Gehrmann Mathias, Stangl Stefan, Foulds Gemma A, Oellinger Rupert, Breuninger Stephanie, Rad Roland, Pockley Alan G, Multhoff Gabriele
Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, United Kingdom.
PLoS One. 2014 Aug 28;9(8):e105344. doi: 10.1371/journal.pone.0105344. eCollection 2014.
We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro.
METHODOLOGY/PRINCIPAL FINDINGS: The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.
CONCLUSIONS/SIGNIFICANCE: This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.
我们之前使用一种独特的小鼠单克隆抗体cmHsp70.1来证明热休克蛋白70（Hsp70）的膜结合形式（memHsp70）选择性地存在于多种白血病细胞以及源自不同实体瘤的单细胞悬液中，但不存在于未转化细胞或来自相应“健康”组织的细胞中。这种抗体可用于体内肿瘤成像，并将肿瘤作为抗体依赖性细胞毒性的靶点。因此，memHsp70的肿瘤特异性表达有可能用于诊疗目的。鉴于肽作为成像和靶向剂的优势，本研究评估了一种14聚体肿瘤穿透肽（TPP；TKDNNLLGRFELSG）是否也可用于体外靶向膜Hsp70阳性（memHsp70+）肿瘤细胞，该肽的序列源自肿瘤细胞表面表达的Hsp70的寡聚化结构域。
方法/主要发现：在Hsp70基因敲除的乳腺肿瘤细胞系统中证实了羧基荧光素（CF-）标记的TPP（TPP）对Hsp70的特异性。TPP特异性结合不同的memHsp70+小鼠和人类肿瘤细胞系，并通过内吞体迅速摄取。与Hsp70膜阳性较低的T47D细胞（29% memHsp70+）相比，在MCF7细胞（82% memHsp70+）和MDA-MB-231细胞（75% memHsp70+）中检测到的CF标记的TPP水平高2至4倍。孵育90分钟后，TPP在memHsp70+肿瘤中与线粒体膜共定位。尽管没有证据表明任何特定的囊泡群体有特异性定位，但荧光团标记的cmHsp70.1抗体和TPP优先聚集在培养细胞贴壁表面附近。这些发现表明膜Hsp70表达与参与黏附、细胞间突触形成和/或膜重组的细胞骨架成分之间可能存在关联。
结论/意义：本研究证明了具有memHsp70+表型的肿瘤细胞对TPP的特异性结合和快速内化。因此，TPP可能有潜力靶向和成像表达memHsp70的大部分肿瘤（约50%）。
