Vladimir Bobek

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.

Enhancement of musculocutaneous nerve reinnervation after vascular endothelial growth factor (VEGF) gene therapy

BackgroundVascular endothelial growth factor (VEGF) is not only a potent angiogenic factor but it also promotes axonal outgrowth and proliferation of Schwann cells. The aim of the present study was to quantitatively assess reinnervation of musculocutaneous nerve (MCN) stumps using motor and primary sensory neurons after plasmid phVEGF transfection and end-to-end (ETE) or end-to-side (ETS) neurorrhaphy. The distal stump of rat transected MCN, was transfected with plasmid phVEGF, plasmid alone or treated with vehiculum and reinnervated following ETE or ETS neurorrhaphy for 2 months. The number of motor and dorsal root ganglia neurons reinnervating the MCN stump was estimated following their retrograde labeling with Fluoro-Ruby and Fluoro-Emerald. Reinnervation of the MCN stumps was assessed based on density, diameter and myelin sheath thickness of regenerated axons, grooming test and the wet weight index of the biceps brachii muscles.ResultsImmunohistochemical detection under the same conditions revealed increased VEGF in the Schwann cells of the MCN stumps transfected with the plasmid phVEGF, as opposed to control stumps transfected with only the plasmid or treated with vehiculum. The MCN stumps transfected with the plasmid phVEGF were reinnervated by moderately higher numbers of motor and sensory neurons after ETE neurorrhaphy compared with control stumps. However, morphometric quality of myelinated axons, grooming test and the wet weight index were significantly better in the MCN plasmid phVEGF transfected stumps. The ETS neurorrhaphy of the MCN plasmid phVEGF transfected stumps in comparison with control stumps resulted in significant elevation of motor and sensory neurons that reinnervated the MCN. Especially noteworthy was the increased numbers of neurons that sent out collateral sprouts into the MCN stumps. Similarly to ETE neurorrhaphy, phVEGF transfection resulted in significantly higher morphometric quality of myelinated axons, behavioral test and the wet weight index of the biceps brachii muscles.ConclusionOur results showed that plasmid phVEGF transfection of MCN stumps could induce an increase in VEGF protein in Schwann cells, which resulted in higher quality axon reinnervation after both ETE and ETS neurorrhaphy. This was also associated with a better wet weight biceps brachii muscle index and functional tests than in control rats.

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.

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.

Site‐specific cytomorphology of disseminated PC‐3 prostate cancer cells visualized in vivo with fluorescent proteins

Circulating tumor cells (CTC) may reach multiple organ sites. However, CTC seeding and growth in distant organs is not random. Each metastatic site may contain a specific subpopulation of the original metastatic tumor capable of growing at that site. The fluorescent orthotopic prostate cancer model (PC‐3‐GFP) model was used for immunomagnetic capture of CTC. The captured CTC were efficiently cultivated in vitro. PC‐3‐GFP cells were also isolated from various metastatic sites, grown in vitro and examined under fluorescence microscopy. The differential morphology was compared of primary tumor cells, CTC and disseminated (DTC) from multiple metastatic sites, from nude mice with orthotopic PC‐3‐GFP. The cultured captured CTC and DTC from various organs have distinctive morphologies. Distinct cancer cell morphologies were observed at different metastatic sites as well as among CTC. The distinct morphologies were maintained during in vitro culture. The results demonstrate extensive tumor heterogeneity that could account for the widely different behavior of cancer cells in a single tumor. Further hetereogeneity testing would be a big promise for personalizing the cancer treatment in the future. Diagn. Cytopathol. 2013.