Immunomodulatory Factors in Primary Endometrial Cell Cultures Isolated from Cancer and Noncancerous Human Tissue–Focus on RAGE and IDO1

Abstract

Background: Immune modulatory factors like indoleamine 2,3-dioxygenase 1 (IDO1) generating kynurenine (Kyn) and receptor for advanced glycation end-products (RAGE) contribute to endometrial and cancer microenvironment. Using adequate experimental models is needed to learn about the significance of these molecular factors in endometrial biology. In this paper we study IDO1 activity and RAGE expression in the in vitro cultured primary human endometrial cells derived from cancerous and noncancerous tissue. Methods: The generated primary cell cultures from cancer and noncancerous endometrial tissues were characterized using immunofluorescence and Western Blot for expression of endometrial and cancer markers. IDO1 activity was studied by Kyn quantification with High Performance Liquid Chromatography with Diode Array Detector. Results: The primary cultures of endometrial cells were obtained with 80% success rate and no major genetic aberrations. The cells retained in vitro expression of markers (mucin MUC1 and HER2) or immunomodulatory factors (RAGE and IDO1). Increased Kyn secretion was associated with cancer endometrial cell culture in contrast to the control one. Conclusions: Primary endometrial cells express immune modulatory factors RAGE and IDO1 in vitro associated with cancer phenotype of endometrium.

The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples

“…Immunohistochemistry (ICH) was performed as described earlier 33 . The tissue was fixed in 4% (v/v) formaldehyde in PBS at the time of surgical removal and was embedded in paraffin (FFPE) for cutting into 4 μm sections.…”

“…Cells were cultured on chamber slides and processed for immunofluorescent staining as described earlier 33 . Briefly, upon 3 washes with PBS, cells were permeabilized with 0.5% Triton X-100 for 15 min at room temperature, and the blocking step was performed using a blocking solution containing 3% NFDM, 1% BSA, 0.1% Triton X-100 in PBS for 30 min at room temperature.…”

“…Collected samples were centrifuged for 10 min at 14 000 × g at 4 °C, and the supernatant was transferred to a new tube, for storage at −20 °C until testing. In addition, to confirm HER2 expression, half of the excised tumor tissue was subjected to immunohistochemistry as described earlier for human tumors 33 .…”

Electrochemical Determination of Kynurenine Pathway Metabolites—Challenges and Perspectives

“…KP metabolites might be also monitored in cells and cell culture medium (Table 2). Kyn is secreted at micromolar amounts into culture medium by various cell types (e.g., cancer cells, monocytes, macrophages, glioma cells, and dendritic cells), while other metabolites are usually not detected [57,75,77]. However, under some conditions such as stimulation with IFN-γ, Trp depletion is accelerated and other downstream KP metabolites are secreted (e.g., 3HKyn, 3HAA, and AA) [56,75,77].…”

“…Kyn is secreted at micromolar amounts into culture medium by various cell types (e.g., cancer cells, monocytes, macrophages, glioma cells, and dendritic cells), while other metabolites are usually not detected [57,75,77]. However, under some conditions such as stimulation with IFN-γ, Trp depletion is accelerated and other downstream KP metabolites are secreted (e.g., 3HKyn, 3HAA, and AA) [56,75,77]. Notably, after IFN-γ treatment, the intracellular concentrations of KP metabolites can be higher than secreted into a culture medium [74].…”