Katarina Kolostova

Essentials of circulating tumor cells for clinical research and practice.

The major cause of death due to cancer is its metastatic deposit in numerous tissues and organs. The metastatic process requires the migration of malignant cells from primary sites to distant environments. Even for tumors initially spreading through lymphatic vessels, hematogenous transport is the most common metastatic pathway. The detachment of cancer cells from a primary tumor into the blood stream is called epithelial-mesenchymal transition (EMT). As these cells circulate further in the bloodstream they are known as circulating tumor cells (CTCs). The CTC population is highly resilient, enabling the cells to colonize a foreign microenvironment. Alternatively, cancer stem cells (CSCs) may arise from differentiated cancer cells through EMT and an embryonic transdifferentiation process. The presence of CTCs/CSCs in blood seems to be a determining factor of metastasis. This paper reviews various methods of clinical cancer detection as well as the biology and molecular characterization of CTCs/CSCs. Our goal was to summarize clinical studies which used CTC/CSCs for prognosis in patients with breast, colorectal, prostate, lung, ovarian, and bladder cancer.

Cimetidine: An anticancer drug? ☆

Cimetidine, H(2) receptor antagonists, is commonly prescribed for gastric and duodenal ulcer disease. Additionally, cimetidine has been shown to have anticancer effects. This review describes the mechanism of antitumor action of cimetidine including its ability to interfere with tumor cell adhesion, angiogenesis and proliferation; its effect on the immune system; as well as inhibition of postoperative immunosuppression. Its anticancer effect is also compared to that of the other H(2) receptor antagonists as well as outcomes of cimetidine in clinical studies in cancer patients.

Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

AbstractThe chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents. Abbreviation: GFP — green fluorescent protein

Cultivation of circulating tumor cells in esophageal cancer

The presence of circulating tumor cells (CTCs) in patients with metastatic carcinoma is generally associated with poor clinical outcome. There have been many investigations showing a possible use of CTCs as minimally invasive predictive and prognostic biomarker in cancer medicine. In this report a size-based method (MetaCell®) for quick and easy enrichment and cultivation of CTCs is presented to enable possible CTCs use in esophageal cancer (EC) management. In total, 43 patients with diagnosed EC, 20 with adenocarcinoma (AdenoCa) and 23 with squamous cell carcinoma (SCC), were enrolled into the adaptive prospective-like study .All the patients were candidates for surgery. The CTCs were detected in 27 patients (62.8%), with a higher rate in adenocarcinoma (75%) than SCC (52%). Finally, there were 26 patients with resectable tumors exhibiting CTCs-positivity in 69.2% and 17 patients with non-resectable tumors with 41.7% CTCs-positivity. Interestingly, in the patients undergoing neoadjuvant therapy, the CTCs were detected at time of surgery in 55.5% (10/18). The overall size-based filtration approach enabled to isolate viable CTCs and evaluate to their cytomorphological features by means of vital fluorescent staining. The CTCs were cultured in vitro for further downstream applications including immunohistochemical analysis. This is the first report of the successful culturing of esophageal cancer CTCs. The detection of CTCs presence could help in the future to guide timing of surgical treatment in EC patients.

In Vitro Culture and Characterization of Human Lung Cancer Circulating Tumor Cells Isolated by Size Exclusion from an Orthotopic Nude-Mouse Model Expressing Fluorescent Protein

In the present study, we demonstrate an animal model and recently introduced size–based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

Assessment of pain threshold in haemophilic patients

Many patients with haemophilia (PWH) live with persistent end‐stage arthritis, as a result of multiple joint haemarthrosis, and experience daily pain. For these people, pain becomes a central aspect of life. The aim of this study was to use mechanical pain thresholds (MPT) to characterize pain perception in different PWH groups. The groups tested were characterized by age, previous bleeding into joints, Hemophilia Joint Health Score (HJHS) and PAIN perception score in the HJHS scoring. A total of 23 PWH (haemophilia A) were included in this study (10 children, 13 adults). A total of 12 PWH suffered from repeated bleeding into some of the tested joints. Data were compared to those collected from 15 age‐matched control subjects. The most significant differences in MPTs were found when the PWH were compared to the controls, based on the differences in PAIN score (PAIN score 1 and 2) in all the tested joints, except for the right knee. Similarly, the difference in MPT in ankle joints was confirmed when PWH with and without bleeding were compared to controls. Summarizing the outcomes, we can emphasize the potential usefulness of MPT as an objective tool in evaluating the pain of PWH.

HLA DRB1, DQB1 and insulin promoter VNTR polymorphisms: interactions and the association with adult‐onset diabetes mellitus in Czech patients

Both the human leucocyte antigen (HLA) DRB1 and the HLA DQB1 gene loci play a role in the development and progression of autoimmune diabetes mellitus (T1DM). Similarly, the insulin promoter variable number tandem repeats (INS‐VNTR) polymorphism is also involved in the pathogenesis of diabetes mellitus (DM). We studied the association between each of these polymorphisms and DM diagnosed in patients older than age 35 years. Furthermore, we analysed possible interactions between HLA DRB1/DQB1 and INS‐VNTR polymorphisms. Based on C‐peptide and GADA levels we were able to distinguish three types of diabetes: T1DM, latent autoimmune diabetes in adults (LADA) and T2DM. INS‐VNTR was genotyped indirectly by typing INS–23HphI A/T polymorphism. The genotype and allele frequencies of INS–23HphI did not differ between each of the diabetic groups and group of healthy subjects. We did, however, observe an association between the INS–23HphI alleles, genotypes and C‐peptide secretion in all diabetic patients: A allele frequency was 86.2% in the C‐peptide‐negative group vs. 65.4% in the C‐peptide‐positive group (Pcorr. < 0.005); AA genotype was found to be 72.4% in the C‐peptide‐negative group vs. 42.6% in the C‐peptide‐positive groups (Pcorr. < 0.01). The HLA genotyping revealed a significantly higher frequency of HLA DRB1*03 allele in both T1DM and LADA groups when compared to healthy subjects: T1DM (25.7%) vs. control group (10.15%), odds ratio (OR) = 3.06, P < 0.05; LADA (27.6%) vs. control (10.15%), OR = 3.37, P < 0.01. The simultaneous presence of both HLA DRB1*04 and INS–23HphI AA genotype was detected in 37.5% of the T1DM group compared to only 9.2% of the healthy individuals group (OR = 5.9, Pcorr. < 0.007). We summarize that in the Central Bohemian population of the Czech Republic, the INS–23HphI A allele appears to be associated with a decrease in pancreatic beta cell secretory activity. HLA genotyping points to at least a partial difference in mechanism, which leads to T1DM and LADA development as well as a more diverse genetic predisposition in juvenile‐ and adult‐onset diabetes. The simultaneous effect of HLA and INS‐VNTR alleles/genotypes predispose individuals to an increased risk of diabetes development.

Association of MHC class I chain related gene-A microsatellite polymorphism with the susceptibility to T1DM and LADA in Czech adult patients.

The results in this study suggest that microsatellite polymorphism within the transmembrane region of MIC‐A gene is associated with genetic susceptibility to adult‐onset of type 1 diabetes mellitus (T1DM), MIC‐A5.1 allele, corrected P = 0.001, whereas it is not associated with latent autoimmune diabetes in adults (LADA) in Czech population. According to our findings, we can hypothesize that adult‐onset T1DM and LADA may have partly different immunogenetic aetiopathogenesis.

Syngeneic lymph-node-targeting model of green fluorescent protein-expressing Lewis lung carcinoma

The Lewis lung tumor has been extensively studied in both syngeneic and allogeneic mouse models. However, its metastatic potential and mechanism are poorly understood. The aim of the present study was to develop a highly metastatic lymph-node targeting, imageable model of the Lewis lung carcinoma in a syngeneic host. We report here a syngeneic model of the Lewis lung carcinoma in which the carcinoma cells are labeled with green fluorescent protein (GFP). The tumor cells were transplanted in the dorsal side of the ear of C57-B16 mice in order to give the tumor cells access to the lymphatic system. This model of the Lewis lung carcinoma extensively metastasized to numerous lymph nodes throughout the body of the animal as well as visceral organs, as visualized by fluorescence microscopy using the bright GFP signal. Twenty-one different metastatic sites, including lymph nodes throughout the body, were identified among the cohort of transplanted animals. The data demonstrate a predilection of the Lewis lung carcinoma for lymphatic pathways for metastasis throughout the animal body. The concomitant macrometastases to the visceral organs observed in this model may be remetastasis from the lymph nodes. This model of the Lewis lung carcinoma should be very useful in defining cellular trafficking and targeting mechanisms of metastasis, in particular those involving lymphatic pathways.

Circulating tumour cells in patients with urothelial tumours: Enrichment and in vitro culture

INTRODUCTION Results of clinical trials have demonstrated that circulating tumour cells (CTCs) are frequently detected in patients with urothelial tumours. The monitoring of CTCs has the potential to improve therapeutic management at an early stage and also to identify patients with increased risk of tumour progression or recurrence before the onset of clinically detected metastasis. In this study, we report a new effectively simplified methodology for a separation and in vitro culturing of viable CTCs from peripheral blood. METHOD We include patients diagnosed with 3 types of urothelial tumours (prostate cancer, urinary bladder cancer, and kidney cancer). A size-based separation method for viable CTC - enrichment from unclothed peripheral blood has been introduced (MetaCell, Ostrava, Czech Republic). The enriched CTCs fraction was cultured directly on the separation membrane, or transferred from the membrane and cultured on any plastic surface or a microscopic slide. RESULTS We report a successful application of a CTCs isolation procedure in patients with urothelial cancers. The CTCs captured on the membrane are enriched with a remarkable proliferation potential. This has enabled us to set up in vitro cell cultures from the viable CTCs unaffected by any fixation buffers, antibodies or lysing solutions. Next, the CTCs were cultured in vitro for a minimum of 10 to 14 days to enable further downstream analysis (e.g., immunohistochemistry). CONCLUSION We demonstrated an efficient CTCs capture platform, based on a cell size separation principle. Furthermore, we report an ability to culture the enriched cells - a critical requirement for post-isolation cellular analysis.