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  • 1
    Online Resource
    Online Resource
    Enzymatic tissue disintegration leads to the loss of immunophenotype of breast cancer stem cells.
    American Society of Clinical Oncology (ASCO) ; 2020
    In:  Journal of Clinical Oncology Vol. 38, No. 15_suppl ( 2020-05-20), p. e13089-e13089
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 38, No. 15_suppl ( 2020-05-20), p. e13089-e13089
    Abstract: e13089 Background: Cancer stem cells (CSC) are a population in the complex hierarchy of tumor tissue responsible for growth, metastasis and resistance to chemotherapy and radiotherapy. The purpose of the study was to reveal the effect of the enzymatic disintegration of breast cancer (BC) tissue on the CSC immunophenotype. Methods: BC tumor samples obtained during surgery were placed for 2 hours in a sterile Hanks’ solution immediately after collection. Samples were fragmented to 1 mm in diameter, the fragments were evenly distributed into 4 Petri dishes with (6 cm diameter) and placed in DMEM medium (Gibco, USA) without collagenase and with the addition of collagenase (Biolot, Russia) to a final concentration of 150, 300 and 450 units/ml. Samples were cultured for 48 hours at 37°C and 5.5% CO 2 . On day 2, the samples incubated in collagenase were grinded by passing through tips of decreasing diameter; the reference sample was grinded in the Medimachine system (BD, USA). The effect of enzymatic tissue disintegration on the preservation of BC SC immunophenotype (CD24 low/- CD44 + ) was evaluated using the BD FacsCanto II flow cytometer (BD, USA) and PE Mouse Anti-Human CD24 (clone ML5; BD, USA) and FITC Mouse Anti-Human CD44 (clone G44-26; BD, USA) antibodies. Results: The total number of isolated cells increased almost exponentially with increasing collagenase concentration. The trypan blue test showed that cell viability did not change significantly with an increase in collagenase concentration and was approximately 50%, being an order of magnitude higher compared to mechanical dissociation (4%). However, 48 h incubation in a collagenase solution led decreased the number of CD44+ cells in proportion to the enzyme concentration by 3 to 10 times in comparison with the control. Similarly, inhibition of CD24 expression was observed compared with the control, inevitably leading to a decreased purity of the isolated subpopulation with the CD24 low/- CD44 + phenotype. Conclusions: Despite an increase in the yield of viable cells, prolonged incubation in a collagenase solution leads to a loss of the immunophenotype of BC SC.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/JCO.2020.38.15_suppl.e13089
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2020
    detail.hit.zdb_id: 2005181-5
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  • 2
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    Online Resource
    Immunohistochemical evaluation of Cyclin D1 and β-Catenin expression in subtypes of triple-negative breast cancer.
    American Society of Clinical Oncology (ASCO) ; 2019
    In:  Journal of Clinical Oncology Vol. 37, No. 15_suppl ( 2019-05-20), p. e12553-e12553
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 37, No. 15_suppl ( 2019-05-20), p. e12553-e12553
    Abstract: e12553 Background: The purpose of the study was to reveal characteristics of Cyclin D1 and β-Catenin expression in subtypes of triple negative breast cancer (TNBC). Methods: The study included 60 patients diagnosed with verified TNBC (T1N1M0/T2N0M0, ER-/PR-/Her2-). Immunohistochemical staining with CK5/6, EGFR, Cyclin D1, β-Catenin antibodies was performed. Basal-like (BL) subtype was identified when 〉 25% cells were CK5/6+ and/or EGFR+ (any staining). The expression of β-Catenin was assessed by its location: the membrane, cytoplasm, nucleus. Cells with negative staining on the membrane and cytoplasmic staining were counted and calculated as a percentage of the total number of tumor cells. Nuclear expression of β-Catenin was considered positive with at least 1 positively stained cell in the field of view at x200 magnification. Parametric statistical methods were used. Significance of the difference between the two means was assessed by the Student's t-test. Results: A number of TNBC samples showed Cyclin D1 overexpression. The loss of β-Catenin on the cell membrane and its abnormal accumulation in the cytoplasm was significantly more frequent in TNBC with Cyclin D1 overexpression. These processes were more pronounced in BL cancers. Loss of membrane expression in BL cancers: 29.6±6.1 and 58±7.4%; accumulation in the cytoplasm: 33.4±5.4 and 63.3±7.2%, with low and high Cyclin D1 respectively. Loss of membrane expression in non-BL cancers: 17.6±3.8 and 33.3±4.3%; accumulation in the cytoplasm: 27.3±6.3 and 49.5±8.2%, with low and high Cyclin D1 respectively. Only BL TNBC demonstrated β-catenin translocation to the nucleus (up to 20 stained cells in the field of view): in 33.3% tumors with Cyclin D1 overexpression and in 12% of tumors with its low expression. Conclusions: Levels of expression of β-catenin and Cyclin D1 in TNBC may have predictive value, and the choice of these biomarkers as targets will improve the treatment with new-generation medications.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/JCO.2019.37.15_suppl.e12553
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2019
    detail.hit.zdb_id: 2005181-5
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  • 3
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    Online Resource
    Immunomagnetic separation in LAK generation in patients with breast cancer.
    American Society of Clinical Oncology (ASCO) ; 2020
    In:  Journal of Clinical Oncology Vol. 38, No. 15_suppl ( 2020-05-20), p. e15220-e15220
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 38, No. 15_suppl ( 2020-05-20), p. e15220-e15220
    Abstract: e15220 Background: The purpose of the study was to evaluate the effect of IL-2 and INF-γ on the proliferation and phenotype of lymphocytes in patients with breast cancer (BC) after T-regs removal from the pool in vitro. Methods: Lymphocytes were isolated from the blood of 11 patients with stage II BC, T-regs were removed by immunomagnetic separation, and samples were cultured for 6 days with cytokines: IL-2 - 0.1/1 μg, INF-γ - 10 IU and their combinations. Results: The level of NK cells in the samples with IL-2 was maintained throughout the experiment, with IFN-γ - gradually decreased, and in the control (samples without stimulation) it decreased sharply. In experimental samples (IL-2, IL-2+IFN-γ), the amount of NKT remained at the initial level by day 6, and in the control it decreased by 2 times (p 〈 0.05). The number of CD335+ NK cells with high cytotoxicity against target cells increased after 3 days in the test samples with IL-2 by 5.2 times, and after 6 days - by 11.6 times in comparison with the control (p 〈 0.05). On day 6, the level of double-positive T cells increased by 1.8 times in co-culturing with IL-2+INF-γ (1 μg/10 IU), (p 〈 0.05). The percentage of double-negative T cells increased in the dynamics of cultivation from days 1 to 6, in some cases statistically significantly (IL-2 - 1 μg: by 2 times; IL-2+INF-γ - 0.1 μg/10 IU: by 1.8 times; p 〈 0.05). The percentage of CD4+CD38+ cells on day 6 of incubation with IL-2 at both doses increased by 1.8-2 times compared with the control, and CD8+CD38+ by 4-6 times. The percentage of CD4+HLA-DR+ cells after 6 days of cultivation with IL-2 increased by 6 times, and CD8+HLA-DR+ by 8-9 times. Cultivation with INF-γ reduced the stimulating effect, and in some cases it did not appear. The combined effect of IL-2 and INF-γ on day 6 led to an increase in the proportion of CD4+CD25+CD127dim cells by 1.8 times in comparison with the control (p 〈 0.05), and when exposed to INF-γ only, the number of these cells remained at the control levels. Conclusions: INF-γ is not an effective inducer of activation of immune effector cells, while the effect of IL-2, both alone and in combination with INF-γ, leads to a significant increase in the proportion of cytotoxic cells, as well as to activation of the T cell unit, including CD4 + CD25 + CD127dim.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/JCO.2020.38.15_suppl.e15220
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2020
    detail.hit.zdb_id: 2005181-5
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  • 4
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    The common gamma-chain cytokines IL-2, IL-7 and IL-15 impact on proliferation of lymphocytes in vitro in breast cancer patients.
    American Society of Clinical Oncology (ASCO) ; 2021
    In:  Journal of Clinical Oncology Vol. 39, No. 15_suppl ( 2021-05-20), p. e13060-e13060
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 39, No. 15_suppl ( 2021-05-20), p. e13060-e13060
    Abstract: e13060 Background: Monotherapy with low doses of cytokines does not provide significant therapeutic results, while treatment with high doses leads to a number of side effects, for example, cytokine release syndrome, etc. Therefore, it is necessary to study the effect of cytokines on immune cells which are involved in the regulation of pro- and antitumor immune response. Thus, the aim of this study was to analyze the dynamics in the proliferation of the total fraction of lymphocytes after expansion and exposure to γc-cytokines: IL-2, IL-7 and IL-15 and their combinations in vitro in patients with diagnosed breast cancer. Methods: The study included 10 patients with locally advanced stage I-III breast cancer. Peripheral blood mononuclear cells (PBMC, ficoll-hypaque) were used as material. After density separation the cells were cultured at a dose of 500 thousand cells / ml in 6-well plates (Biofil, China) in RPMI 1640 (Gibco, USA) supplemented with 10% FBS (HyClone, USA) at 37° C, 5% CO2. Expansion of lymphocytes was performed by kit with anti-biotin magnetic particles MACSiBead to CD2, CD3 and CD28 (Miltenyi Biotec, Germany), according to the manufacturer's protocol. Stimulation of lymphocyte proliferation was performed on days 4, 8 and 12 by introducing recombinant cytokines - IL-2, IL-7, IL-15 (Miltenyi Biotec, Germany) and their combinations. Cytokines were introduced at a concentration of 40 ng / ml each in the following variants: IL-2; IL-2 / IL-15; IL-2 / IL-15 / IL-7; IL-15; IL-15 / IL-7. Plates with cell suspension were cultured at 37 ° C, 5% CO2 for 15 days. Results: The data obtained on the 11th day of incubation demonstrated statistically significant differences in cell viability in samples with the addition of interleukin IL-7 / IL-15 combination (778%) compared to control samples by 1.5 times (p 〈 0.05). The proportion of lymphocytes in samples with the addition of only IL-15 (702%) and IL-7 / IL-15 (756%) combination was 1.47 and 1.6 times higher, respectively, compared with the control (p 〈 0.05) at the 12th day of co-cultivation. Conclusions: Stimulation of lymphocyte proliferation by the IL-7 / IL-15 combination results in a high frequency of viable cell production. Despite the only culture stage, the results of the study may be important for optimizing the use of γc-cytokines in the treatment of patients with breast cancer.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/JCO.2021.39.15_suppl.e13060
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2021
    detail.hit.zdb_id: 2005181-5
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  • 5
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    Online Resource
    Potential use of IL-7 and IL-15 for ex vivo generation of LAK.
    American Society of Clinical Oncology (ASCO) ; 2020
    In:  Journal of Clinical Oncology Vol. 38, No. 15_suppl ( 2020-05-20), p. e15201-e15201
    Details
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 38, No. 15_suppl ( 2020-05-20), p. e15201-e15201
    Abstract: e15201 Background: A review of scientific literature has shown that IL-2 is most often used for the LAK generation, while the potential of other NK-stimulating interleukin cells remains poorly understood. In this study, we investigated the effect of IL-7 and IL-15 on ex vivo LAC generation. Methods: A fraction enriched in NK cells was isolated by magnetic cell sorting with the NK Cell Isolation Kit (#130-092-657, Miltenyi Biotec, Germany) from PBMC in 11 patients with stage II-III breast cancer without treatment. Cells were introduced into a 6-well 3x10 5 plate in RPMI medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA). Cytokines 40 ng/ml were added to the wells in 6 variants: 1) IL-15; 2) IL-2; 3) IL-7; 4) IL-15+IL-7; 5) IL-15+IL-7+IL-2; 6) control without cytokines. Cells were cultured at 5.0% CO 2 and 37°C. Cells were counted with a hemocytometer daily for 5 days and on days 8, 9 and 10 of cultivation. Results: The number of NK cells in control samples gradually decreased: by 2 times on day 5 and by 3 times on day 10. On day 5, the number of NK cells was 1.5 times higher than in the control when cultured with IL-2, and 1.4 times higher when cultured with IL-7+IL-15. After 9 days, a statistically significant increase in the number of cells, compared to the control sample, was observed with the addition of IL-2 (1.6 times); IL-15 and IL-7+IL-15 (1.5 times). On day 10, significant differences from the control were found in most samples: the number of cells was higher in samples cultured with IL-2 and IL-7+IL-15 (1.9 times) and with IL-15 and IL-2+IL-7+IL-15 (1.7 times). IL-7 alone led to a gradual decrease in the number of cells, and on days 8, 9 and 10 it was lower than in the control samples. Conclusions: In general, the introduction of cytokines into the samples enriched with NK cells contributed to the preservation of this subpopulation on days 5-10 of cultivation. However, the use of IL-7 and IL-15, both alone and in combination, did not lead to a significant increase in LAK compared to the use of IL-2.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    URL: Article
    DOI: 10.1200/JCO.2020.38.15_suppl.e15201
    RVK:
    XA 52760
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2020
    detail.hit.zdb_id: 2005181-5
    Library Location Call Number Volume/Issue/Year Availability
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    Online Resource
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