sechenovSechenov Medical JournalСеченовский вестник2218-73322658-3348Сеченовский Университет10.47093/2218-7332.2021.12.1.50-59sechenov-268Research ArticlePATHOLOGICAL PHYSIOLOGYПАТОЛОГИЧЕСКАЯ ФИЗИОЛОГИЯTumour microenvironment markers in spontaneous and induced incubation of breast cancer biopsiesМаркеры опухолевого микроокружения при инкубации биоптатов рака молочной железыhttps://orcid.org/0000-0002-4483-6163ГергенретерЮ. С.GergenreterYu. S.

Гергенретер Юлия Сергеевна, врач-онколог; соискатель кафедры патологической физиологии

Смирновское ущелье, д. 1а, г. Саратов, 410053

Yulia S. Gergenreter, oncologist of the Regional Clinical Oncology Dispensary; Applicant at the Department  of Pathological Physiology

1A, Smirnovsky gorge, Saratov, 410053

https://orcid.org/0000-0001-9410-2240ЗахароваН. Б.ZakharovaN. B.

Захарова Наталия Борисовна, д-р мед. наук, профессор кафедры клинической лабораторной диагностики

ул. Большая Садовая, д. 137, г. Саратов, 410000

Natalya B. Zakharova, Dr. of Sci. (Medicine), Professor, Department of Clinical Laboratory Diagnostics

137, Bolshaya Sadovaya str., Saratov, 410000

https://orcid.org/0000-0003-2453-1319МорозоваО. Л.MorozovaO. L.

Морозова Ольга Леонидовна, д-р. мед. наук, профессор кафедры патофизиологии

ул. Трубецкая, д. 8, стр. 2, г. Москва, 119991

+7 (916) 532-54-81

Olga L. Morozova, Dr. of Sci. (Medicine), Professor, Department of Pathological Physiology

8/2, Trubetskaya str., Moscow, 119991

+7 (916) 532-54-81

morozovaol1971@gmail.comГУЗ «Областной клинический онкологический диспансер»РоссияRegional Clinical Oncology DispensaryRussian FederationФГБОУ ВО «Саратовский государственный медицинский университет им. В.И. Разумовского» Минздрава РоссииРоссияSaratov State Medical University named after V.I. RazumovskyRussian FederationФГАОУ ВО «Первый Московский государственный медицинский университетим. И.М. Сеченова» Минздрава России (Сеченовский Университет)РоссияSechenov First Moscow State Medical University (Sechenov University)Russian Federation2021290420211215059Copyright © Gergenreter Y.S., Zakharova N.B., Morozova O.L., 20212021Гергенретер Ю.С., Захарова Н.Б., Морозова О.Л.Gergenreter Y.S., Zakharova N.B., Morozova O.L.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.sechenovmedj.com/jour/article/view/268Aim

Aim. To study the spontaneous and stimulated production of cytokines in biopsies of breast cancer (BC) depending on the cancer stage.

Materials and methods

Materials and methods. An experimental study was carried out with cell cultures of breast cancer biopsies of stages I–II (group 1, n = 15) and III–IV stages (group 2, n = 15). The control consisted of 6 healthy women who underwent mastopexy. We used enzyme immunoassay method to access spontaneous and induced by a complex of polyclonal activators (PA: phytohemagglutinin 4 μg / ml, concanavalin A 4 μg / ml, lipopolysaccharide 2 μg / ml) concentration of TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF, MCP-1, TGF-β1. The index of the effect of polyclonal activators (IVPA) on cytokine production (induced production / spontaneous production) was calculated. To compare groups, the Mann-Whitney test and the median test, the chi-square test and the Fisher’s exact test were used.

Results

Results. Groups 1 and 2 did not differ in age, histological variant and immunohistochemical type of tumour, predominantly invasive cancer without signs of specificity prevailed. In group 2, a pronounced vascularization was more often observed: in 6 (40%) patients versus 1 (7%) in group 1 (p < 0.05). In both groups, compared with the control, there was a statistically sig-nificant (p < 0.05) increase in spontaneous production of TNF-α by 4.2 and 4.8 times, MCP-1 by 6.7 and 6.3 times, TGF-β1 – 2.2 and 2.5 times, VEGF 11.9 and 14.6 times; GM-CSF 15.6 and 13.4 times, G-CSF 96.8 and 79.5 times, respectively. The concentration of MCP-1 and IFN-γ was higher in group 1 (p < 0.05), VEGF and TGF-β1 – in group 2 (p < 0.05). IVPA in group 2 exceeded similar values   in group 1 for G-CSF, VEGF, TGF-β1 (p < 0.05).

Conclusion

Conclusion. The production of cytokines (TNF-α, MCP-1, GM-CSF, G-CSF, VEGF, TGF-β1) in breast cancer biopsies is significantly higher than in biopsies of the unchanged mammary gland and depends on the stage of the tumour process.

Цель

Цель. Изучить спонтанную и стимулированную продукцию цитокинов в биоптатах рака молочной железы (РМЖ) в зависимости от степени распространенности опухолевого роста.

Материалы и методы

Материалы и методы. Проведено экспериментальное исследование с клеточными культурами биоптатов РМЖ I–II стадии (группа 1, n = 15) и III–IV стадии (группа 2, n = 15). Контроль составили 6 здоровых женщин, которым выполнена мастопексия. Методом иммуноферментного анализа определяли спонтанную и индуцированную ком-плексом поликлональных активаторов (ПА: фитогемагглютинин 4 мкг/мл, конканавалин А 4 мкг/мл, липополисахарид 2 мкг/мл) концентрацию TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF, MCP-1, TGF-β1. Вычисляли индекс влияния поликло-нальных активаторов (ИВПА) на продукцию цитокинов (индуцированная продукция / спонтанная продукция). Для сравнения групп применяли критерий Манна – Уитни и медианный критерий, критерий хи-квадрат и точный критерий Фишера.

Результаты

Результаты. Группы 1 и 2 не различались по возрасту, гистологическому варианту и иммуногистохимическому типу опухоли, преобладал преимущественно инвазивный рак без признаков специфичности. В группе 2 чаще отмеча-лась выраженная степень васкуляризации: у 6 (40%) пациенток против 1 (7%) в группе 1 (р < 0,05). В обеих группах по сравнению с контролем отмечено статистически значимое (р < 0,05) повышение спонтанной продукции: TNF-α – в 4,2 и 4,8 раза, MCP-1 – в 6,7 и 6,3 раза, TGF-β1 – в 2,2 и 2,5 раза, VEGF – в 11,9 и 14,6 раза, GМ-CSF – в 15,6 и 13,4 раза, G-CSF – в 96,8 и 79,5 раза соответственно. Концентрация МСР-1 и IFN-γ была выше в группе 1 (р < 0,05), VEGF и TGF-β1 – в группе 2 (р < 0,05). ИВПА в группе 2 превышал аналогичные значения в группе 1 для G-CSF, VEGF, TGF-β1 (р < 0,05).

Заключение

Заключение. Продукция цитокинов (TNF-α, MCP-1, GМ-CSF, G-CSF, VEGF, TGF-β1) в биоптатах РМЖ значительно выше, чем в биоптатах неизмененной молочной железы, и зависит от стадии опухолевого процесса.

 

 

иммуноонкологические маркерымикроокружение опухолиспонтанная и индуцированная продукциярак молочной железыimmuno-oncological biomarkerscytokines of the tumour microenvironmentgrowth factorscolony-stimulating factorsspontaneous and induced cytokine productionprotective mechanisms of the immune responsebreast cancerindex of the effect of polyclonal activatorsReferencesVafaizadeh V., Barekati Z. Immuno-oncology biomarkers for personalized immunotherapy in breast cancer. Front Cell Dev Biol. 2020; 8: 162. https://doi.org/10.3389/fcell.2020.00162 PMID: 32258038Vafaizadeh V., Barekati Z. Immuno-oncology biomarkers for personalized immunotherapy in breast cancer. Front Cell Dev Biol. 2020; 8: 162. https://doi.org/10.3389/fcell.2020.00162 PMID: 32258038Wen W.X., Leong C.O. Association of BRCA1- and BRCA2- deficiency with mutation burden, expression of PD-L1/PD-1, immune infiltrates, and T cell-inflamed signature in breast cancer. PLoS One. 2019; 14(4): e0215381. https://doi.org/10.1371/journal.pone.0215381 PMID: 31022191Wen W.X., Leong C.O. Association of BRCA1- and BRCA2- deficiency with mutation burden, expression of PD-L1/PD-1, immune infiltrates, and T cell-inflamed signature in breast cancer. PLoS One. 2019; 14(4): e0215381. https://doi.org/10.1371/journal.pone.0215381 PMID: 31022191Gajewski T.F., Schreiber H., Fu Y.X. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 2013; 14(10): 1014–1022. https://doi.org/10.1038/ni.2703 PMID: 24048123Gajewski T.F., Schreiber H., Fu Y.X. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 2013; 14(10): 1014–1022. https://doi.org/10.1038/ni.2703 PMID: 24048123Gnjatic S., Bronte V., Brunet L.R., et al. Identifying baseline immune-related biomarkers to predict clinical outcome of immunotherapy. J Immunother Cancer. 2017; 5: 44. https://doi.org/10.1186/s40425-017-0243-4 PMID: 28515944Gnjatic S., Bronte V., Brunet L.R., et al. Identifying baseline immune-related biomarkers to predict clinical outcome of immunotherapy. J Immunother Cancer. 2017; 5: 44. https://doi.org/10.1186/s40425-017-0243-4 PMID: 28515944Bianchini G., Gianni L. The immune system and response to HER2-targeted treatment in breast cancer. Lancet Oncol. 2014; 15(2): e58–68. https://doi.org/10.1016/S1470-2045(13)70477-7 PMID: 24480556Bianchini G., Gianni L. The immune system and response to HER2-targeted treatment in breast cancer. Lancet Oncol. 2014; 15(2): e58–68. https://doi.org/10.1016/S1470-2045(13)70477-7 PMID: 24480556Spencer K.R., Wang J., Silk A.W., et al. Biomarkers for immunotherapy: current developments and challenges. Am Soc Clin Oncol Educ Book. 2016; 35: e493–503. https://doi.org/10.1200/EDBK_160766 PMID: 27249758Spencer K.R., Wang J., Silk A.W., et al. Biomarkers for immunotherapy: current developments and challenges. Am Soc Clin Oncol Educ Book. 2016; 35: e493–503. https://doi.org/10.1200/EDBK_160766 PMID: 27249758Кунц Т.А., Карпухина К.В., Михайлова Е.С. и др. Цитокиновый профиль супернатантов иммунокомпетентных клеток периферической крови и опухоли при инвазивном протоковом раке молочной железы. Современные проблемы науки и образования. (Электронный журнал) 2016; 5. URL: http://science-education.ru/ru/article/view?id=25296 (дата обращения: 02.01.2021).Kunts T.A., Karpukhina K.V., Mikhaylova E.S., et al. Tsitokinovyi profil’ supernatantov immunokompetentnykh kletok perifericheskoi krovi i opukholi pri invazivnom protokovom rake molochnoi zhelezy [Cytokine pattern of supernatant of blood immunocompetent cells and tumor at invasive ductal carcinoma of the breast.] Modern problems of science and education. 2016; 5 (In Russian). URL: http://science-education.ru/ru/article/view?id=25296 (accessed: 02.01.2021).Li Q., Wang Y., Jia W., et al. Low-dose anti-angiogenic therapy sensitizes breast cancer to pd-1 blockade. Clin Cancer Res. 2020; 26(7): 1712–1724. https://doi.org/10.1158/1078-0432.CCR-19-2179 PMID: 31848190Li Q., Wang Y., Jia W., et al. Low-dose anti-angiogenic therapy sensitizes breast cancer to pd-1 blockade. Clin Cancer Res. 2020; 26(7): 1712–1724. https://doi.org/10.1158/1078-0432.CCR-19-2179 PMID: 31848190Lee S.K., Bae S.Y., Lee J.H., et al. Distinguishing low-risk luminal a breast cancer subtypes with ki-67 and p53 is more predictive of long-term survival. PLoS ONE. 2015; 10(8): e0124658. https://doi.org/10.1371/journal.pone.0124658 PMID: 26241661Lee S.K., Bae S.Y., Lee J.H., et al. Distinguishing low-risk luminal a breast cancer subtypes with ki-67 and p53 is more predictive of long-term survival. PLoS ONE. 2015; 10(8): e0124658. https://doi.org/10.1371/journal.pone.0124658 PMID: 26241661Ribas A., Wolchok J.D. Cancer immunotherapy using checkpoint blockade. Science. 2018; 359(6382): 1350–1355. https://doi.org/10.1126/science.aar4060 PMID: 29567705Ribas A., Wolchok J.D. Cancer immunotherapy using checkpoint blockade. Science. 2018; 359(6382): 1350–1355. https://doi.org/10.1126/science.aar4060 PMID: 29567705Li W., Ma H., Zhang J., et al. Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis. Sci Rep. 2017; 7(1): 13856. Erratum in: Sci Rep. 2018; 8(1): 4276. https://doi.org/10.1038/s41598-017-14364-2 PMID: 29062075Li W., Ma H., Zhang J., et al. Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis. Sci Rep. 2017; 7(1): 13856. Erratum in: Sci Rep. 2018; 8(1): 4276. https://doi.org/10.1038/s41598-017-14364-2 PMID: 29062075Yang X., Zhang K., Zhang C., et al. Accuracy of analysis of cfDNA for detection of single nucleotide variants and copy number variants in breast cancer. BMC Cancer. 2019; 19(1): 465. https://doi.org/10.1186/s12885-019-5698-x PMID: 31101027Yang X., Zhang K., Zhang C., et al. Accuracy of analysis of cfDNA for detection of single nucleotide variants and copy number variants in breast cancer. BMC Cancer. 2019; 19(1): 465. https://doi.org/10.1186/s12885-019-5698-x PMID: 31101027Chakravarthy A., Khan L., Bensler N.P., et al. TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure. Nat Commun. 2018; 9(1): 4692. https://doi.org/10.1038/s41467-018-06654-8 PMID: 30410077Chakravarthy A., Khan L., Bensler N.P., et al. TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure. Nat Commun. 2018; 9(1): 4692. https://doi.org/10.1038/s41467-018-06654-8 PMID: 30410077Mercogliano M.F., Bruni S., Mauro F., et al. Harnessing tumor necrosis factor alpha to achieve effective cancer immunotherapy. Cancers (Basel). 2021; 13(3): 564. https://doi.org/10.3390/cancers13030564 PMID: 33540543Mercogliano M.F., Bruni S., Mauro F., et al. Harnessing tumor necrosis factor alpha to achieve effective cancer immunotherapy. Cancers (Basel). 2021; 13(3): 564. https://doi.org/10.3390/cancers13030564 PMID: 33540543Madhusudan S., Foster M., Muthuramalingam S.R., et al. A phase II study of etanercept (Enbrel), a tumor necrosis factor alpha inhibitor in patients with metastatic breast cancer. Clin Cancer Res. 2004; 10(19): 6528–6534. https://doi.org/10.1158/1078-0432.CCR-04-0730 PMID: 15475440Madhusudan S., Foster M., Muthuramalingam S.R., et al. A phase II study of etanercept (Enbrel), a tumor necrosis factor alpha inhibitor in patients with metastatic breast cancer. Clin Cancer Res. 2004; 10(19): 6528–6534. https://doi.org/10.1158/1078-0432.CCR-04-0730 PMID: 15475440Samarendra H., Jones K., Petrinic T., et al. A meta-analysis of CXCL12 expression for cancer prognosis. Br J Cancer. 2017; 117(1): 124–135. https://doi.org/10.1038/bjc.2017.134 PMID: 28535157Samarendra H., Jones K., Petrinic T., et al. A meta-analysis of CXCL12 expression for cancer prognosis. Br J Cancer. 2017; 117(1): 124–135. https://doi.org/10.1038/bjc.2017.134 PMID: 28535157Carey L.A., Berry D.A., Cirrincione C.T., et al. Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III trial of paclitaxel plus trastuzumab with or without lapatinib. J Clin Oncol. 2016; 34(6): 542–549. https://doi.org/10.1200/JCO.2015.62.1268 PMID: 26527775Carey L.A., Berry D.A., Cirrincione C.T., et al. Molecular heterogeneity and response to neoadjuvant human epidermal growth factor receptor 2 targeting in CALGB 40601, a randomized phase III trial of paclitaxel plus trastuzumab with or without lapatinib. J Clin Oncol. 2016; 34(6): 542–549. https://doi.org/10.1200/JCO.2015.62.1268 PMID: 26527775Hagemann T., Lawrence T., McNeish I., et al. “Re-educating” tumor-associated macrophages by targeting NF-kappaB. J Exp Med. 2008; 205(6): 1261–1268. https://doi.org/10.1084/jem.20080108 PMID: 18490490Hagemann T., Lawrence T., McNeish I., et al. “Re-educating” tumor-associated macrophages by targeting NF-kappaB. J Exp Med. 2008; 205(6): 1261–1268. https://doi.org/10.1084/jem.20080108 PMID: 18490490

The authors declare that there are no conflicts of interest present.