Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers

O’Shannessy et al. Journal of Ovarian Research (2015) 8:29 DOI 10.1186/s13048-015-0156-0 RESEARCH Open Access Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers Daniel J O’Shannessy1*, Elizabeth B Somers1, Li-chong Wang2, Hongwei Wang2 and Ruby Hsu2 Abstract Background: Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues. Methods: RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC. Results: We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells. Conclusions: These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers. Keywords: FOLR1, FOLR2, CD68, CD11b, Epithelial ovarian cancer, Fallopian adenocarcinoma, Tumor microenvironment, Stromal cells Background Ovarian cancer causes more deaths than any other cancer of the female reproductive system [1]. Delayed diagnosis and the presence of widely disseminated disease contribute to the high mortality associated with the disease. Efforts continue in an attempt to understand the underlying molecular mechanisms of the pathogenesis of * Correspondence: 1 Translational Medicine and Diagnostics, Morphotek, Inc, Exton, USA Full list of author information is available at the end of the article ovarian [2–4] and other gynecologic malignancies in order to identify new targets for potential therapy and new modes of diagnosis. It was previously thought that epithelial ovarian cancer (EOC) derived from epithelial cells covering the ovary [5, 6]. However, recent studies have shown that most EOCs do not exhibit characteristics typical of mesodermal epithelium from which the ovaries develop. Therefore, it has been hypothesized that EOCs, particularly those of the serous histotype, originate from the © 2015 O'Shannessy et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. O’Shannessy et al. Journal of Ovarian Research (2015) 8:29 fallopian tubal fimbriae in the form of inclusion cysts which may or may not already exist in a (pre) cancerous state [7–12]. The tubal epithelium, along with other reproductive organs, derives from the müllerian duct, which in turn is derived from the mesoderm. Previous work within our laboratory, based on immunohistochemical detection of folate receptor alpha (FRA), has shown a strong correlation between ovarian serous carcinomas (EOC) and normal fallopian tube as well as fallopian adenocarcinoma [13] with respect to expression of this receptor. In addition, gene expression analysis has demonstrated that numerous genes previously considered to be (over) expressed in EOC (e.g. FOLR1, MSLN, MUC16 and WFDC2) are indeed significantly overexpressed in EOC when compared to normal ovarian tissue yet none of these genes/markers of EOC showed significantly increased expression when compared to either normal fallopian tube or fallopian adenocarcinoma tissues [14]. These data support the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be up-regulated or over expressed in EOC are most likely not of ovarian origin, but fallopian in derivation. The increased serum levels of the protein biomarker products of these genes (FRA, mesothelin, CA125 and HE4) in ovarian cancer may therefore be more a reflection of disease (tissue) burden than either tumor specific markers or over expression. However, it should be noted that expression profiles are most often devoid of architectural context and differences may be partly attributable to, or confounded by, the heterogeneity of cell types, and the relative contributions there from, that are potentially present (e.g. normal epithelia, stromal cells, and tumor) within tissue sections. We therefore undertook an investigation into the expression of FOLR1 (folate receptor alpha, FRA) and FOLR2 (folate receptor beta, FRB) in gynecologic tissues, both normal and malignant, relative to architectural features and cellular specificity using dual-color RNA in situ hybridization. Folate receptors alpha (FRA) and beta (FRB) are glycosylphosphatidylinositol (GPI)-anchored receptors that bind plasma folate (5-methyltetrahydrofolate) with high affinity (KD ~1nM), and transport it into the cell via endocytosis [15]. FRA and FRB exhibit 71 % identity at the amino acid level (Fig. 1) and lie in tandem on chromosome 11q13, along with two other members of this family, folate receptor gamma and folate receptor delta, although much less is known about these two family members. Although closely related both in function and sequence, the tissue distribution and cellular specificity of FRA and FRB are quite distinct. FRA, the most extensively studied isotype, is expressed on several tumors of epithelial origin including ovarian cancer, non-small cell lung adenocarcinoma, breast cancer, renal cancer and endometrial cancer [16–21]. Further, Page 2 of 9 FRA has been shown to be expressed in normal placenta, fallopian tube, kidney, lung, breast and cho (...truncated)