DISCUSSION
Noninvasive imaging techniques are being developed and improved for identifying and evaluating pathophysiological features of tumors in order to assist in the planning of individual patient treatment protocols [10, 11]. Particularly, new imaging approaches that assess tumor vascularization have improved diagnosis and treatment prediction. In this study, we evaluated (i) caliper and contrast enhanced ultrasonography (CEUS) to define tumor dimensions and (ii) contrast enhanced ultrasound (CEUS) and laser speckle contrast imaging (LSCI) to appreciate tumor vascularization.
The murine melanoma model is based on B16F10 cells known for their aggressiveness, high metastatic potential and high expression of the PD-1 receptor [12]. Taken together, these data confirm the value of evaluating the impact of anti-PD1 antibodies in limiting tumor growth. Although the survival data did not allow us to conclude that there was a significant benefit from anti-PD1, they showed that before 7 days of treatment, no mortality was observed, whatever the group of animals, justifying the use of this time frame to evaluate the different imaging techniques.
To determine tumor dimensions, caliper and CEUS were used in this study. Although tumor volumes are similar for both methods, as recently described in non-melanoma skin cancer [13], our data suggested an interest in CEUS because of various biases described with caliper. Firstly, it has been described that caliper often overestimated tumor volume. Furthermore, the caliper measurements were smaller for small tumors compared to greater tumors also relatively seen. Consequently, volume changes measured with caliper in small and large tumors are not comparable and effects of anti-cancer drugs can easily be missed as tumors will tend towards being determined with a greater bias as they grow larger [14].
The value of CEUS has recently been confirmed by Makouei et al. [15]. In a pilot study conducted on mice with a soft tissue tumor, they confirmed that ultrasonography is a feasible and accurate imaging method to assess the tumor volume. However, these authors pointed out a number of limitations. Among these is the failure to take account of vascularization, which is an important factor in the diagnosis and treatment of metastatic tumors. To overcome these limitations, our study was supplemented by microcirculation analyses through LSCI in comparison with CEUS technique.
While CEUS analysis showed a reduction in microcirculation at treatment initiation, no difference was observed after 7 days of treatment suggesting that CEUS is not sufficiently robust for long-term studies. On the other hand, LSCI analysis confirmed a decrease in tumor perfusion over time in mice treated with anti-PD1 compared with control mice. Compared to CEUS, LSCI measurement did not need a direct skin contact which is an advantage in these types of analyses. Indeed, a strongly hold of the ultrasound probe can create a local ischemia or reduce the microcirculatory vascularization and create biased results. Besides, LSCI measure results from local microvascular analysis without a blank analysis, which minimize artefacts. Last, if using the same experimental conditions, LSCI showed a good reliability and reproducibility. These data suggest the potential clinical value of LSCI in improving the diagnosis of metastatic melanoma.