Glumac Paige M, Forster Colleen L, Zhou Hong, Murugan Paari, Gupta Shilpa, LeBeau Aaron M
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota.
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota.
Prostate. 2018 Sep;78(13):981-991. doi: 10.1002/pros.23656. Epub 2018 May 22.
The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines.
A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8 × 10 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC).
The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133 LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133.
We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.
跨膜糖蛋白CD133被认为是成人前列腺干细胞以及癌症干细胞/起始细胞的标志物。由于缺乏用于检测CD133的灵敏且准确的抗体，研究CD133在前列腺正常生物学过程及癌症中的作用变得复杂。在此，我们描述了一种利用人抗体噬菌体展示技术鉴定出的独特抗体的特性，该抗体可在福尔马林固定组织和细胞系中识别CD133。
用具有8×10多样性的人单链可变片段（scFv）抗体噬菌体展示文库针对完全糖基化的重组CD133进行筛选。针对去糖基化的CD133进行反筛选，以选择优先识别不依赖糖基化表位的克隆。通过流式细胞术分析先导scFv，并将其克隆到兔免疫球蛋白支架中用于免疫组织化学（IHC）。
发现命名为HA10的抗体以高亲和力结合CD133肽主链上不依赖糖基化的表位。作为流式细胞术试剂，HA10比常用的市售抗体更准确地检测到CD133。用HA10进行的免疫组织化学分析记录了健康前列腺切片中基底细胞和管腔细胞的染色情况。在腺癌切片中观察到管腔细胞的弱染色，频率非常低。对来自LuCaP患者来源的异种移植组织芯片的检查发现，只有三个LuCaP模型CD133呈阳性。这三个CD133阳性的LuCaP模型均源自具有神经内分泌分化的非雄激素受体（AR）驱动的转移性前列腺癌。随后对一名接受第二代抗雄激素治疗失败患者的肝活检进行检测，发现CD133染色水平很高。然而，该患者最初的经尿道前列腺切除术标本中未检测到CD133。
我们开发了一种新型抗体，能够通过免疫组织化学和流式细胞术检测CD133。使用HA10作为免疫组织化学试剂，我们发现CD133在健康前列腺和腺癌切片中都是一种非常罕见细胞类型的标志物。我们的初步研究还表明，CD133与具有神经内分泌分化的非AR驱动的前列腺癌之间可能存在关联。
