Trophoblast models for tumour studies: understanding the similarities of tumour and trophoblast invasion

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Balahmar, R.M., 2016. Trophoblast models for tumour studies: understanding the similarities of tumour and trophoblast invasion. PhD, Nottingham Trent University.

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Abstract

Cell proliferation, migration and invasion are the important features of tumour metastasis. Interestingly, during embryonic development, trophoblast cells show similar attributes like tumour cells to establish foeto-maternal communications and support normal pregnancy. The invasive nature of tumour as well as trophoblast cells (especially during the first trimester) are believed to be enhanced by a collection of "stem-like cells" (SLCs) called "spheroid bodies". This sub-population of SLCs within tumour or trophoblast cells can proliferate to form a heterogeneous cell groups with different functional attributes. However, tumour SLCs proliferate during invasion; while trophoblast cells proliferate and then invade. Although several previous studies have produced SLCs from tumour cell lines in vitro, limited attempts were made to produce SLCs from trophoblast cells. Therefore, this study firstly aimed to produce and characterise SLCs from transformed first trimester trophoblast cell lines (HTR8sv/neo and TEV-1) and choriocarcinoma (trophoblast tumour cell lines JEG-3 and BeWo) in relation to a non-placental tumour cell line MCF-7. Secondly, to date, the factors that are responsible for there uncontrolled versus controlled invasion are not fully understood. On the other hand, it is worth noting that in pre-eclampsia (PE), the invasion of first trimester trophoblast cells is found to be reduced. Therefore, it was hypothesised that it would be possible to identify the important molecules that may be involved in the controlled trophoblast invasion by comparing the status of different factors that are identified with altered expression in tumour cells between (a) normotensive (NT) and PE placentae, and (b) in the SLCs produced in vitro from trophoblast cells.

SLCs from transformed trophoblast and choriocarcinoma cells were produced by growing in non-attachable or ultra-low attachment flasks with or without doxorubicin (DOX) to produce DOX-resistant and non-resistant spheroids. The “stemness” feature of these spheroids was characterised by comparing the expressions of stem cell markers. The migration and invasive capacities of DOX-resistant and non-resistant spheroids were compared with their parental cells by wound-healing and 2-D/3-D invasion assays. The status and expression of novel factors that may be involved in cell proliferation and invasion was checked by quantitative real-time PCR and western blotting. Also a comparative proteomic (SWATH-MS) analysis was carried out to identify and compare the global changes of the peptide expression during SLCs transformation. In addition the RNA and protein expression of factors that are involved in trophoblast invasion were compared in 13 NT and 12 PE placentae.

Both the transformed trophoblastic cell lines (HTR8/Sv-neo and TEV-1,) and gestational choriocarcinoma cell lines (JEG-3 and BeWo) were able to produce non- spheroidal cells (non-resistant and drug resistant) under 3-D conditions. These spheroids showed increased protein expression of stem cells markers, such as OCT4, SOX2 and NANOG. On the other hand, both trophoblast CDX2 and the cell fate determining transcription factor, NOTCH1, were reduced in spheroidal cells confirming the "stem-like" transformation. Moreover, the 2-D invasion assay showed a statistically significant increase in the invasive potential (number invaded cells) of spheroids. This significance was found to be higher in untreated spheroids from transformed trophoblast cells; P<0.0005 (for both HTR8svneo and TEV-1) and in one of the choriocarcinoma cells JEG-3; whilst the significance between untreated spheroids of BeWo and their parental cells was slightly less (P<0.05).

The 3-D invasion assays have shown a significant time dependent increase in the invasions of non-resistant spheroidal cells in comparison with DOX-resistant counterpart, especially the non-resistant spheroidal cells produced from HTR8/Svneo (p<0.0005) at 48 hours. Spheriodal cell invasion of non-resistant TEV-1 and choricarcinoma cells was significantly higher than DOX-resistant cells (p<0.005). Therefore this study has produced spheroidal cells from (a) transformed first trimester trophoblast (HTR8/Svneo and TEV-1) and (b) choriocarcinoma (JEG-3 and BeWo) cells. Since these cell lines are of trophoblast origin, it is possible to use these spheroids as comparative models to study the effects of chemotherapeutic agents on (a) physiologically rapidly dividing cells (HTR8/Svneo and TEV-1) and (b) tumour models (JEG-3 and BeWo) of similar origins.

Comparisons of mRNA and protein expression between NT and PE placentae have shown a statistically significant increase in the expressions of ALDH3A1, AURK-A, PDGFRα, and TWIST1 in PE placentae (p<0.05); whilst AURK-C and JAG-1 expression was down-regulated. SWATH-MS analysis has also highlighted up-regulation of novel proteins that are associated with proliferation, invasion and cell cycle control in the spheroids produced from these cell lines. These proteins include plasminogen, vitronectin and ALDH1A3. However, the function of most of these factors have not been fully investigated in placenta. In summary the study has generated and characterised "stem-like" spheroids from transformed trophoblast and choriocarcinoma cells. These spheroidal cells may be useful as in vitro toxicological models to study the in vivo cellular effects on rapidly dividing cells.

Item Type:	Thesis
Creators:	Balahmar, R.M.
Date:	June 2016
Rights:	This work is the intellectual property of the author, and may also be owned by the research sponsor(s) and/or Nottingham Trent University. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the first instance to the author.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	27 Oct 2017 08:49
Last Modified:	20 Aug 2021 10:53
URI:	https://irep.ntu.ac.uk/id/eprint/31901