Tumour microenvironment markers in spontaneous and induced incubation of breast cancer biopsies

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

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. 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. 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.

1. Vafaizadeh 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: 32258038

2. Wen 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: 31022191

3. Gajewski 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: 24048123

4. Gnjatic 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: 28515944

5. Bianchini 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: 24480556

6. Spencer 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: 27249758

7. 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).

8. 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: 31848190

9. Lee 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: 26241661

10. Ribas A., Wolchok J.D. Cancer immunotherapy using checkpoint blockade. Science. 2018; 359(6382): 1350–1355. https://doi.org/10.1126/science.aar4060 PMID: 29567705

11. Li 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: 29062075

12. Yang 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: 31101027

13. Chakravarthy 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: 30410077

14. Mercogliano 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: 33540543

15. Madhusudan 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: 15475440

16. Samarendra 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: 28535157

17. Carey 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: 26527775

18. Hagemann 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