter.
• Step14. Heparin (10 IU/Kg) was injected through the microcatheter for heparinization to keep the artery unobstructed and prevent thrombus formation (13).
  Note: It was essential that the catheter remain unobstructed.
• Step 15. Under DSA fluoroscopy, the microcatheter tip was positioned in the upper segment of the abdominal aorta. The micro-guidewire was withdrawn, after which a 1 ml syringe was connected and used to inject contrast agent (ioversol, 0.2–0.3 mL per injection, total ≤1 mL) to determine the location and shape of the celiac trunk opening.
  Note: Non-ionic agents were preferred, and the total amount was minimized to protect renal function.
• Step 16. Under micro-guidewire guidance, the microcatheter was advanced into the celiac trunk, and angiography was again performed to determine the location, number, size, and feeding arteries of the intrahepatic lesions.
• Step 17. The micro-guidewire was used to perform superselective catheterization of the target blood vessels, and angiography was again performed to confirm the position of the microcatheter.
  Note: If encountered, resistance was avoided during microcatheter advancement to prevent vasospasm, dissection, or the rupture of the vessel being imaged.
• Step 18. A 1 mL syringe was connected to the microcatheter, and an appropriate amount of iodized oil chemoembolization agent was injected under DSA fluoroscopy to embolize the lesion.
  Note: In this study, an iodized oil and idarubicin emulsion (dose ≤2 mg/kg) was used.
• Step 19. After embolization, the microcatheter was flushed, and angiography was re-performed as necessary based on the experimental purpose.
• Step 20. The microcatheter was withdrawn, the proximal end of the femoral artery was ligated, and hemostasis was confirmed at the incision site.
  Note: Slight tightening of the proximal sutures reduced bleeding during microcatheter withdrawal.
• Step 21. The subcutaneous tissue and skin were sutured layer by layer.
• Step 22. The nude rats were observed in a heat-preserving box, and feeding was resumed after full recovery from anesthesia.

Outcome measures and statistical description

Procedure durations were recorded, including: duration from skin incision to femoral artery exposure (T1); time required for successful femoral artery cannulation (T2); time required for successful hepatic artery catheterization (T3); and total duration from skin incision to wound closure (T4). Intraoperative and postoperative adverse events were documented. Technical success was defined as the successful completion of the following procedural steps: (I) femoral artery cannulation; (II) abdominal aortography identifying celiac trunk origin; (III) selective celiac trunk catheterization; (IV) superselective catheterization of hepatic artery branches; and (V) injection of chemoembolic agent without procedure-related mortality or major complications. The statistical analysis was performed using SPSS 26.0 (Chicago, IL, USA). Continuous data are presented as the mean ± standard deviation (minimum, maximum).

Technical performance and safety outcomes

Technical success was achieved in 100% of cases (6/6). The 1.2 Fr microcatheter (FloatSky^TM, RICOTON) was successfully advanced through the femoral artery of the nude rats. After local lidocaine application, the femoral artery diameter slightly exceeded 0.4 mm. All six nude rats underwent successful celiac arteriography, selective catheterization, and TACE (Figure 4). No anesthesia-related complications, major bleeding, or deaths occurred during the procedures. Due to anatomical variations, the celiac trunk originated from the right anterior aspect of the abdominal aorta, exhibiting “U”, “C”, or “L” shapes under fluoroscopy, projecting between the inferior border of the L1 vertebral body and the upper L2 vertebral body (Figure 5). The mean procedural times were as follows: T1: 4.1±1.2 min (range, 2.5–6.0 min); T2: 4.5±1.5 min (range, 3.0–7.5 min); T3: 4.5±1.5 min (range, 2.5–6.4 min); and T4: 39.3±7.1 min (range, 29.0-48.0 min) (Table 1). Within seven days postoperatively, one nude rat exhibited decreased skin temperature in the right hindlimb, while the others showed no signs of limb ischemia. No TACE-related mortality occurred.

Figure 4 Celiac trunk angiography and superselective TACE in nude rats (A-F), demonstrating tumor staining (black arrows). CHA, common hepatic artery; GDA, gastroduodenal artery; LGA, left gastric artery; PHA, proper hepatic artery; SA, splenic artery; TACE, transcatheter arterial chemoembolization.

Figure 5 DSA fluoroscopic projections of CT origins (A-F). CT, celiac trunk; DSA, digital subtraction angiography; L1, first lumbar vertebral body; L2, second lumbar vertebral body; SMA, superior mesenteric artery.

Comments

In vivo animal models, particularly immunodeficient mouse models, serve as crucial platforms for studying the pathogenesis, progression, and treatment of HCC (14). Compared to nude mice, nude rats are larger in size, facilitating surgical manipulation and monitoring, making them ideal xenograft models for HCC research (15). For TACE in HCC, establishing PDX orthotopic HCC models and simulating human TACE procedures hold significant value for translational research, including vascular-targeted therapies, drug screening, treatment efficacy evaluation, and preclinical studies of novel materials (e.g., drug-eluting beads, radiopaque microspheres, and biodegradable embolic agents).

Previous studies using Sprague-Dawley rats weighing 500–580 g showed the feasibility of transcaudal artery angiography in rat vascular intervention models, and provided a reference for vascular interventions (13). However, to date, no studies have described TACE in Foxn1^rnu nude rat orthotopic PDX HCC models. In TACE procedures performed in rat models, vascular diameter and catheter compatibility are critical limiting factors, with body weight directly influencing vascular anatomy. Hepatic artery diameter is strongly correlated with body weight (12). While larger rats allow the use of commercially available interventional devices, small-sized and low-weight rats present technical challenges due to narrower vessels, limiting the applicability of clinical-grade materials in murine TACE models. This study successfully performed human-simulated TACE via the transfemoral approach in small-sized, low-weight nude rat PDX HCC models using commercial interventional devices, achieving a 100% technical success rate without anesthesia-related complications, major bleeding, or procedure-related mortality.

Key technical considerations for transfemoral TACE include: (I) topical lidocaine application to prevent vasospasm after femoral artery exposure; (II) distal artery ligation to facilitate arteriotomy and cannulation; (III) gentle traction on the distal suture during microcatheter advancement to improve navigation; (IV) the use of pre-shaped guidewires to reduce celiac artery cannulation time, and the use of minimal contrast volume to reduce renal toxicity; and (V) intermittent, fractional chemoembolic agent injection to prevent non-target embolization from catheter residue.

The technical proficiency of the operator and the anatomical variations of rat blood vessels directly affected TACE duration. Prolonged T2 (femoral artery cannulation time) was primarily associated with vasospasm during initial attempts. Extended T3 (hepatic artery catheterization time) was correlated with celiac trunk anatomical variations, particularly “U-shaped” configurations requiring additional manipulation. The longest T4 (48.0 min in Rat 4) was attributed to challenging celiac trunk anatomy combined with initial learning curve effects. These observations highlight the importance of anatomical familiarity and microcatheter maneuvering skills in reducing the procedural time.

As interventional radiologists engaged in daily interventional practice, we fully recognize the importance of performing human-like TACE procedures in animal models for clinical liver cancer research. Previous studies of TACE in liver cancer animal models have largely employed laparotomy with hepatic arterial drug injection. Although widely adopted, this approach has several limitations. Compared to open laparotomy with hepatic arterial injection for TACE, the transfemoral arterial approach for TACE has several advantages: (I) minimally invasive: it improves animal welfare, and ensures greater compliance with animal ethics; (II) unparalleled clinical relevance: this represents its greatest value. It not only delivers the therapeutic agent but also fully simulates the entire clinical pathway of “arterial puncture → catheter navigation → selective cannulation → angiographic confirmation → chemoembolization”. Consequently, it provides highly translatable data on embolic materials, drug efficacy, drug resistance, and imaging evaluation; and precision treatment: it enables superselective embolization of tumor-feeding arteries using microcatheters, maximizing tumor destruction while preserving normal liver tissue and function. However, this method also has several drawbacks: (I) a high technical threshold requiring specialized expertise; operators must have a solid foundation in both clinical and imaging knowledge, resulting in a steep learning curve; (II) dependence on expensive specialized equipment (e.g., digital subtraction angiography systems) and exposure to radiation; and (III) prolonged procedure duration and potential risk of vascular injury.

This study had several limitations. First, as a technical feasibility study, it did not evaluate treatment efficacy endpoints such as the tumor necrosis rate or pathological response. Imaging assessments of tumor response at different time points post-TACE and histological analyses were not performed. Although human-simulated TACE was successfully performed in Foxn1^rnu nude rat PDX HCC models, standardized protocols for embolic agents and chemotherapeutic dosages in rats are lacking. Future studies should optimize embolization regimens to provide a theoretical basis for subsequent research. Additionally, attempts at transcaudal artery access were limited by the small vessel diameters of these rats. A comparative study of transfemoral versus transcaudal TACE in rat HCC models is warranted to further validate the transvascular preclinical research techniques.

Conclusions

TACE via the transfemoral approach in small-sized, low-body-weight nude rat orthotopic PDX HCC models is reproducible and technically feasible.

Acknowledgments

We sincerely acknowledge the support and assistance provided by the Department of Interventional Radiology and the Molecular Imaging Laboratory at The Third Affiliated Hospital of Sun Yat-sen University throughout this study.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1248/rc

Funding: This study was supported by the National Natural Science Foundation of China (Grant No. 82072035).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-1248/coif). M.H. reports grant from the National Natural Science Foundation of China (grant No. 82072035) awarded to the institution. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (Nos. II2025-036-01 and SYSU-IACUC-2024-002867) granted by the Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University and the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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