It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. H Circulation cytometry analysis of EpCAM expression of HT-29 with EpCAM-FITC antibody. Black: unfavorable control; Blue: DT3 aptamer (A, C, E, G); Red: Ep23 aptamer (A, C, E, G); Purple: EpCAM antibody (B, D, MM-589 TFA F, H). I: EpCAM expression was confirmed by Western analysis using the 323/A3 antibody; J: Relative expression of EpCAM was compared to -actin.(TIF) pone.0057613.s001.tif (8.2M) GUID:?CBC1A9A7-F888-4F65-938A-D2F8F0625224 Physique S2: Representative images of positive and negative control slides for chromogenic staining. HT-29 and U118MG tissue sections were stained with either EpCAM antibody or Ep23 aptamer as part of each MM-589 TFA staining experiment of clinical breast cancer cases to confirm specificity of each staining reaction.(TIF) pone.0057613.s002.tif (4.3M) MM-589 TFA GUID:?6F78201C-D34D-4685-9137-48CAEB200192 Abstract EpCAM is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70C90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this Rabbit Polyclonal to Collagen I is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not strong enough to be able to detect EpCAM expression in all pathological tissues. Here we statement a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded main breast cancers. This new aptamer, together with our previously explained aptamer, showed no non-specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast malignancy xenograft where an anti-EpCAM antibody (323/A3) showed limited or no reactivity. Our results demonstrated a more strong detection of EpCAM using RNA aptamers over antibodies in clinical samples with chromogenic staining. This shows the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy. Introduction Surgical pathology has become the platinum standard for the diagnosis of tumors MM-589 TFA [1]. When morphological features of the tumor are not adequate for any definitive diagnosis, immunohistochemistry (IHC) may be of benefit [2]. Indeed, antibodies have become an integral part of the pathology laboratory in the last 40 years [3]. Biomarkers are recognized through the use of antibodies and molecular techniques, and help to identify particular characteristics of each tumor including those related to prognosis [4]. In addition to being an integral part of the diagnostic arsenal, IHC is also now being used to identify or differentiate those patients who are likely to benefit from certain directed or targeted therapies [5], [6]. Such is the case in HER2 positive breast malignancy patients who benefit from treatment with trastuzumab [7]. EpCAM is a type I glycosylated membrane protein expressed at low levels in a variety of human epithelial tissues, but overexpressed in most solid cancers [8]. Indeed, its expression has been shown to be inversely related to the prognosis of malignancy patients [8]. The detection of EpCAM on the surface of malignancy cells is becoming increasingly important with the introduction of anti-EpCAM immunotherapy. However, studies have shown that there is heterogeneity in the reactivity of antibodies against EpCAM, which could be due to different conformational says when the epitopes are differentially glycosylated [9], [10]. Antibody production is a time-consuming process that still relies greatly on the use of animals for their production, and while monoclonal antibodies are generally more real than polyclonal antibodies, they may be contaminated by antibodies other than the one of interest when the ascites fluid is collected from your host animal [11]. An additional problem is usually that there may be batch-to-batch variance, with antibodies from different batches directed against the same epitope showing discrepancies in their staining [11]. Therefore, MM-589 TFA an alternative to antibodies that does not rely on production, and thus eliminates these variables would be highly advantageous. The generation of chemical antibodies, also known as aptamers, as an alternative to conventional antibodies is an area of research that shows promise in expanding the diagnostic and clinical arsenal in malignancy diagnosis.