The method used for detection of enriched CTCs varies considerably between studies.
Many are based on surface markers or other cytological features if using flow cytometry, immunostaining, or FISH for detection (as with CellSpotter, Cytospin, and other custom techniques). Cells are then counted by cell sorting or microscopic analysis. Alternatively, many enrichments are followed by qPCR for detection of CTCs by mRNA quantifications and consist of many different markers as this is still a rapidly evolving field and consensus has not been reached. The advantage of this method is its potential to characterize many different CTCs while avoiding subjectivity in classification of a CTC due to observed cellular features. The drawback is that the cells are not counted in the process.
Here, we used a multi-marker mRNA panel comprised of epithelial and EMT markers to cover a variety of CTC-subtypes and to allow for further characterization of the CTC population. The specific markers were chosen based on the documented function of their transcripts and their performance in preliminary testing. EPCAM, keratins, breast cancer specific markers, EMT transcription factors, and novel markers were investigated.
To arrive at this marker panel, a preliminary list of potential markers was created (as described in Sections 2.2.10.1 and 3.2) that included markers investigated in other studies and markers found to be differentialy expressed in breast and WBCs in the SAGE database. Among 13 other common markers, two new markers (LUM and CCDC80) were selected, based on this analysis. The selected markers were first validated by measuring their levels in cancer cell lines. From this, only one marker (KRT16) was excluded. Of all the markers, it exhibited the lowest expression across all four cell lines tested. In addition, it was one of four keratins considered, so after exclusion there still remained three others for testing (KRT7,KRT8 andKRT19). LUM was kept for further study in the tissues despite the average low expression in cell lines. The expression of the markers among the different cell lines was variable and seemed to follow the subtypes of breast cancer that they reflect. By transcriptional profiling, the cell lines fall into the following subtypes: MCF-7 as luminal A, ZR-75-1 as luminal B, and MDA-MB-231 as claudin-low [117]. It comes as no surprise then that the most agressive cell line type, MDA-MB-231 (claudin-low or triple-negative), expressed EMT markers at a higher level together with the mesothelioma line, SDM103T2. MDA-MB-231 was also presented by Holliday et al. to express E-cadherin at a lower level, further supporting an EMT phenotype [117].
For further validation, the breast tumor samples and control blood samples from healthy volunteers were analyzed. This provided essential data for the final determination of the
Chapter 4. Discussion 65
multi-marker panel as the cell lines did not reflect real tumor heterogeneity. The breast tumors demonstrated this heterogeneity, with wide variations of expression seen for each marker (Figure3.4). The differential expression among tissues and between markers can be seen in more detail in Appendix C.KRT7 had overall lower expression in the tissues compared to the other keratins and was thus excluded. While SNAIL, SLUG, and TWIST did not look very promising in the tumor samples, they were included due to their documented EMT marker potential. These solid tumor samples do have limited value as they may not share the same properties as CTCs with regard to loss of epithelial expression.
Additionally, the background expression of the markers in normal blood was a very important point to consider if CTC expression was to be detected over normal expression.
For some markers, the control expression in normal blood cells was too high to include them in the final panel (TFF3 and ZEB1). The normal control blood samples in this early validation experiment came from 3 healthy persons. In hindsight, this was not a large enough sample to reflect the variability of background expression and use the expression for initial selection. In future screening experiments, many more samples should be analyzed for preliminary validation of marker expression levels in PBMCs.
To note, a larger cohort of 30 healthy volunteers was recruited for the PBCB patient analysis.
With the final 10 markers chosen for analysis, there were a wide-range of characteristics and functions covered (Table 4.1). Many of the markers used already have extensive use in the CTC field. Most prevalent of course being EPCAM. This is due to its im-portant function as a cellular adhesion molecule in epithelial cells. KRT19 and KRT8 are also commonly used due to the prevalence of keratins as CTC markers. The role of these two are in maintenance of cell structure and integrity [118]. Many other ker-atins exist and have complex expression patterns in both solid tumors [119] and CTCs [42], but we were limited in scope and chose to have a equal or greater focus on EMT markers. Mammoglobin A (SCGB2A2) andHER2 (ERBB2) were were chosen for their high expression in breast tumors and clinical relevance, respectively. Mammoglobin A, a secretoglobin, is only expressed in the mammary gland and is often over-expressed in breast cancer tissue and cell lines [120]. It serves as a useful marker, but its function is largely unknown [120]. Possible roles of the secretoglobin protein include signalling, im-mune response, chemotaxis, and steroid hormone transport [121]. Due to the exclusively epithelial source, it was categorized as an epithelial marker. Since ERBB2 is involved in epithelial processes (Table 4.1), it was also considered an epithelial marker. The role of SNAIL, SLUG, and TWIST as EMT-initiating trasncription factors solidified their use as EMT markers [40,50,122,123], as well as extensive evidence of their expression in both CTCs and DTCs [45,68,78,79,93,124].
The two new markers (LUM and CCDC80) were retained in the panel from results of the preliminary tests. Both had higher expression in the more mesenchymal-like cell
Table 4.1: Marker Gene Ontology, NCBI.[118] *Inferred from Electronic Annota-tion (IEA). **Traceable Author Statement (TAS). ***Non-traceable Author Statement
(NAS). ****Inferred from Physical Interaction (IPI).
Chapter 4. Discussion 67
Figure 4.1: CCDC80 (DRO1) molecular interactions. Reprinted with permission from Springer: Current Colorectal Cancer Reports, copyright 2015 [1]
lines and variable expression among the breast tumor samples, coupled with low control expression. They are novel in breast cancer CTCs with promising use as markers. The CCDC80 (coiled-coil domain containing 80) gene codes for a presently uncharacterized protein involved in extra-cellular matrix (ECM) organization [118].
It has been implicated as a JAK2-binding protein[125], a downstream effector of the hedgehog pathway and fibronectin binding protein [126], involved in Wnt/β-catenin pathway [127] and adipogenesis [128] (Figure4.2). It is considered by some to be a tu-mor suppressor as it has been found to promote cell adhesion, apoptosis, and E-cadherin expression in thyroid and colorectal cancer [1, 129]. On the other hand, CCDC80 has also been shown to be over-expressed in response to estrogen with a potential carcino-genic role in the breast [130] and differentially expressed in single pancreatic CTCs along with other ECM genes (i.e. SPARC) [131]. The other ECM genes were investigated and were not found in the epithelial cells of the tumor, but expressed higher in CTCs and the stroma of tumors, colocalized with keratins at the epithelial-stromal border [131].
Lumican is encoded byLUM and is a protein that joins decorin, biglycan, fibromodulin, keratocan, epiphycan, and osteoglycin as a small, leucine-rich proteoglycan [118]. It has a well-established role in extracellular matrix assembly, specifically collagen fibril assembly and stability, and mediating cell-matrix interactions, cell migration, prolifer-ation, tissue repair, and tumor growth [132–134]. The expression of lumican stromal cells has been associated with tumor invasiveness, progression, and shorter survival [134–139]. Inhibition of cancer growth by lumican has also been documented, however, in pancreatic cancer and melanoma [2, 140] and longer survival was documented with lumican-expressing cancer cells (versus stromal expression) [136]. The only investigation
Figure 4.2: LUM molecular interactions. Reprinted with permission from John Wiley and Sons: FEBS Journal, copyright 2013[2]
into its role in breast cancer was by Paniset al. who found increased lumican expression in cells was associated with advanced disease [138].
Both genes serve functions in ECM organization and have lower expression in normal epithelial cells. Conversely, their higher expression in the stroma mean that excretion by fibroblasts or mesenchymal cells are their main source. This along with their associations with EMT-pathways and molecules (fibronectin, integrin, E-cadherin, Wnt/β-catenin, JAK/STAT3, TGF-β/AKT), and expression patterns in the cell lines, make them likely EMT markers. The research and findings are conflicting in both cases nonetheless and their use in this study is prospective. They may serve a role in EMT, but much more research is needed to substantiate that hypothesis and elucidate their role in breast cancer and CTCs specifically.
Multi-marker qPCR detection and characterization of CTCs is widespread in the field (Table 4.2) Multiple studies have shown the use of multiple markers is beneficial and confers greater sensitivity and detection over single marker analysis [60,61, 76]. These panels have the potential to select for different CTCs, but can leave others undetected.
Ideally, a set of markers would be found that could include all CTCs. As this is unverifi-able, a panel with 100% sensitivity in regards to the metrics being measured would have to serve as proxy. These metrics could be based on basic cancer diagnosis or prognostic and response classifications.
Chapter 4. Discussion 69
Table 4.2: Genes used in other studies of multi-marker detection of CTCs. *Adna measures EPCAM, MUC1, HER2. **AdnaBC measures GA733-2, MUC1, ERBB2,
β-actin. EMT, epithelial-mesenchymal transition. SC, stem cell.
Study Marker Panel
Mikhitarian et al. 2008 [58]
SCGB PIP CEA PSE KRT19 MUC1 EPCAM
Aktas et al. 2009 [48] Adna, EMT: TWIST1,Akt2,PI3Kα; SC:ALDH1 Shenet al. 2009 [59] Survivin,hTERT andSCGB
Obermayr et al. 2010 [60] CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC and SLC6A8;EPCAM,SCGB
Van der Auwera et al.
2010 [61]
Adna, KRT19,MAM
Markou et al. 2011 [66] KRT19, ERBB2, SCGB, MAGEA3, TWIST1, PBGB
Molloyet al. 2011 [67] KRT19,p1B,EGP and SCGB
Strati et al. 2011 [68] KRT19, MAGEA3, ERBB2, TWIST1, hTERT a+b+,SCGB
De Albuquerque et al.
2011 [69]
KRT19,SCGB,MUC1,EPCAM,BIRC5 ERBB2
Giodrano et al. 2012 [78] TWIST1,SNAI1,ZEB1, and TG2
Strati et al. 2013 [76] KRT19,ERBB2,MAGEA3,PBGB, AdnaBC Markiewicz et al. 2014
[79]
KRT19, MGB1, VIM, TWIST1, SNAIL, SLUG, HER2,CXCR4 and uPAR
Vishnoi et al. 2015 [65] 83 genes in qPCR array
Kuniyoshi et al. 2015 [82] KRT19,ERBB2, Oncotype genes