Ultrasound imaging-guided protocol for monitoring tumor growth in orthotopic rat model of hepatocellular carcinoma

doi:10.4254/wjh.v17.i10.109517

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Oct 27, 2025 (publication date) through Oct 27, 2025

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World Journal of Hepatology

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1948-5182

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Basic Study

World J Hepatol. Oct 27, 2025; 17(10): 109517
Published online Oct 27, 2025. doi: 10.4254/wjh.v17.i10.109517

Ultrasound imaging-guided protocol for monitoring tumor growth in orthotopic rat model of hepatocellular carcinoma

Aswathy R Devan, Sithara Manakkaparambil Sasidharan, Kannoth Panicker Sreekumar, Ayalur Kodakara Kochugovindan Unni, Sabitha Mangalathillam, Abna Ansar, Ashok R Unni, Lekshmi R Nath

Aswathy R Devan, Lekshmi R Nath, Department of Pharmacognosy, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Science Campus, Ponekkara PO, Kochi 682041, Kerala, India

Sithara Manakkaparambil Sasidharan, Kannoth Panicker Sreekumar, Ayalur Kodakara Kochugovindan Unni, Ashok R Unni, Central Lab Animal Facility, Department of Veterinary Medicine, Amrita Institute of Medical Sciences, Kochi 682041, Kerala, India

Sabitha Mangalathillam, Department of Pharmaceutics, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Science Campus, Kochi 682041, Kerala, India

Abna Ansar, Department of Pharmacy Practice, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Science Campus, Kochi 682041, Kerala, India

Co-first authors: Aswathy R Devan and Sithara Manakkaparambil Sasidharan.

Co-corresponding authors: Ashok R Unni and Lekshmi R Nath.

Author contributions: Nath LR and Unni AR contributed to conceptualization, design and methodology development, designated as co-corresponding authors; Unni AR, Sasidharan SM, Devan AR performed imaging analysis; Devan AR and Sasidharan SM contributed equally in data curation, data organization, writing of the original manuscript and designated as co-first authors; Nath LR performed funding acquisition, project administration and supervision; Nath LR and Unni AR contributed in reviewing and editing of the manuscript; Ansar A provided statistical expertise and contributed to data interpretation; Sreekumar KP, Unni AKK and Mangalathillam S performed formal analysis, review and editing; All authors have read and agreed to the published version of the manuscript. Devan AR performed the data collection, analysis and prepared the first draft of the manuscript. Sasidharan SM contributed in laparotomy surgery, histopathological evaluation and data analysis. Both authors made equal contribution towards the completion of the project and thus qualified as the co-first authors of the paper. Both Nath LR and Unni AR have played important roles in experimental design, methodology, data interpretation and manuscript preparation. Nath LR conceptualized, designed and supervised the whole process of the project. Nath LR applied for and obtained the funds for this research project. She reviewed and edited the manuscript and was responsible for submission of the article. Unni AR was instrumental in laparotomy procedures for tumor induction, ultrasound and micro-CT analysis. He contributed to the interpretation of imaging data and optimization of the tumor induction protocol. Both authors have made crucial and indispensable contributions towards the completion of the project and thus qualified as the co-corresponding authors of the paper.

Supported by Amrita Vishwa Vidyapeetham Seed Grant, No. K-PHAR-24-722; and DST INSPIRE Fellowship, No. IF190226.

Institutional animal care and use committee statement: The experimental protocol was approved by the IAEC with reference number [IAEC/2023/1/10]. All animal procedures were performed in accordance with the guidelines set by Committee for the Control and Supervision of Animal Experiments (CCSEA) with the approval of the Institutional Animal Ethics Committee (IAEC) of Amrita Vishwa Vidyapeetham.

Conflict-of-interest statement: All authors declare no competing interests.

ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.

Data sharing statement: The authors confirms that the data supporting the finding of this study are available within the manuscript.

Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/

Corresponding author: Lekshmi R Nath, PhD, Associate Professor, Department of Pharmacognosy, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Science Campus, Ponekkara PO, Kochi 682041, Kerala, India. lekshmirnath@aims.amrita.edu

Received: May 14, 2025
Revised: June 20, 2025
Accepted: September 25, 2025
Published online: October 27, 2025
Processing time: 167 Days and 1.2 Hours

Abstract

BACKGROUND

Syngeneic orthotopic tumor models offer an optimal functional tumor–immune interface for hepatocellular carcinoma research. Yet, unpredictable growth kinetics and spontaneous regression pose major obstacles. Efficient induction protocols and continuous monitoring are therefore essential. Routine exploratory surgeries are ethically untenable, making non-invasive imaging modalities attractive alternatives. High-resolution magnetic resonance imaging and microcomputed tomography deliver detailed insights but incur substantial equipment costs, radiation risks, time demands, and require specialized expertise—challenges that limit their routine use. In contrast, ultrasound (US) imaging emerges as a cost-effective, radiation-free, and rapid approach, facilitating practical and ethical longitudinal assessment of tumor progression in preclinical studies.

AIM

To optimize the orthotopic hepatocellular carcinoma model and evaluate the potential of US imaging for accurate and cost-effective tumor monitoring.

METHODS

Hepatocellular carcinoma was induced in 28 Sprague Dawley rats by implanting 5 × 10⁶ N1S1 cells into the left lateral hepatic lobe. Tumor progression was monitored weekly via US. Upon reaching 100-150 mm³, an experimental group (n = 14) received Sorafenib (40 mg/kg) orally on alternate days for 28 days; efficacy was compared to untreated controls. US accuracy was validated against micro-computed tomography, gross caliper measurements and histopathological analysis. Reliability and operator proficiency in US assessment were also evaluated.

RESULTS

US images procured 7-day post-surgery revealed a well-defined hypoechoic nodule at the left liver lobe tip, confirming successful tumor induction (mean volume 130 ± 39 mm³). Only three animals exhibited spontaneous regression by week 2, underscoring the model’s stability. Sorafenib treatment elicited a marked tumor reduction (678 ± 103 mm³) vs untreated control (6005 ± 1760 mm³). US assessment demonstrated robust intra and interobserver reproducibility with high sensitivity and specificity for tumor detection. Moreover, US derived volumes correlated strongly with gross caliper measurements, histopathological analysis, and microcomputed tomography imaging, validating its reliability as a non-invasive monitoring tool in preclinical hepatocellular carcinoma studies.

CONCLUSION

The results demonstrate that US imaging is a reliable, cost-effective, and animal sparing approach with an easy to-master protocol, enabling monitoring of tumor progression and therapeutic response in orthotopic liver tumor models.

Keywords: Hepatocellular carcinoma; Syngeneic N1S1 orthotopic model; Ultrasound imaging; Tumor growth monitoring; Therapeutic response; Cost-effective imaging tool; Inter-observer reproducibility; Receiver operating characteristics analysis

Core Tip: Orthotopic rat tumor models are valuable for replicating human tumor microenvironments, yet tumor regression complicates consistent growth assessment, underscoring the need for reliable imaging modalities. While advanced techniques like magnetic resonance imaging and micro-computed tomography deliver high-resolution data, their expense, effects and demands limit routine implementation. We present an ultrasound (US) imaging protocol that achieves accuracy and sensitivity for regular tumor growth monitoring. Our results demonstrate US’s potential for early detection of orthotopic liver tumors and robust evaluation of antitumor drug efficacy, offering a cost-effective, accessible alternative strategy in preclinical research.