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Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

BACKGROUND The incidence and number of circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients are lower than in other cancer types,which may point to a particular biology of colorectal cancer affecting CTC detection. METHODS We detected CTCs in the peripheral and mesenteric blood of colorectal cancer patients by use of 2 independent technologies on the basis of different biological properties of colon cancer cells. Seventy-five patients diagnosed with localized (M0,n = 60) and metastatic (M1,n = 15) colorectal cancer were included. Peripheral and mesenteric blood samples were collected before tumor resection. We performed CTC enumeration with an EpCAM-independent enrichment method followed by the Epispot assay that detected only viable CK19-releasing CTCs. In parallel,we used the FDA-cleared EpCAM-dependent CellSearch® as the reference method. RESULTS The enumeration of CK19-releasing cells by the CK19-Epispot assay revealed viable CTCs in 27 of 41 (65.9%) and 41 of 74 (55.4%) (P = 0.04) patients in mesenteric and peripheral blood,respectively,whereas CellSearch detected CTCs in 19 of 34 (55.9%) and 20 of 69 (29.0%) (P = 0.0046) patients. In mesenteric blood,medians of 4 (range 0-247) and 2.7 CTCs (range 0-286) were found with Epispot and CellSearch (P = 0.2),respectively,whereas in peripheral blood,Epispot and CellSearch detected a median of 1.2 (range 0-92) and 0 CTCs (range 0-147) (P = 0.002). CONCLUSIONS A considerable portion of viable CTCs detectable by the Epispot assay are trapped in the liver as the first filter organ in CRC patients. View Publication

Ovarian cancer is the most lethal gynaecologic cancer,in large part because of its early dissemination and rapid development of chemotherapy resistance. Spheroids are clusters of tumor cells found in the peritoneal fluid of patients that are thought to promote this dissemination. Current models suggest that spheroids form by aggregation of single tumor cells shed from the primary tumor. Here,we demonstrate that spheroids can also form by budding directly as adherent clusters from a monolayer. Formation of budded spheroids correlated with expression of vimentin and lack of cortical E-cadherin. We also found that compared to cells grown in monolayers,cells grown as spheroids acquired progressive resistance to the chemotherapy drugs Paclitaxel and Cisplatin. This resistance could be completely reversed by dissociating the spheroids. Our observations highlight a previously unappreciated mode of spheroid formation that might have implications for tumor dissemination and chemotherapy resistance in patients,and suggest that this resistance might be reversed by spheroid dissociation. View Publication

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted,yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence,in this study,we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2),mild dysplasia (DOK),severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44,p75(NTR),CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44,p75(NTR),CD24 antigens and ALDH activity (ALDEFLUOR(®) assay),with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer,increased FOXA1 and decreased FOXA2 expression correlated with disease severity,but OCT3/4,Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. View Publication

Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study,we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR),a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand-receptor and also receptor-promoter interactions. Here,we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4,a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity,another stem cell-associated factor,enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1,which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G(0) cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation,parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together,our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. View Publication

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However,because most HES patients lack FIP1L1-PDGFRA,we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G,L507P,I562M,H570R,H650Q,N659S,L705P,R748G,and Y849S). When cloned into 32D cells,N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation,clonogenic growth,and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity,we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion,our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients. View Publication

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM,but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study,we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However,these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis,a potent,selective,and bioavailable HDAC6 inhibitor,WT161,was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress,followed by caspase activation and apoptosis. More importantly,this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells,which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM. View Publication