AIM: To investigate the feasibility of separation and cultivation of circulating tumor cells (CTCs) in pancreatic cancer (PaC) using a filtration device.

METHODS: In total, 24 PaC patients who were candidates for surgical treatment were enrolled into the study. Peripheral blood samples were collected before an indicated surgery. For each patient, approximately 8 mL of venous blood was drawn from the antecubital veins. A new size-based separation MetaCell^® technology was used for enrichment and cultivation of CTCs in vitro. (Separated CTCs were cultured on a membrane in FBS enriched RPMI media and observed by inverted microscope. The cultured cells were analyzed by means of histochemistry and immunohistochemistry using the specific antibodies to identify the cell origin.

RESULTS: CTCs were detected in 16 patients (66.7%) of the 24 evaluable patients. The CTC positivity did not reflect the disease stage, tumor size, or lymph node involvement. The same percentage of CTC positivity was observed in the metastatic and non-metastatic patients (66.7% vs 66.7%). We report a successful isolation of CTCs in PaC patients capturing proliferating cells. The cells were captured by a capillary action driven size-based filtration approach that enabled cells cultures from the viable CTCs to be unaffected by any antibodies or lysing solutions. The captured cancer cells displayed plasticity which enabled some cells to invade the separating membrane. Further, the cancer cells in the “bottom fraction”, may represent a more invasive CTC-fraction. The CTCs were cultured in vitro for further downstream applications.

CONCLUSION: The presented size-based filtration method enables culture of CTCs in vitro for possible downstream applications.

The lethal nature of cancer is caused by its invasive character, and spread via blood and lymphatic system to distant locations generating metastatic disease. Moreover, pancreatic cancer (PaC) counts to the solid tumors with the shortest overall survivals. The aggressiveness of the disease is demonstrated in clinic by very early metastatic disease and chemoresistance[1-3].

Prognostic value of tumor cells disseminated to the blood and bone marrow has been shown for various types of solid tumors. Circulating tumor cells (CTCs) are cells shed from primary tumor and metastatic sites to the peripheral blood.

Large patient series of breast, prostate, lung, colon cancer have been tested for CTCs, but no complete results have been reported so far in pancreatic cancer clinical trials[4-6].

The limitation of the recently available tumor markers in PaC could be overcome by CTC.

The analytical methods developed to identify CTCs in PaC include direct and indirect CTCs- detection. Analytical assays based on antibodies against EpCAM antigen expressed on the cells surface count to the direct CTC- isolation methods together with size based separations. The polymerase chain reaction-based assays analyzing DNA and RNA count for indirect detection methods[7,8].

Characterization of CTCs in PaC including enumeration could be an important part of the diagnostic process. CTCs detection aims to reveal the tumor recurrence risk, chemo and radiotherapy resistance markers[9].

Moreover, the conventional prognostic indicators to predict patient outcome are often imperfect, owing mainly to tumor plasticity and subjective assessment criteria. Therefore, there is an urgent need for the establishment of new sensitive prognostic methods capable of identifying patients with a worse prognosis or those who will progress quickly.

In the present study, we have employed size-based separation method to detect CTCs. Our goal was to create an accurate assay that would improve the both detection and cultivation of CTCs/disseminated tumor cells (DTCs) of pancreatic cancer patients avoiding false-positive results and to allow for the personalization of therapy regimens.

The frequency of the CTCs positivity is summarized for different patient subgroups in Figure 4. CTCs were detected in 16 patients of 24 patients (66.7 %), (Figure 4A), with comparable frequencies in operable and inoperable patients (60% vs 77.8%). Due to the low number of patients in the different disease stage groups, CTC-positivity does not reflect the disease stage (Figure 4B), tumor size (Figure 4C), or lymph node involvement (Figure 4D). The same percentage of CTC positivity was observed in the metastatic and nonmetastatic patients (66.7% vs 66.7%) (Figure 4E). Interestingly 88.9% patients with tumor grade 3 were affected by the spread of the pancreatic disease defined by CTCs (Figure 4F). The CTC - positivity reported for each patient individually is shown in the Table 2. We are not able to report any correlation of CTC-abundance and histology tumor subtype.

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Figure 4 Circulating tumor cells positivity reported for patient subgroups. A: Circulating tumor cells (CTCs) in PaC patients, reflecting operability of the primary tumor; B: CTCs in PaC patients in different disease stages; C: CTCs in PaC patients, grouped according the tumor size; D: CTCs in PaC patients, grouped according the lymph node involvement; E: CTCs in PaC patients grouped according the metastasis; F: CTCs in PaC patients, grouped according to tumor grade.

We evidence successful CTCs isolation in patients with pancreatic cancer, capturing viable cells with proliferation potential. The cells captured by size-based filtration approach are enriched with good fitness, what enables the culture of the CTCs unaffected by any antibodies or lysing solutions. The CTCs were cultured in vitro for further downstream applications. The confluent cell growth was reached in the majority of the cultivated CTC cases.

The size of the captured cells guided us in the cancer cell identification process even without any additional staining (e.g., May-Grűnwald -MGG). This standard staining protocol (MGG) has enabled us to analyze the nuclei including nucleoli. Generally the nucleus was bigger than 10 μm itself and the cells did not present much of cytoplasm immediately after separation process. The nuclear-cytoplasmatic ratio is relatively high in cancer cells, but not in the in vitro cultured CTCs. The CTCs get big and long in the culture, changing the nuclear-cytoplasmatic ratio. The cytoplasm of CTCs is rather pale than condensed.

Due to the cell size (15 μm), nucleus size (10 μm),shape, and nucleoli visualized by MGG or simple DAPI-stain in the formerly fixed cells, cancer cells are detected on the separating membrane (Figure1A), and also on the plastic bottom of the 6-well plate (Figure 1B-D). These results indicate that the captured cancer cells display plasticity enabling to grow through the separating membrane. Concerning the shape of the cancer cells in the “bottom fraction”, these cells could present a more invasive CTC-fraction with a spindle cell-like shape. The immunohistochemical analysis has shown the abundance of the cytokeratin-18 in the “membrane” fraction as well as in the “bottom” fraction proving the carcinoma origin. We see an enormous potential in the gene expression analysis, which could reveal the epithelial - mesenchymal character of the detected cancer cells. The mesenchymal like type phenotype of CTCs after an epithelial- mesenchymal transition (EMT), was described by expression of CK7, CEA and vimentin (Figure 3).

The presented study aimed to successful isolation of CTCs from patients with PaC using a simple size-based separation device. The gentle antibody independent isolation process allows the subsequent CTC-cultivation in vitro.

The antibody independence could be of advantage because the detection of CTCs cannot be based only on the expression of epithelial makers (EpCAM) or cytokeratins due to their lost within EMT process[11-13].

The epithelial markers can be down regulated during tumor cell dissemination, affecting the detection rates of CTCs[14-16]. Relatively low CTC-detection rates were reported using the isolation methods relying on EpCAM expression (e.g., CellSearch^®, Adnagen^®) in the early disease stages suggesting, EpCAM-based isolation processes may not be efficient for CTC-detection[17-19].

Two platforms for CTC- enumeration based on different isolation principles (CellSearch^® and ISET) were compared in the study of pancreatic cancer by Khoja et al[20] prospectively. CellSearch^® works on the antibody dependent principle of CTCs-separation. All CTCs expressing epithelial markers should be captured by immunomagnetic separation. ISET is asize-based, blood filtration device. It has been reported that CTCs were detected in 93% of patients via ISET and in 40 % by CellSearch^®.

We detected CTCs in the 66.7% cases. If the patients are subcategorized, the CTC- positivity reaches 80% in some subgroups (e.g., T3-stage). In discussing the results of the CTC-frequency in the pre-defined patient subgroups we should report the relationship between the CTCs and operability of the PaC patients. The CTCs were detected in the 77.8 % of inoperable patients. In the future the CTCs-tests could be used for pre-screening patients before operation. Stage III encompasses patients whose tumors can be surgically removed which represents more than a half of the patients. The CTCs positivity results show, that also in the resectable cases, the CTC-positivity number is very high (60%). There is a possibility to pre-treat the CTC-positive patients within neoadjuvant therapy (chemotherapy or radiotherapy) combined regimens. The survival analysis of the PaC patients in the tested group is not available yet to show how different the patient performance and therapy resulted following CTC testing.

We have to acknowledge the potential of CTCs in understanding and discovering the biological principles of cancer dissemination. The information on CTCs should help to manage patients according the CTC- presence. The in vitro proliferation of CTCs in PaC could enable an implementation of new analytical methodologies such as genome profiling, microRNA studies, protein expression testing, chemoresistance analysis, and discovery of new therapeutic regimens and targets. To use the CTCs as biomarker monitoring the therapeutic efficiency in PaC could be of advantage in near future. Subsequently, CTC-testing is helping to improve patient stratification for new targeted drugs regimens[21-25].

Nevertheless, due to the lack of treatment possibilities in PaC, it is not possible yet to identify by help of CTCs new “druggable targets”.But it is possible to identify in CTCs susceptible genes, which could be used for gene therapy development and immunotherapy optimalization in individual patients[7].

CTCs may help to identify differences between primary tumor and metastasis on the phenotype and genotype level. The clonal heterogeneity of the primary tumor could explain differences between populations of disseminated cells and the evolution of their new genotypes and phenotypes[26]. To identify the differences on the gene expression level between the primary tumor cells and CTCs should be a base for improving the therapeutical approaches. To answer all the questions discussed above one needs larger number of CTCs. There are several ways how to obtain larger numbers of cells: (1) have more precise and specific separation techniques; (2) To analyze bigger blood volumes; or (3) culture CTCs in vitro enabling their proliferation and further downstream analysis. This could be reached by separation technologies as presented here. The newly reported successful in vitro CTCs-culture could be a tool to overcome the low-number CTCs limits, enabling any further research e.g., on protein expression level.