Estudo translacional da expressão de IL-33 e seu papel prognóstico no câncer de mama triplo negativo: polarização de macrófagos M1

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype that primarilyaffects women under the age of 50. It is characterized by the absenceof estrogen andprogesterone receptors and little to nooverexpressionof the human epidermal growth factorreceptor 2 (HER2) gene. Interleukin 33 (IL-33) is a pleiotropic cytokine that signals throughthe ST2 receptor.Theidentification of new prognostic markers is essential for improving therapeutic outcomes. Objective: To evaluate the association between IL-33/ST2 expressionand clinicopathological parameters in TNBC and its prognostic significance, as well as toinvestigate in vitro macrophage polarization after exposure to tumor cells incubated withchemotherapeutics. Methodology:The study was divided into clinical and experimentalcomponents. The clinical study was retrospective and cross-sectional, using clinicopathologicaldata from 73 patients treated at the Haroldo Juaçaba Hospital. IL-33 and ST2 expression wasanalyzed by Indirect Immunofluorescence (IFI) and Immunohistochemistry (IHC) in corebiopsy samples. In the experimental part, the IL-33/ST2 pathway was investigated in TNBCcell lines (MDA-MB-231) and luminal cell lines (MCF-7), incubated with or withoutdoxorubicin (DOX) and/or paclitaxel (PTX) by IFI. To evaluate macrophages, the RAW 264.7cell line was exposed to the supernatant of tumorcells, incubated or not with chemotherapeutics,for 24 hours. The supernatant was collected for cytokine measurement (IL-33, TNF-alpha, IL-10, and IL-18). IL-33 levels were also measured in tumor cell lines. Additionally, the expressionof iNOS (inducible nitric oxide synthase, M1 phenotype) and Arginase-1 (M2 phenotype) wasinvestigated by IFI, and gene expression for M1 (Tnf-alpha, inos, Il-18, and Tgf-beta) and M2(Mmp9, Cd206, Arg1) was analyzed by qPCR. Nitrite levels were measured by the Griess assay.Results: Most patients were ≥50 years old (63.01%), with a mean age of 53.27 ± 11.86 years.A family history of breast cancer was reported in 41.10% of the cases. The Ki-67 proliferationindex (>20%) was the most prevalent (84.93%). Low IL-33 expression was found in 56.45%of the samples (p=0.0068 vs. highexpression), with Ki-67>20% (p=0.0068) and tumorrecurrence (p=0.0440) associated with TNBC. No significant difference in overall survival wasobserved between patients with low and high IL-33 expression (p=0.84). No significantassociations were found with ST2 (p>0.05). In vitro assays showed an increase in IL-33expression in tumor cell lines, especially in groups incubated with DOX or DOX/PTX (p<0.05).In LPS-activated RAW 264.7 cells, increased nitric oxide (NO) production was observed afterincubation with the tumor cell supernatant. NO production was also observed in other groups,even without LPS stimulation. TNF-alpha levels (p<0.05) and gene expression of Tnf-alpha, Il-18, and inos were increased, suggesting polarization toward the M1 phenotype. Conclusion:Low IL-33 expression is associated with a high Ki-67 proliferation index and tumor recurrence(p<0.05). Its presence in the TNBC tumor microenvironment may negatively regulate tumorbiology. The conditioned medium from tumor cells incubated with chemotherapeutics inducesmacrophage polarization toward the M1 phenotype, which may influence the tumor immuneresponse.