Identifying an ovarian cancer cell hierarchy regulated by bone morphogenetic protein 2

Whether human cancer follows a hierarchical or stochastic model of differentiation is controversial. Furthermore, the factors that regulate cancer stem-like cell (CSC) differentiation potential are largely unknown. We used a novel microfluidic single-cell culture method to directly observe the differentiation capacity of four heterogeneous ovarian cancer cell populations defined by the expression of the CSC markers aldehyde dehydrogenase (ALDH) and CD133. We evaluated 3,692 progeny from 2,833 cells. We found that only ALDH⁺CD133⁺ cells could generate all four ALDH^+/−CD133^+/− cell populations and identified a clear branched differentiation hierarchy. We also observed a single putative stochastic event. Within the hierarchy of cells, bone morphologenetic protein 2 (BMP2) is preferentially expressed in ALDH⁻CD133⁻ cells. BMP2 promotes ALDH⁺CD133⁺ cell expansion while suppressing the proliferation of ALDH⁻CD133⁻ cells. As such, BMP2 suppressed bulk cancer cell growth in vitro but increased tumor initiation rates, tumor growth, and chemotherapy resistance in vivo whereas BMP2 knockdown reduced CSC numbers, in vivo growth, and chemoresistance. These data suggest a hierarchical differentiation pattern in which BMP2 acts as a feedback mechanism promoting ovarian CSC expansion and suppressing progenitor proliferation. These results explain why BMP2 suppresses growth in vitro and promotes growth in vivo. Together, our results support BMP2 as a therapeutic target in ovarian cancer.The cancer stem cell (CSC) hypothesis postulates that cancers are composed of hierarchically organized cell subpopulations with distinct phenotypes and tumorigenic capacities. CSCs are rare cells within the tumor that can not only proliferate to maintain tumorigenic potential, but also asymmetrically divide to generate other, phenotypically distinct cells with diminished tumorigenic potential. CSCs have therefore been suggested as a source of disease recurrence; residual CSCs after primary therapy can proliferate and differentiate to recreate a tumor (1).Although there is very strong support in the literature for the existence of cancer stem-like cells, the CSC hypothesis remains controversial. Supporting the CSC hypothesis, murine studies using lineage-tracing experiments identified subsets of cells with CSC-like characteristics in tumor precursor lesions (2, 3). Furthermore, in overt malignancy, a subset of rare chemotherapy-resistant CSC-like cells were found to be responsible for hierarchical tumor cell regrowth after chemotherapy treatment (4). However, other studies indicate stochastic events in which non–stem-like cells can acquire stem-like characteristics under the influence of the right environmental/genetic stresses (5–8). Studies to define a hierarchical vs. stochastic model with human tumor specimens are complicated by a small but real contamination rate associated with both magnetic- and FACS-based cell purification.Little is known about a potential ovarian cancer cell hierarchy. Although some reports suggest that ovarian cancer may not follow a hierarchical model (9), numerous potential CSC populations, defined by various cell markers, have been reported (1, 10). Aldehyde dehydrogenase enzymatic activity (ALDH) and the stem cell marker CD133, either alone or in combination, are perhaps the best supported ovarian CSC (OvCSC) markers (11–16). We recently reported that ALDH and CD133 can be used to define distinct heterogeneous populations of ovarian cancer cells (17). We found that both ALDH⁺CD133⁺ cells and ALDH⁺CD133⁻ primary human ovarian tumor cells can act as OvCSCs, with the ability to initiate and passage tumors in mice that recapitulate the primary human tumor. ALDH⁺CD133⁺ OvCSCs demonstrated the greatest engraftment potential and generated tumors within 2–4 months whereas ALDH⁺CD133⁻ primary human ovarian cancer cells have lower engraftment potential and longer growth requirements (6–12 months). ALDH⁻CD133⁻ cells from primary samples are unable to initiate tumors. Clinical observations also support ALDH and CD133 as identifying at least one population of OvCSCs; the presence of increased numbers of ALDH⁺CD133⁺ cells in primary tumor debulking samples is associated with poor patient outcome (11); and ALDH and CD133 (and CD44) are enriched in patient tumors and human PDX tumors immediately after chemotherapy (14, 18). ALDH and CD133 (along with LGR5) were also found to identify a population of normal ovarian stem cells, suggesting a potential role of ALDH⁺CD133⁺ cells in the ovarian cancer cell of origin (19).Based on these data, we hypothesized that ALDH and CD133 can be used to define a hierarchy of OvCSC differentiation; highly tumorigenic ALDH⁺CD133⁺ CSCs give rise to somewhat less tumorigenic ALDH⁺CD133⁻ CSC/progenitors, which in turn give rise to less tumorigenic/nontumorigenic ALDH⁻CD133⁻ cells. Here, we report a using a microfluidic device (20) to directly interrogate the differentiation potential of the distinct ALDH^+/−CD133^+/− cell populations in ovarian cancer and to define a branched differentiation lineage. We show that BMP2 is differentially expressed in the distinct ovarian cancer cell populations, with low expression in ALDH⁺CD133⁺ cells and highest expression in ALDH⁻CD133⁻ cells. Furthermore, we find that BMP2 promotes expansion of the ALDH⁺CD133⁺ CSC cell population and inhibits the proliferation of progenitors. In vivo treatment with BMP2 results in increased tumor growth and chemotherapy resistance. Inhibition of BMP2 signaling with Noggin or BMP2 knockdown is associated with a decrease in the number of ALDH+CD133+ and ALDH-CD133+ cells, reduced tumor initiation capacity, and a reduction in tumor growth.     

Changes of immunological markers after autologous peripheral blood stem cell transplantation

Recently high-dose chemotherapy with peripheral blood stem cell transplantation (PBSCT) has become an important treatment for hematological and solid tumors. Methods: Immunological parameters were examined before and after PBSCT in 9 patients with lung cancer and 13 patients with malignant lymphoma. Findings were compared with those for bone marrow transplantation (BMT). Peripheral blood cells were analyzed for phenotype and the levels of cytokines and soluble factors were measured. Results: After PBSCT, activated T cells (CD3⁺HLA-DR⁺ cells, CD8⁺HLA-DR⁺ cells) and suppressor/cytotoxic T cells (CD8⁺CD11b⁻ cells) were significantly higher in the patients with lung cancer than in those with malignant lymphoma. Serum levels of interleukin-4 and soluble interleukin-2 receptor were also significantly higher in the patients with lung cancer than in those with lymphoma. On the other hand, the serum levels of interferon γ, tumor necrosis factor α, interleukin-6, soluble human leukocyte antigen class 1, and soluble thrombomodulin were significantly increased after bone marrow transplantation. The transfused peripheral stem cells of lung cancer and lymphoma patients had a similar number of granulocyte/macrophage-colony-forming units, but lung cancer patients had significantly more CD34-positive cells. Conclusion: By reinfusing large numbers of autologous immune cells, PBSCT may accelerate immune reconstitution, with T cells being likely to have a marked therapeutic potential. The changes after PBSCT were greater in patients with lung cancer than in lymphoma patients. These blood cells are potent mediators of anticancer activity and could play an important role in the elimination of autologous malignant cells. Received: 20 April 1998 / Accepted: 24 August 1998     

TGF-β1 Secreted by Hepatocellular Carcinoma Induces the Expression of the Foxp3 Gene and Suppresses Antitumor Immunity in the Tumor Microenvironment

Aim

The purpose of this study was to explore the mechanisms of TGF-β1 mediated immunosuppression in tumor stroma.

Methods

The expression of TGF-β1 was investigated in Huh7, Hep 3B, SGC-7901, Eca-109 and Hepa1-6 cell lines using immunofluorescence. Knocked-down TGF-β1 of the Hepa1-6 cell line was established through lentivirus-based RNA interference. The interference efficiency of the TGF-β1 gene was tested by real-time PCR and ELISA; the expression of Foxp3, IFN-γ and CD83 in CD4⁺, CD8⁺ or dendritic cells was examined via flow cytometry; and the tumorigenic ability of the cancer cells was investigated in the animal experiments.

Results

The diverse digestive cancer cells were found to secrete TGF-β1, mRNA of which was knocked down by 78 % thanks to lentivirus-based interference in Hepa1-6 cells. Flow cytometry showed that CD4⁺CD25⁺Foxp3⁺ regulatory T cells significantly increased in hepatocellular carcinoma patients when compared with those in the healthy controls. The supernatant from Hepa1-6 cells and recombinant TGF-β1 significantly induced the expression of Foxp3 gene in vitro, while that from sh TGF-β1 Hepa1-6 cells restored it. Hepa1-6 cells inhibited IFN-γ and CD83 expression in CD8⁺ or dendritic cells by secreting TGF-β1. The animal experiments indicated that the knockdown TGF-β1 gene impaired the tumorigenic ability of Hepa1-6 cells.

Conclusion

TGF-β1, expressed in cancer cells, might be a potential therapeutic target for cancer treatment.     

A tumor hypoxic niche protects human colon cancer stem cells from chemotherapy

Purpose

Hypoxia has been found to play an important role in regulating the biological characteristics of cancer stem cells (cCSCs). In this study, we tested whether a tumor hypoxic niche serves to the chemotherapeutic resistance of colon cCSCs.

Methods

Each of 23 fresh samples of human colon adenocarcinoma was transplanted into nude mice. The tumor-bearing mice randomly and equally received (A) saline, (B) 5-fluorouracil (15 mg/kg), (C) oxaliplatin (10 mg/kg), and (D) oxaliplatin plus 5-fluorouracil when xenografts reached 250 mm³ (n = 10). After 2-week treatment, tumor cells were quantified by flow cytometry for expression of CD133 and the hypoxic proportion of CD133⁺ and CD133^? cells which were also sorted and detected for ki67 and pimonidazole via immunofluorescence.

Results

The hypoxic subpopulation of CD133⁺ and CD133^? cells was 66.5 and 26.4 %, respectively. Although there was no marked change for the hypoxic subpopulation of CD133⁺ cells after treatment, the hypoxic fraction of proliferative CD133⁺ cells was increased by 14.62, 16.45, and 20.46 % in groups B, C, and D, respectively. Furthermore, proliferative cells in CD133⁺ and CD133^? cells were reduced by 29.93 and 62.5 % in group C, and by 25.26 and 68.22 % in group D; in group B, however, the proliferative CD133⁺ cells were increased by 37.09 %; the CD133^? cells were unchanged.

Conclusions

Most CD133⁺ cCSCs are located in a hypoxic niche, where cCSCs are better at retaining proliferating property under chemotherapy. Oxaliplatin, rather than 5-FU, inhibits proliferation of cCSCs, which may be the mechanism underlying a better outcome by oxaliplatin in colon cancer patients.     

Expression of CD147 in advanced non-small cell lung cancer correlated with cisplatin-based chemotherapy resistance

CD147, a widely expressed cell surface glycoprotein in cancer, is associated with tumor invasiveness and chemotherapy resistance. Recently, CD147 is also regarded as a potential therapeutic target for cancer therapy. The aim of the study was to investigate CD147 expression in non-small cell lung cancer (NSCLC), and evaluate its correlation with cisplatin-based chemotherapy resistance. In this study, we examined immunohistochemically the expression of CD147 in 118 advanced NSCLC cases treated with cisplatin-based chemotherapy, and then the association of CD147 expression with clinicopathological characteristics was analyzed. Furthermore, RNA interference approach was used to silence CD147 expression in a cisplatin-resistant human lung cancer cell line A549/DDP, and the inhibition effect of cisplatin on tumor cells was assayed by MTT. In the overall series, positive CD147 expression was observed in 101/118 (85.6%) cases. A membranous CD147 pattern was identified in 76/101 (75.2%) of CD147 positive tumors. CD147 membranous expression,but not the overall CD147 expression, was associated with poor response to cisplatin-based chemotherapies and a poor prognosis in advanced NSCLC patients. In vitro results showed that silencing CD147 increased the proliferation inhibitory effect of cisplatin to A549/DDP cells. In conclusion, our study indicated that membranous CD147 expression is a predictive factor of the response to cisplatin-based chemotherapies, and the use of CD147-targeted therapeutic adjuvants might be considered in the treatment of advanced NSCLC patients.     

CD24<Superscript>+</Superscript>/CD38<Superscript>-</Superscript> as new prognostic marker for non-small cell lung cancer

Background

Lung cancer is the leading cause of death among cancers in the world. The annual death toll due to this disease exceeds the combined deaths caused by colon, breast, prostate, and pancreatic cancers. As a result, there has been a tremendous effort to identify new biomarkers for early detection and diagnosis of lung cancer.

Methods

In this study we report the results of screening a panel of eight non-small cell lung cancer (NSCLC) cell lines originating from different subtypes of lung cancer in an attempt to identify potential biomarkers unique to this disease. We used real-time polymerase chain reaction and flow cytometry techniques to analyze the expression of ALDHA1, EpCAM, CD133, CD24, and CD38 in this panel.

Results

We demonstrate for the first time that the majority of NSCLC cells do not express levels of CD38 that would qualify it as a new biomarker for the disease. In contrast, we found that CD24 is over-expressed in 6 out of 8 of the cell lines. The combined CD24⁺/CD38^-/low phenotype was detected in 50% of the cell lines that are also positive for CD133 and EpCAM.

Conclusions

We report that CD24⁺/CD38^-/low signature could potentially be used as a new biomarker for the early detection of NSCLC.

     

The utility of hyperthermic intra-abdominal chemotherapy with gemcitabine for the inhibition of tumor progression in an experimental model of pancreatic peritoneal carcinomatosis,in relation to their behavior with pancreatic cancer stem cells CD133+ CXCR4+

ObjectiveThe origin of pancreatic cancer has been identified as a population of malignant pancreatic stem cells CD133+ CXCR4+ immunophenotype. These cells have high capacity for early locoregional invasion, being responsible for early recurrence and high mortality rates of pancreatic cancer. We propose a study for decreasing tumor progression of pancreatic cancer by reducing the volume and neoplastic subpopulation of pancreatic cancer stem cells CD133+ CXCR4+. Therefore, we develop a new therapeutic model, characterized by the application of HIPEC (Hyperthermic Intraperitoneal Chemotherapy) with gemcitabine.DesignPancreatic tumor cell line: human cell line BxPC-3. The animal model involved 18 immunosuppressed rats 5 weeks weighing 150–200 gr. The implantation of 13 × 10⁶ cells/mL was performed with homogeneous distribution in the 13 abdominopelvic quadrants according to the peritoneal carcinomatosis index (PCI) and were randomized into three treatment groups. Group I (4 rats) received intravenous saline. Group II (6 rats) received intravenous gemcitabine. Group III (8 rats) received HIPEC at 41 °C for 30 min with gemcitabine + gemcitabine IV. A histological study confirmed pancreatic cancer and immunohistochemical quantification of pancreatic cancer stem cells CD133+ CXCR4+ tumor cells.ResultsThere was a population decline of pancreatic cancer stem cells CD133+ CXCR4+ in the HIPEC group with respect to the other two groups (p < 0.001). There was a decrease in PCI between treatment groups (p < 0.05).ConclusionThe initial results are encouraging since there is a declining population of cancer stem cells CD133+ CXCR4+ in the HIPEC group and decreased tumor volume compared to the other two treatment groups. All the conclusions are only valid for BxPC3 cell line, and the effects HIPEC on Kras-driven pancreatic tumors remain to be determined.     

Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44⁺/CD24^−/low cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44⁺/CD24^−/low and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44⁺/CD24^−/low-MS signature. The CD44⁺/CD24^−/low-MS signature was found mainly in human breast tumors of the recently identified “claudin-low” molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44⁺/CD24^−/low-MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin (VIM) in cytokeratin-positive epithelial cells metalloproteinase 2 (MMP2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.     

Comparison of mammosphere formation from breast cancer cell lines and primary breast tumors

Background

Breast cancer stem cells (BCSCs) can be enriched by culturing of cells in non-adherent non-differentiating conditions. However, culturing mammospheres from primary breast tumors are costly and difficult to control. In order to overcome problems associated with using primary human tissues, continuous breast cancer cell lines have been developed from various sources.

Methods

In this study, a luminal subtype breast cancer cell line MCF-7 and a basal subtype cell line MDA-MB-231 were chosen. We explored the optimal culturing system for BCSCs from the two cell lines and primary breast tumors. Then, mammosphere formation efficiency (MFE), CD44⁺/CD24^–/lowESA⁺Lin^– cell proportion in mammospheres, and tumorigenecity of mammospheres generated from the two breast cancer cell lines and primary breast tumors were compared.

Results

Enzymatic digestion of 60 mins and the addition of B27 to the culture medium were optimal for mammosphere culturing. Mammospheres could be formed in all the three cells, in which MCF-7 had the highest MFE. After 3 weeks culture, CD44⁺/CD24^–/lowESA⁺Lin^– cell proportion in mammospheres from MCF-7, MDA-MB-231 cells and primary breast tumors was 95.0%±2.5%, 82%±22% and 21.5%±1.0%, respectively. A total of 1,000 cells from MCF-7, MDA-MB-231 mammospheres but not primary mammospheres were tumorigenic.

Conclusions

This study validates the use of breast cancer cell lines as models to elucidate the nature of BCSCs.     

Population alterations of l-arginase- and inducible nitric oxide synthase-expressed CD11b+/CD14−/CD15+/CD33+ myeloid-derived suppressor cells and CD8+ T lymphocytes in patients with advanced-stage non-small cell lung cancer

Background

Immune aberrations have been demonstrated in tumorogenesis, and myeloid-derived suppressor cells (MDSC) have shown to play a pivotal role in mediating immune suppression in animal models of human tumors. In the present study, we explored the clinical relevance of CD11b⁺/CD14^?/CD15⁺/CD33⁺ MDSCs and the association of MDSCs with CD8⁺ cytotoxic T lymphocytes in patients with non-small-cell lung cancer (NSCLC).

Patients and methods

The population of CD11b⁺/CD14^? cells in peripheral blood mononuclear cells (PBMNC) was determined in 173 patients with NSCLC and 42 control subjects. The expression of CD15, CD33, IL-4R, INF-γR, iNOS and l-arginase were analyzed. Cocultures with CD8⁺ T lymphocytes and Jurkat cells were developed to determine the impact of MDSCs on the expression of CD3ζ of CD8⁺ T lymphocytes.

Results

Patients with treatment-naïve, advanced-stage NSCLC (n = 87) had an increased subpopulation of CD11b⁺/CD14^?/CD15⁺/CD33⁺ cells in the PBMNCs with characteristics of MDSCs (P < 0.0001). The CD11b⁺/CD14^? cells in PBMNC also express IL-4R and INF-γR and can suppress CD3ζ expression in CD8⁺ T lymphocytes. The subpopulation of CD11b⁺/CD14^? cells in PBMNC was decreased in the advanced-stage NSCLC patients who had responsiveness to chemotherapy (n = 41, P < 0.0001) and in the early-stage NSCLC patients after removal of tumor (n = 8, P = 0.0391). Notably, a negative association existed between the population of CD11b⁺/CD14^? cells in PBMNC and the frequency of CD8⁺ T lymphocytes (n = 48, r = ?0.3141, P = 0.0297).

Conclusions

Our study provided evidence of an increased pool of CD11b⁺/CD14^?/CD15⁺/CD33⁺ MDSCs in the peripheral blood of NSCLC patients. For the suppressive effect of the cells on CD8⁺ T lymphocytes, these findings suggest the important role of the CD11b⁺/CD14^?/CD15⁺/CD33⁺ MDSCs in mediating immunosuppression in NSCLC.     

Microenvironmental modulation of asymmetric cell division in human lung cancer cells

Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell–cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133⁺ lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.     

The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations

Characterization of the molecular pathways that are required for the viability and maintenance of self-renewing tumor-initiating cells may ultimately lead to improved therapies for cancer. In this study, we show that a CD133⁺/CD44⁺ population of cells enriched in prostate cancer progenitors (PCaPs) has tumor-initiating potential and that these progenitors can be expanded under nonadherent, serum-free, sphere-forming conditions. Cells grown under these conditions have increased in vitro clonogenic and in vivo tumorigenic potential. mRNA expression analysis of cells grown under sphere-forming conditions, compared with long-term monolayer cultures, revealed preferential activation of the PI3K/AKT signaling pathway. PI3K p110α and β-protein levels were higher in cells grown under sphere-forming conditions, and phosphatase and tensin homolog (PTEN) knockdown by shRNA led to an increase in sphere formation as well as increased clonogenic and tumorigenic potential. Similarly, shRNA knockdown of FoxO3a led to an increase in tumorigenic potential. Consistent with these results, inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 led to growth inhibition of PCaPs. Taken together, our data strongly suggest that the PTEN/PI3K/Akt pathways are critical for prostate cancer stem-like cell maintenance and that targeting PI3K signaling may be beneficial in prostate cancer treatment by eliminating prostate cancer stem-like cells.     

Prognostic impact of 18F-FDG uptake on PET in non-small cell lung cancer patients with postoperative recurrence following platinum-based chemotherapy

BackgroundWhether fluorine-18-fluorodeoxyglucose (¹⁸F-FDG) uptake within tumor cells differs between primary and recurrent lung cancers is unknown. The aim of this study was to investigate the prognostic significance of ¹⁸F-FDG uptake by comparing that measured preoperatively at the primary site to that measured postoperatively at sites of non-small cell lung cancer (NSCLC) recurrence. Only patients with postoperative recurrences who received platinum-based chemotherapy as the initial treatment after recurrence were included in the study.MethodsFifty-two patients underwent ¹⁸F-FDG positron emission tomography (PET) examinations before thoracotomy and at the time of recurrence after curative surgery. All recurrences were treated with platinum-based chemotherapy.Results¹⁸F-FDG uptake in the preoperative primary tumors was significantly higher than that in the recurrent tumors (p=0.028), demonstrating a statistically significant correlation (Pearson's correlation coefficient γ=0.482, p<0.001), especially in adenocarcinoma (AC) patients. Low ¹⁸F-FDG avidity within the primary tumor significantly correlated with the presence of epidermal growth receptor factor (EGFR) mutations. ¹⁸F-FDG uptake in the primary tumors was an independent prognostic factor for predicting outcome in NSCLC patients receiving platinum-based chemotherapy for the treatment of postoperative recurrence.ConclusionsIn NSCLC patients treated by chemotherapy for recurrence, preoperative measurements of ¹⁸F-FDG uptake may be a more powerful surrogate marker for predicting outcome when measured preoperatively at the primary tumor site rather than postoperatively at sites of recurrence.     

CD133 marks a stem cell population that drives human primary myelofibrosis

Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133⁺ stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133⁺ but not CD34⁺CD133⁻ cells, with a variable burden for JAK2^V617F mutation, had multipotent cloning capacity in vitro. CD133⁺ cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F⁺ myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F⁺ megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133⁺ cells harboring EZH2^D265H but lacking a secondary JAK2^V617F mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms.     

Strong correlation between N-cadherin and CD133 in breast cancer: role of both markers in metastatic events

Purpose

Epithelial mesenchymal transition is a major mechanism to explain metastatic events in breast cancer. Another important aspect is that cells with stem cell properties are able to become resistant against chemotherapeutics. Our main goal was to investigate the role of the EMT marker, N-cadherin, and of the stem cell marker, CD133, in breast cancer.

Methods

The expressions of N-cadherin and CD133 were assessed by immunohistochemistry in 307 primary tumors from breast cancer patients and for 30 patients, in the related recurrences and/or metastases. We studied the correlation between both markers, their associations with known clinicopathological parameters and their role as predictive markers for survival time. Different expressions of both markers in primary tumor and recurrences or metastases were examined.

Results

N-cadherin and CD133 expressions correlated positively in the 261 primary tumor samples (p = 0.000) and in the 45 primary tumor, recurrence or metastasis samples (p = 0.010). In patients without lymph node metastases, the 10-year survival time was significantly lower when the tumor was N-cadherin-positive (p = 0.042). Expression of N-cadherin was also significantly higher in metastases than in the related primary tumors (p = 0.039).

Conclusion

N-cadherin and CD133 expressions are strongly correlated and N-cadherin appears as a potential metastases marker in a specific patient subpopulation.     

Identification and characterization of tumorigenic liver cancer stem/progenitor cells

BACKGROUND & AIMS: Recent efforts in stem cell biology suggest that tumors are organized in a hierarchy of heterogeneous cell populations and that the capability to maintain tumor formation/growth specifically resides in a small population of cells called cancer stem cells (CSCs). The aim of this study is to identify, isolate, and characterize the CSC population that drives and maintains hepatocellular carcinoma (HCC) growth and metastasis. METHODS: Normal stem cells involved in liver regeneration were identified using a severe partial hepatectomy model. Purified HCC cells, with or without expression of the identified normal stem cell phenotype, were evaluated, based on their tumorigenic potential and exhibition of defined stem/progenitor cell-like properties, to determine whether liver CSCs can be or partly be identified by this surface marker. RESULTS: We report the identification and isolation of a population of CSCs expressing a CD133 surface phenotype from human liver cell lines. CD133(+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. These cells are endowed with characteristics similar to those of progenitor cells including the expression of "stemness" genes, the ability to self-renew, and the ability to differentiate into nonhepatocyte-like lineages. Furthermore, CD133 is found to represent only a minority of the tumor cell population in human HCC specimens. CONCLUSIONS: We report the identification of a CSC population in HCC characterized by their CD133 phenotype. The identification of tumorigenic liver CSCs could provide new insight into the HCC tumorigenic process and possibly bear great therapeutic implications.     

Changes of lymphocyte subsets in peripheral blood before and after operation of patients with advanced ovarian carcinoma

Summary Comparison was made between lymphocyte subsets in peripheral blood from patients with benign ovarian tumor and those with advanced ovarian carcinoma. In addition, changes of lymphocyte subsets of patients with ovarian carcinoma before and after operation were also examined. The percentage and absolute number of CD3^–/HLA-DR⁺ (B cells) in peripheral blood from patients with advanced ovarian carcinoma were significantly lower than values from patients with benign ovarian tumor, whereas both percentage and absolute number of CD3^–/HLA-DR^– (null cells) cells in patients with advanced ovarian carcinoma were significantly higher. Although there was no significant difference in natural killer (NK) cell subsets (CD57⁺CD16^– and CD57⁺ CD16⁺ cells) between patients with benign ovarian tumor and ovarian carcinoma, the percentage and absolute number of CD57^–/CD16⁺ (highly differentiated NK cells) cells in patients with ovarian carcinoma were significantly higher than those in patients with benign ovarian tumor. Both the absolute number and percentage of CD3⁺/HLA-DR⁺ (activated T cells) cells in ovarian cancer patients with minimal residual tumors after operation were significantly increased, compared to the levels before operation, while the values in the patients with large residual tumors were significantly decreased. In addition, the percentage and absolute number of CD3^–/HLA-DR^– (null cells) cells in the patients with minimal residual tumors were significantly decreased after operation, while values in the patients with large residual tumors remained unchanged before and after operation. The patients with minimal residual tumors after operation were characterized by a significant increase in the percentage of CD57^–CD16⁺ (highly differentiated NK cells) cells. On the other hand, in the patients with large residual tumors no change of the NK cell subsets was observed before and after operation.Abbreviation NK natural killer - FITC fluorescein isothiocyamate - PE phycoerythrin     

Dynamics of endothelial progenitor cells in patients with advanced hepatocellular carcinoma

Background and aimsCirculating endothelial progenitor cells (EPC) predict tumor vascularization and disease progression, but limited information is available on their dynamics in hepatocellular carcinoma (HCC) undergoing systemic treatment.MethodsWe prospectively analyzed different populations of EPC in 16 patients with advanced HCC receiving sorafenib. Patients were studied before therapy (T0, n = 16) and after two (T2, n = 12) and eight weeks (T8, n = 8), using high-performance flow-cytometry. The tumor response at T8 was categorized as progressive disease (PD) or clinical benefit (CB, all other responses).ResultsAt T0, higher levels of CD34⁺CD133⁺KDR⁺ and CD34⁺KDR⁺ were observed in patients with alpha-fetoprotein ≥400 ng/ml or non-viral liver disease, whereas CD34⁺CD133⁺KDR⁺ cells were virtually absent in patients with vascular invasion. CD34⁺KDR⁺ and CD34⁺CD133⁺KDR⁺ were directly correlated with platelet count. Frequencies of all populations of EPC declined in patients receiving sorafenib. Levels of CD34⁺CD133⁺ were higher at T0 in patients with CB compared to patients with PD. In patients belonging to the CB group CD34⁺KDR⁺ cells at T0 were directly correlated to platelet count.ConclusionIn patients with advanced HCC, EPC are directly correlated with platelet count, suggesting a common activation of selected bone marrow pathways. Levels of a CD34⁺KDR⁺ are higher at baseline in patients responding to sorafenib.     

Glioblastoma and stem cells

This review presents compelling evidence that human glioblastoma is a heterogenous tumor composed from tumor cells and small portion of cancer stem cells - tumor-initiating cells, which have a high tumorigenic potential and a low proliferation rate. Glioma cancer stem cells are phenotypically similar to the normal stem cells, they express CD133 gene and other genes characteristic of neural stem cells and posses the self-renewal potential. Cancer stem cells derived from glioblastoma are capable recapitulate original polyclonal tumors when xenografted to nude mice. They are chemoresistant and radioresistant and therefore responsible for tumor progression and recurrence after conventional glioblastoma therapy. Cancer stem cells contribute to glioma radioresistance by an increase of DNA repair capacity through preferential activation of the DNA damage response checkpoints. Potential therapies that modulate or target cancer stem cells are also reviewed. Mesenchymal stem cells and/or neural stem cells were shown to target brain tumors therefore these cells are considered as an effective delivery system to target and disseminate therapeutic agents to brain tumors. Stem cell-based gene therapies for glioblastoma were shown in experiments to be effective way to target brain tumors. Effects of bone morphogenetic protein (BMP4) on glioma cancer stem cells are also reviewed. BMP4 reduces effectively proliferation of CD133 positive cells in vitro and the tumor growth in vivo. BMP4 may act as a key inhibitory regulator of cancer initiation and therefore may be used in combined stem cell-based therapy as a non-cytotoxic therapeutic agent. Key words: Glioblastoma, cancer stem cells, CD113 marker, chemoresistance, radio-resistance, rat glioma models, vascular niche, mesenchymal stem cells, stem cell-based gene therapy, BMP4.     

CD133在肺癌中的研究与进展

肺癌是世界范围内死亡率最高的恶性肿瘤之一,也是我国常见的恶性肿瘤之一,恶性程度高,发展迅速,治疗困难,总体疗效不理想。肿瘤干细胞是肿瘤组织中一小部分具有自我更新、无限增殖和多向分化能力的肿瘤细胞,它在肿瘤组织中所占比例虽然很少,却与肿瘤起源、发展与转移关系密切,因此肿瘤干细胞被看作是一个根除癌症的潜在目标。CD133是...