Folate Receptor Beta Targeting for In Vivo Optical Imaging of Head and Neck Squamous Cell Carcinoma

OBJECTIVE: The folate receptor (FR) is a high-affinity folic acid binding endocytic receptor uncommonly expressed in normal tissues. The α isoform (FR-α) is overexpressed in a variety of epithelial neoplastic cells. In contrast, functional expression of the β isoform (FR-β) is limited to activated macrophages. Importantly, in many malignancies FR serves as a target for the delivery of tumor specific drugs and imaging markers. Folic acid conjugated fluorescent dyes have been used to guide tumor resection in mouse models and humans. However, their potential utility in head and neck squamous cell carcinoma (HNSCC) is unclear due an incomplete characterization of FR expression in such tumors. We hypothesized that tumor infiltrating macrophages expressing FR-β could allow fluorescent visualization of HNSCC tumors using folate conjugated dyes even when FR expression in cancer cells is low. SUBJECTS AND METHODS: Immunohistochemistry was performed on a tissue microarray (TMA) containing primary tumor tissue and matched tumor free surgical margins from 22 patients who underwent HNSCC resection. Primary tumor sites included the oral tongue, base of tongue, tonsil, supraglottic larynx, glottic larynx and hypopharynx. We evaluated the expression of FR-α, FR-β, TGF-β, CD68 and arginase-1. To examine the use of folate targeting for image guided surgery, orthotopic xenograft HNSCC tumor models were generated from nude mice. The mice received 0.8 mg/kg intravenous injections of fluorescein isothiocyanate conjugated folate (Folate-FITC) and were imaged for fluorescent emission under 495nm light two hours later. RESULTS: No FR-α expression was observed in any TMA tumor specimen. All tumor samples demonstrated positive FR-β expression. Cellular morphology and CD68 expression identified the FR-β expressing cells as tumor infiltrating macrophages. No association was observed between FR-β staining and either TGF-β or arginase-1 staining. In tumor xenograft mouse models, tumors showed strong fluorescence in vivo after folate-FITC injection. Normal salivary glands and surrounding neck muscles did not demonstrate significant fluorescence. Histologic examination of the xenografts revealed that fluorescence within the tumors was confined to areas of inflammatory cell infiltration, consistent with our TMA data. Conclusion: HNSCC tumors contain a significant population of FR-β expressing macrophages. In contrast to many other carcinomas, the HNSCC tumor cells in our TMA did not express FR-α. By targeting tumor infiltrating macrophages, the folate linked delivery of fluorescent dyes can facilitate image guided HNSCC resection even when the tumor cells themselves do not express FR.