BACKGROUND: Dendritic cell (DC) therapy counts to the promising strategies how to weaken and eradicate cancer disease. We aimed to develop a good manufacturing practice (GMP) protocol for monocyte-derived DC (Mo-DC) maturation using circulating tumor cells lysates with subsequent experimental T-cell priming in vitro. METHODS: DC differentiation was induced from a population of immunomagnetically enriched CD14 + monocytes out of the leukapheresis samples (n = 6). The separation was provided automatically, in a closed bag system, using CliniMACS Prodigy® separation protocols (Miltenyi Biotec). For differentiation and maturation of CD14 + cells, DendriMACs® growing medium with supplements (GM-CSF, IL-4, IL-6, IL-1B, TNFa, PGE) was used. Immature Mo-DCs were loaded with autologous circulating tumor cell (CTCs) lysates. Autologous CTCs were sorted out by size-based filtration (MetaCell®) of the leukapheresis CD14-negative fraction. A mixture of mature Mo-DCs and autologous non-target blood cells (NTBCs) was co-cultured and the activation effect of mature Mo-DCs on T-cell activation was monitored by means of multimarker gene expression profiling. RESULTS: New protocols for mMo-DC production using automatization and CTC lysates were introduced including a feasible in vitro assay for mMo-DC efficacy evaluation. Gene expression analysis revealed elevation for following genes in NTBC (T cells) subset primed by mMo-DCs: CD8A, CD4, MKI67, MIF, TNFA, CD86, and CD80 (p ≤ 0.01). CONCLUSION: Summarizing the presented data, we might conclude mMo-DCs were generated using CliniMACS Prodigy® machine and CTC lysates in a homogenous manner showing a potential to generate NTBC activation in co-cultures. Identification of the activation signals in T-cell population by simple multimarker-qPCRs could fasten the process of effective mMo-DC production.

• Keywords
• Circulating tumor cells, Dendritic cells, Immunotherapy, MetaCell, Personalized medicine, T cells,
• MeSH
• Dendritic Cells * metabolism   MeSH
• Granulocyte-Macrophage Colony-Stimulating Factor pharmacology   MeSH
• Interleukin-4 pharmacology   MeSH
• Interleukin-6 pharmacology   MeSH
• Humans MeSH
• Monocytes * metabolism   MeSH
• Neoplastic Cells, Circulating * metabolism   MeSH
• Prostaglandins E pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Granulocyte-Macrophage Colony-Stimulating Factor MeSH
• Interleukin-4 MeSH
• Interleukin-6 MeSH
• Prostaglandins E MeSH

INTRODUCTION: This study analyzes peripheral blood samples from breast cancer (BC) patients. CTCs from peripheral blood were enriched by size-based separation and were then cultivated in vitro. The primary aim of this study was to demonstrate the antigen independent CTC separation method with high CTC recovery. Subsequently, CTCs enriched several times during the treatment were characterized molecularly. METHODS: Patients with different stages of BC (N = 167) were included into the study. All patients were candidates for surgery, surgical diagnostics, or were undergoing chemotherapy. In parallel, 20 patients were monitored regularly and in addition to CTC presence, also CTC character was examined by qPCR, with special focus on HER2 and ESR status. RESULTS: CTC positivity in the cohort was 76%. There was no significant difference between the tested groups, but the highest CTC occurrence was identified in the group undergoing surgery and similarly in the group before the start of neoadjuvant treatment. On the other hand, the lowest CTC frequencies were observed in the menopausal patient group (56%), ESR+ patient group (60%), and DCIS group (44.4%). It is worth noting that after completion of neoadjuvant therapy (NACT) CTCs were present in 77.7% of cases. On the other hand, patients under hormonal treatment were CTC positive only in 52% of cases. DISCUSSIONS: Interestingly, HER2 and ESR status of CTCs differs from the status of primary tumor. In 50% of patients HER2 status on CTCs changed not only from HER2+ to HER2-, but also from HER2- to HER2+ (33%). ESR status in CTCs changed only in one direction from ESR+ to ESR-. CONCLUSIONS: Data obtained from the present study suggest that BC is a heterogeneous disease but CTCs may be detected independently of the disease characteristics in 76% of patients at any time point during the course of the disease. This relatively high CTC occurrence in BC should be considered when planning the long-term patient monitoring.

• Keywords
• Breast cancer, CTCs, Circulating tumor cells, Cultivation, Gene expression, In vitro, MetaCell,
• MeSH
• Estrogen Receptor alpha genetics   MeSH
• Adult MeSH
• Genetic Heterogeneity * MeSH
• Middle Aged MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   MeSH
• Neoplastic Cells, Circulating pathology   MeSH
• Breast Neoplasms blood   genetics   pathology   MeSH
• Receptor, ErbB-2 genetics   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Aged MeSH
• Neoplasm Staging MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Estrogen Receptor alpha MeSH
• ERBB2 protein, human MeSH Browser
• ESR1 protein, human MeSH Browser
• Biomarkers, Tumor MeSH
• Receptor, ErbB-2 MeSH

The focus of the study was to implement a new workflow for circulating tumor cells (CTCs) characterization that would allow the analysis of CTCs on a cytomorphological and molecular level in patients with diagnosed gynecological cancer. Our findings may be useful in future cancer patient management. The study introduces a size-based enrichment (MetaCell(®)) method for the separation of viable CTCs, followed by CTCs culturing in vitro and gene expression characterization. It is based on the observation of CTCs and DTCs (Disseminated Tumor Cells) in several case studies of ovarian, endometrial and cervical cancer by means of cytomorphology and gene expression profiling. The viability of the enriched CTCs was estimated using vital and lethal fluorescence nuclear staining. This type of staining may be predictive for the success rate of subsequent CTC growth in vitro. To identify CTCs in the enriched CTC fraction, cytomorphological evaluations based on vital fluorescence staining were followed by gene expression analysis of tumor-associated (TA) genes. Cytokeratin expression (KRT7, KRT19) was analyzed in combination with MUC1, MUC16, CD24, CD44 and ALDH1. Gene expression analysis has shown that short-term in vitro culture enhanced the differentiation process of the captured CTCs growing on a membrane. On the other hand, redundant white blood cells captured on the membrane were eliminated during a short-term culture. The most frequently elevated genes in ovarian cancer (serous type) are EPCAM, KRT19 and MUC1. It has been demonstrated that CTC presence revealed by cytomorphological evaluation may be usefully complemented by TA-gene expression analysis, to increase the sensitivity of the analysis.

• Keywords
• CTCs, cervical cancer, cultivation, endometrial cancer, gynecological cancers, in vitro, ovarian cancer,
• Publication type
• Journal Article MeSH