[Expression of immune checkpoints PD-L1, CTLA4, LAG3 in the microenvironment of colon adenocarcinoma depending on MMR status]. / Ekspressiya immunnykh kontrol'nykh tochek PD-L1, CTLA4, LAG3 v mikrookruzhenii adenokartsinomy tolstoi kishki v zavisimosti ot MMR-statusa.

OBJECTIVE: Study of the features of expression of immune checkpoint proteins PD-L1, CTLA4 and LAG3 in the microenvironment of colon adenocarcinoma depending on MMR status. MATERIAL AND METHODS: The study group consisted of 32 patients with a morphologically confirmed diagnosis of colon cancer; all of them underwent surgical treatment in the form of hemicolonectomy or resection. The work assessed samples of tumor tissue obtained as a result of surgery, the study was carried out in 3 stages: morphological examination of histological slides of colon tumors at the light-optical level, immunohistochemistry examination of tumor samples to determine the dMMR/pMMR status of carcinoma using a panel of antibodies to proteins of the unpaired nucleotide repair system MLH1, MSH2, MSH6 and PMS2, multiplex analysis of PD-L1, CTLA4, LAG3, CD3+, CD8+, CD163+ markers using the Vectra 3.0.3 tissue scanning system (Perkin Elmer, USA). RESULTS: Significant differences in the expression of PD-L1, CTLA4, LAG3 in the area of the invasive tumor margin were revealed between the dMMR and pMMR groups of colon adenocarcinomas in patients comparable in clinical and morphological characteristics and treatment. In the group of tumors with dMMR status, an increase in the expression of all studied markers was noted. The number of CD3+ TILs was also significantly higher in the invasive margin of tumors with dMMR status. Similarly, in this group of colon carcinomas, a large number of CD163+ macrophages were noted both in the center and in the invasive margin zone. No statistically significant differences were found in the expression of immune checkpoints and the composition of TILs in the central zone of tumors with different MMR status. CONCLUSION: A study using multiplex immunohistochemical analysis showed that MMR-deficient colon adenocarcinomas are characterized by more pronounced immune infiltration and increased expression of immune checkpoints in microenvironmental cells, mainly in the area of invasive tumor growth. The data obtained may be important for understanding the mechanisms of immune-mediated control of tumor growth and the choice of immunotherapy tactics depending on MMR status.

The Rarity of Metastasis to the Spleen - a Phenomenon with an Unknown Mechanism.

The review analyses the frequency of malignant tumors metastasizing to the spleen. The facts are presented of a higher frequency of metastasis to the spleen in the presence of multiple metastases to other organs and the extreme rarity of isolated metastases to the spleen. Despite the rarity of spleen metastases, their frequency varies depending on the nosological form of the malignancy. The data about clinical manifestations of spleen metastases and positive effects of splenectomy in these cases are presented. The hypotheses explaining the rarity of metastases to the spleen are analyzed. Emphasis is placed on the multiple immune functions of the spleen, including the development of immunogenesis and tolerance, and the possible role of these processes in inhibiting the development of spleen metastases. However, to date, there is no complete understanding of the mechanisms of spleen metastasis inhibition. The spleen is an area where antimetastatic microenvironment is naturally formed. Understanding of the mechanisms inhibiting the development of metastases in the spleen and underlying the failure of this function in cases where metastases do occur could arm oncologists with a new strategy to prevent metastasis to any organ. Targeted research in this field is required.

Breast tumour cell subpopulations with expression of the MYC and OCT4 proteins.

The MYC and OCT4 genes are known factors associated with maintaining pluripotency and are linked with a more aggressive course, progression, and resistance to therapy in cancer. Determining the subpopulations of tumour cells expressing the Myc and Oct4 proteins will provide an opportunity to understand which tumour cell subpopulations expressing MYC and OCT4 are associated with metastasis and resistance and which subpopulations can be targeted by anti-MYC and anti-OCT4 therapy. The study included paraffin-embedded tissue from tumours from 27 patients with luminal B breast cancer obtained after neoadjuvant chemotherapy (NACT). Immunofluorescence staining was used to identify subpopulations of tumour cells expressing Myc, Oct4 and Snai2 (Opal™ 7-Color Kit (PerkinElmer, Hopkinton, MA). The following tumour cell subpopulations were identified with the Myc and Oct4 proteins and the Snai2 EMT marker: stem/progenitor tumour cells with/without Myc, Oct4 or Snai2 expression; differentiated tumour cells with/without Myc, Oct4 or Snai2 expression; and other nontumour cells (CK7-EpCAM-CD44+/-Myc+/-(Oct4, Snai2)+/-) within the inflammatory infiltrate in the tumour parenchyma and stroma. The circulating tumour cell subpopulations with Oct4 protein expression in the bloodstream were studied by flow cytometry. It was found that in patients with partial regression (PR) in response to NACT, the frequency of tumour stem cells was 3.6-fold increased (p = 0.038) in the non-EMT state (CK7+EpCam+CD44+Snai2-). In patients with metastases, there was a statistically significant 2.5-fold increase in the frequency of differentiated tumour cells with Myc expression (CK7+EpCam+CD44-Myc+) and a 2.7-fold increase in the frequency of cells with Oct4 expression (CK7+EpCam+CD44-OCT4+). In the next stage, the frequencies of subpopulations with expression of the Oct4 protein and signs of EMT among circulating tumour cells (CTCs) were determined. In patients with metastases, the frequency of tumour stem cells in the EMT state (CD326+CD44+CD24-CD325+) (p = 0.015) was more than fourfold increased, and the frequency of progenitor tumour cells with expression of the Oct4 stem protein (CD326+CD44+CD24+Oct4+) (p = 0.016) was almost sixfold higher than that in patients without metastases. Nonstem (differentiated) tumour cells with expression of the stemness proteins Myc and Oct4 were present in the breast tumour. Their content was significantly higher in residual tumours after NACT in patients who subsequently developed metastases compared with that in patients without metastases. Such cells are a new in situ marker of metastasis.

Intravasation as a Key Step in Cancer Metastasis.

Intravasation is a key step in cancer metastasis during which tumor cells penetrate the vessel wall and enter circulation, thereby becoming circulating tumor cells and potential metastatic seeds. Understanding the molecular mechanisms of intravasation is critically important for the development of therapeutic strategies to prevent metastasis. In this article, we review current data on the mechanisms of cancer cell intravasation into the blood and lymphatic vessels. The entry of mature thymocytes into the circulation and of dendritic cells into the regional lymph nodes is considered as example of intravasation under physiologically normal conditions. Intravasation in a pathophysiological state is illustrated by the reverse transendothelial migration of leukocytes into the bloodstream from the sites of inflammation mediated by the sphingosine 1-phosphate interaction with its receptors. Intravasation involves both invasion-dependent and independent mechanisms. In particular, mesenchymal and amoeboid cell invasion, as well as neoangiogenesis and vascular remodeling, are discussed to play a significant role in the entry of tumor cells to the circulation. Special attention is given to the contribution of macrophages to the intravasation via the CSF1/EGF (colony stimulating factor 1/epidermal growth factor) paracrine signaling pathway and the TMEM (tumor microenvironment of metastasis)-mediated mechanisms. Other mechanisms including intravasation of tumor cell clusters surrounded by the vessel wall elements, cooperative intravasation (entry of non-invasive tumor cells to the circulation following invasive tumor cells), and intravasation associated with the vasculogenic mimicry (formation of vascular channels by tumor cells) are also discussed. Novel intravasation-specific mechanisms that have not yet been described in the literature are suggested. The importance of targeted therapeutic strategies to prevent cancer intravasation is emphasized.

Different morphological structures of breast tumors demonstrate individual drug resistance gene expression profiles.

AIM: To identify gene expression profiles involved in drug resistance of different morphological structures (tubular, alveolar, solid, trabecular, and discrete) presented in breast cancer. MATERIAL AND METHODS: Ten patients with luminal breast cancer have been included. A laser microdissection-assisted microarrays and qRT-PCR were used to perform whole-transcriptome profiling of different morphological structures, to select differentially expressed drug response genes, and to validate their expression. RESULTS: We found 27 differentially expressed genes (p < 0.05) encoding drug uptake (SLC1A3, SLC23A2, etc.) and efflux (ABCC1, ABCG1, etc.) transporters, drug targets (TOP2A, TYMS, and Tubb3), and proteins that are involved in drug detoxification (NAT1 and ALDH1B1), cell cycle progression (CCND1, AKT1, etc.), apoptosis (CASP3, TXN2, etc.), and DNA repair (BRCA1 and USP11). Each type of structures showed an individual gene expression profile related to resistance and sensitivity to anticancer drugs. However, most of the genes (19/27; p < 0.05) were expressed in alveolar structures. Functional enrichment analysis showed that drug resistance is significantly associated with alveolar structures. Other structures demonstrated the similar number (10-13 out of 27) of expressed genes; however, the spectrum of resistance and sensitivity to different anticancer drugs varied. CONCLUSION: Different morphological structures of breast cancer show individual expression of drug resistance genes.

Types of Immune-Inflammatory Responses as a Reflection of Cell-Cell Interactions under Conditions of Tissue Regeneration and Tumor Growth.

Inflammatory infiltration of tumor stroma is an integral reflection of reactions that develop in response to any damage to tumor cells including immune responses to antigens or necrosis caused by vascular disorders. In this review, we use the term "immune-inflammatory response" (IIR) that allows us to give an integral assessment of the cellular composition of the tumor microenvironment. Two main types of IIRs are discussed: type 1 and 2 T-helper reactions (Th1 and Th2), as well as their inducers: immunosuppressive responses and reactions mediated by Th22 and Th17 lymphocytes and capable of modifying the main types of IIRs. Cellular and molecular manifestations of each IIR type are analyzed and their general characteristics and roles in tissue regeneration and tumor growth are presented. Since inflammatory responses in a tumor can also be initiated by innate immunity mechanisms, special attention is given to inflammation based on them. We emphasize that processes accompanying tissue regeneration are prototypes of processes underlying cancer progression, and these processes have the same cellular and molecular substrates. We focus on evidence that tumor progression is mainly contributed by processes specific for the second phase of "wound healing" that are based on the Th2-type IIR. We emphasize that the effect of various types of immune and stroma cells on tumor progression is determined by the ability of the cells and their cytokines to promote or prevent the development of Th1- or Th2-type of IIR. Finally, we supposed that the nonspecific influence on the tumor caused by the cytokine context of the Th1- or Th2-type microenvironment should play a decisive role for suppression or stimulation of tumor growth and metastasis.

Effect of small and radical surgical injury on the level of different populations of circulating tumor cells in the blood of breast cancer patients.

Circulating tumor cells (CTCs) constitute a heterogeneous population. Some tumor cells are cancer stem cells (CSCs), while others are in the process of the epithelial-mesenchymal transition (EMT); however, most CTCs are neither stem cells nor in the EMT. This prospective study of 22 patients with nonspecific-type invasive carcinoma of the breast identified different populations of CTCs by flow cytometry in the blood of patients before biopsy, after biopsy and after surgical tumor removal without neoadjuvant chemotherapy. The results showed that minor surgical injury (biopsy) was accompanied by a significant increase in the blood levels of CTCs without signs of the EMT or stemness (Epcam+CD45-CD44-CD24-Ncadh-) and CTCs with signs of stemness and without signs of the EMT (Epcam+CD45-CD44+CD24-Ncadh-). Our results suggest that minor surgical injury to a tumor contributes to the release of CTCs into the bloodstream, including a population of stem cells.

ROLE OF INNATE LYMPHOID CELLS DURING CANCER.

It is well known that inflammatory infiltrates in many cases present in the tumor tissue. In addition, to date, convincing evidence for its predictive value for different types of cancer. To the cells of the adaptive immune system, until recently, received more attention but in the last decade has seen a growing number of publications on the participation of innate immunity in cancer processes. This interest is partly due to the fact that in a separate class of innate lymphoid cells were isolated relatively recently. However, when analyzing the literature becomes apparent lack of information about the significance of lymphocytes in vivo innate immunity in the tumor microenvironment formation, angiogenesis, tumor cells motility acquisition and invasive properties, but also for the prediction of the occurrence other forms of progression. This review attempts to structure the available data on the lymphoid cells of the innate immune system, give the brief descriptive characteristics of each subclass, light the question of plasticity of these cells and interaction with the adaptive immune system. Special attention is given to the analysis of the role of the ILC in reparative regeneration as physiological prototype of events that underlie in tumor progression.

The effects of galectin-1 on the gene expression of the transcription factors TBX21, GATA-3, FOXP3 and RORC.

CD4(+) T cells orchestrate the immune response by differentiating into T helper (Th) or regulatory (Treg) cell subsets that secrete distinct sets of cytokines. They also play a critical role in the pathogenesis of autoimmunity, asthma, allergy and, likely, cancer. The mechanisms involved in the regulation of CD4(+) T cell homeostasis by galectin-1 remain poorly characterized. To investigate whether galectin-1 modulates the differentiation of CD4(+) T cells, the effects of galectin-1 on the mRNA expression levels of TBX21, GATA-3, FOXP3 and RORC in activated peripheral blood mononuclear cells were examined. The expression levels of GATA-3 and FOXP3 mRNA were up-regulated after treatment with 1.0 µg/ml galectin-1 and were unchanged (for GATA-3) or slightly elevated (for FOXP3) compared with untreated cells when 2.0 µg/ml galectin-1 was added. At the same time, at both concentrations of galectin-1, we observed reduced TBX21 and RORC mRNA expression levels. These findings support the concept that galectin-1 skews the differentiation of CD4(+) T cells towards Th2 and Treg cells.

Effect of galectin-1 on apoptosis of CD4(+) lymphocytes differentiated in vitro towards regulatory T cells.

We studied the effect of galectin-1 on apoptosis of CD4(+) lymphocytes intact and in vitro differentiated towards regulatory T cells. An increase in the content of apoptotic CD4(+) lymphocytes was observed after exposure of intact cells with 15 ng/ml galectin-1 and after exposure of regulatory T cells with 10 and 15 ng/ml galectin-1. Apoptosis of regulatory T cells induced by galectin-1 was accompanied by an increase in the content of proapoptotic protein Bad.

Nitric oxide donor NOC-5 increases XIAP and Aven level in Jurkat cells.

Mitochondrial permeabilisation after NO donor application did not activate caspase-9. We have studied the X-linked apoptosis inhibitor (XIAP) and Aven protein content in NO-treated Jurkat cells. The level of both proteins increased in NO-treated cells. Thus the increase in XIAP and Aven content could be the cause of the lack of caspase-9 activity after mitochondrial permeabilisation in NO-treated Jurkat cells.

[Regulation of the genes expression of CD4+ T-lymphocytes differentiation transcription factors by galectin-3 in vitro].

Now a number of CD4+ T-lymphocytes, known as Th1, Th2, Treg and Th17, is currently identified and well- studied. The methods basing on the targeted regulation of differentiation process of the Th-lymphocytes that carry out the immune response polarization attract an attention of scientists dealing with a correction of immune-mediated. In the present study, endogenous beta-galactoside-binding protein of the lectin family, galectin-3, was investigated as a regulator of T-cell homeostasis. A galectin-3 is known to be actively produced by tumor cells in malignant transformation and able to influence the processes of signal transduction, cell-cell cooperation and the implementation of programmed death. As cell differentiation processes are directly connected with the regulation of gene expression, we investigated the effect of recombinant galectin-3 on expression of mRNA of transcription.factors, which guide the differentiation of CD4+ lymphocytes. The study was performed on peripheral blood mononuclear cells of healthy individuals. The gene expression levels were evaluated by a real-time PCR. In the experiments in vitro, it has been first found the recombinant galectin-3 (0.5 mg/mL) up-regulating the expression of transcription factors Gata-3 and Rorc mRNAs and down-regulating the mRNA expression of transcription factors T-bet and FoxP3. Up to a concentration of 1 mg/mL recombinant galectin-3 stimulates Th-cells by dose-dependent manner, whereas at higher concentrations stimulating effect weakens, and inhibiting action starts prevailing. Thus, one can suppose that galectin-3 through regulation of lymphocytes differentiation promote development of allergic, autoimmune and neoplastic diseases that allows us to consider the galectin-3 as a.potential target for therapy of these diseases.

[Intracellular targets of proapoptotic influence of gaseous transmitters].

Main molecular targets of nitric oxide, hydrogen sulfide and carbon monoxide proapoptotic action in Jurkat cells were determined in this study. Decrease of mitochondrial transmembrane potential was shown during all three gases action. Reason of this event is the Bcl-2 family members disbalance. Proapoptotic proteins release after mitochondrion membranes permeabilisation could be abolished by protein xIAP inhibition of caspase -9 and -3 activity during NO and CO application.

[Intracellular gaseous messengers, nitric oxide, carbon monoxide and hydrogen sulfide participate in apoptosis regulation].

In this paper, participation of gases, nitric oxide, carbon monoxide and hydrogen sulfide, in cell apoptosis regulation has been analyzed according to the literature data and our own findings. Different mechanisms of nitric oxide influence on apoptotic reaction including modulation of transcription factors activity and increase in mitochondrion membrane permeabilisation are described. Brief description of the generation and signal transduction pathways of carbon monoxide is presented. Pro- and antiapoptotic mechanisms of hydrogen sulfide influence on cell fate are analyzed.