Introduction
Breast and prostate cancer mutually represent the most commonly occurring malignancies worldwide in women and men, respectively. The mutative state, recurrence capacity, resistance to conventional chemotherapy, low success rate of surgery and risks associated with radiotherapy confound the management of both these malignancies. There are several similarities between breast and prostate cancer, like growth hormone dependence and similar chemotherapeutic interventions. Therapy based on radiopharmaceuticals targeting the prostate-specific membrane antigen (PSMA) is proving to be a cutting-edge theranostics intervention for prostate cancer. Clinical positron emission tomography (PET) scans have located anti-PMSA binding sites in breast cancer in vivo. This indicates possible non-prostatic expression of PSMA, therefore research focused on understanding the cellular kinetics, PSMA expression profiles using two breast cancer adenocarcinoma cell lines as breast cancer models. This approach was to assess PSMA as a biomarker molecule that can aid in development of more selective, effective and safe diagnostic and therapeutic alternatives for breast cancer. This study was aimed at evaluating PSMA expression of MCF-7 or MDA-MB-231 mammary adenocarcinoma cell lines in comparison to a known high PSMA expressing LNCaP prostate carcinoma and EA.hy926 hybrid vascular endothelial cell line.
Methods
In vitro cultures of LNCaP’s , a prostatic adenocarcinoma cell line, MCF-7 and MDA-MB-231 breast adenocarcinoma cell lines and endothelial EA.hy926 cells were tested for expression of PSMA by flow cytometry. The LNCaP cells were used a positive control. Cellular localisation of PSMA was achieved utilising confocal microscopy and fluorescently-tagged antibodies in all the cell lines tested. PSMA was quantified in all the cell lines utilising ELISA. Prior to experimentation, a pilot study was undertaken to optimise cell detachment methods. Trypsinisation was compared to mechanical scraping to evaluate a cell detachment method that allowed optimal downline experimentation.
Results
Findings from three supporting and complementary techniques demonstrate positive PSMA identification, localisation and quantification in all the probed cell lines despite three cell types not having a prostrate origin. Quantitatively, LNCaP cells reported the highest concentration of PSMA followed by the malignant MDA-MB-231 cells, then the MCF-7 cell line and least in EA.hy926 cells. The difference in fluorescence between LNCaP cells and all three investigational cell lines was statistically significant however the difference in fluorescence between the three investigational cell lines was not statistically significant. The PSMA antigen was localised on the cell membrane and diffused within the cytosol in LNCaP cells. The MDA-MB-231, MCF-7 and EA.hy926 cells all exhibited a differential expression pattern of PSMA. These cells showed diffuse cytosolic accumulation and intense circular region accumulation apparently bordering the cell membrane and the cell nucleus. The quantification of PSMA reported the highest concentration as being in LNCaP cells. The MDA-MB-231 cells were second, then the MCF-7 cells and the lowest concentration. Significant differences were seen between the positive control and the investigation cell lines. The difference in concentration between the investigational cell lines was not significant. Finally, cryotome sections of biopsies of tumours from two breast cancer patients were found to show detectable PSMA presence.
Discussion
Fluorescence is directly proportional to concentration. The high fluorescence of PSMA exhibited by LNCaP cells in the flow cytometry results can be equated to concentration. A fundamental point of departure from which PSMA expression in the breast carcinoma cell lines could be investigated was established. Expression of PSMA is associated with cancer aggression, metastatic progression and increased malignancy. These clinicopathological characteristics support the expression of PSMA seen in MDA-MB-231. Contrastingly, the same characteristics aren’t seen in MCF-7 cells but expression of PSMA was observed. The expression is not entirely dismissible as other luminal A cell lines have also been shown to express PSMA. The EA.hy926 cells are somatic hybrids that are made up of lung A549 cells and HUVEC’s. Lung cancer has been shown to also express PSMA when probed utilising histology. The expression of PSMA in EA.hy926 is the first of its kind but may be attributable to its lung carcinoma makeup. The pattern of expression in the LNCaP confocal microscopy images can be expected. The PSMA antigen is a transmembrane receptor and as such intense fluorescence was seen on the membrane. Expression of PSMA in the cytoplasm has been reported and was equally observed in the LNCaP cells. The investigation cell line showed accumulation of green fluorescence in vesicular bodies bordering the cell membrane and in juxtanuclear positions. The expression of PSMA has been reported in the mitochondria and the green fluorescence at the nucleus could be mitochondrial. The Golgi apparatus and endoplasmic reticulum have also been recognised as potential location of PSMA expression. The localisation of both these organelles at the nucleus along with the expression of PSMA seen close to the nucleus could be associated. Worth noting is the internalisation properties of PSMA. The antigen has an internalisation signal that can be induced by ligand binding or in the absence of a ligand. Upon internalisation the receptors are vesicled and transported either for degradation of for recycling. The vesicular expression seen close to the membrane in the investigational cell lines could be PSMA that is being trafficked for recycling or degradation upon internalisation. The ELISA quantification revealed the levels of PSMA in the positive control are 100-fold greater than those in the investigation cell lines. The ability to translate PSMA targeting in clinical settings is questionable when considering the difference in concentration values. The probing of PSMA in histological slices was positive and showed patterns that are similar to those seen in the monolayer cultures. This shows continuity between two-dimensional cultures and heterogeneous tissue samples. The premise for investigation of PSMA as a potential theranostic target was established through positive identification, localisation and quantification across three independents methods.
Conclusion
This study is the first of its kind to report reproducible expression of PSMA in the two-dimensional cultures of breast adenocarcinoma MDA-MB-231 and MCF-7 cell lines as well as in the hybrid endothelial EA.hy926 cell line. The results were confirmed by three different techniques where different antibodies were used for the ELISA showing reproducibility in the findings. Moreover, the generated results support the apparent localisation of PSMA in breast cancer patients utilising PSMA targeting radionuclides in PET imaging in a clinical setting. The potential application of this study’s result is stimulating. The success being realised in prostate cancer theranostics through PSMA targeting, may conceivably be realised in other carcinomas, particularly breast carcinoma theranostics. / Dissertation (MSc)--University of Pretoria, 2020. / https://doi.org/10.25403/UPresearchdata.14885454 / Pharmacology / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/79132 |
| Date | January 2020 |
| Creators | Tshabalala, Malvin Thabani |
| Contributors | Cromarty, Allan Duncan, mtshabalala7t@gmail.com, Serem, June Cheptoo, Ebenhan, Thomas |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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