Identification of specific genetic and phenotypic changes occurring in CTC could result in a better understanding of the metastatic process and lead to effective therapeutic strategies
. To our knowledge, for the first time global gene expression profiles were generated from blood enriched for CTC by a negative depletion technique. Depletion of dead (7AAD staining) and non-epithelial cell types (such as leukocytes (CD45+) and other endothelial/leukocyte progenitor cells (CD34+) results in a more unbiased enrichment of CTC both in spiking experiments and in vivo compared to a priori positive selection using epithelium-specific antibodies.
An important issue in small-sample gene expression microarray analyses is the quality of the final RNA sample. Indeed, all methods requiring additional steps (e.g. tissue biopsies from resection specimens, density centrifugation, FACS) to acquire very small amounts of cells bear the risk of RNA degradation. Moreover, the pancreas is known for its high concentration in RNases, leading to quick RNA degradation. In an effort to minimize this effect, periods of warm ischemia between removal of the surgical specimen and sample processing were kept to a strict minimum. Also, tissue samples were immediately submerged into a RNA stabilizing agent (RNAlater®) and care was taken to store these samples under optimal conditions. Despite these stringent precautions moderate degradation was observed in a substantial number of samples. Nevertheless, moderate RNA degradation probably disturb microarray results only modestly
, and the final number of samples with high-quality RNA was sufficient to draw sound conclusions from the present study. Microarray analyses showed differential gene expression profiles between the CTC, T, P and G fractions. In contrast to other reports, we found only 6 genes (C19orf33, ECT2, IL1RN, S100P, SFN, TUBA4A) to be significantly upregulated in the primary PDAC (T) as compared to the non-tumoural pancreatic tissue (P) samples, whilst no genes were found to be downregulated. This finding can be explained by the fact that the ductal epithelial cells, of which PDAC originates, make up only a small proportion of the bulk surrounding pancreas or tumour. Indeed, expression profiles often predominantly reflect the proportion of cell types and obscure more meaningful data. Another explanation may be that we finally retained only 8,152 probe sets out of 46,467 probes initially tested, because Robust Multichip Average (RMA) was applied only to probe sets with a present detection call in at least 4 out of 6 CCD samples.
In our stringent comparison of the various cell fractions (CTC vs. T, AND CTC vs. P, AND CTC vs. G), the top molecular and cellular functions expressed in CTC were cell-to-cell signalling and interaction, cellular development, and cellular movement. The pathway with the highest expression ratio in CTC was p38 MAPK signalling, which is known to be involved in biological processes such as inflammation, apoptosis, and cell differentiation
. More recently, the p38 MAPK signalling cascade was also shown to play a pivotal role in cancer cell migration, and could be stimulated by S100A8
. Moreover, potential secretion of TNFα, VEGF-A and TGF-β1 from primary tumours induced S100A8 and S100A9, which in turn stimulated the enrichment of other inflammatory chemo-attractants. These findings suggest that an increased p38 MAPK activity in CTC could occur in response to S100A8 produced not only in homing organs (such as lungs, liver) but also in CTC. Stimulation of p38 MAPK by S100A8 has been shown to induce formation of pseudopodia important to dynamic cell movement in the process of invasion and extravasation
. Therefore, we used the gene ontology application software AmiGO at other genes that were overexpressed in CTC involved in the same process and in p38 MAPK signalling. Other genes mediating many of the component processes of invasion include Talin-1 and ELMO1. Both these genes were found to be upregulated in our CTC. Besides these genes, we found another 7 genes, i.e. ALOX12, AMFR, ARHGEF2, CCL5, STAT3, TPM4, and VCL. These 9 genes were termed CTC motility panel for ease of formulation.
In the p38 MAPK pathway TGF-β1, cPLA2, and MAX were significantly upregulated in CTC. TGF-β1 regulates a spectrum of cellular events, including cell proliferation, differentiation, and migration. In addition to the canonical Smad pathway, TGF-β1 can also activate MAPK, phosphatidylinositol 3-kinase (PI3K)/Akt and small GTPases in a cell-specific manner. Activation of ERK or p38 MAPK is required for both TGF-beta-induced epithelial-mesenchymal transition (EMT) and cell migration
. Reversible and local activation of TGFβ/Smad-signalling in breast cancer cells has recently been shown to cause a switch from cohesive movement to single cells motility and to promote haematogenous metastasis
A clear association has been described between the CCL5/CCR5 axis and the directional migration and invasion of human cancer cells. In vitro, this has been shown to involve specific upregulation of matrix-metalloproteinase-9 (MMP-9)
. STAT3 is a transcription factor that has been linked with metastasis in different tumourtypes e.g. lung, colon adenocarcinoma, and PDAC
. Besides increasing cell motility and invasion, STAT3 is also thought to upregulate the expression of anti-apoptotic and growth-promoting genes and to facilitate the colonisation of the liver by tumour cells
[20, 21]. Therefore, STAT3 could be another new target for prevention of metastasis formation by CTC and for the treatment of established metastasis. STAT3 inhibitors are already available and currently undergo testing in various animal cancer models
Our study provides a valuable source for researchers to explore. Using a negative depletion technique we were capable of isolating CTC, without using a priori selection markers. We have identified several candidate genes in CTC from PDAC, which can be used for various purposes. First, a combination of these genes may distinguish between benign and malignant disease of the pancreas. This was beyond the scope of this study. Also, as these genes represent the molecular signature of CTC in peripheral blood they may be useful for disease monitoring, prediction of survival, and response to therapy. Therefore we determined the expression level of our cell motility gene signature in 78 independent PDAC samples using the nCounter system. This system uses digital technology based on direct multiplexed measurement of gene expression, and offers high levels of sensitivity and precision
. We found that the combination of expression of our CTC motility panel and TGF-β1 identified a subgroup of primary PDAC at high risk of early recurrence and worse overall survival. Some of these genes may also be interesting therapeutic targets and need further exploration.However, we should remain cautious as measurement of mRNA levels to infer events at the protein level can be misleading, as discordances may exist between mRNA levels and encoded protein levels.