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Pro-carboxypeptidase B2 (pro-CPB2) or thrombin-activatable fibrinolysis inhibitor (TAFI) is a glycoprotein encoded by the CPB2 gene and deregulated in several cancer types, including breast cancer. Thrombin binding to thrombomodulin (TM), encoded by THBD, is important for TAFI activation. CPB2 gene expression is influenced by genetic polymorphism and cytokines such as interleukin 10 (IL-10). Our previous results showed that tumor infiltrating monocytes/macrophages (CD14/CD16) isolated from inflammatory breast cancer (IBC) patients' secrete high levels of IL-10. The aim of the present study is to test genetic polymorphism and expression of CPB2 in healthy breast tissues and carcinoma tissues of non-IBC and IBC patients. Furthermore, to investigate whether IL-10 modulates the expression of CPB2 and THBD in vivo and in-vitro. We tested CPB2 Thr325Ile polymorphism using restriction fragment length polymorphism, (RFLP) technique in healthy and carcinoma breast tissues. The mRNA expression of CPB2, THBD and IL10 were assessed by RT-qPCR. Infiltration of CD14 cells was assessed by immunohistochemistry. In addition, we investigated the correlation between infiltration of CD14 cells and expression of IL10 and CPB2. Furthermore, we correlated IL10 expression with the expression of both CPB2 and THBD in breast carcinoma tissues. Finally, we validated the role of recombinant IL-10 in regulating the expression of CPB2 and THBD using different breast cancer cell lines. Our results showed that CPB2 genotypes carrying the high-risk allele [Thr/Ile (CT) and Ile/Ile (TT)] were more frequent in both IBC and non-IBC patients compared to control group. CPB2 genotypes did not show any statistical correlation with CPB2 mRNA expression levels or patients' clinical pathological properties. Interestingly, CPB2 and IL10 expression were significantly higher and positively correlated with the incidence of CD14 cells in carcinoma tissues of IBC as compared to non-IBC. On the other hand, THBD expression was significantly lower in IBC carcinoma versus non-IBC tissues. Based on molecular subtypes, CPB2 and IL10 expression were significantly higher in triple negative (TN) as compared to hormonal positive (HP) carcinoma tissues of IBC. Moreover, CPB2 expression was positively correlated with presence of lymphovascular invasion and the expression of IL10 in carcinoma tissues of IBC patients. Furthermore, recombinant human IL-10 stimulated CPB2 expression in SUM-149 (IBC cell line) but not in MDA-MB-231 (non-IBC cell line), while there was no significant effect THBD expression. In conclusion, carcinoma tissues of IBC patients are characterized by higher expression of CPB2 and lower expression of THBD. Moreover, CPB2 positively correlates with IL10 mRNA expression, incidence of CD14 cells and lymphovascular invasion in IBC patients. IL-10 stimulated CPB2 expression in TN-IBC cell line suggests a relevant role of CPB2 in the aggressive phenotype of IBC.

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Background: Many studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations and changes in tumor microenvironment (TME). IBC characterized by significantly higher infiltration of tumor associated macrophages (TAMs). TAMs contribute to the metastatic process in IBC patients via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. To understand the role of TAMs in IBC, there is a need to firstly understand how IBC TME affect the polarization of tumor infiltrated monocytes via using sensitive and accurate analytical tool. Synchrotron FTIR microspectroscopy (SR-µFTIR) is a highly advanced analytical approach has the ability to detect any biochemical changes even prior to any morphological changes. Herein we will use SR-µFTIR to study the biochemical changes in tumor infiltrating monocytes after stimulation by the secretome of non-IBC and IBC breast cancer cells. Methods: ten breast cancer patients were enrolled in this study (5 IBC and 5 non-IBC). tumor infiltrated monocytes were isolated from patient’s blood samples which collected during modified radical mastectomy surgery. The isolated monocytes were stimulated with the secretome of MCF7, MDA-MB-231 and SUM149 cell line. Synchrotron FTIR Microspectroscopy (SR-FTIRM) single cell analysis was done at SESAME light source. The obtained raw spectral data were processed and analyzed using OMNIC©, PeakFit© V4.12, and Unscrambler© X v.10.4 software. Results: The obtained FTIR spectra showed very intense absorption bands in the region of 1700- 1500 cm-1 attributed to the amide I (ν(C=O), & νAS (C-N), δ (N-H) and amide II ν(C-N), δ (N-H) proteins bands, with a strong broad signal in the 3600-3200 cm-1 related to the νN-H Amides A and B. In addition, three peaks of different intensity and area were detected in the lipids region of the vCH2 and vCH3 stretching modes positioned within the 3000 - 2800 cm-1 range. Curve fitting assignment clearly demonstrate some differences between the three stimulated monocytes in both amide I and II, and lipids regions. The Principal Component Analysis (PCA) scores plot of the second derivative spectra of the amide region explain 77% of the variance, with 63% explained by PC1 and 14% explained by PC2. On the other hand, the PCA scores plot of the lipid region explain a total of 92% of the variance with PC1 explaining 72% and PC2 explaining 20%. Conclusion: The obtained results showed that there is different effect of non-IBC and IBC cells on the polarization of tumor infiltrating monocytes even with the cell surface markers look the same and from this, we may start to understand different roles of TAMs in IBC microenvironment. Citation Format: Hossam T. Mohamed, Gihan Kamel, Aya A. El-Sharkawy, Noura El-Husseiny, Mohamed El-Shinawi, Mona M. Mohamed. Synchrotron Fourier-transform infrared microspectroscopy: Characterization of tumor infiltrating monocytes stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6132.