Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature

Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44⁺ cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positve and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44⁺ CSC-like cells invade Matrigel through an EMT, while in contrast, CD44⁻ cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44⁺CD24⁻ prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA methyltransferase 1 is essential for initiation of the colon cancers

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1^−/−). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR⁺) and CD44-positive and CD24-positive (CD44⁺CD24⁺) cell rates were lower in DNMT1^−/− cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1^−/− cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1^−/− cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.     

Ovarian cancer stem cells promote tumour immune privilege and invasion via CCL5 and regulatory T cells

Emerging evidence indicates a link between the increased proportion of regulatory T cells (T_regs) and reduced survival in patients who have been diagnosed with cancer. Cancer stem cells (CSCs) have been indicated to play a vital role in tumour initiation, drug resistance and recurrence. However, the relationship between T_regs and CSCs remains largely unknown. Here, we sorted out ovarian cancer stem‐like side population (SP) cells and CD133⁺ cells to investigate the influence of ovarian CSCs on T_regs. Among the various immune‐related molecules that we assessed, C‐C motif chemokine ligand 5 (CCL5) was the most elevated in ovarian CSCs relative to that in the non‐CSCs. The expression of its receptor, C‐C motif chemokine receptor 5 (CCR5), was also increased on the surface of T_regs in ovarian cancer patients. This receptor‐ligand expression profile indicated that ovarian CSCs recruit T_regs via CCL5–CCR5 interactions. We further assessed the expression of interleukin (IL)‐10 in T_regs cultured with different cancer cells. T_regs cultured in conditioned medium (CM) from ovarian CD133⁺ cells expressed a higher level of IL‐10 than T_regs cultured in CM from CD133^– cells, indicating that T_regs exert pronounced immune‐inhibitory functions in CSC‐rich environments. Furthermore, co‐culture with ovarian cancer cell lines induced the expression of matrix metalloproteinase‐9 (MMP9) in T_regs which, in turn, enhanced the degradation of the extracellular matrix and enabled the invasion of tumour cells, thereby facilitating tumour metastasis. For the first time, to our knowledge, our findings describe the relationship between ovarian CSCs and T_regs, and demonstrated that these two cell populations co‐operate to promote tumour immune tolerance and enhance tumour progression.     

Clinicopathologic correlation of cancer stem cell markers CD44, CD24, VEGF and HIF-1α in ductal carcinoma in situ and invasive ductal carcinoma of breast: an immunohistochemistry-based pilot study

CD24^−/lowCD44⁺ cells have been identified as putative cancer stem cells (CSCs) in breast cancer. However, the expression of these markers, as well as their association with clinical parameters or tumor microenvironment of breast cancer, remains largely unknown. In the present study, we examined the expression of CD44, CD24, VEGF, and HIF-1α in human breast tumor tissues and assessed their clinicopathological correlations. We investigated tissue samples, including 117 cases of invasive ductal carcinoma (IDCa), 14 cases of ductal carcinoma in situ (DCIS), and 15 cases of intraductal hyperplasia (IDH) from breast tissues. The expression of CD44, CD24, HIF-1α, and VEGF was evaluated using immunohistochemical staining. CD24, CD44, HIF-1α, and VEGF were expressed in 49 (41.9%), 51 (43.6%), 32 (27.4%), and 97 cases (82.9%), respectively, in IDCa. CD24^−/lowCD44⁺ cells were noted in 48 (41.3%) cases. The levels of CD24 and VEGF expression correlated positively with tumor malignancy (P < 0.05). Meanwhile, the expression of CD24, CD44, and VEGF correlated significantly positively with increasing tumor grade (P < 0.05). In addition, associations between CD44 and VEGF, CD24 and VEGF, HIF-1α and VEGF, CD24^−/lowCD44⁺ and VEGF, CD24^−/lowCD44⁺ and HIF-1α were also observed (P < 0.05). The HIF-1α expression level was relatively higher in early stage breast cancer patients with CD24^−/lowCD44⁺ cells. Taken together, our results suggest that CD24 and VEGF may play important roles in breast tumorigenesis and progression, while HIF-1α may play a role in the early stage of breast carcinogenesis.     

IL-22 producing NKp46+ innate lymphoid cells can differentiate from hematopoietic precursor cells

The IL-22 NKp46⁺ innate lymphoid cells, NCR22 cells, are very important for the early host defense against microbial pathogens. We show here that NCR22 cells were differentiated from Lin⁻CD127⁺CD117⁺ cells that were derived from hematopoietic precursor cells (HPCs) of mouse bone marrow cells. The combination of low concentrations of IL-23 and IL-15 induced differentiation of NCR22 cells from Lin⁻CD127⁺CD117⁺ cells. NCR22 cells expressed a large amount of IL-22 and RORγt, and they had poor cytolytic activity and produced little IFN-γ. Lin⁻CD127⁺CD117⁺ cells were very similar to intestinal lamina propria LTi-like cells; both cells dominantly expressed RORγt and IL-22. Meanwhile, Lin⁻CD127⁻CD117⁺ cells that were also derived from HPCs did not express RORγt and IL-22, and they developed into conventional NK cells, not into NCR22 cells. These findings revealed that NCR22 cells can be differentiated from Lin⁻CD127⁺CD117⁺ cells which are derived from HPCs.     

Inhibition of cancer stem cell-like properties and reduced chemoradioresistance of glioblastoma using microRNA145 with cationic polyurethane-short branch PEI

Glioblastomas (GBMs) are the most common primary brain tumors with poor prognosis. CD133 has been considered a putative marker of cancer stem cells (CSCs) in malignant cancers, including GBMs. MicroRNAs (miRNAs), highly conserved small RNA molecules, may target oncogenes and have potential as a therapeutic strategy against cancer. However, the role of miRNAs in GBM-associated CSCs remains mostly unclear. In this study, our miRNA/mRNA-microarray and RT-PCR analysis showed that the expression of miR145 (a tumor-suppressive miRNA) is inversely correlated with the levels of Oct4 and Sox2 in GBM-CD133⁺ cells and malignant glioma specimens. We demonstrated that miR145 negatively regulates GBM tumorigenesis by targeting Oct4 and Sox2 in GBM-CD133⁺. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic-delivery vehicle, PU-PEI-mediated miR145 delivery to GBM-CD133⁺ significantly inhibited their tumorigenic and CSC-like abilities and facilitated their differentiation into CD133⁻-non-CSCs. Furthermore, PU-PEI-miR145-treated GBM-CD133⁺ effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation and temozolomide. Finally, the in vivo delivery of PU-PEI-miR145 alone significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic GBM-CD133⁺-transplanted immunocompromised mice. Therefore, PU-PEI-miR145 is a novel therapeutic approach for malignant brain tumors.     

CD133<Superscript>+</Superscript> single cell-derived progenies of colorectal cancer cell line SW480 with different invasive and metastatic potential

Single cell progenies (SCPs) inherit biological properties from their isogenetic mother cells. If a single cancer cell can give rise to progenies, which can be passaged sustainably in vitro and produce tumor in xenotransplantation, the cell should be cancer initiating cell. CD133 (Prominin-1, Prom1) is the marker of human colorectal cancer (CRC) stem cells and probably a marker of metastatic cancer stem cells (CSCs). Thirty-three SCPs of CRC cell line SW480 were isolated by limited dilution methods, thirty of which are CD133 positive and three negative. All of the CD133⁺ SCPs are tumorigenic, and the subcutaneous tumors expanded rapidly, while only 1 of 3 CD133⁻ SCPs developed a minimal tumor in nude mice. Orthotopic transplantation experiments showed that CD133⁺ SCPs possessed heterogeneity in intestinal wall invasion, lymph node and liver metastases. CD133⁺ SCPs varied in cell growth, invasive ability, epithelial-mesenchymal-transition and expression of CSCs markers (CD133, CD44, and CXCR4) associated with metastatic potential. CD133⁻ SCPs did not produce secondary transplanted tumor, intestinal invasion and metastasis. The results indicated CD133⁺ subpopulation of SW480 SCPs bear heterogeneous invasive and metastatic ability, and CRC-CSCs might be a heterogeous subpopulation.     

Role of ursolic acid chalcone,a synthetic analogue of ursolic acid,in inhibiting the properties of CD133+ sphere-forming cells in liver stem cells

The expression of CD133 decreases with differentiation of tumor cell, indicating that CD133 is a specific marker for isolation and identification of CSCs. In the present study the effect of Ursolic acid chalcone (UAC) on CD133⁺ hepatocellular carcinoma cell (HCC CSCs) differentiation, their self-renewal, tumorigenic capacity and sensitivity to chemotherapeutic drugs was studied. The results demonstrated that UAC inhibits the expression of CD133⁺ in a dose and time-dependent manner in PLC/PRF/5 and Huh7 HCC cells. The inhibition was significant at 50 μM and on day 8. The percentage of CD133⁺ cells decreased from an initial 59.3% in PLC/PRF/5 to 37.1% and 78.2% in Huh7 to 59.2% on treatment with UAC. There was inhibition of Oct4, Tert, Bmi1, β-catenin, ABCG2, and tumor sphere-related gene Ep300. In addition it also decreased number of CK19-positive cells and increased number of CK8/18-positive cells. UAC treatment caused a decrease in self-renewal capability and increase in sensitivity to doxorubicin and vincristine drugs in CD133⁺ HCC CSCs. Therefore, UAC can be a potent therapeutic agent to target differentiation of CSC in HCC.     

CD28 CD8 T cells are significantly reduced and correlate with disease duration in juveniles with type 1 diabetes

Type 1 diabetes (T1D) is a chronic disease caused by autoimmune destruction of insulin-producing pancreatic β-cells. T1D is typically diagnosed in children, but information regarding immune cell subsets in juveniles with T1D is scarce. Therefore, we studied various lymphocytic populations found in the peripheral blood of juveniles with T1D compared to age-matched controls (ages 2–17). One population of interest is the CD28⁻ CD8⁺ T cell subset, which are late-differentiated cells also described as suppressors. These cells are altered in a number of disease states and have been shown to be reduced in adults with T1D. We found that the proportion of CD28⁻ cells within the CD8⁺ T cell population is significantly reduced in juvenile type 1 diabetics. Furthermore, this reduction is not correlated with age in T1D juveniles, although a significant negative correlation between proportion CD28⁻ CD8⁺ T cells and age was observed in the healthy controls. Finally, correlation analysis revealed a significant and negative correlation between the proportion of CD28⁻ CD8⁺ T cells and T1D disease duration. These findings show that the CD28⁻ CD8⁺ T cell population is perturbed following onset of disease and may prove to be a valuable marker for monitoring the progression of T1D.     

CD2 and CD8α define porcine γδ T cells with distinct cytokine production profiles

γδ T cells are a remarkably prominent T-cell subset in swine with a high prevalence in blood. Phenotypic analyses in this study showed that CD2⁻ γδ T cells in their vast majority had a CD8α⁻SLA-DR⁻CD27⁺ phenotype. CD2⁺ γδ T cells dominated in spleen and lymph nodes and had a more heterogeneous phenotype. CD8α⁺SLA-DR⁻CD27⁺ γδ T cells prevailed in blood, spleen and lymph nodes whereas in liver a CD8α⁺SLA-DR⁺CD27⁻ phenotype dominated, indicating an enrichment of terminally differentiated γδ T cells. γδ T cells were also investigated for their potential to produce IFN-γ, TNF-α and IL-17A. Within CD2⁺ γδ T cells, IFN-γ and TNF-α single-producers as well as IFN-γ/TNF-α double-producers dominated, which had a CD8α⁺CD27^+/− phenotype. IL-17A-producing γδ T cells were only found within CD2⁻ γδ T cells, mostly co-produced TNF-α and had a rare CD8α⁺CD27⁻ phenotype. However, quantitatively TNF-α single-producers strongly dominated within CD2⁻ γδ T cells. In summary, our data identify CD2 and CD8α as important molecules correlating with functional differentiation.     

Regulatory function of cytomegalovirus-specific CD4CD27CD28 T cells

CMV infection is characterized by high of frequencies of CD27⁻CD28⁻ T cells. Here we demonstrate that CMV-specific CD4⁺CD27⁻CD28⁻ cells are regulatory T cells (T_R). CD4⁺CD27⁻CD28⁻ cells sorted from CMV-stimulated PBMC of CMV-seropositive donors inhibited de novo CMV-specific proliferation of autologous PBMC in a dose-dependent fashion. Compared with the entire CMV-stimulated CD4⁺ T-cell population, higher proportions of CD4⁺CD27⁻CD28⁻ T_R expressed FoxP3, TGFβ, granzyme B, perforin, GITR and PD-1, lower proportions expressed CD127 and PD1-L and similar proportions expressed CD25, CTLA4, Fas-L and GITR-L. CMV-CD4⁺CD27⁻CD28⁻ T_R expanded in response to IL-2, but not to CMV antigenic restimulation. The anti-proliferative effect of CMV-CD4⁺CD27⁻CD28⁻ T_R significantly decreased after granzyme B or TGFβ inhibition. The CMV-CD4⁺CD27⁻CD28⁻ T_R of HIV-infected and uninfected donors had similar phenotypes and anti-proliferative potency, but HIV-infected individuals had higher proportions of CMV-CD4⁺CD27⁻CD28⁻ T_R. The CMV-CD4⁺CD27⁻CD28⁻ T_R may contribute to the downregulation of CMV-specific and nonspecific immune responses of CMV-infected individuals.     

CD133-Targeted Niche-Dependent Therapy in Cancer: A Multipronged Approach

Cancer treatment continues to be challenged by the development of therapeutic resistances and relapses in the clinical setting, which are largely attributed to tumor heterogeneity, particularly the existence of cancer stem cells (CSCs). Thus, targeting the CSC subpopulation may represent an effective therapeutic strategy. However, despite advances in identifying and characterizing CD133⁺ CSCs in various human cancers, efforts to translate these experimental findings to clinical modalities have been slow in the making, especially in light of the growing awareness of CSC plasticity and the foreseeable pitfall of therapeutically targeting CSC base sorely on a surface marker. We, and others, have demonstrated that the CD133⁺ CSCs reside in complex vascular niches, where reciprocal signaling between the CD133⁺ CSCs and their microenvironment may govern niche morphogenesis and homeostasis. Herein, we discuss the multifaceted functional role of the CD133⁺ cells in the context of their niche, and the potential of targeting CD133 as a niche-dependent approach in effective therapy.CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2014 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Solid tumors and hematopoietic cancers are morphologically and functionally heterogeneous, which is believed to be attributed to subpopulations of tumor cells with stem cell–like properties, termed cancer stem cells (CSCs). Such as their normal stem cell counterparts, CSCs are primitive cell entities able to self-renew and asymmetrically divide to maintain a stem cell pool, as well as differentiated cell progenies.¹ On the basis of these criteria, CSCs have been isolated or enriched from numerous tissue types and demonstrated to exhibit increased resistance to conventional cancer treatments, including chemotherapy and radiotherapy, which can be partially explained by their quiescence, up-regulation of efflux transporters, and increased ability for DNA damage repair.¹ Taken together, CSCs may explain cancer relapses and metastases, which have led to the argument that targeting CSCs may yield improved survival and treatment outcomes for patients.The identification and characterization of CSCs is largely owed to the use of cell-surface markers. Although this approach has been widely applied for a range of solid tumors and hematopoietic cancers from varying tissue types, there is evidence that the CSC phenotype may be acquired or induced in cancer cells lacking CSC markers, such as glioma or pancreatic cancer cells.^2,3 These observations challenge the hierarchical CSC hypothesis and propose a stochastic model, whereby non-CSCs can acquire stem cell properties through mutations or external cues.² Thus, stem cell properties of tumor cells are dynamic, and strategies that solely target CSCs may not be effective because CSCs may be replenished through phenotypic switching from non-CSCs.CD133 is an approximately 120-kDa pentaspan transmembrane glycoprotein that is primarily localized to the plasma membrane, and its cell-surface epitope, AC133, has been highly used as a cell-surface marker to isolate and enrich for CSCs from a wide range of tissue types.⁴ Consistent with its role as a CSC marker, CD133 expression has been associated with increased chemoresistance and radioresistance, as well as a poor prognosis in various cancers.^4,5 However, its utility as a CSC marker has been the subject of controversy because CD133 has been demonstrated to be expressed in differentiated cell populations,⁴ and the CD133^– fractions have been shown to initiate tumor in vivo.^6,7 We, and others, have partially addressed this concern by demonstrating that the AC133 epitope is regulated by specific CD133 N-glycosylation processing,^8,9 as well as alternative splicing.¹⁰ Indeed, cell-surface AC133 appears to be specific to stem cell populations and is lost during differentiation, whereas CD133 protein and mRNA expression can be maintained.⁸ Furthermore, the tumorigenic property of CD133⁻ tumor subsets is believed to arise in cancer cells that either harbor masked CD133 epitopes¹¹ or subsequently induced CD133 because of environmental cues.^2,3 Compelling evidence suggests that certain CD133 glycoforms not only serve as a marker for CSCs, but also contribute to cell survival, maintenance of stemness, and tumorigenesis.^12–14 In addition, recent findings have linked CD133 to tumor vascularization, because it appears to be required not only for angiogenesis, such as in brain tumors,¹⁵ but also vasculogenic mimicry (VM) in melanoma,¹² beast,¹⁶ and brain¹⁷ cancers. Despite these advances, the functions of CD133 in various cancer types at the cellular and molecular levels remain an underdeveloped area of research. In this review, we will discuss the known and hypothesized roles of CD133-expressing subsets in the vascular niche and their potential as targets for niche-dependent therapy.     

Immunological Peculiarities of CD-56-Positive Serous Ovarian Adenocarcinoma

Here we present the results of evaluation of the expression of neural cell adhesion molecules CD56 (NCAM) in serous ovarian adenocarcinoma. The expression was detected in 48.5% cases. Infiltration of tumor stroma and parenchyma with CD8⁺ и CD4⁺ lymphocytes was significantly less pronounced in tumors expressing neural cell adhesion molecules; CD3⁺CD4⁺CD25⁺ predominate among CD4⁺ lymphocytes in CD56⁺ tumors. CD56⁺ tumors were lower in size (5.2±0.6, 7.9±0.8 and 10.3±1.5 cm in monomorphic, mosaic, and negative phenotypes, respectively (p=0.05) and were characterized by the absence of cystic component (p=0.012), larger disseminations in the peritoneum (4.2±1.1 and 2.7±0.5 cm; p=0.05), and larger volume of the residual tumor (p=0.018) after surgical treatment. NCAM phenotype of the tumor does not correlate with the stage and differentiation degree of serous ovarian adenocarcinoma.     

Maintenance of naïve CD8 T cells in nonagenarians by leptin, IGFBP3 and T3

Research into the age-associated decline in the immune system has focused on the factors that contribute to the accumulation of senescent CD8 T cells. Less attention has been paid to the non-immune factors that may maintain the pool of naïve CD8 T cells. Here, we analyzed the status of the naïve CD8 T-cell population in healthy nonagenarians (≥90-year-old), old (60-79-year-old), and young (20-34-year-old) subjects. Naïve CD8 T cells were defined as CD28⁺CD95⁻ as this phenotype showed a strong co-expression of the CD45RA⁺, CD45RO⁻, and CD127⁺ phenotypes. Although there was an age-associated decline in the percentage of CD28⁺CD95⁻ CD8 T cells, the healthy nonagenarians maintained a pool of naïve CD28⁺CD95⁻ cells that contained T-cell receptor excision circles (TREC)⁺ cells. The percentages of naïve CD28⁺CD95⁻ CD8 T cells in the nonagenarians correlated with the sera levels of insulin-like growth factor binding protein 3 (IGFBP3) and leptin. Higher levels of triiodothyronine (T3) negatively correlated with the accumulation of TREC⁻CD28⁻CD95⁺ CD8 T cells from nonagenarians. These results suggest a model in which IGFBP3, leptin and T3 act as non-immune factors to maintain a larger pool of naïve CD8 T cells in healthy nonagenarians.     

Highly enriched CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> stem-like cells with CD133<Superscript>+</Superscript>CD44<Superscript>high</Superscript> metastatic subset in HCT116 colon cancer cells

Stem-like cancer cells (SLCCs) are distinct cellular subpopulation in colon cancer that is essential for tumor maintenance. Previous studies indicated that SLCCs accounted for only a minor subset in a given cancer model. However, we found that SLCCs frequency varied among a panel of colon cancer cell lines, with HCT116 cells composed mainly of SLCCs, as demonstrated by colonosphere forming capability and CD133 expression. Indeed, flow cytometric analysis revealed more than 60% HCT116 cells co-expressed the putative SLCCs markers CD133 and CD44. Compared with non-CD133⁺CD44⁺ cells, FACS sorted CD133⁺CD44⁺ cells were undifferentiated, endowed with extensive self-renewal and epithelial lineage differentiation capacity in vitro. CD133⁺CD44⁺ exhibited enhanced tumorigeneicity in NOD/SCID mice. One thousand CD133⁺CD44⁺ cells initiated xenograft tumors efficiently (3/6) while 1 × 10⁵ non-CD133⁺CD44⁺ cells could only form palpable nodule with much slower growth rate (1/6). More interestingly, long-term cultured self-renewing CD133⁺CD44⁺ cells enriched CD133⁺CD44^high subset, which expressed epithelial to mesenchymal transition marker, were more invasive in vitro and responsible solely for liver metastasis in vivo. In conclusion, these data demonstrated for the first time that CD133⁺CD44⁺ SLCCs were highly enriched in HCT116 cells and that metastatic SLCCs resided exclusively in a CD133⁺CD44^high subpopulation.     

A stromal cell free culture system generates mouse pro‐T cells that can reconstitute T‐cell compartments in vivo

T‐cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage^?Sca1⁺c‐kit⁺ BM hematopoietic progenitors very efficiently differentiate into pro‐T cells. This culture system consists of plate‐bound Delta‐like 4 Notch ligand and the cytokines SCF and IL‐7. The pro‐T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D–J rearranged. They could be expanded for over 3 months and used to reconstitute the T‐cell compartments of sublethally irradiated T‐cell‐deficient CD3ε^?/? mice or lethally irradiated WT mice. Pro‐T cells generated in this system could partially correct the T‐cell lymphopenia of pre‐Tα^?/? mice. However, reconstituted CD3ε^?/? mice suffered from a wasting disease that was prevented by co‐injection of purified CD4⁺ CD25^high WT Treg cells. In a T‐cell‐sufficient or T‐lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro‐T cells for transplantation and possibly with clinical applications.     

Profiles of Cancer Stem Cell Subpopulations in Cholangiocarcinomas

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13⁺ CSCs characterized mixed-intrahepatic, whereas LGR5⁺ characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13⁺ or CD90⁺ CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90⁺ and CD13⁺ cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell–based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy and arises from the neoplastic transformation of cells in the cholangiocytic lineage.¹ CCA is associated with a very bad prognosis with virtually no response to current chemotherapeutics or radiation therapies.¹ CCA is classified as intrahepatic (IHCCA), perihilar (pCCA), or distal, characterized by significant differences in terms of epidemiology, pathobiology, and molecular biology.¹ Recent studies reveal that IHCCA comprises two different forms: mucin-IHCCA constituted by pure mucin-secreting cells and displaying similarities with pCCA, and mixed-IHCCA comprising areas of hepatocytic differentiation and neoplastic ductular reaction.²The cancer stem cell (CSC) hypothesis has been validated recently by the identification of a subpopulation of self-renewing stem cells that give rise to maturational lineages with a hierarchical organization and are able to divide symmetrically and asymmetrically to generate the tumor mass.^3,4 CSCs, also referred to as tumor-initiating cells or tumor-propagating cells, are tumorigenic, metastatic, resistant to chemoradio therapies, and responsible for tumor recurrence.^3,4 For all these reasons, CSCs represent a primary therapeutic target.^3,4Recently, several CSC markers have been reported in human CCA, including CD133,⁵ epithelial cell adhesion molecule (EpCAM),⁶ CD44,⁷ CD13,⁸ and CD90.⁹ In addition, most cells in human CCAs have been demonstrated to coexpress cytokeratin (K)19 and albumin, a feature characterizing hepatobiliary stem/progenitor cells.¹⁰ Recent reports support further investigations on the role of CSCs in CCA. Unfortunately, very little information exists with respect to CSCs in CCA and its subtypes.Our aim was to analyze CSCs in different human CCA subtypes, primary cultures obtained from human CCA, and established CCA cell lines.     

Islet allograft tolerance in the absence of invariant natural killer T cells

The invariant NKT cells are involved in both immunity and immune tolerance. However, their roles in transplant models remain controversial. We studied the role of NKT cells in the allograft response using two different strains of NKT deficient mice (CD1d−/− and Jα18−/− mice), and found that CD1d−/− and Jα18−/− mice rejected islet allografts with a similar kinetics as wild type B6 mice. Treatment of CD1d−/− and Jα18−/− mice with donor specific transfusion and anti-CD154 induced donor specific tolerance, which was identical to similarly treated wt B6 mice. The islet allograft tolerance requires Foxp3⁺ Tregs. In the periphery, Foxp3⁺ Tregs in CD1d−/−, Jα18−/−, and wt B6 mice were comparable both phenotypically and functionally. In addition, CD1d−/− and Jα18−/− CD4⁺ T cells (non-Tregs) could be readily converted to Foxp3⁺ Tregs by TGF-β in vitro. Our data suggest that islet allograft tolerance can be successfully established without invariant NKT cells.     

Regulatory T cells generated during cytomegalovirus in vitro stimulation of mononuclear cells from HIV-infected individuals on HAART correlate with decreased lymphocyte proliferation

HIV-infected patients fail to fully recover cell-mediated immunity despite HAART. To identify regulatory factors, we studied the phenotype and function of in vitro cytomegalovirus (CMV)-stimulated T cells from HAART recipients. CFSE-measured proliferation showed CD4⁺ and CD8⁺ cells dividing in CMV-stimulated cultures. Compared with healthy controls, CMV-stimulated lymphocytes from HAART recipients had lower ³H-thymidine incorporation; lower IFNγ and TNFα production; higher CD4⁺CD27⁻CD28⁻ and CD8⁺CD27⁻CD28⁻ frequencies; lower CD4⁺CD25^hi; and higher FoxP3 expression in CD8⁺CD25^hi cells. CMV-specific proliferation correlated with higher IFNγ, TNFα and IL10 levels and higher CD4⁺perforin⁺ and CD8⁺perforin⁺ frequencies. Decreased proliferation correlated with higher CD4⁺CD27⁻CD28⁻ frequencies and TGFβ1 production, which also correlated with each other. Anti-TGFβ1 neutralizing antibodies restored CMV-specific proliferation in a dose-dependent fashion. In HIV-infected subjects, decreased proliferation correlated with higher CMV-stimulated CD8⁺CD25^hi frequencies and their FoxP3 expression. These data indicate that FoxP3- and TGFβ1-expressing regulatory T cells contribute to decreased immunity in HAART recipients.     

Characterisation of simian immunodeficiency virus-infected cells in pigtail macaques

Defining which cells become infected with simian immunodeficiency virus (SIV) in vivo should assist in unravelling the pathogenesis of human immunodeficiency virus (HIV)/SIV infection. HIV/SIV infection of CD4⁺ T cells resulted in down-regulation of CD3 and CD4 surface molecules in vitro, however this phenomenon is poorly characterised in vivo. Intracellular SIV p27 was studied by flow cytometry in serial blood samples and lymph node samples during acute infection of 17 SIVmac-infected pigtail macaques. Two weeks after infection, a mean of 56±6.8% the p27⁺ cells were lymphocytes negative for surface CD4 and CD3, and indeed the highest proportion of SIV infected cells were found in the small subset of CD3^LoCD4⁻CD8⁻ lymphocytes, indicating that infection has lead to down-regulation of these markers in vivo. Furthermore, the relative amount of SIV p27 within lymphocytes (based of mean fluorescence intensity) was higher in CD3^LoCD4⁻ and CD3⁻ infected cells than in CD3⁺ or CD4⁺ p27⁺ populations, consistent with greater viral production in CD4⁺ T cells down-regulating CD3 and CD4 molecules. The CD3⁻CD4⁻ infected cells expressed T cell markers CD2 and CD5 and were negative for monocyte, NK and B cell markers. The majority of infected cells were CD28⁺CD95⁺ central memory T cells. Surprisingly, p27⁺ blood lymphocytes were mostly negative for activation markers CD25 and CD69, but most of the infected lymph nodes cells were activated. Our results characterise productively-infected macaque lymphocytes in vivo. The high proportion of SIV-infected lymphocytes that are CD3⁻CD4⁻ has important implications for the in vivo study of pathogenesis of SIV/HIV infections.