(P 108) EFFECT OF TUMOUR-TARGETED HYPERTHERMIA AND CHEMOTHERAPY ON THE IMMUNE PROFILE OF A MURINE MODEL OF EMBRYONAL RHABDOMYOSARCOMA

Objective: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Prognosis remains poor in patients with recurrent/relapsed RMS, and treatment-associated toxicity is prevalent. Current standard of care chemotherapy includes vincristine, while second line chemotherapy includes doxorubicin which is associated with dose-limiting cardiotoxicity. Thermosensitive liposomal doxorubicin (TLD) releases doxorubicin at a temperature of approximately 40-42°C. To heat the drug in vivo, magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) can produce tumour-localized hyperthermia (HT) to target doxorubicin release to the site of disease. HT+TLD treatment resulted in improved survival and reduced off-target toxicity in an immunocompetent embryonal RMS (ERMS) mouse model. HT has also been shown to modulate the immune response, which may allow for a synergistic effect with immunotherapies. The objectives of this project are to characterize the immune profile of a murine model of ERMS over time and to determine what effect chemotherapy and/or HT has on immune infiltrates.

Methods: C57BL/6 mice were intramuscularly injected with a murine ERMS cell line into the right hind limb to generate tumours. Mice were divided into 2 groups: HT or normothermia (NT). Tumours of HT mice were heated to 40.5°C for 21.5 minutes, while NT mice received no heat treatment. Drugs tested included vincristine (VINC), free doxorubicin (FD), and TLD resulting in 8 treatment groups: control (untreated), VINC, FD, TLD, HT, HT+VINC, HT+FD, and HT+TLD. Tumours were collected at 24 and 168 hours post-treatment (hpt). Immunohistochemistry for CD11b (myeloid immune cells), CD3 (T cells), and B220 (B cells) was conducted in n=99 tumours and quantified using HALO image analysis software. Imaging mass cytometry (IMC) for a panel of 17 markers was conducted for central and peripheral 1mm2 regions of interest in a subset of 3 tumours: untreated, HT 24 hpt, and HT 168 hpt, along with a mouse spleen used as a control.

Results: Of the three immunohistochemical markers, CD11b+ cells were the most abundant, followed by T cells, and very few B cells, which have been associated with increased survival and immunotherapy response in sarcoma. TLD alone decreased CD11b+ and T cells within tumours at 24 hpt (p=0.0032, 0.0137 respectively) (Figure 1). Vincristine also decreased CD11b+ cells within tumours at 168 hpt (p=0.0338) (Figure 1). HT increased CD11b+ cells in TLD treated tumours at 24 hpt, and increased B cells in tumours treated with no drug at 168 hpt (p=0.0101, 0.0489 respectively) (Figure 2). IMC revealed that the most abundant immune-related markers within tumours were MHC II, Sirpa, CD11b, and CD64, while the least abundant were CD8a, B220, CD22, and Ly6G (Figure 3). Comparison of IMC between differently treated tumour samples suggested central clustering of MHC II, increased CD31, increased Ly6G, increased peripheral CD64, and decreased dendritic cells (Cd11c+, CD64-) in the HT 24 hpt tumour compared to the untreated tumour (Figure 4).

Conclusion: The tumour microenvironment in this murine model of ERMS is macrophage dominated, with very few cytotoxic T cells, B cells, and neutrophils. Drug treatment alone can impact the immune microenvironment, with both TLD and VINC causing decreases in myeloid immune cell and T cell quantity in tumours at either 24 or 168 hpt. The mechanism behind this is under investigation. HT may be stimulating an immune response, particularly with respect to increased macrophages, neutrophils, and endothelial cells at 24 hpt, increased B cells at 168 hpt, and decreased dendritic cells (potentially due to migration to the lymph nodes for antigen presentation) at 24 hpt. Ultimately, this data will help us understand how we can harness HT-related immune stimulation from an already promising therapy (HT+TLD) to create an even greater synergistic effect with immunotherapy in order to increase treatment efficacy while reducing toxicity in RMS patients.