Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.

Clinical and immunological characteristics of sarcomas patients with clonogenic tumors.

Tumorigenesis is related to the generation of heterogeneous tumor cell population, which is the result of genetic and epigenetic alterations followed by clonal selections and subsequent expansion. In basic studies genetic, histological and morphological diversity of different clones within a patient's neoplasm and specifics of their interrelation with patient's immune system are investigated mostly on the models of tumors of epithelial origin. Mesenchymal tumors such as soft tissue and bone-derived sarcomas (STBS) have been poorly studied in this regard. The molecular genetic methods used to examine intratumoral heterogeneity do not currently provide insight into which portion of the identified subclones are able to grow autonomously. Limiting dilution cloning demonstrates the existence of self-regulating tumor cells in the population and can serve as an independent prognostic predictor of poor prognosis. Intratumoral heterogeneity results not only in differences in growth dynamics, gene expression, and phenotypic markers, but also in the resistance to treatment, especially immunotherapy, thus causing tumor eluding immune escape. The changes that accompany this process can be affected by the cellular immune system, resulting in an imbalance between populations. The variations in the population composition of immune system cells are now widely debated as a predictor of response to immunotherapy, which is of obvious interest for sarcomas, where the effectiveness of chemotherapy is low and the prognosis is unfavorable, especially in case of metastatic disease development. The search for new predictive markers of disease prognosis and treatment efficacy is an important task, to which this study is focused. Our results demonstrate that clonogenic tumor characteristics such as clonogenic potential is independent predictor of unfavorable prognosis in cases of cancer and correlate with the clinical characteristics of the tumor such as overall survival (OS) and progression free survival (PFS). It was found that patients with clonogenic sarcomas had a lower content of activated cytotoxic T-lymphocytes (CTL) with the CD3+CD8+HLA-DR+ phenotype and an increased number of natural NK killers (p < 0.05) compared to nonclonogenic tumors. In addition, according to our data, a high neutrophil to lymphocyte ratio (NLR), a low value of major T-lymphocyte populations, and a higher number of natural killer cells (NK) in the blood can be negative prognostic factors for the immunotherapy of this disease.

Biological features of tissue and bone sarcomas investigated using an in vitro model of clonal selection.

The malignancy progression is an evolutionary process in which tumor clones are selected and competed for the duration of the disease. Intratumor heterogeneity is one of the key problems in the development of treatment methods for cancer patients. In this study we obtained metastatic soft tissue and bone sarcomas (STBSs) cultures from 54 patients, performed in vitro cloning and randomly selected 83 clones. Cloning was successful in 22 cases (40.7%). STBSs cultures with a high clonogenic potential (CP) were characterized by greater proliferative activity and increased Aldehyde dehydrogenase (ALDH) expression. We studied the transcription activity of the following cancer-testis genes (CTG): MAGE, NY-ESO-1, PRAME, GAGE, SSX1, HAGE1, PASD1, SCP1, SEMG1, SLLP1 and SPANXA1. The SEMG1 expression wasn't registered in any studied case. CTG activity wasn't observed in 10 cases out of 52 (19,2%) STBS cultures. We observed CTG activation and increased transcription activity in 82 STBSs clones. Clustering by the gene profile has revealed three different patterns: 1 st - with low expression CTG, 2nd - with co-expression GAGE1, PASD1 and PRAME, 3d - with co-expression SLLP1 and GAGE1. The last two clusters included most cloned cell lines and their clones. CP of STBSs cell lines was associated with the parameters of patients overall survival (OS) at comparable progression-free survival (PFS). Among patients with STBSs with the high CP, median OS was 7.6 months (min 0.7 - max 11.0 months). In the group with the low CP, OS did not reach the median value by the end of the five-year observation period. PFS was 5.6 months (min 0.2 - max 19.2 months) in the first group and 3.2 months (min 0.3- max 71.3 months) in the second group. Resistance to therapeutic doses of chemotherapy drugs was correlated with CP cultures STBSs. We suggest that chemotherapy-resistant clones are pre-existing in the tumor rather than being formed under the influence of chemotherapy. Highly aggressive metastatic sarcomas may be a promising candidate for immunotherapy against cancer-testis antigens (CTAs).

Exploring bioactivity potential of polyphenolic water-soluble lignin derivative.

Many natural substances exhibit anti-inflammatory activity and considerable potential in prophylaxis and treatment of allergies. Knowing exact molecular targets, which is required for developing these as medicinal products, is often challenging for multicomponent compositions. In the present study we examined novel polyphenolic substance, a water-soluble fraction of wood lignin (laboratory code BP-Cx-1). In our previous study, a number of polyphenolic components of BP-Cx-1 (flavonoids, sapogenins, phenanthrenes etc.) were identified as the major carriers of biological activity of BP-Cx drug family, and several molecular targets involved in cancer and/or inflammation signaling pathways were proposed based on the results of the in vitro and in silico screening studies. In the present study, half maximal inhibitory concentration (IC50) of BP-Cx-1 was established with a radioligand method and a range of IC50 values between 22.8 and 40.3 µg/ml were obtained for adenosine receptors A1, A2A and prostaglandin receptors EP2, IP (PGI2). IC50 for serotonin 5-HT1 and for glucocorticoid GR receptors were 3.0 µg/ml and 12.6 µg/ml, respectively, both being within the range of BP-Cx-1 concentrations achievable in in vivo models. Further, distribution of [3H] labelled BP-Cx-1 in NIH3T3 murine fibroblasts and MCF7/R carcinoma cells was studied with autoradiography. [3H]-BP-Cx-1 (visualized as silver grains produced by tritium beta particles) was mainly localized along the cell membrane, in the perinuclear region and in the nucleus, suggesting ability of BP-Cx-1 to enter cells and bind to membrane or cytosol receptors. In our experiment, we observed the effect of BP-Cx-1 on maturation of dendritic cells (DCs): downregulation of expression of the lipid-presentation molecule CD1a, co-stimulatory molecules CD80, CD83 and CD 40, decreased production of pro-inflammatory cytokines IL-4 and TNF-α and increased production of anti-inflammatory cytokine IL-10. It is hypothesized that [3H]-BP-Cx-1 detectable in the nucleus is part of the activated GR complex, known to be involved in regulation of transcription of genes responsible for the anti-inflammatory response. Based on IC50, cell distribution data and results of the experiment with DCs it is suggested that the in vivo effects of BP-Cx-1 are mediated via GR and 5-HT1 receptors thus promoting development of tolerogenic effector function in dendritic cells.

Effects of Azospirillum brasilense Sp7 lectin and Phaseolus vulgarus phytohemagglutinin on cytokine activity of lymphocytes in vitro.

We studied the effect of fucose-specific lectin from Azospirillum brasilense Sp7 bacteria and Phaseolus vulgarus phytohemagglutinin (PHA) on activity of IFN-γ, IFN-α, IL-1α, IL-4, IL-8, and TNF-α in human blood in vitro. During the experiment, IFN-γ production increased from 25 pg/ml in controls to 103 and 56 pg/ml under the influence of lectin and PHA, respectively. Agglutinins had similar effects on IFN-α production. Bacterial lectin increased IL-1α level from 2.65±0.08 to 8.45±0.41 pg/ml. PHA also increased IL-1α levels, but this effect was by 1.5-2 times less pronounced. Bacterial lectin and PHA increased production IL-4 by almost 2 times and production of IL-8 by 6 and 5 times, respectively, compared to the control. No differences were found in the effects of bacterial lectin and PHA on TNF-α synthesis. Experiments showed that immunostimulatory and immunomodulatory effects of bacterial lectin are more pronounced than those of PHA.