(P 124) EFFECT OF MAGNETIC RESONANCE GUIDED HIGH INTENSITY FOCUSED ULTRASOUND AND CHEMOTHERAPY ON THE IMMUNE PROFILE OF A MURINE MODEL OF RHABDOMYOSARCOMA

Objective: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma (STS), for which vincristine, actinomycin D, and cyclophosphamide (VAC) chemotherapy remains the backbone. In cases of recurrent or advanced RMS, alternate lines of chemotherapy include doxorubicin which, although effective, 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) combines MRI and ultrasound to produce targeted and controlled hyperthermia (HT). Previous unpublished work from our team demonstrates a survival advantage in RMS mice treated with TLD and HT compared to standard therapy. The objectives of this project are to characterize the immune profile of a murine model of embryonal RMS (ERMS) by investigating the quantity and location of immune cells within tumours, determining if HT enriches immune infiltration, and analyzing the immune response in RMS tumours over time post-treatment.

Methods: A syngeneic, immunocompetent ERMS mouse model previously developed by the Gladdy lab was employed. p53 heterozygous mice were intramuscularly injected with ~10⁴ p53 null, FGFR4 ^V550E mutant murine myoblast cells into the right hind limb at 4-6 weeks of age (Figure 1). Mice were divided into 2 groups: HT vs. normothermia (NT). The HT group underwent localized heating of tumours with MRgHIFU at 40°C for 20 minutes followed by intravenous chemotherapy. The drugs included free doxorubicin (FD), and TLD resulting in six treatment groups: control (untreated) HT, FD, HT+FD, TLD, and HT+TLD (Figure 1). Tumours were collected via necropsy at 24 and 168 hours post-treatment and immunohistochemistry was performed on tumour sections with antibodies against CD3 (T cells), CD11b (macrophages, monocytes, natural killer cells, granulocytes), and B220 (B cells) (Figure 1). Positively stained cells in whole tumour sections were counted using HALO image analysis software and sample tumour sections were manually counted and verified by a sarcoma pathologist.

Results: Data from n=65 tumours demonstrated that CD11b+ cells were the most abundant cell population of the three markers in the murine RMS tumours (900-9000 cells/mm2), while B lymphocytes, associated with increased survival and immunotherapy response in sarcoma, were sparse (Figure 2, 0-140 B220+ cells/mm2). CD3+ cells were present in the range of 0-800 cells/mm2 with the exception of outliers. There were more CD11b+ cells in the HT+TLD treated tumours compared to the TLD treated tumours at 24 hours post-treatment, and CD3+ cells were significantly lower in the HT+FD treated tumours compared to the FD treated tumours at 24 hours post-treatment, p=0.0019 and p=0.034 respectively (Figure 2). Overall, HT treated tumors (n=23) had significantly increased B220+ cells at 168 hours post-treatment compared to all NT tumors (n=19) (Figure 3), p=0.019, while CD3 (24 and 168h), B220 (24h), and CD11b (24 and 168h)+ cells showed no significant difference between HT and NT groups. Samples sizes were small in this pilot project, and interpretation may be confounded by variable tumor size at treatment initiation, tumour necrosis, and thermal artifacts. However, additional replicates (10 mice per group, total n=120), statistical evaluation on the effect of tumour size, and counting optimization (taking into account necrosis and thermal artifacts) are underway to ensure a representative, controlled data set is profiled.

Conclusion: Characterization of immune infiltrates in RMS was feasible in this immunocompetent mouse model. The relative density of CD11b+ and B220+ cells was similar to that of macrophages and B lymphocytes in human RMS indicating that this murine model is capable of modeling the human immune response. There were significantly more B lymphocytes in HT treated tumours compared to NT tumours at 168 hours post treatment, and further analysis of individual treatment groups warrants investigation. We will expand our mouse cohort for further characterization of immune infiltrate in this murine ERMS model. Additional studies will include performance of imaging mass cytometry (IMC) on tumour samples to visualize sub-populations of immune cells in the tumour microenvironment (TME).

CTOS Figure 1 June 27.pdf

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