Pancreatic Cancer Cells Resistant to Chemoradiotherapy Rich in “Stem-Cell-Like” Tumor Cells

Background  

Tumor resistance to chemoradiation therapy is partly attributed to the presence of apoptosis-resistant cancer stem cells (CSCs). Chemoradiation therapy can enrich CSCs by killing apoptosis-susceptible cancer cells.     

Chimeric 5/35 adenovirus-mediated Dickkopf-1 overexpression suppressed tumorigenicity of CD44+ gastric cancer cells via attenuating Wnt signaling

Background

Gastric cancer stem cells (CSCs), which require activation of Wnt signaling to maintain their self-renewal and tumorigenicity, are proposed to be critical targets for effective therapy of gastric carcinomas. Gene therapies that are delivered by adenovirus of serotype 5 (Ad5) or chimeric 5/35(Ad5/35) adenovirus have shown promise for treating various cancers. Here we aimed to develop a gene therapy strategy that targeted gastric CSCs (CD44⁺ cells).

Methods

CD44⁺ cells were isolated by fluorescence activated cell sorting from both primary gastric cancer cells and cell lines. Expression of adenovirus receptors was examined in CD44⁺ and CD44^? cells. A potent Wnt antagonist Dickkopf-1 (DKK1) was delivered into CD44⁺ cells using Ad5/35 (Ad5/35-DKK1). The therapeutic outcomes were evaluated.

Results

Expression of Coxsakievirus adenovirus receptor for Ad5 was significantly reduced, while abundance of CD46, the receptor for Ad5/35, was slightly higher in CD44⁺ cells. Accordingly, CD44⁺ cells were sensitive to Ad5/35 infection, but not to Ad5. Ad5/35-DKK1 introduced DKK1 into CD44⁺ cells and deactivated endogenous Wnt/β-catenin signaling efficiently. Overexpression of DKK1 inhibited survival, anchorage-independent colony formation, and invasion of CD44⁺ cells, which were restored by a GSK-3 specific inhibitor BIO-acetoxime. More importantly, introduction of DKK1 abrogated the tumorigenicity of CD44⁺ cells in vivo. However, Ad5/35-DKK1 only showed minimal cytotoxicity to normal tissue-derived cells, L-02 and GES-1.

Conclusions

We developed, for the first time, a novel Ad5/35-DKK1-based approach to abrogate Wnt signaling in CSCs and demonstrated that gastric CSC-targeting gene therapy was effective in preclinical experiments.     

Breast cancer adaptive resistance: HER2 and cancer stem cell repopulation in a heterogeneous tumor society

Purpose

The lethal effects of cancer are associated with the enhanced tumor aggressiveness in recurrent and metastatic lesions that show resistant phenotype to anti-cancer therapy, a major barrier to improving overall survival of cancer patients. The presence of heterogeneous populations of cancer cells within a specific tumor including the tumor-initiating cells or so-called cancer stem cells (CSCs) has linked the acquired resistance (AR, or adaptive resistance). Herein, we discuss the CSC-mediated tumor repopulation in AR of breast cancer in this review.

Methods

We emphasize a dynamic feature of gene induction in tumor cells that undergo long-term treatment, and describe a specific HER2-NF-κB-HER2 pro-survival pathway that can be initiated in breast CSCs upon radiation therapy.

Results

Elucidation of HER2-induced pro-survival networks, specifically the force driving tumor repopulation due to radioresistant CSCs during anticancer therapies, will have a significant impact on the generation of new diagnostic and therapeutic targets to control of recurrent and metastatic breast tumors.     

Low expression of chemokine receptor CCR5 in human colorectal cancer correlates with lymphatic dissemination and reduced CD8+ T-cell infiltration

Background  

Chemokines and their receptors have been proposed to distinctly contribute to tumor growth, dissemination, and local immune escape. The aim of this study was to evaluate the relevance of the chemokine receptor CCR5 expression for the progression of human colorectal cancer.     

Noninvasive positron emission tomography and fluorescence imaging of CD133+ tumor stem cells

A technology that visualizes tumor stem cells with clinically relevant tracers could have a broad impact on cancer diagnosis and treatment. The AC133 epitope of CD133 currently is one of the best-characterized tumor stem cell markers for many intra- and extracranial tumor entities. Here we demonstrate the successful noninvasive detection of AC133⁺ tumor stem cells by PET and near-infrared fluorescence molecular tomography in subcutaneous and orthotopic glioma xenografts using antibody-based tracers. Particularly, microPET with ⁶⁴Cu-NOTA-AC133 mAb yielded high-quality images with outstanding tumor-to-background contrast, clearly delineating subcutaneous tumor stem cell-derived xenografts from surrounding tissues. Intracerebral tumors as small as 2–3 mm also were clearly discernible, and the microPET images reflected the invasive growth pattern of orthotopic cancer stem cell-derived tumors with low density of AC133⁺ cells. These data provide a basis for further preclinical and clinical use of the developed tracers for high-sensitivity and high-resolution monitoring of AC133⁺ tumor stem cells.Cancer stem cells (CSCs) are highly undifferentiated tumor cells with characteristics similar to normal stem cells. These characteristics include long-term replication, self-renewal, and aberrant differentiation (1, 2). Based on these characteristics, it has been hypothesized that only CSCs are able to propagate tumors for long periods of time and to initiate relapses or metastases. Furthermore, CSCs are considered to be more resistant to conventional radio- and chemotherapy than more differentiated tumor cells (3–5). Hence, elimination of CSCs is challenging but necessary for successful tumor eradication. The stem cell hypothesis of cancer development and progression is conceptually attractive and is supported by many preclinical (1, 2, 5–7) and some clinical studies (4, 8). However, larger clinical trials investigating the role of CSCs in patients have been hampered by the lack of techniques to detect, localize, and quantify the presence of CSCs noninvasively. Specifically, successful noninvasive imaging of unmanipulated CSCs with clinically relevant imaging probes (e.g., antibodies or other ligands binding CSC-specific cell-surface proteins) has not yet been reported (9–11).AC133 is an N-glycosylation–dependent epitope of the second extracellular loop of CD133/prominin-1, a cholesterol-binding protein of unknown function that locates to plasma membrane protrusions (12–14). Postnatally, the CD133 protein is expressed by certain epithelial and nonepithelial cells, by stem and progenitor cells of various organs, and by CSCs of many different types of malignant tumors (15). With a few exceptions, recognition of the AC133 epitope by the AC133 mAb appears to be limited to cells harboring stem cell properties, and the AC133 epitope—but not necessarily the CD133 protein—is down-regulated upon differentiation, presumably because of changes in glycosylation (12, 13, 15).AC133⁺ tumor stem cells have been described for glioblastoma multiforme (the most common and most aggressive primary brain tumor in adults), various pediatric brain and central nervous system tumors (medulloblastoma, ependymoma, pineoblastoma, teratoid/rhabdoid tumors, and retinoblastoma), brain metastases, many different types of carcinomas including colon, pancreatic, lung, liver, and ovarian cancer, melanoma, sarcomas, and different types of leukemia. Although AC133⁻ tumor stem cells also exist (16–18), AC133⁺ cells found in these and other tumor types have been shown to be able to self-renew, to differentiate, and to recreate the original tumors when injected into immunocompromised mice (8, 17, 19–22). Both, stemness and highly agressive malignant tumors often are associated with hypoxia (23), and hypoxia can promote the expansion of CD133⁺ cells (24). Therefore the frequent expression of AC133 on CSCs may reflect, in part, their common localization in a hypoxic environment (25).We previously reported the successful noninvasive detection of the AC133 epitope by antibody-based near-infrared fluorescence molecular tomography (NIR FMT) in mice with s.c. xenografts of CD133-overexpressing tumor cells or traditional tumor cell lines naturally displaying AC133 (26). However, we did not investigate patient-derived CSCs with the above-mentioned stem cell characteristics in that study, and NIR fluorescence, although penetrating tissues more deeply (2–4 cm) than visible-light fluorescence, has limited importance for clinical whole-body imaging (27).We report here the successful noninvasive detection of tumor-associated AC133 by PET, using a radiolabeled AC133-specific mAb in mice xenografted with tumor cell lines overexpressing CD133 or with patient-derived AC133⁺ CSCs. PET is highly sensitive and is widely used for clinical whole-body diagnostic imaging. As a PET nuclide, we used ⁶⁴Cu (t_1/2 = 12.7 h), which allows long-term tracking for at least 48 h, to follow the tumoral accumulation of relatively large molecules such as antibodies, that exhibit relatively slow tumor penetration (28). We chose S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA, hereafter abbreviated as NOTA) as the ⁶⁴Cu chelator, because high labeling efficiencies and high in vivo stability have been reported for ⁶⁴Cu-NOTA-antibody conjugates (29). In addition, we report the successful noninvasive detection of AC133⁺ CSCs with fluorescently labeled AC133 mAb and NIR imaging, a modality that is important for whole-body small-animal imaging and for intraoperative and endoscopic imaging and imaging of superficial tumors in humans (27, 30). In addition to imaging s.c. growing tumors, we report the successful antibody-mediated imaging of orthotopic xenografts initiated from AC133⁺ glioblastoma stem cells in the brain of immunocompromised mice, emphasizing the feasibility of noninvasive antibody-mediated imaging of brain tumors.     

Tumorspheres derived from prostate cancer cells possess chemoresistant and cancer stem cell properties

Purpose

Prostate cancer (PCa) becomes lethal when cancer cells develop into castration-resistant PCa, which remains incurable because of the poor understanding of their cell origin and characteristics. We aim to investigate the potential role of cancer stem cells (CSCs) in PCa progression.

Methods

Human PCa cell lines (LNCaP, 22RV1, DU145 and PC-3) were plated in serum-free suspension culture system allowed for tumorsphere forming. To evaluate the CSC characteristics of tumorspheres, the self-renewal, chemoresistance, tumorigenicity of the PCa tumorsphere cells, and the expression levels of stemness-related proteins in the PCa tumorsphere cells were assessed, comparing with the parental adherent cells.

Results

Tumorsphere cells from PCa cell lines displayed enhanced self-renewal, chemoresistance and tumor-initiating capacity when compared with the adherent cells. Additionally, these cells overexpressed CSC marker CD44. Also, the tumorsphere cells expressed high levels of “stemness” genes Gli1, ABCG2 and Bmi-1.

Conclusions

Collectively, these data demonstrated that tumorspheres derived from PCa cells possess chemoresistant and CSC properties. Our study suggests that the identification of PCa CSCs could provide new insight into the lethal phenotype of PCa and therapeutic implications.     

Multi-institutional study of risk factors of liver metastasis from colorectal cancer: correlation with CD10 expression

Background  

The risk factors for liver metastasis from colorectal cancer are still unclear. We therefore evaluated the relationships between various clinicopathological factors, including CD10 expression, liver metastasis, and survival, in patients with colorectal cancer.     

Clinical significance of pAKT and CD44v6 overexpression with breast cancer

Purpose  

CD44v6 contributes to apoptosis resistance and metastasis via upregulated activity of the PI3K/AKT pathway. The purpose of this study was to investigate the expression patterns and predictive value of phosphorylated AKT (pAKT) and CD44v6 in breast cancer tissues.     

CD133 expression is a potential prognostic indicator in intrahepatic cholangiocarcinoma

Background and aim  

CD133 is one of the most important cancer-initiating (stem) cell markers and was confirmed to be expressed in solid cancers such as colon cancer. However, no one has investigated the role of CD133 in intrahepatic cholangiocarcinoma (IHCC). The aim of this study was to clarify the clinical role of CD133 expression in IHCC.     

Attenuated adipose tissue and skeletal muscle inflammation in obese mice with combined CD4+ and CD8+ T cell deficiency

Objectives

High-fat diet (HFD) feeding in mice is characterized by accumulation of αβ T cells in adipose tissue. However, the contribution of αβ T cells to obesity-induced inflammation of skeletal muscle, a major organ of glucose uptake, is unknown. This study was undertaken to evaluate the effect of αβ T cells on insulin sensitivity and inflammatory state of skeletal muscle and adipose tissue in obesity. Furthermore, we investigated whether CD4+IFNγ+ (T_H1) cells are involved in skeletal muscle and adipose tissue metabolic dysfunction that accompanies obesity.

Methods

Mice lacking αβ T cells (T cell receptor beta chain-deficient [TCRb−/−] mice) were fed HFD for 12 weeks. Obesity-induced skeletal muscle and adipose tissue inflammation was assessed by flow cytometry and quantitative RT-PCR. To investigate the effect of T_H1 cells on skeletal muscle and adipose tissue inflammation and metabolic functions, we injected 5 × 10⁵ T_H1 cells or PBS weekly over 12 weeks into HFD-fed TCRb−/− mice. We also cultured C2C12 myofibers and 3T3-L1 adipocytes with T_H1-conditioned medium.

Results

We showed that similar to adipose tissue, skeletal muscle of obese mice have higher αβ T cell content, including T_H1 cells. TCRb−/− mice were protected against obesity-induced hyperglycemia and insulin resistance. We also demonstrated suppressed macrophage infiltration and reduced inflammatory cytokine expression in skeletal muscle and adipose tissue of TCRb−/− mice on HFD compared to wild-type obese controls. Adoptive transfer of T_H1 cells into HFD-fed TCRb−/− mice resulted in increased skeletal muscle and adipose tissue inflammation and impaired glucose metabolism. T_H1 cells directly impaired functions of C2C12 myotubes and 3T3-L1 adipocytes in vitro.

Conclusions

We conclude that reduced adipose tissue and skeletal muscle inflammation in obese TCRb−/− mice is partially attributable to the absence of T_H1 cells. Our results suggest an important role of T_H1 cells in regulating inflammation and insulin resistance in obesity.     

Expression and prognostic relevance of CD44v6 in stage I non-small cell lung carcinoma

Purpose  

Expression of CD44 and its variants has been shown to be relevant to tumor progression in various human malignancies. We evaluated the expression of CD44v6 in the primary lesions of stage I non-small cell lung cancer (NSCLC) and correlated the expression level to its prognosis.     

Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells

Background

The clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3⁺ T-regulatory cells may contain interleukin-17–producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3⁺ T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy.

Design and Methods

T-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability.

Results

We found that CD4⁺CCR6⁺CD161⁺ T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a “Th17-favorable” environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed.

Conclusions

These data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.     

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

Background

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

Methods

Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.

Results

The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

Conclusions

Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.     

Stem cell factor/c-kit signaling mediated cardiac stem cell migration via activation of p38 MAPK

Objective

It was reported that there are cardiac stem cells (CSCs) in the rat heart, and they could reconstitute well-differentiated myocardium that are formed by blood-carrying new vessels and myocytes. However, how do the CSCs migrate into the peri-infarcted areas after myocardial infarction (MI)? It remains entirely unknown about the signal transduction involved in the migration of CSCs.

Methods and results

Rat heart MI was induced by left coronary artery ligation. Both immunohistochemical staining and Western blotting analysis was performed to detect the expression of SCF protein, and RT-PCR was conducted for the expression of SCF mRNA. Cardiac stem cells were isolated from rat hearts, and a cardiac stem cell migration assay was performed using a 48-well chemotaxis chamber system. On day 5 after MI in rats, the expression of stem cell factor (SCF) mRNA and protein was significantly increased in the peri-infarcted area, which was matched with more accumulation of CSCs in the region and improvement of cardiac function, which was blocked by p38 MAPK selective inhibitor SB203580. In in vitro experiments, SCF induced CSC migration in a concentration-dependent manner, and the antibody against SCF receptor (c-kit) blocked the SCF-induced CSC migration. Western blot analysis showed that the phosphorylated p38 MAPK (Phospho-p38 MAPK) was highly increased in the SCF-treated CSCs, and the inhibition of p38 MAPK activity significantly attenuated SCF-induced the migration of CSCs.

Conclusion

It demonstrated that SCF/c-kit signaling may mediate the migration of CSCs via activation of p38 MAPK.

     

Impact of long-term viral suppression in CD4+ recovery of HIV-children on Highly Active Antiretroviral Therapy

Background  

The effects of HAART may differ between children and adults because children have a developing immune system, and the long-term immunological outcome in HIV-infected children on HAART is not well-known. A major aim of our study was to determine CD4+ evolution associated with long-term VL control during 4 years of observation on HAART.     

Impaired suppression of synovial fluid CD4+CD25− T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells

Objective

Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25− effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells.

Methods

The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in ³H‐thymidine proliferation assays. Cytokine levels were analyzed using Bio‐Plex Pro assay.

Results

Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA–DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells.

Conclusion

Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

Fanconi anemia genes are highly expressed in primitive CD34<Superscript>+</Superscript>hematopoietic cells

Background  

Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells.