CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44^high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44^high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44^high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44^high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.     

A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

Mortality in head and neck squamous cell carcinoma (HNSCC) is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs) or cells with an epithelial-mesenchymal transition (EMT) phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR), both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44^high/EGFR^low, CD44^high/EGFR^high and CD44^low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44^high/EGFR^low population showed a spindle-shaped EMT-like morphology, while the CD44^low population was dominated by cobblestone-shaped cells. The CD44^high/EGFR^low population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44^high/EGFR^low cells in comparison to CD44^low cells. Moreover, CD44^high/EGFR^low cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44^high/EGFR^high and CD44^low counterparts. In conclusion, our results show that the combination of CD44 (high) and EGFR (low) cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.     

Sensitization of Chemo-Resistant Human Chronic Myeloid Leukemia Stem-Like Cells to Hsp90 Inhibitor by SIRT1 Inhibition

Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44^high K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44^high K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44^high cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.     

Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1)

The antibody trastuzumab (Herceptin) has substantially improved overall survival for patients with aggressive HER2-positive breast cancer. However, about 70% of all treated patients will experience relapse or disease progression. This may be related to an insufficient targeting of the CD44^highCD24^low breast cancer stem cell subset, which is not only highly resistant to chemotherapy and radiotherapy but also a poor target for trastuzumab due to low HER2 surface expression. Hence, we explored whether the new antibody-drug conjugate T-DM1, which consists of the potent chemotherapeutic DM1 coupled to trastuzumab, could improve the targeting of these tumor-initiating or metastasis-initiating cells. To this aim, primary HER2-overexpressing tumor cells as well as HER2-positive and HER2-negative breast cancer cell lines were treated with T-DM1, and effects on survival, colony formation, gene and protein expression as well as antibody internalization were assessed. This revealed that CD44^highCD24^lowHER2^low stem cell-like breast cancer cells show high endocytic activity and are thus particularly sensitive towards the antibody-drug conjugate T-DM1. Consequently, preexisting CD44^highCD24^low cancer stem cells were depleted by concentrations of T-DM1 that did not affect the bulk of the tumor cells. Likewise, colony formation was efficiently suppressed. Moreover, when tumor cells were cocultured with natural killer cells, antibody-dependent cell-mediated cytotoxicity was enhanced, and EMT-mediated induction of stem cell-like properties was prevented in differentiated tumor cells. Thus our study reveals an unanticipated targeting of stem cell-like breast cancer cells by T-DM1 that may contribute to the clinical efficacy of this recently approved antibody-drug conjugate.     

Activated breast stromal fibroblasts exhibit myoepithelial and mammary stem cells features

BackgroundActive breast cancer-associated fibroblasts (CAFs) promote tumor growth and spread, and like tumor cells they are also heterogeneous with various molecular sub-types and different pro-tumorigenic capacities.MethodsWe have used immunoblotting as well as quantitative RT-PCR to assess the expression of various epithelial/mesenchymal as well as stemness markers in breast stromal fibroblasts. Immunofluorescence was utilized to assess the level of different myoepithelial and luminal markers at the cellular level. Flow cytometry allowed to determine the proportion of CD44- and ALDH1-positive breast fibroblasts, while sphere formation assay was used to test the ability of these cells to form mammospheres.ResultsWe have shown here that IL-6-dependent activation of breast and skin fibroblasts promotes mesenchymal-to-epithelial transition and stemness in a STAT3- and p16-dependent manner. Interestingly, most primary CAFs isolated from breast cancer patients exhibited such transition and expressed lower levels of the mesenchymal markers N-cadherin and vimentin as compared to their adjacent normal fibroblasts (TCFs) isolated from the same patients. We have also shown that some CAFs and IL-6-activated fibroblasts express high levels of the myoepithelial markers cytokeratin 14 and CD10. Interestingly, 12 CAFs isolated from breast tumors showed higher proportions of CD24^low/CD44^high and ALDH^high cells, compared to their corresponding TCF cells. These CD44^high cells have higher abilities to form mammospheres and to enhance cell proliferation of breast cancer cells in a paracrine manner relative to their corresponding CD44^low cells.ConclusionTogether, the present findings show novel characteristics of active breast stromal fibroblasts, which exhibit additional myoepithelial/progenitor features.     

Identification and characterization of autoantibody-producing B220 B (B-1) cells appearing in malarial infection

Mice with malaria showed unique immunological responses, including the expansion of NK1.1⁻TCR^int cells (extrathymic T cells). Since TCR^int cells with autoreactivity and autoantibody-producing B cells (B-1 cells) are often simultaneously activated under autoimmune conditions, it was examined whether B-1 cells were activated in the course of malarial infection. From days 14 after infection, B220^low B-1 cells appeared in the liver and spleen. The number of B220^low B cells was highest at day 14, but the ratio was highest at days 28-35. In parallel with the appearance of B220^low cells, autoantibodies against HEp-2 cells and double-stranded DNA were detected in sera. These B220^low cells had phenotypes of CD44^high, CD23⁻ and CD62L⁻. In sharp contrast, conventional B220^high B cells (B-2 cells) were CD44^low, CD23⁺ and CD62L⁺. These results suggested that malaria immune responses were not mediated by conventional T and B cells but resembled the responses during autoimmune diseases.     

Hypoxia Regulates CD44 and Its Variant Isoforms through HIF-1α in Triple Negative Breast Cancer

Background

The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization.

Methods and Findings

The roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of ¹²⁵I-radiolabeled CD44 antibody.

Conclusions

Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.     

Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133^high/CD44^high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133^low/CD44^low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133^high/CD44^high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.     

CD49fhigh Cells Retain Sphere-Forming and Tumor-Initiating Activities in Human Gastric Tumors

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f^high cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f^low cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f^high cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f^high sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.     

Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

Collective detachment of cancer cells at the invading front could generate efficient metastatic spread. However, how cancer cell clusters shed from the leading front remains unknown. We previously reported that the dynamic expression of CD44 in breast cancers (BrCas) at collectively invading edges was associated with tumor-associated macrophages (TAMs). In this study, we first observed that the highly expressed CD44 (CD44^high) cancer cell clusters were located in the BrCa circulating vessels, accompanied by CD206⁺ TAMs. Next, we identified that the cancer cell clusters can be converted to an invasive CD44^high state which was induced by TAMs, thus giving rise to CD44-associated signaling mediated cohesive detachment. Then, we showed that disrupting CD44-signaling inhibited the TAMs triggered cohesive detaching using 3D organotypic culture and mouse models. Furthermore, our mechanistic study showed that the acquisition of CD44^high state was mediated by the MDM2/p53 pathway activation which was induced by CCL8 released from TAMs. Blocking of CCL8 could inhibit the signaling cascade which decreased the CD44-mediated cohesive detachment and spread. Our findings uncover a novel mechanism underlying collective metastasis in BrCas that may be helpful to seek for potential targets.Subject terms: Breast cancer, Cancer microenvironment     

Cytotoxicity of natural products and derivatives toward MCF-7 cell monolayers and cancer stem-like mammospheres

Although cancer stem-like cells (CSCs) are rare, they can enter a non-proliferative or dormant state and resist therapy. Furthermore, quiescent CSCs are responsible for metastases that can appear after curative surgical treatment of a primary tumor. Because of drug resistance of CSCs, the development of novel therapies is urgently required that specifically target CSCs.PurposeThe aim of the present study was to investigate the potential of a panel of natural products and derivatives to inhibit CSC-enriched mammospheres of MCF-7 breast cancer cells.MethodsCD44^high/CD24^low cells were identified by flow cytometry and maintained as mammospheres. As a control, we used two clinically established anticancer drugs (5-fluorouracil and docetaxel). A panel of natural products, shikonin, two cajanin stilbene acid (CSA) derivatives and artesunate were tested by resazurin assay on CSC-enriched mammospheres and MCF-7 monolayer cells. Besides, cellular shikonin uptake experiments were performed by flow cytometry.ResultsWe found two CSA derivatives (Nos. 6 and 19) to be active cancer stem-like MCF-7 mammospheres. Especially, CSA derivative No. 19 clearly showed collateral sensitivity in mammospheres compared to monolayer cells.ConclusionPhytochemicals which provoke collateral sensitivity in cancer-stem like cells are worth more detailed investigations in the future, since there is a great potential for improved chemotherapy to eradicate tumors and prolong cancer patients’ survival times.     

Functional and Phenotypical Analysis of Subsets of Rat CD4+ T Cells

Rat CD4⁺ T cells were divided into two distinct subsets by a monoclonal antibody RTH-1 recognizing a unique epitope on rat CD45R. Cellular distribution of OX-22- and RTH-1-defined antigens was the same. However, OX-22 and RTH-1 recognized different epitopes that exist on rat CD45R. The expression of IL-4 gene was detected only in RTH-1^low CD4+ T cell subset upon various stimulations. In contrast, the expression of IL-2 and IFN-γ gene varied depending upon the nature of stimuli. The increased cell surface expression of CD44 was detected in RTH-1^high CD4+ T cell subset. Conversely the increased expression of CD2 was detected in RTH-1^low CD4+ T cell subset. The expression of CD3 and LFA-1 was not significantly different between RTH-1^high and RTH-1^low subsets.     

CD8low T cells expanded following acute Trypanosoma cruzi infection and benznidazole treatment are a relevant subset of IFN-γ producers

CD8 T cells are regarded as pivotal players in both immunoprotection and immunopathology following Trypanosoma cruzi infection. Previously, we demonstrated the expansion of CD8⁺ T lymphocytes in the spleen of T. cruzi-infected mice under treatment with benznidazole (N-benzyl-2-nitroimidazole acetamide; Bz), a drug available for clinical therapy. This finding underlies the concept that the beneficial effects of Bz on controlling acute T. cruzi infection are related to a synergistic process between intrinsic trypanocidal effect and indirect triggering of the active immune response. In the present study, we particularly investigated the effect of Bz treatment on the CD8⁺ T cell subset following T. cruzi infection. Herein we demonstrated that, during acute T. cruzi infection, Bz treatment reduces and abbreviates the parasitemia, but maintains elevated expansion of CD8⁺ T cells. Within this subset, a remarkable group of CD8^low cells was found in both Bz-treated and non-treated infected mice. In Bz-treated mice, early pathogen control paralleled the lower frequency of recently activated CD8^low cells, as ascertained by CD69 expression. However, the CD8^low subset sustains significant levels of CD44^highCD62L^low and CD62L^lowT-bet^high effector memory T cells, in both Bz-treated and non-treated infected mice. These CD8^low cells also comprise the main group of spontaneous interferon (IFN)-γ-producing CD8⁺ T cells. Interestingly, following in vitro anti-CD3/CD28 stimulation, CD8⁺ T cells from Bz-treated T. cruzi-infected mice exhibited higher frequency of IFN-γ⁺ cells, which bear mostly a CD8^low phenotype. Altogether, our results point to the marked presence of CD8^low T cells that arise during acute T. cruzi infection, with Bz treatment promoting their significant expansion along with a potential effector program for high IFN-γ production.     

Selective Loss of Early Differentiated,Highly Functional PD1high CD4 T Cells with HIV Progression

The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127^high CD4 T cells within the early/intermediate differentiated (EI) (CD27^highCD45RA^low) T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load). A significant loss of circulating PD-1^highCTLA-4^low CD4 T cells was found specifically in the CD127^highCD27^highCD45RA^low compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1^high compared to PD-1^low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1^low EI CD4 T cells. In line with our previous findings, PD-1^high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.     

Relationship between FOXP3 positive populations and cytokine production in systemic lupus erythematosus

In this work we studied CD4⁺FOXP3⁺ populations in systemic lupus erythematosus (SLE) and the relationship with Th cytokine production. We found an increment in CD25^?FOXP3⁺ population in SLE associated with CD4⁺ downregulation and disease progression. CD25^low cells were also upregulated and showed increased percentages of FOXP3⁺ and CD127^?/low cells, supporting the activated status of SLE lymphocytes. Despite the normal levels of CD25^highFOXP3⁺ cells, the negative correlations observed in controls with the frequency of IFNγ, TNFα and IL-10 secreting cells were disrupted in patients, supporting a defective Treg function. Also, CD25^high cells showed an altered balance in the production of these cytokines. In addition, CD25^highFOXP3⁺ cells correlated directly with IL-17A and IL-8 but not with TGFβ in SLE. The increased proportion of IL-17⁺ cells among the CD25^high subset and the positive correlation between IL-17 levels and Treg cells suggest a trans-differentiation of Treg into Th17 cells in SLE.     

High Production Rates Sustain In Vivo Levels of PD-1high Simian Immunodeficiency Virus-Specific CD8 T Cells in the Face of Rapid Clearance

Programmed Death 1 (PD-1) expression by human/simian immunodeficiency virus (HIV/SIV)-specific CD8 T cells has been associated with defective cytokine production and reduced in vitro proliferation capacity. However, the cellular mechanisms that sustain PD-1^high virus-specific CD8 T cell responses during chronic infection are unknown. Here, we show that the PD-1^high phenotype is associated with accelerated in vivo CD8 T cell turnover in SIV-infected rhesus macaques, especially within the SIV-specific CD8 T cell pool. Mathematical modeling of 5-bromo-2′ deoxyuridine (BrdU) labeling dynamics demonstrated a significantly increased generation rate of PD-1^high compared to PD-1^low CD8 T cells in all memory compartments. Simultaneous analysis of Ki67 and BrdU kinetics revealed a complex in vivo turnover profile whereby only a small fraction of PD-1^high cells, but virtually all PD-1^low cells, returned to rest after activation. Similar kinetics operated in both chronic and acute SIV infection. Our data suggest that the persistence of PD-1^high SIV-specific CD8 T cells in chronic infection is maintained in vivo by a mechanism involving high production coupled with a high disappearance rate.     

Interleukin‐4 induces a CD44high/CD49bhigh PC3 subpopulation with tumor‐initiating characteristics

Pro‐ and anti‐inflammatory cytokines may influence proliferation, migration, invasion, and other cellular events of prostate cancer (PCa) cells. The hyaluronan receptor CD44, which is regulated by Interleukin (IL)‐4, is a prostate basal cell marker. CD44_high/CD49b_high expressing cells have been demonstrated to have tumor‐initiating characteristics. Here, we aimed to analyze the effects of long‐term IL‐4 treatment on CD44/CD49b expression, migration, proliferation, and clonogenic potential of basal‐like PCa cells. To this end PC3 cells were treated over 30 passages with 5 ng/mL IL‐4 (PC3‐IL4) resulting in an increased population of CD44_high expressing cells. This was concurrent with a clonal outgrowth of cuboid‐shaped cells, with increased size and light absorbance properties. Flow cytometry revealed that the PC3‐IL4 CD44_high expressing subpopulation corresponds to the CD49b_high population. Isolation of the PC3‐IL4 CD44_high/CD49b_high subpopulation via fluorescence‐associated cell sorting showed increased migrative, proliferative, and clonogenic potential compared to the CD44_low/CD49b_low subpopulation. In conclusion, IL‐4 increases a PC3 subpopulation with tumor‐initiating characteristics. Thus, IL‐4, similar to other cytokines may be a regulator of tumor‐initiation and hence, may present a suitable therapy target in combination with current treatment options.     

Quantitative Trait Locus Analysis Implicates CD4+/CD44high Memory T Cells in the Pathogenesis of Murine Autoimmune Pancreatitis

The mouse strain MRL/MpJ is prone to spontaneously develop autoimmune pancreatitis (AIP). To elucidate the genetic control towards the development of the phenotype and to characterize contributions of immunocompetent cell types, MRL/MpJ mice were interbred with three additional strains (BXD2/TYJ, NZM2410/J, CAST/EIJ) for four generations in an advanced intercross line. Cellular phenotypes were determined by flow cytometric quantification of splenic leukocytes and complemented by the histopathological evaluation of pancreatic lesions. An Illumina SNP array was used for genotyping. QTL analyses were performed with the R implementation of HAPPY. Out of 41 leukocyte subpopulations (B cells, T cells and dendritic cells), only three were significantly associated with AIP: While CD4⁺/CD44^high memory T cells and CD4⁺/CD69⁺ T helper (Th) cells correlated positively with the disease, the cytotoxic T cell phenotype CD8⁺/CD44^low showed a negative correlation. A QTL for AIP on chromosome 2 overlapped with QTLs for CD4⁺/CD44^high and CD8⁺/CD44^high memory T cells, FoxP3⁺/CD4⁺ and FoxP3⁺/CD8⁺ regulatory T cells (Tregs), and CD8⁺/CD69⁺ cytotoxic T cells. On chromosome 6, overlapping QTLs for AIP and CD4⁺/IL17⁺ Th17 cells and again FoxP3⁺/CD8⁺ Tregs were observed. In conclusion, CD4⁺/CD44^high memory T cells are the only leukocyte subtype that could be linked to AIP both by correlation studies and from observed overlapping QTL. The potential role of this cell type in the pathogenesis of AIP warrants further investigations.     

Phagocytized Beads Reduce the α5β1 Integrin Facilitated Invasiveness of Cancer Cells by Regulating Cellular Stiffness

Cell invasion through the extracellular matrix (ECM) of connective tissue is an important biomechanical process, which plays a prominent role in tumor progression. The malignancy of tumors depends mainly on the capacity of cancer cells to migrate and metastasize. A prerequisite for metastasis is the invasion of cancer cells through connective tissue to targeted organs. Cellular stiffness and cytoskeletal remodeling dynamics have been proposed to affect the invasiveness of cancer cells. Here, this study investigated whether highly invasive cancer cells are capable of invading into dense 3D-ECMs with an average pore-size of 1.3 or 3.0 μm when phagocytized beads (2.7 and 4.5 μm diameter) increased their cellular stiffness and reduced their cytoskeletal remodeling dynamics compared to weakly invasive cancer cells. The phagocytized beads decreased the invasiveness of the α5β1^high cancer cells into 3D-ECMs, whereas the invasiveness of the α5β1^low cancer cells was not affected. The effect of phagocytized beads on the highly invasive α5β1^high cells was abolished by specific knock-down of the α5 integrin subunit or addition of an anti-α5 integrin blocking antibody. Furthermore, the reduction of contractile forces using MLCK and ROCK inhibitors abolished the effect of phagocytized beads on the invasiveness of α5β1^high cells. In addition, the cellular stiffness of α5β1^high cells was increased after bead phagocytosis, whereas the bead phagocytosis did not alter the stiffness of α5β1^low cells. Taken together, the α5β1 integrin dependent invasiveness was reduced after bead phagocytosis by altered biomechanical properties, suggesting that the α5β1^high cells need an appropriate intermediate cellular stiffness to overcome the steric hindrance of 3D-ECMs, whereas the α5β1^low cells were not affected by phagocytized beads.     

ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma

Background

Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends.

Methodology/Principal Findings

We have isolated populations of cells with high ALDH activity (ALDH^high) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDH^high and ALDH^low cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice.

Conclusions/Significance

We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133⁺ and ALDH^low cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH^high cell populations. ALDH^high/CD44⁺/CD24⁺ or ALDH^low/CD44⁺/CD24⁺ phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.