Background Tumor cells can shed from the tumor, enter the circulation and travel to distant organs, where they can seed metastases. These cells are called circulating tumor cells (CTCs). The ability of CTCs to populate distant tissues and organs has led us to believe they are the primary cause of cancer metastasis. The biological properties and interaction of CTCs with other cell types during intravasation, circulation in the bloodstream, extravasation and colonization are multifaceted and include changes of CTC phenotypes that are regulated by many signaling molecules, including cytokines and chemokines. Considering a sample is readily accessible by a simple blood draw, monitoring CTC levels in the blood has exceptional implications in oncology field. A method called the liquid biopsy allows the extraction of not only CTC, but also CTC products, such as cell free DNA (cfDNA), cell free RNA (cfRNA), microRNA (miRNA) and exosomes. Conclusions The clinical utility of CTCs and their products is increasing with advances in liquid biopsy technology. Clinical applications of liquid biopsy to detect CTCs and their products are numerous and could be used for screening of the presence of the cancer in the general population, as well as for prognostic and predictive biomarkers in cancer patients. With the development of better CTC isolation technologies and clinical testing in large prospective trials, increasing clinical utility of CTCs can be expected. The understanding of their biology and interactions with other cell types, particularly with those of the immune system and the rise of immunotherapy also hold great promise for novel therapeutic possibilities.

Calcium electroporation (CaEP) is a novel anti-tumour treatment that induces cell death by internalization of large quantities of calcium. The anti-tumour effectiveness of CaEP has been demonstrated in vitro, in vivo, and in preliminary clinical trials; however, its effects on the vasculature have not been previously investigated. Using a dorsal window chamber tumour model, we observed that CaEP affected to the same degree normal and tumour blood vessels in vivo, as it disrupted the vessels and caused tumour eradication by necrosis. In all cases, the effect was more pronounced in small vessels, similar to electrochemotherapy (ECT) with bleomycin. In vitro studies in four different cell lines (the B16F1 melanoma, HUVEC endothelial, FADU squamous cell carcinoma, and CHO cell lines) confirmed that CaEP causes necrosis associated with acute and severe ATP depletion, a picture different from bleomycin with electroporation. Furthermore, CaEP considerably inhibited cell migratory capabilities of endothelial cells and their potential to form capillary-like structures. The finding that CaEP has anti-vascular effects and inhibits cell migration capabilities may contribute to the explanation of the high efficacy observed in preclinical and clinical studies.

Delivery of electric field pulses, i.e. electroporation (EP), to tissues has been shown to have a blood flow modifying effect. Indeed, the diameter of blood vessels exposed to EP is immediately reduced resulting in blood flow abrogation, followed by an increase in vascular permeability. The main cause of the increased permeability remains unknown. The aim of this study was to determine whether the in vivo effects of EP on permeability of blood vessels are linked to the permeabilization of endothelial cells' membrane (EC) and/or disruption of cell-to-cell junctions. We used a dorsal window chamber model in C57Bl/6 mice coupled with multiphoton microscopy and fluorescently labelled antibodies against PECAM-1 (CD31) to visualize endothelial cell-to-cell junctions. Clinically validated EP parameters were used and behavior of cell-to-cell junctions, in combination with leakage of 70 kDa fluorescein isothiocyanate labelled dextran (FD), was followed in time. After EP, a constriction of blood vessels was observed and correlated with the change in the shape of ECs. This was followed by an increase in permeability of blood vessels for 70 kDa FD and a decrease in the volume of labelled cell-to-cell junctions. Both parameters returned to pretreatment values in 50% of mice. For the remaining 50%, we hypothesize that disruption of cell-to-cell junctions after EP may trigger the platelet activation cascade. Our findings show for the first time in vivo that alterations in cell-to-cell junctions play an important role in the response of blood vessels to EP and explain their efficient permeabilization.

Several studies have shown that different control plasmids may cause antitumor action in different murine tumor models after gene electrotransfer (GET). Due to the differences in GET protocols, plasmid vectors, and experimental models, the observed antitumor effects were incomparable. Therefore, the current study was conducted comparing antitumor effectiveness of three different control plasmids using the same GET parameters. We followed cytotoxicity in vitro and the antitumor effect in vivo after GET of control plasmids pControl, pENTR/U6 scr and pVAX1 in B16.F10 murine melanoma cells and tumors. Types of cell death and upregulation of selected cytosolic DNA sensors and cytokines were determined. GET of all three plasmids caused significant growth delay in melanoma tumors; nevertheless, the effect of pVAX1 was significantly greater than pControl. While DNA sensors in vivo were not upregulated significantly, cytokines IFN β and TNF α were upregulated after GET of pVAX1. In vitro, the mRNAs of some cytosolic DNA sensors were overexpressed after GET; however, with no significant difference among the three plasmids. In summary, although differences in antitumor effects were observed among control plasmids in vivo, no differences in cellular responses to plasmid GET were detected in tumor cells in vitro. Thus, the tumor microenvironment as well as some plasmid properties are most probably responsible for the antitumor effectiveness.

Circulating tumor cell (CTC) count is an independent prognostic factor in early breast cancer. CTCs can be found in the blood of 20% of patients prior to neoadjuvant therapy. We aimed to assess the suitability of magnetic-activated cell separation (MACS) technology for isolation and cytological characterization of CTCs. In the preclinical part of the study, cell lines were spiked into buffy coat samples derived from healthy donors, and isolated using MACS. Breast cancer cells with preserved cell morphology were successfully isolated. In the clinical part, blood for CTC isolation was drawn from 44 patients with early and locally advanced breast cancer prior to neoadjuvant chemotherapy. Standard Giemsa, Papanicolaou and pancytokeratin staining was applied. 2.3% of samples contained cells that meet both the morphological and immunocytochemical criteria for CTC. In 32.6% of samples, partially degenerated pancytokeratin negative cells with morphological features of tumor cells were observed. In 65.1% of samples, CTCs were not found. In conclusion, our results demonstrate that morphologically intact tumor cells can be isolated using MACS technology. However, morphologically intact tumor cells were not detected in the clinical part of the study. At present, MACS technology does not appear suitable for use in a clinical cytopathology laboratory.