Head and neck squamous cell carcinoma (HNSCC) is a source of significant morbidity and mortality worldwide with risk factors including HPV status, tobacco and alcohol. For HPV-negative tumors, no prognostic assays have been widely accepted. We previously analyzed the Cancer Genomic Atlas (TCGA) which contains the largest collection of patient HNSCC tumor specimens. In HPV negative tumors, reduced survival outcomes associated with tumor protein 53 (TP53) mutation only occur in combination with loss of chromosome 3p with reduction in median survival from 5 years for TP53 mutations to 1.7 years for a double hit (TP53 and 3p). The reasons for worse outcomes are still unclear with potential explanations including decreased radiosensitivity of double-hit tumors and/or role of infiltrating host immune cells/macrophages. Previous in vitro experiments have shown that combined genomic events of TP53 mutation and FHIT deletion (as a proxy for 3p deletion) correlate with decreased radiotherapy sensitivity in HNSCC cell lines¹. Previous studies have also shown that “double-hit/ Cal27” xenografts haves higher matrix metalloproteinase (MMP) activity, as evidenced by in vivo ratiometric cell penetrating peptide (RACPP) imaging compared to “single-hit/ SCC4”. Here, we aimed to evaluate the role of infiltrating immune cells in worse clinical outcomes with double-hit TCGA murine tumor model. A perfluorocarbon (PFC) emulsion was used to tag macrophages in situ with high specificity and sensitivity in 19F MRI with no background. Briefly, when PFC is administered intravenously, the nanoemulsion droplets are taken up by cells of the reticuloendothelial system and when labeled cells accumulate in sufficient amounts at the site of inflammation, they become detectable by 19F MRI. Approximately 5x10⁶ cells of two different cell lines, single-hit/ SCC4 and double-hit/ Cal27, were injected in bilateral flanks of 10 mice (n=5 in each group). After visible tumor growth, a single 0.2 ml of PFC emulsion (VS-1000, Celsense, Inc., Pittsburgh, PA) was injected IV and mice were longitudinally imaged with 19F and proton MRI on Day 2 & 10 post-injection using methods described ². Longitudinal MRI data was analyzed using software package Voxel Tracker (Celsense) to measure apparent tumor associated macrophage burdening each tumor over time. Tumors were then excised for histology to evaluate immune cell recruitment.The average number of 19F spins within the tumors were approximately double for the Cal27 group compared to the SCC4 group (3.94x10¹⁹ compared to 1.98x10¹⁹ 19F / tumor) on Day 2 post injection, signifying increased tumor associated macrophage burden in the double hit tumors. Prior studies have shown that the 19F number is linearly proportional to macrophage burden ³.The number of 19F spins per tumor decreased in both groups over the course of 8 days but not significantly. Even at Day 10, the 19F spins in group 1 (Cal27) tumors were significantly higher than group 2 (SCC4). Tumors were then excised on Day 10 for histology to evaluate immune cell recruitment. Histology results are still pending but we except correlation of 19F signal with tumor associated macrophage accumulation.While cancer therapy is making strides in the clinic, the mechanisms of action and reasons for failure are not yet well-understood. There is a need to develop quantitative methods to assess efficacy. In this study we succeeded to use 19F MRI as a method for quantifying immune cells in tumor microenviorment which aids in tumor distinction because double and single hit head and neck tumors differ in patient prognosis and radiosenstivity. The double hit tumors demonstrated twice more macrophage accumulation which was evident on combined 19F/proton MRI images and easily quantified using Voxel Tracker software. This tumor distinction using noninvasive MRI will help in pre-operative planning to provide radiotherapy to the appropriate cohort of double hit tumor patients. Further investigations will include assessment of the effect of radiation on immune cell aggregation in single and double hit head and neck tumors.