Category: Formulation and Quality
Purpose: Studies have demonstrated that low intensity focused ultrasound (LOFU) can be an effective method to increase tumor vascular permeability and neoantigen expression, resolving barriers to drug delivery and immune cell filtration, respectively. Mechanical sound waves disrupt tumor cells, causing tumors to express stress signals and initiate an anti-tumor immune response. This effect is further enhanced by microbubbles (MBs), 2-10 μm lipid-shelled gas vesicles, which rapidly expand and contract in response to acoustic pressure. This phenomenon, known as cavitation, generates additional mechanical stress when acting upon cells. Specifically, cavitation causes tumor cells to express stress signals and recruit antigen-presenting cells that present neoantigens to CD4+ and CD8+ T cells. However, tumor cells can express PD-L1 receptors that inhibit and exhaust T cell response. Checkpoint inhibitors such as anti-PD1 agents, namely pembrolizumab, aim to prevent T cell exhaustion and enhance the immune response. Although pembrolizumab has had some success in treating patients with melanoma, the lack of drug and immune cell infiltration has prevented interpatient success. We hypothesize that mechanical stimulation by LOFU and MBs will cause implanted B16F10 melanoma cells to release stress signals, which will in turn recruit CD8+, CD4+, macrophages and dendritic cells to the tumor. (Figure 1). These results will inform future studies to combine ultrasound and immunotherapy for melanoma treatment.
Methods: 35 female C57BL6/J mice were injected subcutaneously with 1 x 106 B16F10 murine melanoma cells. After approximately 11 days, the tumors were treated with a Therapy and Imaging Probe System (TIPS, Philips Research, Briarcliff NY, USA), which has a focused transducer consisting of an eight-element annular array. Immediately prior to ultrasound treatment, a bolus injection of 7 x 107 size-isolated, lipid shell microbubbles from Advanced Microbubbles Laboratories, LLC was administered over a few seconds. Pulsed LOFU was then raster scanned along a grid, covering approximately 1 mm outside of the tumor. Relevant parameters include: 1 MHz frequency, 0.5 MPa amplitude, 10% duty cycle, 0.13 W power, 1 ms pulse width, with a total treatment time of about 20 minutes. Mice were then placed into one of three groups to be sacrificed 2 days, 1 week or 2 weeks post-treatment. Figure 2 summarizes the treatment scheme. Tumors, spleen and tumor draining lymph nodes were harvested and analyzed by flow cytometry to determine immune cell populations.
Results: Our data demonstrates that the greatest immune response occurs within 48 hours of treatment, the most noteworthy being a 3X increase in F4/80 macrophages in the tumor-draining lymph node (TDLN), indicating an inflammatory response (Figure 3). Also, in the TDLN, we found a significant decrease in the CD4+ population, which could represent CD4+ activation and migration through the lymphatic system to the tumor. There was a slight yet insignificant increase of CD4+ population in the tumor at the final timepoint of 10 days after treatment, potentially signifying an adaptive response. The percentage of cytotoxic CD8+ T cells in the tumor increased from 7% (control) to 11% (treatment) (Figure 3). There were no significant changes of immune cells in the spleen (not shown).
Conclusion: These preliminary results demonstrate that low intensity, pulsed therapeutic ultrasound in combination with microbubble contrast agents can enhance tumoral immune cell infiltration. Importantly, the heightened immune response after 48 hours informs a future study to combine this treatment with commercial anti-PD-1 checkpoint immunotherapy for the treatment of melanoma. Later time points are also needed to further elucidate the adaptive effect of LOFU and MBs.