Diagnostic and prognostic methods for estrogen-induced cancers
A technology for estrogen and cancer, applied in the fields of biochemical equipment and methods, disease diagnosis, and microbial determination/examination, which can solve the problems of poor prognosis, lack of practicability, and limitations in early cancer
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Embodiment 1
[0127] Example 1 - Experimental procedure
[0128] Cell Lines and Culture Conditions
[0129] at 5% CO 2 MEM (HyClone) medium (supplemented with 5% heat-inactivated FBS (Bovogen Biologics), 2mmol / L L-glutamine (HyClone) and antibiotics (50U / mL penicillin, 50mg / The human endometrial adenocarcinoma cell line Ishikawa (Sigma #99040201) was cultured in L streptomycin; Gibco). Cell line identification was performed by short tandem repeat (STR) DNA typing (DNA profiling) method, and MycoAlert TM Plus Mycoplasma Detection Kit (Lonza) routinely detects mycoplasma contamination in cells.
[0130] clinical samples
[0131] Human Endometrial Carcinoma Patient Samples: Collected at the John Hunter Hospital from patients undergoing tumor resection or surgical debulking using a protocol approved by the University of Newcastle Human Research Ethics Committee Human endometrial cancer tissue samples and adjacent normal tissue samples. Fresh tissue samples were shipped to the laboratory,...
Embodiment 2
[0152] Example 2 - ALPPL2 as a Secreted Protein in Human Endometrial Carcinoma Organoids
[0153] To identify potential tumor biomarkers for the diagnosis of endometrial cancer patients, the inventors characterized the secretome of endometrial cancer cells. To characterize the secretome of human endometrial cancer cells, the inventors three-dimensionally (3D) cultured Ishikawa cells on an extracellular matrix to form organoids. In contrast to conventional two-dimensional culture, organoid culture is beneficial because organoids are self-organizing, stable and resemble the tissue of origin. Endometrial organoids mimic the body's uterine glands, which respond to steroid signals and secrete components of "uterine milk."
[0154]Endometrial cancer organoids formed in 3D typically start from single cells to organize into associated multicellular polarized (shown by GM130 on the apical side) and glandular (shown by actin filament alignment) structures. Compared to other proteins (...
Embodiment 3
[0155] Example 3 - Organoid Growth and ALPPL2 Expression of Endometrial Carcinoma Show Similar Trends to Estrogen and Progesterone
[0156] The inventors here demonstrate that the size and proliferation of Ishikawa organoids increase after administration of estrogen (E2), and that progesterone (P4) counteracts the mitogenic effect of E2 ( figure 2 A and 2B). As endometrial cancer cells secrete ALPPL2, the inventors wondered whether E2-mediated proliferation of endometrial cancer cells also stimulates ALPPL2 expression. Real-time quantitative PCR analysis of ALPPL2 was performed in Ishikawa organoids grown in E2 alone, P4 alone, or in both E2 and P4. In E2-treated organoids, ALPPL2 mRNA expression was significantly upregulated, whereas P4 suppressed this effect ( figure 2 C). Overexpression of ALPPL2 was also confirmed by Western blot analysis of total cell lysates of Ishikawa cells and organoids treated with E2 and / or P4 ( figure 2 D and 2E). The results concluded that...
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