Mohamed, H. T., M. El-Shinawi, and M. M. Mohamed, "Editorial: Inflammatory tumor microenvironment: role of cytokines and virokines in breast cancer progression and metastasis", Front Cell Dev Biol., vol. 12, issue 1414734, 2024.
Tarek, A., H. T. Mohamed, A. A. El-Sharkawy, S. K. El-Sayed, J. M. Hirshon, W. A. Woodward, M. El-Shinawi, and M. M. Mohamed, "Differential gene expression of Fresh Tissue and Patient-Derived Explants' Matricellular Proteins Augment Inflammatory Breast Cancer Metastasis: The Possible Role of IL-6 and MCP-1.", QJM : monthly journal of the Association of Physicians, 2023. Abstract

BACKGROUND: Matricellular proteins comprising matrisome and adhesome are responsible for structure integrity and interactions between cells in the tumour microenvironment of breast cancer. Changes in the gene expression of matrisome and adhesome augment metastasis. Since inflammatory breast cancer (IBC) is characterized by high metastatic behavior. Herein we compared the gene expression profile of matrisome and adhesome in non-IBC and IBC in fresh tissue and ex-vivo patients derived explants (PDEs), we also compared the secretory inflammatory mediators of PDEs in non-IBC and IBC to identify secretory cytokines participate in cross-talk between cells via interactions with matrisome and adhisome.

METHODS: Fifty patients (31 non-IBC; 19 IBC) were enrolled in the present study. To test their validation in clinical studies, PDEs were cultured as an ex-vivo model. Gene expression and cytokine array were used to identify candidate genes and cytokines contributing to metastasis in the examined fresh tissues and PDEs. Bioinformatics analysis was applied on identified differentially expressed genes (DEGs) using GeneMANIA and Metascape gene annotation and analysis resource to identify pathways involved in IBC metastasis.

RESULTS: Normal and cancer fresh tissues and PDEs of IBC were characterized by overexpression of CDH1 and MMP14 and downregulation of CTNNA1 and TIMP1 compared to non-IBC. The secretome of IBC cancer PDEs is characterized by significantly high expression of interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared to non-IBC.

CONCLUSION: Genes expressed by adhisome and matrisome play a significant role in IBC metastasis and should be considered novel target therapy.

Mohamed, H. T., G. Kamel, N. El-Husseiny, A. A. El-Sharkawy, A. A. El-Sherif, M. El-Shinawi, and M. M. Mohamed, "Synchrotron Fourier-Transform Infrared Microspectroscopy: Characterization of in vitro polarized tumor-associated macrophages stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells.", Biochimica et biophysica acta. Molecular cell research, vol. 1870, issue 1, pp. 119367, 2023. Abstract

Studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations accompanied by specific cellular and molecular mechanisms in the IBC tumor microenvironment (TME). IBC is characterized by significantly higher infiltration of tumor-associated macrophages (TAMs) that contribute to its metastatic process via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. Thus, there is a need to first understand how IBC-TME modulates the polarization of TAMs to better understand the role of TAMs in IBC. Herein, we used gene expression signature and Synchrotron Fourier-Transform Infrared Microspectroscopy (SR-μFTIR) to study the molecular and biochemical changes, respectively of in vitro polarized TAMs stimulated by the secretome of IBC and non-IBC cells. The gene expression signature showed significant differences in the macrophage's polarization-related genes between stimulated TAMs. FTIR spectra showed absorption bands in the region of 1700-1500 cm attributed to the amide I ν(C=O), & ν (CN), δ (NH), and amide II ν(CN), δ (NH) proteins bands. Moreover, three peaks of different intensities and areas were detected in the lipid region of the νCH and νCH stretching modes positioned within the 3000-2800 cm range. The PCA analysis for the second derivative spectra of the amide regions discriminates between stimulated IBC and non-IBC TAMs. This study showed that IBC and non-IBC TMEs differentially modulate the polarization of TAMs and SR-μFTIR can determine these biochemical changes which will help to better understand the potential role of TAMs in IBC.

Ibrahim, A. S., M. El-Shinawi, S. Sabet, S. A. A. Ibrahim, and M. M. Mohamed, "Role of adipose tissue-derived cytokines in the progression of inflammatory breast cancer in patients with obesity.", Lipids in health and disease, vol. 21, issue 1, pp. 67, 2022. Abstract

BACKGROUND: Inflammatory breast cancer (IBC) represents a deadly aggressive phenotype of breast cancer (BC) with a unique clinicopathological presentation and low survival rate. In fact, obesity represents an important risk factor for BC. Although several studies have identified different cellular-derived and molecular factors involved in IBC progression, the role of adipocytes remains unclear. Cancer-associated adipose tissue (CAAT) expresses a variety of adipokines, which contribute to tumorigenesis and the regulation of cancer stem cell (CSC). This research investigated the potential effect of the secretome of CAAT explants from patients with BC on the progression and metastasis of the disease.

METHODS: This study established an ex-vivo culture of CAAT excised from IBC (n = 13) vs. non-IBC (n = 31) patients with obesity and profiled their secretome using a cytokine antibody array. Furthermore, the quantitative PCR (qPCR) methodology was used to validate the levels of predominant cytokines at the transcript level after culture in a medium conditioned by CAAT. Moreover, the impact of the CAAT secretome on the expression of epithelial-mesenchymal transition (EMT) and cells with stem cell (CSC) markers was studied in the non-IBC MDA-MB-231 and the IBC SUM-149 cell lines. The statistical differences between variables were evaluated using the chi-squared test and unpaired a Student's t-test.

RESULTS: The results of cytokine array profiling revealed an overall significantly higher level of a panel of 28 cytokines secreted by the CAAT ex-vivo culture from IBC patients with obesity compared to those with non-IBC. Of note, interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemo-attractant protein 1 (MCP-1) were the major adipokines secreted by the CAAT IBC patients with obesity. Moreover, the qPCR results indicated a significant upregulation of the IL-6, IL-8, and MCP-1 mRNAs in CAAT ex-vivo culture of patients with IBC vs. those with non-IBC. Intriguingly, a qPCR data analysis showed that the CAAT secretome secretions from patients with non-IBC downregulated the mRNA levels of the CD24 CSC marker and of the epithelial marker E-cadherin in the non-IBC cell line. By contrast, E-cadherin was upregulated in the SUM-149 cell.

CONCLUSIONS: This study identified the overexpression of IL-6, IL-8, and MCP-1 as prognostic markers of CAAT from patients with IBC but not from those with non-IBC ; moreover, their upregulation might be associated with IBC aggressiveness via the regulation of CSC and EMT markers. This study proposed that targeting IL-6, IL-8, and MCP-1 may represent a therapeutic option that should be considered in the treatment of patients with IBC.

Mohamed, H. T., A. A. El-Sharkawy, M. El-Shinawi, R. J. Schneider, and M. M. Mohamed, "Inflammatory Breast Cancer: The Secretome of HCMV Tumor-Associated Macrophages Enhances Proliferation, Invasion, Colony Formation, and Expression of Cancer Stem Cell Markers.", Frontiers in oncology, vol. 12, pp. 899622, 2022. Abstract

Inflammatory breast cancer (IBC) is a highly aggressive phenotype of breast cancer that is characterized by a high incidence early metastasis. We previously reported a significant association of human cytomegalovirus (HCMV) DNA in the carcinoma tissues of IBC patients but not in the adjacent normal tissues. HCMV-infected macrophages serve as "mobile vectors" for spreading and disseminating virus to different organs, and IBC cancer tissues are highly infiltrated by tumor-associated macrophages (TAMs) that enhance IBC progression and promote breast cancer stem cell (BCSC)-like properties. Therefore, there is a need to understand the role of HCMV-infected TAMs in IBC progression. The present study aimed to test the effect of the secretome (cytokines and secreted factors) of TAMs derived from HCMV monocytes isolated from IBC specimens on the proliferation, invasion, and BCSC abundance when tested on the IBC cell line SUM149. HCMV monocytes were isolated from IBC patients during modified radical mastectomy surgery and tested for polarization into TAMs using the secretome of SUM149 cells. MTT, clonogenic, invasion, real-time PCR arrays, PathScan Intracellular Signaling array, and cytokine arrays were used to characterize the secretome of HCMV TAMs for their effect on the progression of SUM149 cells. The results showed that the secretome of HCMV TAMs expressed high levels of IL-6, IL-8, and MCP-1 cytokines compared to HCMV TAMs. In addition, the secretome of HCMV TAMs induced the proliferation, invasion, colony formation, and expression of BCSC-related genes in SUM149 cells compared to mock untreated cells. In addition, the secretome of HCMV TAMs activated the phosphorylation of intracellular signaling molecules p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK in SUM149 cells. In conclusion, this study shows that the secretome of HCMV TAMs enhances the proliferation, invasion, colony formation, and BCSC properties by activating the phosphorylation of p-STAT3, p-AMPKα, p-PRAS40, and p-SAPK/JNK intracellular signaling molecules in IBC cells.

Tarek, A., S. K. El-Sayed, W. A. Woodward, M. El-Shinawi, J. M. Hirshon, and M. M. Mohamed, "Inflammatory Breast Cancer: The Cytokinome of Post-Mastectomy Wound Fluid Augments Proliferation, Invasion, and Stem Cell Markers.", Current issues in molecular biology, vol. 44, issue 6, pp. 2730-2744, 2022. Abstract

Inflammatory breast cancer (IBC) is an aggressive phenotype with a high recurrence and low survival rate. Approximately 90% of local breast cancer recurrences occur adjacent to the same quadrant as the initial cancer, implying that tumor recurrence may be caused by residual cancer cells and/or quiescent cancer stem cells (CSCs) in the tumor. We hypothesized that wound fluid (WF) collected after modified radical mastectomy (MRM) may activate cancer cells and CSCs, promoting epithelial mesenchymal transition (EMT) and invasion. Therefore, we characterized the cytokinome of WF drained from post-MRM cavities of non-IBC and IBC patients. The WF of IBC patients showed a significantly higher expression of various cytokines than in non-IBC patients. In vitro cell culture models of non-IBC and IBC cell lines were grown in media conditioned with and/without WF for 48 h. Afterwards, we assessed cell viability, the expression of CSCs and EMT-specific genes, and tumor invasion. Genes associated with CSCs properties and EMT markers were regulated in cells seeded in media conditioned by WF. IBC-WF exhibited a greater potential for inducing IBC cell invasion than non-IBC cells. The present study demonstrates the role of the post-surgical tumor cavity in IBC recurrence and metastasis.

Bassiony, H., A. A. El-Ghor, T. A. salah eldin, S. Sabet, and M. M. Mohamed, "Tissue Distribution, Histopathological and Genotoxic Effects of Magnetite Nanoparticles on Ehrlich Solid Carcinoma.", Biological trace element research, 2022. Abstract

Nanoparticles can potentially cause adverse effects on cellular and molecular level. The present study aimed to investigate the histopathological changes and DNA damage effects of magnetite nanoparticles (MNPs) on female albino mice model with Ehrlich solid carcinoma (ESC). Magnetite nanoparticles coated with L-ascorbic acid (size ~ 25.0 nm) were synthesized and characterized. Mice were treated with MNPs day by day, intraperitoneally (IP), intramuscularly (IM), or intratumorally (IT). Autopsy samples were taken from the solid tumor, thigh muscle, liver, kidney, lung, spleen, and brain for assessment of iron content, histopathological examination, and genotoxicity using comet assay. The liver, spleen, lung, and heart had significantly higher iron content in groups treated IP. On the other hand, tumor, muscles, and the liver had significantly higher iron content in groups treated IT. MNPs induced a significant DNA damage in IT treated ESC. While a significant DNA damage was detected in the liver of the IP treated group, but no significant DNA damage could be detected in the brain. Histopathological findings in ESC revealed a marked tumor necrosis, 50% in group injected IT but 40% in group injected IP and 20% only in untreated tumors. Other findings include inflammatory cell infiltration, dilatation, and congestion of blood vessels of different organs of treated groups in addition to appearance of metastatic cancer cells in the liver of non-treated tumor group. MNPs could have an antitumor effect but it is recommended to be injected intratumorally to be directed to the tumor tissues and reduce its adverse effects on healthy tissues.

Senousy, Z., M. M. Abdelsamea, M. M. Mohamed, and M. M. Gaber, "3E-Net: Entropy-Based Elastic Ensemble of Deep Convolutional Neural Networks for Grading of Invasive Breast Carcinoma Histopathological Microscopic Images.", Entropy (Basel, Switzerland), vol. 23, issue 5, 2021. Abstract

Automated grading systems using deep convolution neural networks (DCNNs) have proven their capability and potential to distinguish between different breast cancer grades using digitized histopathological images. In digital breast pathology, it is vital to measure how confident a DCNN is in grading using a machine-confidence metric, especially with the presence of major computer vision challenging problems such as the high visual variability of the images. Such a quantitative metric can be employed not only to improve the robustness of automated systems, but also to assist medical professionals in identifying complex cases. In this paper, we propose Entropy-based Elastic Ensemble of DCNN models (3E-Net) for grading invasive breast carcinoma microscopy images which provides an initial stage of explainability (using an uncertainty-aware mechanism adopting entropy). Our proposed model has been designed in a way to (1) exclude images that are less sensitive and highly uncertain to our ensemble model and (2) dynamically grade the non-excluded images using the certain models in the ensemble architecture. We evaluated two variations of 3E-Net on an invasive breast carcinoma dataset and we achieved grading accuracy of 96.15% and 99.50%.

Ahmed, S., H. T. Mohamed, N. El-Husseiny, M. M. El Mahdy, G. Safwat, A. A. Diab, A. A. El-Sherif, M. El-Shinawi, and M. M. Mohamed, "IL-8 secreted by tumor associated macrophages contribute to lapatinib resistance in HER2-positive locally advanced breast cancer via activation of Src/STAT3/ERK1/2-mediated EGFR signaling.", Biochimica et biophysica acta. Molecular cell research, vol. 1868, issue 6, pp. 118995, 2021. Abstract

Locally advanced breast cancer (LABC) is an aggressive disease characterized by late clinical presentation, large tumor size, treatment resistance and low survival rate. Expression of EGFR/HER2 and activation of intracellular tyrosine kinase domains in LABC are associated with poor prognosis. Thus, target therapies such as the anti-receptor tyrosine kinases lapatinib drug have been more developed in the past decade. The response to lapatinib involves the inhibition of RTKs and subsequently signaling molecules such as Src/STAT3/Erk1/2 known also to be activated by the cytokines in the tumor microenvironment (TME). The aim of the present study is to identify the major cytokine that might contribute to lapatinib resistance in EGFR+/HER2+ LABC patients. Indeed, tumor associated macrophages (TAMs) are the main source of cytokines in the TME. Herein, we isolated TAMs from LABC during modified radical mastectomy (MRM). Cytokine profile of TAMs revealed that IL-8 is the most prominent highly secreted cytokine by TAMs of LABC patients. Using in-vitro cell culture model we showed that recombinant IL-8 (50 and 100 ng/mL) at different time intervals interfere with lapatinib action via activation of Src/EGFR and signaling molecules known to be inhibited during treatment. We proposed that to improve LABC patients' response to lapatinib treatment it is preferred to use combined therapy that neutralize or block the action of IL-8.

Mohamed, H. T., E. A. El-Ghonaimy, M. El-Shinawi, M. O. H. A. M. E. D. HOSNEY, M. Götte, W. A. Woodward, T. El-Mamlouk, and M. M. Mohamed, "IL-8 and MCP-1/CCL2 regulate proteolytic activity in triple negative inflammatory breast cancer a mechanism that might be modulated by Src and Erk1/2.", Toxicology and applied pharmacology, vol. 401, pp. 115092, 2020. Abstract

Inflammatory breast cancer (IBC) is a highly metastatic and lethal breast cancer. As many as 25-30% of IBCs are triple negative (TN) and associated with low survival rates and poor prognosis. We found that the microenvironment of IBC is characterized by high infiltration of tumor associated macrophages (TAMs) and by over-expression of the cysteine protease cathepsin B (CTSB). TAMs in IBC secrete high levels of the cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared to non-IBC patients. Herein, we tested the roles of IL-8 and MCP-1/CCL2 in modulating proteolytic activity and invasiveness of TN-non-IBC as compared to TN-IBC and addressed the underlying molecular mechanism(s) for both cytokines. Quantitative real time PCR results showed that IL-8 and MCP-1/CCL2 were significantly overexpressed in tissues of TN-IBCs. IL-8 and MCP-1/CCL2 induced CTSB expression and activity of the p-Src and p-Erk1/2 signaling pathways relevant for invasion and metastasis in TN-non-IBC, HCC70 cells and TN-IBC, SUM149 cells. Dasatinib, an inhibitor of p-Src, and U0126, an inhibitor of p-Erk1/2, down-regulated invasion and expression of CTSB by HCC70 and SUM149 cells, a mechanism that is reversed by IL-8 and MCP-1/CCL2. Our study shows that targeting the cytokines IL-8 and MCP-1/CCL2 and associated signaling molecules may represent a promising therapeutic strategy in TN-IBC patients.

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