技术资料

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines,we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples,72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P textless 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies,where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly,tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations,but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer,ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy,significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P textless 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients,and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced,but not absolute,tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

BACKGROUND: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy,a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS: Cancer stem cells were defined as CD44+/CD24�?� cells that could self-renew (ie,generate cells with the tumorigenic CD44+/CD24�?� phenotype),differentiate,invade,and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells,weakly tumorigenic parental MCF-7 cells,and MCF-7/MDR,an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry,with in vitro invasion assays,and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg,CD44,TGFB1,and SNAI1). MCF-7/ADR cells were highly invasive,formed mammospheres,and were tumorigenic in mice. In contrast to parental MCF-7 cells,more than 30% of MCF-7/ADR cells had a CD44+/CD24�?� phenotype,could self-renew,and differentiate (ie,produce CD44+/CD24�?� and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1,CCNE1,and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field,difference = 6.69 cells per field,95% confidence interval = 4.82 to 8.55 cells per field,P textless .001). No enrichment in the CD44+/CD24�?� or CD133+ population was detected in MCF-7/MDR. CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. View Publication

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24(low/�?�)). Since then,CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties,including aldehyde dehydrogenase (ALDH) activity,have also been used to isolate CSCs from malignant tissues. Recently,we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24,and CD133. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic,but ALDH+ cells are relatively more invasive. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,a fluorescent substrate of ALDH. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells. View Publication

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system,we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes,including TMPRSS2-ERG fusion,SPOP mutation,SPINK1 overexpression,and CHD1 loss. Whole-exome sequencing shows a low mutational burden,consistent with genomics studies,but with mutations in FOXA1 and PIK3R1,as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. View Publication

Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here,we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells,resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival,respectively. Thus,our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment. View Publication

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system,an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points,CTCs were detected in 37{\%},54.9{\%} and 58.7{\%} of patients using CellSearch,CellCollector and EPISPOT,respectively. The cumulative positivity rate of the three CTC assays was 81.3{\%} (87/107) with 21.5{\%} (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66{\%} before RP to 34{\%} after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p {\textless} 0.0001) and clinical tumor stage (p = 0.04),while the other assays showed no significant correlations. In conclusion,CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer. View Publication

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines,causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified textgreater 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM,whereas no cytotoxicity against EGF-R-negative leukemia cells was observed,even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival,19 days) and all control mice had tumors that rapidly progressed to reach an average size of textgreater 500 mm3 by 58 days,40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size textgreater 50 mm3. Thus,targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication