Reducing tumor growth and angiogenesis using a triple therapy measured with Contrast-enhanced ultrasound (CEUS)

Background: To evaluate the in vivo response by detecting the anti-angiogenic and invasion-inhibiting effects of a triple-combination-therapy in an experimental-small-animal-squamous-cell-carcinoma-model using the "flash-replenishment" (FR) method to assess tissue hemodynamics via contrast-enhanced-ultrasound (CEUS). Methods: Human hypopharynx-carcinoma-cells were subcutaneously injected into the left flank of 22-female-athymic-nude-rats. After seven days of subcutaneous tumor growth, FR-measurements were performed on each rat. Treatment-group and control-group were treated every day for a period of one week, with the treatment-group receiving solvents containing a triple therapy of Upamostat (R), Celecoxib (R) and Ilomastat (R) and the control-group solvents only. On day seven, follow-up measurements were performed using the same measurement protocol to assess the effects of the triple therapy. VueBox (R) was used to quantify the kinetic parameters and additional immunohistochemistry analyses were performed for comparison with and validation of the CEUS results against established methods (Proliferation/Ki-67, vascularization/CD31, apoptosis/caspase3). Results: Compared to the control-group, the treatment-group that received the triple-therapy resulted in a reduction of tumor growth by 48.6% in size. Likewise, the immunohistochemistry results showed significant decreases in tumor proliferation and vascularization in the treatment-group in comparison to the control-group of 26%(p <= 0.05) and 32.2%(p <= 0.05) respectively. Correspondingly, between the baseline and follow-up measurements, the therapy-group was associated with a significant(p <= 0.01) decrease in the relative-Blood-Volume(rBV) in both the whole tumor(wt) and hypervascular tumor(ht) areas (p <= 0.01), while the control-group was associated with a significant (p <= 0.01) increase of the rBV in the wt area and a non-significant increase (p <= 0.16) in the ht area. The mean-transit-time (mTT) of the wt and the ht areas showed a significant increase (p <= 0.01) in the follow-up measurements in the therapy group. Conclusion: The triple-therapy is feasible and effective in reducing both tumor growth and vascularization. In particular, compared with the placebo-group, the triple-therapy-group resulted in a reduction in tumor growth of 48.6% in size when assessed by CEUS and a significant reduction in the number of vessels in the tumor of 32% as assessed by immunohistochemistry. As the immunohistochemistry supports the CEUS findings, CEUS using the "flash replenishment"(FR) method appears to provide a useful assessment of the anti-angiogenic and invasion-inhibiting effects of a triple combination therapy.