The Effect of Synergistic Immuno-Photothermal-Nanotherapy (SYMPHONY) on Immune Response at Distant Bladder Cancer Tumor Sites Using Murine Window Chamber Model
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Bladder cancer has been ranked as one of the top ten and top twenty most commonly occurring cancers in men and women, respectively, with approximately half of the diagnoses being late stage and/or metastatic disease. The current standard-of-care treatment for metastatic bladder cancer is cisplatin-based chemotherapy, but only about 60% of patients qualify for this treatment option and the remaining cohort have few alternatives available. In fact, the only widely considered alternative is immune checkpoint blockades, or immunotherapies, which work to reactivate inhibited functions of immune cells. Unfortunately, these alone have not proven effective against metastatic malignancies. We believe that we can enhance the effects of clinically available immunotherapies with the addition of nanostar mediated photothermal therapy to the primary tumor. In fact, this combination of anti-PD-L1 immune checkpoint blockade and gold nanostar mediated photothermal therapy, henceforth called SYnergistic iMmuno PHOtothermal NanotherapY (SYMPHONY), was previously tested in a pilot study where C57BL/6 mice were injected with MB49 bladder cancer cells at two locations. One of the sites was treated with one of five treatments and the second remained untreated. Both tumor volumes were measured over time and the survival of the mice was also documented. This study resulted in one of the five SYMPHONY mice having complete tumor control after treatment and no other treatment group had this outcome. Upon rechallenge of the same tumor cell line, a tumor did not grow suggesting long-term immunity to this cancer. Along with a proof of concept, these studies were successful in identifying that macrophages and T-cells are associated with the tumor eradication. However, there is still little known about the quantification of the immune response at the distant tumor site after treatment. Our long-term goal is to develop an effective alternative treatment option for patients with metastatic bladder cancer. The overall objective of this application was to quantify the immune response of transgenic mice with fluorescent reporter genes on monocytes, natural killer cells, and dendritic cells undergoing one of four treatments (SYMPHONY, photothermal therapy alone (GNS), immunotherapy alone (anti-PD-L1) or no treatment (control)) as well as identify time points within the week following therapy in which additional studies could be conducted. Our central hypothesis was that mice treated with SYMPHONY would exhibit an elevated immune cell infiltration at the site of the distant tumor within ~48 hours post treatment, and that SYMPHONY will induce a greater immune response at the distant tumor site compared to anti-PD-L1 alone. This hypothesis was tested by implanting a primary flank tumor and a smaller, untreated distant tumor in 14 mice. The distant tumor cells were first stained with a far-red DiD fluorescent dye before injection into the center of a dorsal skinfold window chamber. The primary tumors were treated with one of the four treatment modalities and the window chamber was imaged using intravital microscopy for 7 days after the treatment. From this study, we found that the greatest change in the immune signal for any group occurred in the SYMPHONY group on the day of treatment and this immune signal remained elevated throughout the 7-day imaging period. This change was greater than all other treatment groups, including the anti-PD-L1 group, therefore, we accept our hypothesis. Further work should focus on assessing the immune response in mice on the day of treatment, looking at other immune cell types and by quantifying the effects of laser treatment on this day. It is noted that using the immune signal surrounding the distant tumor to predict an immune response within the tumor requires imaging at intervals more frequent than every 24 hours because of estimated macrophage travel velocity in tissue, where a more suitable frequency would be every 1.5-2 hours.
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