Ultrasound imaging in orthotopic hepatocellular carcinoma models: Promise, practicality, and points for refinement

Abstract

The study by Devan et al presents an ultrasound-based protocol for monitoring tumor growth in a syngeneic orthotopic rat model of hepatocellular carcinoma (HCC). This approach is commendable for its reproducibility, cost-effectiveness, and alignment with ethical imperatives, particularly in reducing the need for invasive assessments. The strong correlation of ultrasound-based volumes with histology and therapeutic response highlights its translational promise. However, certain considerations merit further discussion. Ultrasound imaging, while accessible, is inherently operator-dependent, and its accuracy may decline with irregular or heterogeneous tumor morphology. Moreover, the exclusive reliance on the rat hepatoma cell line (N1S1) cells raises questions about generalizability to other HCC models with differing immune interactions. Future refinements should standardize training protocols, incorporate multimodal validation, and explore diverse tumor settings. Despite these limitations, the study provides a useful approach, and its broader integration could democratize preclinical oncology research, especially in resource-constrained environments.

Key Words: Hepatocellular carcinoma; Ultrasound imaging; Histology; Tumor growth monitoring; Tumor volumetry

Core Tip: High frequency ultrasound is a rapid, low-cost, minimally invasive tool for serial volumetric monitoring of orthotopic hepatocellular carcinoma that correlates well with terminal histology and can match micro computed tomography (CT)/magnetic resonance imaging (MRI) for gross tumor size, but it lacks cellular resolution and remains operator-dependent. To increase translational impact, future work should standardize acquisition and training, perform multimodal validation in subset cohorts (histology, micro CT/MRI), and adopt contrast-enhanced/photoacoustic imaging, along with automated radiomics/artificial intelligence segmentation to reduce bias and improve sensitivity.

TO THE EDITOR

Devan et al[1] describe an ultrasound-based protocol for longitudinal monitoring of tumor growth in a syngeneic orthotopic rat model of hepatocellular carcinoma (HCC). The authors demonstrate that high-frequency ultrasound yields reproducible volumetric measurements that strongly correlate with terminal histology and with observed therapeutic responses, enabling serial, noninvasive assessment of tumor kinetics. The protocol is low-cost, scalable, and reduces reliance on invasive endpoints, aligning with 3R (replacement, reduction, and refinement) principles. By validating imaging against histopathology and treatment outcomes, the study advances preclinical HCC models and offers a practical route to broaden access to longitudinal efficacy testing, particularly in resource-limited settings, and accelerate translational research[1].

EXPERIMENTAL APPROACH AND ANALYTICS

A syngeneic orthotopic HCC model was established in male Sprague Dawley rats by subcapsular implantation of the rat hepatoma cell line (N1S1) hepatoma cells into the left lateral liver lobe. Tumor-bearing rats were randomized into control and sorafenib-treated groups, and tumor progression was monitored longitudinally using high-frequency ultrasound. Volumetric estimates were validated against micro computed tomography (CT), gross, and histopathological measurements to confirm accuracy. Reproducibility was assessed through multi-observer analysis, and a structured training protocol was used to evaluate the learning curve of novice operators. Ultrasound performance metrics, including correlation with histology and diagnostic accuracy, were analyzed statistically to establish the reliability and translational applicability of this noninvasive monitoring approach[1].

MAJOR INSIGHTS AND BIOLOGICAL SIGNIFICANCE

Devan et al[1] show that high-frequency ultrasound provides reproducible longitudinal volumetry in a syngeneic orthotopic rat HCC model, correlating strongly with terminal histopathology and detecting treatment-related changes in tumor kinetics. The technique is low-cost and minimally invasive, allowing serial assessments in the same subjects and reducing animal use and invasive procedures. Its scalability and high throughput make it suitable for repeated efficacy testing, even in resource-limited laboratories[2,3]. Key limitations of the study include operator dependence and reduced accuracy for very small, irregular, or highly heterogeneous lesions. In addition, reliance on a single N1S1 cell line constrains the generalizability of findings across the diverse phenotypes of HCC. To translate broadly, standardized training and quality assurance, multimodal validation with magnetic resonance imaging (MRI), micro CT, and histology, contrast-enhanced or photoacoustic augmentation, and artificial intelligence (AI)-driven automated segmentation are recommended, and prospective multi-center preclinical validation efforts as mentioned in Table 1[1-12].

Table 1 Comparative overview of imaging modalities utilized in preclinical hepatocellular carcinoma models.

Imaging modality	Advantages	Limitations	Ref.
High-frequency ultrasound	Real-time, non-invasive, affordable; enables serial monitoring and correlates well with histology or MRI	Limited soft-tissue contrast for small or heterogeneous lesions; accuracy may decrease for deep or poorly defined tumors	Devan et al[1], Roth et al[2], Molière et al[3]
Histology	Gold standard for cellular detail; assessment of necrosis, vessels and immune cells	Requires tissue harvest; terminal procedure; not suitable for longitudinal studies	Herrero de la Parte et al[5], Choi et al[10]
Micro computed tomography	High-resolution three dimensional anatomy and vasculature; volume quantification	Involves radiation exposure and need for contrast agents; limited soft-tissue contrast	Singh et al[8], Cigliano et al[11]
MRI	Excellent soft-tissue contrast; quantitative; reproducible; translational	Expensive; long scan time; limited accessibility in small animal settings	Rojas et al[4], Renzulli and Giampalma[12]
Photoacoustic imaging	Combines optical and ultrasound contrast to visualize vascular oxygenation and perfusion	Restricted penetration depth; often relies on exogenous optical contrast agents; specialized setup required (operator dependent)	Nyayapathi et al[7]
Super-resolution ultrasound	Allows visualization of microvascular changes beyond conventional resolution	Requires contrast microbubbles and operator dependent complex data processing; limited availability and standardization	Hoyt[6], Riberdy et al[9]

Comparison of major imaging modalities used in preclinical hepatocellular carcinoma models, highlighting their respective advantages and limitations. MRI: Magnetic resonance imaging.

EMERGING TRENDS AND OPPORTUNITIES

Devan et al[1] reinforce a pivotal shift: High-frequency ultrasound is evolving from a practical imaging tool into a versatile, translational platform for longitudinal HCC research. Their demonstration of reproducible, treatment-sensitive volumetry complements benchmark studies showing ultrasound outperforms caliper-based measures and closely approximates MRI for gross tumor size[2,4,5]. Practical workflow innovations, rapid, high-throughput scanning protocols, and ultrasound-based orthotopic implantation further reduce inter-sample variability and increase experimental efficiency[3,13]. At the technological frontier, super-resolution ultrasound and microvascular mapping enable detection of vascular remodeling and perfusion changes that are mechanistically linked to antiangiogenic and immunomodulatory therapies[6]. Combining photoacoustic imaging with contrast-enhanced ultrasound helps overcome the limitations of standard brightness mode ultrasound and improves detection of tissue differences and blood flow[7]. Ultrasound serves both diagnostic and therapeutic roles, as ultrasound-mediated drug delivery and sonoporation enable integrated imaging and treatment approaches well-suited for testing in orthotopic HCC models[14].

Recent technological advances are improving methodological rigor. Studies comparing ultrasound findings with histopathology and other imaging techniques such as micro CT, positron emission tomography (PET)-CT, MRI, bioluminescence etc. strengthen external validity and support the use of ultrasound-based measures as reliable surrogate endpoints when properly validated[4,8]. At the same time, radiomics and AI-based image segmentation are helping reduce operator dependence, extract detailed quantitative biomarkers (such as texture and vascular features), and develop predictive imaging signatures of treatment response[9]. Comprehensive immune profiling and evaluation of common syngeneic models, like N1S1, help define the limits of single-model studies and emphasize the need for cross-model validation to better capture HCC heterogeneity[10,11]

Overall, these advances offer a practical pathway to expand longitudinal HCC research: Low-cost high-frequency ultrasound enables widespread use in resource-limited settings, while contrast techniques, multimodal validation, and AI analytics improve sensitivity, objectivity, and translational potential[12]. Combining Devan et al’s reproducible protocol[1] with these technological and methodological improvements can accelerate reliable, ethical, and clinically informative preclinical oncology studies.

FUTURE DIRECTIONS

To realize ultrasound’s full translational potential for orthotopic HCC, coordinated efforts must prioritize standardization, multimodal validation, technological augmentation, and broad biological testing. Validated acquisition standard operating procedures for high-frequency ultrasound, operator training curricula, and quality assurance checklists should be developed and disseminated to reduce inter-operator variability and enable multicenter comparability[3]. Routine validation of ultrasound-derived volumetry and functional indices against histopathology and at least one orthogonal imaging modality (MRI, CT, PET-CT or bioluminescence) in representative subsets will establish external validity across tumor morphologies and growth kinetics[2,4,8]. Integrating super-resolution, contrast-enhanced, and photoacoustic imaging can enhance detection of microvascular changes vital for targeted therapies[6,7]. Open imaging datasets and AI/radiomics tools will improve automation and biomarker discovery[9]. Validating ultrasound workflows across diverse HCC models; including syngeneic, carcinogen-induced, and patient-derived xenograft systems, will ensure broader translational relevance[10,11].

CONCLUSION

Devan et al’s ultrasound-based protocol[1] establishes high-frequency ultrasonography as a practical, animal-sparing tool for longitudinal volumetric assessment in orthotopic HCC, demonstrating strong concordance with histopathology and treatment response. When combined with standardized training, multimodal validation (MRI/micro CT/histology), and emerging contrast and AI-driven analytics, ultrasound can deliver scalable, cost-effective imaging endpoints for preclinical oncology. Broad adoption and systematic cross-model validation will be essential to translate these advances into reproducible, clinically informative pipelines that accelerate HCC therapeutic discovery and improve patient-centric outcomes, as illustrated in Figure 1[1].

Figure 1 Representative high-frequency ultrasound–based monitoring of orthotopic hepatocellular carcinoma established using the rat hepatoma cell line (N1S1). Tumor volume measurements obtained by ultrasound demonstrate an excellent correlation with reference histological and volumetric assessments (r = 0.998, P < 0.001; Devan et al[1]), underscoring the accuracy, reproducibility, and experimental utility of this imaging approach for preclinical tumor evaluation. CT: Computed tomography; HCC: Hepatocellular carcinoma; 3D: Three dimensional; 3R: Replacement, reduction, and refinement. Created in BioRender (www.biorender.com).