The diagnositic value of dynamic contrast-enhanced ultrasound for evaluation of tissue oxygen status in rat hepatoma model

Abstract

Background: Hypoxia is a characteristic of solid tumors, but whether significant hypoxia exists in the hepatocellular carcinoma remains unclear. This animal study aims to explore the value of dynamic contrast-enhanced ultrasound (CEUS) quantitative parameters to evaluate the oxygen status in two rat hepatoma models.

Materials and methods: N1S1 and McA-RH7777 S-D rat orthotopic hepatoma models were established. Once the tumors reached a diameter of 10-15 mm, CEUS and oxygen partial pressure (pO2) polarography were performed. Immunohistochemical staining for HIF-1α and pimonidazole was conducted after euthanizing the rats. Correlation between quantitative CEUS parameters, pO_2, and the immunohistochemical integrated optical density (IOD) was analyzed to assess the predictive ability of CEUS quantitative parameters for the tissue oxygen environment.

Result: Eleven N1S1 models and ten McA-RH7777 models were established successfully. There was no significant difference in pO₂ (35.5 mmHg vs 32.2 mmHg, P = 0.917), IOD of HIF-1α (13.4 vs 20.0, P = 0.159) and pimonidazole (0.70 vs 1.30, P = 0.926) between the tumor and the peritumoral liver tissue. The pO₂ values were correlated with CEUS quantitative parameters including mean time-intensity curves (mTIC) (P = 0.003), peak intensity (PKI) (P = 0.010), area under the curve (AUC) (P = 0.009), area under the wash-in curve (WiAUC) (P = 0.006), and arrival time (AT) (P = 0.033). The IOD of HIF-1α correlated with AUC (P = 0.022), WiAUC (P = 0.009), ascending slope (AS) (P = 0.044), and falling time (FT) (P = 0.009). Multiple linear regression indicated that the "short AT" was an independent protective factor for hypoxia (β = -2.347, 95% CI: -4.948, -0.394, P = 0.022), and CEUS had the ability to predict the tumor pO₂ (P = 0.003).

Conclusion: No evidence of significant hypoxia was identified in two rat orthotopic hepatoma models. Quantitative CEUS parameters correlated with the oxygen status of the tumor, which could be utilized to predict the tumor tissue pO₂.

Keywords: Hypoxia; Liver neoplasm; Ultrasonography.

Fig. 1

The schematic diagram shows the experimental procedure in this study. A Animal modeling and monitoring. Two orthotopic hepatoma (N1S1 and McA-RH7777) Sprague–Dawley rat models were established. After models were established, B-mode ultrasound was used to monitor the growth of the hepatoma; B Dynamic CEUS image acquisition and quantitative CEUS parameter analysis; C Tissue oxygen partial pressure measurement by polarography method. Under the guidance of ultrasound, the oxygen electrode was inserted into the corresponding position of CEUS observation. The pO₂ value of hepatoma and peritumoral liver parenchyma was recorded; D Pathological and immunohistochemical examination. After the hepatoma was harvested, WSI was obtained according to the section of CEUS observation. CEUS: contrast-enhanced ultrasound; IOD was analyzed WSI: whole-slide images; IOD: integrated optical density

Fig. 2

Examples of ultrasound image acquisition, quantitative parameter analysis, and oxygen partial pressure measurement. A The length and height of the tumors were measured by two-dimensional ultrasound; B The width of the tumor was measured by two-dimensional ultrasound; C CDFI showed abundant blood flow signals in the tumor; D The peritumoral liver parenchyma, the whole tumor area and different subregions of the tumor were delineated, and their parametric imaging, enhancement curves were shown. E CEUS quantitative parameters were shown. F Under the guidance of ultrasound, the oxygen electrode was inserted into the superficial layer of the tumor, corresponding to the green circle area (Fig. 3E). CDFI: Color Doppler flow imaging; CEUS: contrast-enhanced ultrasound

Fig. 3

The violin plot illustrates the distribution of oxygen partial pressure values in the different models. A The pO₂ value of all hepatoma and peritumoral liver parenchyma; B The pO₂ value of N1S1 tumor and peritumoral liver parenchyma; C The pO₂ value of McA-RH7777 tumor and peritumoral liver parenchyma

Fig. 4

Correlation between the IOD of hypoxia markers and CEUS quantitative parameters. A Linear correlation between IOD of HIF-1α and AUC; B Linear correlation between IOD of HIF-1α and WiAUC; C Linear correlation between IOD of HIF-1α and AS; D Linear correlation between IOD of HIF-1α and FT; E HIF-1α was weekly expressed in the perinecrotic part of the tumor; F Pimonidazole was weekly expressed in the perinecrotic part of the tumor; G HIF-1α was not obviously expressed in tumors without necrosis; H Pimonidazole was not obviously expressed in tumors without necrosis