Chemotherapy changes cytotoxic activity of NK-cells in cancer patients

In recent years, it has been shown that under certain conditions cytostatic agents (chemotherapy and radiotherapy) can restore the functioning of the immune system impaired by malignancy burden. The modifications of biological properties by cytostatics acting make cancer cells visible for the immune system recognition and elimination. Eighteen patients diagnosed with primary local breast (8) and gastric (10) cancer between 2014 and 2016 were enrolled in the investigation. The phenotypic features of NK were assessed by flow cytometry using mAb (BD Pharmingen) against CD45 (common leukocyte antigen) and CD56 (NK-marker) for surface staining, CD107a (LAMP-1), Perforin (PF) and Gransime B (GB) for intracellular staining. We examined NK populations in the peripheral blood of cancer patients before treatment and in 5 days after second course of NACT. We found that NK populations produced PF in cancer patents, which were absent before treatment, increased after NACT. Their emergence can be associated with the immunoactivating effects of chemotherapy, realized by the modification of tumor cells or elimination of immunosuppressive cells

Macrophage polarization markers of blood monocytes in patients with breast cancer

Polarized monocytes can have diagnostic value in breast cancer, including a triple negativ

Фенотипический профиль клеток моноцитарно-макрофагального ряда в зависимости от состояния респираторного эпителия

The mechanism of the relationship between pretumor changes in the bronchial respiratory epithelium and the risk of progression of non-small cell lung cancer (NSCLC) remains unclear. It has been suggested that the relationship between reactive changes in the bronchial mucosa and NSCLC progression may be caused by the functional status of monocytic-macrophage cells as important participants in infammation, which determines both the risk of premalignant changes in the epithelium and malignant progression. The purpose of the study was to investigate the phenotypic profle of peripheral blood monocytes and macrophages induced from monocytes in vitro depending on the state of respiratory epithelium in NSCLC patients. Material and Methods. The study included 39 patients with newly diagnosed NSCLC. Based on the morphological examination of small bronchi taken at the distance of 3–5 cm from the tumor, patients were divided into the following groups depending on the type of pretumor changes: no pretumor changes (n=6), isolated basal cell hyperplasia (BCH) (n=13), combination of BCH and squamous metaplasia (SM) (n=3), combination of unchanged epithelium and focal BCH (n=17). The phenotypic features of peripheral blood monocytes and in vitro-induced macrophages were assessed before treatment using fow cytometry. Results. The state of the respiratory epithelium in NSCLC patients prior to the start of anticancer treatment was associated with the phenotypic features of peripheral blood monocytes, but not with the profle of macrophages induced from them. Distortion of the response of induced macrophages to the polarizing stimuli was observed in NSCLC patients: the cultured cells responded to both M1 and M2 inducers (LPS and IL-4, respectively) with a phenotype shift to M2, while the CD206 marker expression varied depending on the presence and type of pretumor changes. Conclusion. The phenotypic profle of peripheral blood monocytes was associated with the state of the respiratory epithelium in NSCLC patients before anti-tumor treatment, but not with the phenotypic features of induced macrophages.Механизм взаимосвязи предопухолевых изменений в респираторном эпителии бронхов с риском прогрессирования немелкоклеточного рака легкого (НМРЛ) остается неясным. Предполагается, что связь между реактивными изменениями в слизистой бронхов и прогрессированием НМРЛ может быть обусловлена функциональным статусом клеток моноцитарно-макрофагальной линии, являющихся важными участниками процессов воспаления, которое определяет как риск предопухолевых изменений в эпителии, так и прогрессирование злокачественного новообразования. Цель исследования – изучение фенотипического профиля моноцитов периферической крови и макрофагов, индуцированных из моноцитов in vitro, в зависимости от состояния респираторного эпителия у больных НМРЛ. Материал и методы. В исследование включено 39 больных с впервые диагностированным НМРЛ. На основании результатов морфологического исследования мелких бронхов, взятых на расстоянии 3–5 см от опухоли, в зависимости от варианта предопухолевых изменений больные были разделены на следующие группы: отсутствие предопухолевых изменений (n=6), изолированная базальноклеточная гиперплазия (БКГ) (n=13), сочетание БКГ и плоскоклеточной метаплазии (ПМ) (n=3), сочетание неизмененного эпителия и очаговой БКГ (n=17). Оценка фенотипических особенностей моноцитов периферической крови и индуцированных in vitro макрофагов проводилась до начала противоопухолевого лечения больных методом проточной цитометрии. Результаты. Состояние респираторного эпителия у больных НМРЛ до начала противоопухолевого лечения ассоциировано с фенотипическими особенностями моноцитов периферической крови, но не с профилем индуцированных из них макрофагов. У больных НМРЛ отмечено искажение ответа индуцированных макрофагов на поляризующие стимулы: как на М1-, так и на М2-индукторы (LPS и IL-4, соответственно) культивируемые клетки отвечали сдвигом фенотипа в М2-сторону, при этом экспрессия маркера CD206 варьировала в некотором диапазоне в зависимости от наличия и варианта предопухолевых изменений. Заключение. Фенотипический профиль моноцитов периферической крови ассоциирован с состоянием респираторного эпителия у больных НМРЛ до начала противоопухолевого лечения, но не с фенотипическими особенностями индуцированных макрофагов.

Изменение функционального профиля моноцитов крови при раке молочной железы

The purpose of the study was to identify functional features of circulation monocytes in patients with nonmetastatic breast cancer.Material and Methods. The study cohort consisted of 10 breast cancer patients treated at Tomsk Cancer Research Institute. 7 healthy female volunteers were enrolled as a control group. CD14+16-, CD14+16+ and CD14-16+ monocytes subsets were obtained from blood by sorting. Whole transcriptome profling was provided in monocytes from patients and healthy females. Macrophages were differentiated from the obtained monocytes under in vitro conditions. The ability of conditioned media obtained from macrophages to infuence apoptosis and proliferation of MDA-MB 231 cell line was evaluated.Results. Transcriptomic profling revealed signifcant changes in monocytes of breast cancer patients. CD14+16- subset showed higher expression of transporters ABCA1 and ABCG1; chemokines CCR1, CRRL2, CXCR4; maturation and differentiation factors Mafb and Jun; endocytosis mediating factors CD163 and Siglec1; proteases and tetrasponins ADAM9, CD151, CD82, and growth factor HBEGF in patient group. Macrophages derived from monocytes of breast cancer patients produced factors that supported proliferation of the MDA-MB 231 cell line, which was not observed for monocytes from healthy volunteers.Conclusion. Thus, breast carcinoma has a systemic effect on peripheral blood monocytes, programming them to differentiate into macrophages with tumor supporting capacity. Цель исследования ‒ оценить особенности функционального профиля моноцитов периферической крови у больных неметастатической формой рака молочной железы.Материал и методы. В исследование вошли 10 больных раком молочной железы II–III стадии (T1–3N0–2M0). В качестве контроля была обследована группа из 7 здоровых женщин. Моноциты были получены из периферической крови путем сортировки популяций с фенотипом CD14+16-, CD14+16+ и CD14-16+. Проведено полнотранскриптомное профилирование полученных моноцитов от больных раком молочной железы и здоровых женщин. Из полученных моноцитов in vitro были дифференцированы макрофаги. Проведена оценка способности полученных от макрофагов кондиционных сред влиять на апоптоз и пролиферацию клеток линии MDA-MB 231.Результаты. Показано, что транскриптомный профиль моноцитов больных РЖЖ имеет выраженные отличия по сравнению со здоровыми женщинами. Моноциты пациенток с раком молочной железы отличаются повышенной экспрессией мРНК белков-транспортеров ABCA1, ABCG1; хемокинов CCR1, CRRL2, CXCR4; факторов созревания и дифференцировки моноцитов Mafb и Jun; факторов, опосредующих эндоцитоз CD163, Siglec1; протеаз и тетраспонинов ADAM9, CD151, CD82 и ростового фактора HBEGF. Макрофаги, полученные в результате культивирования моноцитов больных раком молочной железы в условиях in vitro, продуцировали факторы, которые позволили поддерживать пролиферацию клеточной линии опухолевых клеток, чего не наблюдалось для моноцитов здоровых доноров.Заключение. Опухоль молочной железы оказывает системное влияние на моноциты периферической крови, программируя их к дифференцировке в макрофаги с проопухолевой функциональной активностью.

Поляризация макрофагов: механизмы, маркеры и факторы индукции

Macrophages  are  key  components  of  the  innate  immune  system. The  variability  of  the  macrophage’s participation in tumor progression, determined by their functional polarization, opens up a wide prospect for modulating their functional profile, primarily in the direction of increasing antitumor activity. The purpose of the study was to provide up-to-date data on the process of macrophage polarization, mechanisms of its regulation, polarization markers and induction factors. Material and methods. A search was made for available literature sources published in Web of Science, Scopus and other databases. more than 160 sources devoted to the study of the process of macrophage polarization were found, of which 121 were included in this review. Results. This review presents data on the molecular mechanisms and gene signatures associated with M1 and M2 polarization of macrophages. We displayed information on metabolic, phenotypic characteristics and cytokine profile of M1- and M2-macrophages, as well as highlighted data on polarization factors and targets of their action. Conclusion. The information presented in the review can serve as an information base for the development of experimental and clinical approaches for editing the functional profile of macrophages in order to control their involvement in various pathological processes.Макрофаги являются ключевыми компонентами врожденной иммунной системы. Вариативность участия макрофагов в различных патологических процессах, определяемая их функциональной поляризацией, открывает широкую перспективу для модуляции их функционального профиля для использования в терапевтических целях. Цель исследования – предоставить современные данные о процессе поляризации макрофагов, механизмах его регуляции, маркерах поляризации и факторах индукции. Материал и методы. Проведен поиск доступных литературных источников, опубликованных в базах данных Web of Science, Scopus и др. Найдено более 160 источников, посвященных изучению процесса поляризации макрофагов, из которых 121 включен в данный обзор. Результаты. В обзоре представлены данные о молекулярных механизмах и генных сигнатурах, ассоциированных с М1- и М2- поляризацией макрофагов, информация о метаболических, фенотипических признаках и цитокиновом профиле М1- и М2-макрофагов, а также освещены данные о факторах поляризации и мишенях их воздействия. Заключение. Представленные в обзоре сведения могут послужить информационной базой для разработки экспериментальных и клинических подходов для редактирования функционального профиля макрофагов с целью управления патологическими процессами с их участием

T93

Risk of metastasis formation is provided by both tumor cell biological characteristics and the microenvironment features within the primary tumor along with local and systemic conditions for metastatic niche formation. The inflammatory infiltration has been shown to strongly impact on tumor progression (Whiteside, 2013). Dronca et al. (2011) showed that immunosuppressive factors in the tumor microenvironment may impair not only local immune responses but also disturb systemic immunity. Zitvogel et al. anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.To study the impact of immune system on clinical response to neoadjuvant chemotherapy and metastasis-free survival in breast cancer patients. Materials and methods: 350 patients with newly diagnosed invasive breast cancer treated with neoadjuvant chemotherapy (NAC) were enrolled into the study. The procedures were made in accordance with the Helsinki Declaration. Clinical response to chemotherapy, the 5-year metastasis-free survival and all major clinical and morphological parameters were determined. The original method of multidimensional data visualization was applied to present the immune system state as integral entirety in visual image for classification of patients with different risk of metastasis (NovoSpark Corporation, Canada). Copy number aberrations (CNA) of cytokine gene regions in tumor specimens were tested using high-density microarray platform CytoScanTM HD Array (Affymetrix, USA). Cytokine gene polymorphism was analyzed. Subpopulations of lymphocytes and macrophages were determined within the primary tumors by IHC. Results: We found, that favorable clinical immediate response to preoperative chemotherapy was related to the high levels of IL-1beta, TNF-alpha and IL-10 production by peripheral mononuclear cells before the treatment. This correlation was further confirmed by data from the study on association between cytokine gene functional polymorphism and response to NAC. We used NovoSpark Corporation visualization approach allowing the representation the immune system state as integral unit and to discriminate breast cancer patients with high and low risk of haematogeneic metastasis. When estimated before cancer treatment, 95% of breast cancer patients had risk of metastasis. The neoadjuvant chemotherapy and surgical tumor removal reduced the risk of tumor progression to 62–71%. However, in a year after adjuvant chemo- and radiotherapy, the patient group with high risk of metastases increased to 81% again. Thus, the cancer treatment can change the primarily estimated outcome prognosis in breast cancer patients, and the monitoring of immune system is a promising approach to predict the risk of cancer progression or resistance to the therapy. We have found the connection between the profile of intra-tumor inflammatory elements and chemotherapy efficacy.Cytokine gene expression may be influenced by the chromosome anomalies (CNA – Copy Number Aberration) – deletion and amplification – of cytokine gene loci in tumor cells. We found the close relation between the clinical response to NAC and gain of function of IL-10 and CHI3L1 (YKL40) genes. In contrast, loss of TNF-alpha and IL-17 gene function due to corresponding CNA was associated with good response to NAC. Metastasis-free survival of breast cancer patients was shown to be closely related to CNA. Conclusion: The parameters of the activation of systemic and intra-tumoral immune system by growing tumor and its dissemination have to be validated in order to identify the new prognostic markers for the efficiency of the neoadjuvant chemotherapy

P10

Tumour-associated macrophages (TAM) derived from peripheral blood monocytes are a key immune regulatory component of tumour microenvironment (Qian, Pollard, 2010). Advanced clinically applicable strategy for TAM targeting is their functional reprogramming (Wynn et al., 2013). Monocytes/macrophages are heterogeneous and versatile cells. Two major macrophage subpopulations with different functions which represent extreme of a continuum in a universe of activation states, including classically activated/inflammatory (M1) and alternatively activated/regenerative (M2) macrophages, have long been recognized (Zhou et al., 2014). CD68 and CD163 are the general markers of M1 and M2 subsets respectively (Hoene et al., 2015, Tedesco et al., 2014). However, there are different markers expressed on surface of monocytes/macrophages that reflected some special functional activities of these cells. Thus, expression of CD119(IFN-γR) or CD124(IL-4R) reflects the predisposition to M1 or M2-polarisation by suitable cytokines IFN-γ and IL-4 (Tedesco et al., 2014). The pro-inflammatory marker CD282 (TLR2) is associated with antibacterial function. Anti-inflammatory CD36+ cells take part in scavenging of apoptotic remains and inducing IL-10 production. Although monocytes/macrophages could undergo their phenotypically/functionally dynamic switch in response to the microenvironment signals there is some predisposition of their status in peripheral blood to polarization in tissues. Modulation of monocyte peripheral status before their infiltration is an attractive target for cancer therapeutic approaches. The aim is to study phenotypic subsets of peripheral blood monocytes in breast and gastric cancer patients. Materials and methods: 8 breast cancer and 8 gastric cancer patients T1N0-3M0 treated in Tomsk Cancer Institute were enrolled in investigation. The diagnosis was histologically verified. Phenotypic features were assessed by flow cytometry using mAb CD119(IFN-γRα), CD124(IL-4R), CD282 (TLR2), CD36, CD68, CD163 (BD Pharmingen) before treatment and in 5 days after surgery of tumour. Results: We found that amounts of monocytes expressing main surface markers of M-1 or M2-type activation in peripheral blood in cancer patients wasn’t large: CD68+ and CD163+ cells were 4.18 ± 3.41% and 7.64 ± 3.75% of whole pool of monocytes respectively. This finding supports the fact that in general M1/M2-polarization of monocytes/macrophages occurs in local tissues by specific micro- environmental conditions [Ambarus et al., 2012, Zhou et al., 2014]. But high levels of CD119(IFN-γR)+monocytic cells (87.36 ± 12.08%) in peripheral blood showed that predisposition to M1-polarization of circulating monocytes exists in cancer patients, because it’s known IFN-γ significantly increased the fraction of surface CD68 – expressing cells and down-regulated expression of the M2 markers in vitro [Tedesco et al., 2014]. Expression of CD124 (IL-4R) was detected only in 26.81 ± 8.11% of monocytes in cancer patients. The subset of pro-inflammatory CD282+ (TLR2) circulating monocytes amounted 64.38 ± 9.47% of total count of monocytic cells. Anti-inflammatory CD36+ monocytes were 35.66 ± 7.00%. It is noteworthy this subset was 48.43 ± 8.13% in breast cancer patients and only 22.88 ± 9.86% in gastric cancer patients (p < 0.05). On the 5-th day after surgery, we detected decrease in CD282+ cells from 80.90 ± 8.55% to 38.70 ± 9.47% of total monocytes count in breast cancer patients (p < 0.05). Another noticeable feature of monocytes pool after operation was increasing of anti-inflammatory CD36+monocytes as in breast (1.69-fold) and gastric (1.97 fold) cancer patients. Conclusion: There were a small accounts of M1 (CD68+) or M2 (CD163+) polarized monocytes in peripheral blood in breast and gastric cancer patients. INF-γ(CD119)+ and CD282(TLR2)+ pro-inflammatory subsets were dominated in cancer patients. The high levels of CD36+monocytes was the feature of breast cancer patients as compared with gastric cancer ones. Surgery removal of tumour was associated with increasing of CD36+ monocytes in both breast and gastric cancer and with decreasing of CD282 (TLR2)+ monocytes in breast cancer