Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment

Isolation and characterization of tumor cells from the ascites of ovarian cancer patients: Molecular phenotype of chemoresistant ovarian tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions

X-Linked Lissencephaly With Absent Corpus Callosum and Abnormal Genitalia: An Evolving Multisystem Syndrome With Severe Congenital Intestinal Diarrhea Disease

X-linked lissencephaly with abnormal genitalia is a rare and devastating syndrome. The authors present an infant with a multisystem phenotype where the intestinal manifestations were as life limiting as the central nervous system features. Severe chronic diarrhea resulted in failure to thrive, dehydration, electrolyte derangements, long-term hospitalization, and prompted transition to palliative care. Other multisystem manifestations included megacolon, colitis, pancreatic insufficiency hypothalamic dysfunction, hypothyroidism, and hypophosphatasia. A novel aristaless-related homeobox gene mutation, c.1136G>T/p.R379L, was identified. This case contributes to the clinical, histological, and molecular understanding of the multisystem nature of this disorder, especially the role of ARX in the development of the enteroendocrine system

Tumorigenic properties of NAD and AD cells purified from the ascites of CR patients.

<p>(<b>A</b>) A phase contrast microscope image of NAD cells adhered to plastic before preparing the cell suspension for i.p. injection; (<b>B</b>) H and E staining of agarose embedded patient sample before injection; (<b>C</b>) image of solid tumor obtained from a mouse fourteen weeks after i.p. injection of NAD cells (5×10⁶); (<b>D</b>) H and E staining of mouse ascites NAD cells embedded on agarose; (<b>E</b>) Flow cytometric comparison of the expression of CA125, EpCAM and CD44 between the patient’s and mouse ascites cells. Results are representative of two independent samples. The filled histogram in each figure represents control IgG, black lines indicate protein expression in human cells, broken lines indicate the expression of the protein in mouse ascites cells.</p

mRNA expression of epithelial, mesenchymal and CSC markers in isolated ascites cells.

<p>qPCR was performed on purified NAD and AD populations as described in the Methods and Materials. Yields were converted to femtograms based on the standard curve for each PCR product, and the resultant mRNA levels were normalized to the 18S mRNA level per sample. The data were calculated from the results of eight independent samples assessed in triplicate. Significantly different in AD versus NAD cells *(p&lt;0.05) and **(p&lt;0.01).</p

Expression and immunolocalization of CA125 and CSC markers by immunofluorescence.

<p>Purified NAD and AD cells were evaluated by immunofluorescence using mouse monoclonal antibody (green) as described in the Methods and Materials. Cellular staining was visualized using the secondary Alexa 488 (green) fluorescent labeled antibody, and nuclei were detected by DAPI (blue) staining. Images are representative of three independent samples. Magnification was 200×; scale bar = 50 µm.</p

Distribution of markers in isolated cells obtained from the ascites of ovarian cancer patients.

<p>The antigens have been scored as high expression (⁺⁺⁺), moderate expression (⁺⁺), low expression (⁺), and no expression (⁻).</p

Cellular assessment of NAD and AD cells obtained from CN and CR patients.

<p>Percentage distribution of total cells in the NAD and AD populations of ascites of CN (n = 5) and CR (n = 5) patients was determined by Trypan Blue Exclusion assay. Results are mean±SEM of five independent samples assessed in triplicate. Significantly different in CR versus CN samples, **(p&lt;0.01).</p

A model of tumor cell progression in the ascites of ovarian cancer patients post-chemotherapy.

<p>Most ovarian cancer patients at diagnosis (stage IIIc/IV) present with ascites (CN) which consists of fibroblast-like stromal cells and a very few tumor cells. The majority of these patients (∼80%) after surgery and first line of chemotherapy return with recurrent cancer associated with ascites (CR). During the course of chemotherapy treatment and subsequent relapses, the percentage of stromal cells in the ascites is gradually decreased and the patient with recurrent cancer presents with ascites that consists mostly of CA125⁺⁺⁺/EpCAM⁺⁺⁺/STAT3⁺⁺⁺chemoresistant epithelial NAD tumor cells. These CA125⁺⁺⁺/EpCAM⁺⁺⁺/STAT3⁺⁺⁺ rich tumor cells are the eventual source of extraovarian peritoneal adhesions. These adhesions are the ultimate cause of patient mortality.</p