Synchrotron Fourier-transform infrared microspectroscopy: Characterization of tumor infiltrating monocytes stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells.

Abstract 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.