Trop-2 protein, named for its location in trophoblast cells, is a membrane glycoprotein and the closest homologue to the well-known EpCAM protein (Trop1, CD326). Under healthy (normal) conditions, this protein is involved in embryonic development, maintenance of the epithelial barrier, and in the case of damage, also in tissue regeneration (for example, airway epithelium in the lungs during Chronic Obstructive Pulmonary Disease - COPD). Trop2 belongs among the markers of stem and progenitor cells such as mesenchymal stromal cells, prostate basal cells with stem cell characteristics, bladder and intestinal progenitor cells, and hepatic oval cells. Various signaling pathways and interaction partners have been reported in association with Trop2 action, but it is still not completely described how Trop2 affects different processes and to what extent its role is essential (1).
From gelatinous drop-like corneal dystrophy, human disease in which Trop2 is mutated, it has been learnt that Trop-2 is instrumental for the correct localization of Claudins 1 and 7. Trop-2 is overexpressed in multiple carcinomas and increased expression has mostly unfavorable effect. Still, the prognostic value of Trop2 expression seems to be dependent both on type of cancer and the involved signaling pathway (PI3K/Akt, NF-kB, Wnt, etc). Moreover, inter- and intratumoral heterogeneity are common for Trop-2 expression in malignancies such as those of prostate and breast. In general, Trop-2 is thought to be associated with the epithelial phenotype of cancer cells and many studies have reported that epithelial markers positively correlate with its expression, whereas mesenchymal markers typically exert negative correlation (2). In recent years, interest in Trop-2 has focused primarily on its use as a target for antibody-drug conjugate (ADC) therapy for the treatment of cancer. In this case, the Trop-2 antibody is used as a ligand that can induce internalization of the Trop-2-ADC complex and thus mediate cellular uptake of cytotoxic drugs (3). To understand the efficiency of such treatment, the knowledge of Trop-2 role in carcinogenesis as well as in healthy tissues needs to be further expanded.
The figure below demonstrates the use of the new anti-Trop2 antibody - TrMab-6 in some of the models that we routinely use (4). Validation of the antibody revealed its universal applicability in various methods of protein detection. Western blot shows Trop-2 in negative and positive cell populations sorted from the parental cell line, in the Trop-2-deficient cells, and in the Trop-2-overexpressing cells. For western blot, it is appropriate to use 5% BSA to dilute the antibody. Flow cytometry and immunocytochemistry also showed the specificity of the TrMab6 antibody, for fixed samples it was enough to use a standard protocol for fixation and permeabilization (fixation with 4% PFA for 15 minutes, permeabilization with 0,1% Triton X-100 in 1% BSA). TrMab-6 antibody was also able to detect Trop-2 in the airway epithelium of human and pig lungs using standard paraffin sections.
Figure 1. Detection of Trop-2 by TrMab-6.
A) Western blot analysis of Trop-2 in various cell models by TrMab-6 antibody
B) Native ICC staining of Trop-2 by TrMab-6 antibody. MDA-MB-231 Trop-2 WT (empty vector control) and MDA-MB-231 Trop-2 OE (overexpressing) cells were stained in native or fixed condition.
C) Flow cytometry analysis of Trop-2 by TrMab-6 antibody. MCF7, MDA-MB-231 Trop-2 WT (empty vector control) and MDA-MB-231 Trop-2 OE (overexpressing) cells were stained in native or fixed condition. IgG2b isotype control was used for detection of non-specific binding. T-47D and MDA-MB-231 Trop-2 KO and OE clones and respective control clones were kindly provided by Dr. Petr Beneš (Masaryk University).
D) Immunohistochemical detection of Trop-2 by TrMab-6 antibody in paraffin sections of human and pig lung tissues. Human/Pig lungs – positive in basolateral parts of airway epithelium. Scale bar represents 100 μm.
1 Lenárt S, Lenárt P, Šmarda J, Remšík J, Souček K, Beneš P. Trop2: Jack of All Trades, Master of None. Cancers (Basel). 2020;12(11):3328. Published 2020 Nov 11. doi:10.3390/cancers12113328
2 Remšík J, Binó L, Kahounová Z, et al. Trop-2 plasticity is controlled by epithelial-to-mesenchymal transition. Carcinogenesis. 2018;39(11):1411-1418. doi:10.1093/carcin/bgy095
3 Cardillo TM, Govindan SV, Sharkey RM, et al. Sacituzumab Govitecan (IMMU-132), an Anti-Trop-2/SN-38 Antibody-Drug Conjugate: Characterization and Efficacy in Pancreatic, Gastric, and Other Cancers. Bioconjug Chem. 2015;26(5):919-931. doi:10.1021/acs.bioconjchem.5b00223
4 Sayama Y, Kaneko MK, Kato Y. Development and characterization of TrMab 6, a novel anti TROP2 monoclonal antibody for antigen detection in breast cancer. Mol Med Rep. 2021;23(2):92. doi:10.3892/mmr.2020.11731
Vacek O.1,2,3, Pelková V.4 , Marečková K. 4, Hampl A.2,4 , Souček K.1,2,3
1 Department of Cytokinetics, Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic
2 International Clinical Research Center, St. Anne’s University Hospital, Brno, Czech Republic
3 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
4 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
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