Abstract
Seventypercentofovariancancerpatientsdieduetoconsecutiveepisodesofrecurrencesresultingfromthere-growth of ovarian tumor cells resistant to conventional chemotherapies. In an effort to identify chemoresistance mechanisms, we comparedtheexpressionofgenesintumorcellsisolatedfromtheascitesofadvanced-stageserousovariancancerpatientsprior to(chemonaive,CN)andafterchemotherapytreatments(chemoresistant/recurrent,CR).Anovel,recentlypublishedmethodwas used for the isolation of tumor cells from the ascites of CN and CR patients. Illumina HT-12 platform was used to assess the differentialexpressionofgenes(DEGs)betweentheisolatedtumorcellsfromtheascitesofCNandCRpatients.Theidentification ofDEGswasachievedbycomparingthegeneticsignaturesofCNversusCRsamplesbyameanexpressionratio(foldchange)of 2 and P < 0.05. Validation of selected genes was performed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The dominant canonical pathways in the CR versus CN tumor cells were determined by Ingenuity Pathway Analysis. Gene expressionanalysisrevealeddifferentialexpressionof414genes,with179genesupregulatedand235downregulatedintheCR group. There were significant differences in gene expressions encoding for proteins involved with cancer stem cells, cell-cell adhesion, embryonic development, tumor suppression, immune surveillance, retinoic acid and energy metabolism in tumor cells isolated from CR compared to CN patients. Pathway analysis revealed that changes in cell cycle pathways, prominently those involved with mitosis and polo-like kinase (PLK1), G2/M DNA damage and proteins linked with cell cycle checkpoint regulation associated with chemoresistance. This preliminary molecular profiling, on a small number of patient samples, suggests an importantdiscriminationofgenesintheisolatedtumorcellsderivedfromtheascitesofCNandCRpatients.Thistypeofstudyon a larger cohort of samples may have important clinical implications for the development of therapeutic strategies to overcome chemoresistance and associated recurrences in ovarian cancer patients.
Highlights
Ovarian cancer represents 3% of all the new cancer cases in American women, but accounts for 5% of all the cancerrelated deaths [1]
Morphology of cells collected from the ascites of ovarian cancer patients Ascites cells derived from both chemotherapy treatment (CN) (n 1⁄4 4) and CR patients (n 1⁄4 3) were assessed by phase contrast microscopy after seeding on low attachment plates for 24 h
Two distinct populations of cells were observed: (i) multicellular aggregates that floated as three-dimensional structures in the growth medium without attachment (Figure 1A), and (ii) spindle shaped fibroblast-like single cells that adhered to the low attachment plates (Figure 1C)
Summary
Ovarian cancer represents 3% of all the new cancer cases in American women, but accounts for 5% of all the cancerrelated deaths [1]. Microscopic inspection of ascites display a complex heterogeneous picture of cellular environment constituting single cells, floating multicellular aggregates of non-adherent cells, cancer-associated fibroblasts, myeloid cells, activated mesothelial cells and cancer stem cells (CSCs) [10,11,12]. Extensive seeding of these floating cells on the uterus, sigmoid colon and omentum is frequently encountered in advanced-stage and recurrent patients and leads to disruption of major organs and eventually death [7]. The presence of floating cellular aggregates, commonly known as spheroids, in the ascites of ovarian cancer patients is strongly associated with recurrence, and there is an urgent need to study these spheroids in the ascites in order to establish the mechanisms of recurrence
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