Tumor genetic heterogeneity analysis of chronic sun‐damaged melanoma

Chronic sun‐damaged (CSD) melanoma represents 10%–20% of cutaneous melanomas and is characterized by infrequent BRAF V600E mutations and high mutational load. However, the order of genetic events or the extent of intra‐tumor heterogeneity (ITH) in CSD^high melanoma is still unknown. Ultra‐deep targeted sequencing of 40 cancer‐associated genes was performed in 72 in situ or invasive CMM, including 23 CSD^high cases. In addition, we performed whole exome and RNA sequencing on multiple regions of primary tumor and multiple in‐transit metastases from one CSD^high melanoma patient. We found no significant difference in mutation frequency in melanoma‐related genes or in mutational load between in situ and invasive CSD^high lesions, while this difference was observed in CSD^low lesions. In addition, increased frequency of BRAF V600K, NF1, and TP53 mutations (p < .01, Fisher's exact test) was found in CSD^high melanomas. Sequencing of multiple specimens from one CSD^high patient revealed strikingly limited ITH with >95% shared mutations. Our results provide evidence that CSD^high and CSD^low melanomas are distinct molecular entities that progress via different genetic routes.     

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.     

Competitively disrupting the neutrophil-specific receptor–autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177^pos/mPR3^high and CD177^neg/mPR3^low populations. The CD177^pos/mPR3^high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)–associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 “blockers”) as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177^pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177^pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177^pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3–ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3–ANCAs provoked significantly more superoxide production in CD177^pos/mPR3^high than in CD177^neg/mPR3^low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177^pos cells to the level of the CD177^neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3–ANCA diseases.     

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.     

Identification of Tumor Endothelial Cells with High Aldehyde Dehydrogenase Activity and a Highly Angiogenic Phenotype

Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDH^high TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDH^high/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDH^low TECs, ALDH^high TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDH^high TECs than that in ALDH^low TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDH^high TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.     

Hydrazinocurcumin Encapsuled Nanoparticles “Re-Educate” Tumor-Associated Macrophages and Exhibit Anti-Tumor Effects on Breast Cancer Following STAT3 Suppression

Tumor-associated macrophages (TAMs) are essential cellular components within tumor microenvironment (TME). TAMs are educated by TME to transform to M2 polarized population, showing a M2-like phenotype, IL-10^high, IL-12^low, TGF-β^high. STAT3 signaling triggers crosstalk between tumor cells and TAMs, and is crucial for the regulation of malignant progression. In our study, legumain-targeting liposomal nanoparticles (NPs) encapsulating HC were employed to suppress STAT3 activity and “re-educate” TAMs, and to investigate the effects of suppression of tumor progression in vivo. The results showed that TAMs treated by HC encapsuled NPs could switch to M1-like phenotype, IL-10^low, IL-12^high, TGF-β^low, and the “re-educated” macrophages (M1-like macrophages) considerably demonstrated opposite effect of M2-like macrophages, especially the induction of 4T1 cells migration and invasion in vitro, and suppression of tumor growth, angiogenesis and metastasis in vivo. These data indicated that inhibition of STAT3 activity of TAMs by HC-NPs was able to reverse their phenotype and could regulate their crosstalk between tumor cells and TAMs in order to suppress tumor progression.     

Monocyte populations as markers of response to adalimumab plus MTX in rheumatoid arthritis

Introduction

The treatment of rheumatoid arthritis (RA) patients with anti-tumor necrosis factor alpha (TNFα) biological drugs has dramatically improved the prognosis of these patients. However, a third of the treated patients do not respond to this therapy. Thus, the search for biomarkers of clinical response to these agents is currently highly active. Our aim is to analyze the number and distribution of circulating monocytes, and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets in methotrexate (MTX) non-responder patients with RA, and to determine their value in predicting the clinical response to adalimumab plus MTX treatment.

Methods

This prospective work investigated the number of circulating monocytes, and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets, in 35 MTX non-responder patients with RA before and after three and six months of anti-TNFα treatment using multiparametric flow cytometry. The number of circulating monocytes in an age- and sex-matched healthy population was monitored as a control.

Results

Non-responder patients with RA show an increased number of monocytes and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets after three months of adalimumab plus MTX treatment that remained significantly increased at six months. In contrast, significant normalization of the numbers of circulating monocytes was found in responders at three months of adalimumab plus MTX treatment that lasts up to six months. CX3CR1 expression is increased in monocytes in non-responders. At three months of anti-TNFα treatment the number of circulating monocytes and their subsets was associated with at least 80% sensitivity, 84% specificity and an 86% positive predictive value (PPV) in terms of discriminating between eventual early responders and non-responders.

Conclusions

The absolute number of circulating monocytes and of their CD14^+highCD16^-, CD14^+highCD16⁺ and CD14^+lowCD16⁺ subsets at three months of adalimumab plus MTX treatment, have a predictive value (with high specificity and sensitivity) in terms of the clinical response after six months of anti-TNFα treatment in patients with RA.     

The relationship between airway immunoglobulin activity and eosinophils in COPD

In chronic obstructive pulmonary disease (COPD), the effects of inhaled corticosteroids are predicted by blood eosinophil counts. We previously briefly reported increased immunoglobulin (Ig)A and IgM levels in bronchoalveolar lavage (BAL) of COPD patients with higher (eosinophil^high) compared to lower (eosinophil^low) blood eosinophils (>250/μL versus < 150/μL), suggesting differences in adaptive immune function. An inverse relationship exists between eosinophil counts and airway pathogenic bacteria levels. The mechanistic reasons for these associations between eosinophils, corticosteroids and pathogenic bacteria are unclear. IgA, IgM and IgG levels were assessed in BAL, bronchial biopsies and epithelium collected from eosinophil^high (n = 20) and eosinophil^low (n = 21) patients. Bronchial B-cell numbers were measured by immunohistochemistry. B-cell activity was assessed in bronchial samples and following exposure to BAL from eosinophil^high and eosinophil^low patients. BAL levels of non-typeable Haemophilus influenza (NTHi)-specific immunoglobulins were quantified. Results showed airway expression of IgA, IgG1 and IgM were lower in eosinophil^low compared to eosinophil^high patients, with lower levels of NTHi-specific IgA and IgM. Bronchial B-cell numbers were similar in both groups, but B-cell activity was lower in eosinophil^low patients. In conclusion, COPD eosinophil^low patients show differences in adaptive immune function compared to COPD eosinophil^high patients. These differences may cause different microbiomes in these COPD phenotypes.     

Exploring Functional β-Cell Heterogeneity In Vivo Using PSA-NCAM as a Specific Marker

Background

The mass of pancreatic β-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous β-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of β-cells and investigated their physiological relevance in increased insulin demand conditions in rats.

Methods

Two rat β-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. β^high and β^low-cells. Insulin release, Ca²⁺ movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, β^high and β^low-cell distribution and functionality were investigated in animal models with decreased or increased β-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat.

Results

We show that β-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike β^low-cells, β^high-cells express functional β-cell markers and are highly responsive to various insulin secretagogues. Whereas β^low-cells represent the main population in diabetic pancreas, an increase in β^high-cells is associated with gain of function that follows sustained glucose overload.

Conclusion

Our data show that a functional heterogeneity of β-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in β-cell defects in type 2 diabetes.     

Establishment of Self-Renewable GM-CSF-Dependent Immature Macrophages In Vitro from Murine Bone Marrow

Macrophages play a key role in the innate immune system. Macrophages are thought to originate from hematopoietic precursors or the yolk sac. Here, we describe the in vitro establishment of self-renewable GM-CSF-dependent immature macrophages (GM-IMs) from murine bone marrow (BM). GM-IMs grow continuously in vitro in conditioned medium containing GM-CSF. The immunophenotype of GM-IMs is F4/80^high CD11b^high CD11c^low Ly6C^low. By comparing gene expression in GM-IMs and BM dendritic cells, we found that GM-IMs expressed lower levels of chemokines, cytokines and their receptors. GM-IMs are round in shape, attach loosely to non-coated culture dishes and have a marked phagocytic capacity. These results indicate that GM-IMs are macrophage precursor cells. Following stimulation with LPS, monocyte-like GM-IMs converted to flat macrophage-like cells that tightly adhered to non-coated culture dishes and produced pro-inflammatory cytokines TNFα, IL-6 and IL-1β. These results indicated that GM-IMs differentiated to M1 pro-inflammatory macrophages. This was confirmed by stimulation of GM-IMs with IFNγ, an inducer of M1 markers. GM-IMs showed enhanced expression of M2 macrophage markers such as Arg1 and Retnla following stimulation by Th2 cytokines IL-4 and IL-13. When GM-IMs were injected into mice at sites of wounding, wound repair was enhanced. These results indicate that GM-IMs can differentiate to M2 macrophages. When GM-IMs were injected into clodronate-treated mice, they induced resident macrophage proliferation by producing M-CSF. In conclusion we have established self-renewable GM-CSF-dependent immature macrophages in vitro from murine BM, which differentiate to M1 or M2 macrophages.     

Aldehyde dehydrogenase 1, a potential marker for cancer stem cells in human sarcoma

Tumors contain a small population of cancer stem cells (CSC) proposed to be responsible for tumor maintenance and relapse. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in different cancer types. This study used the Aldefluor® assay and fluorescence-activated cell sorting (FACS) analysis to isolate ALDH1^high cells from five human sarcoma cell lines and one primary chordoma cell line. ALDH1^high cells range from 0.3% (MUG-Chor1) to 4.1% (SW-1353) of gated cells. Immunohistochemical staining, analysis of the clone formation efficiency, and xCELLigence microelectronic sensor technology revealed that ALDH1^high cells from all sarcoma cell lines have an increased proliferation rate compared to ALDH1^low cells. By investigating of important regulators of stem cell biology, real-time RT-PCR data showed an increased expression of c-Myc, β-catenin, and SOX-2 in the ALDH1^high population and a significant higher level of ABCG2. Statistical analysis of data demonstrated that ALDH1^high cells of SW-982 and SW-1353 showed higher resistance to commonly used chemotherapeutic agents like doxorubicin, epirubicin, and cisplatin than ALDH1^low cells. This study demonstrates that in different sarcoma cell lines, high ALDH1 activity can be used to identify a subpopulation of cells characterized by a significantly higher proliferation rate, increased colony forming, increased expression of ABC transporter genes and stemness markers compared to control cells. In addition, enhanced drug resistance was demonstrated.     

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.     

Cross‐communication between histone H3 and H4 acetylation and Akt‐mTOR signalling in prostate cancer cells

Molecular tumour targeting has significantly improved anti‐cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti‐tumour potential, but also to prevent resistance development seen under mono‐drug therapy. This investigation was designed to evaluate whether cross‐communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU‐145 prostate cancer cells were treated with insulin‐like growth factor (IGF) to activate the Akt‐mTOR cascade or with the HDAC‐inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock‐down blocked the Akt‐mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up‐regulated histone acetylation, but also activated mTOR‐Akt signalling. HDAC1 and 2 knock‐down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC‐mTOR communication, therefore, is apparent whereby tumour‐promoting (Akt/mTOR^high, aH3/aH4^low) and tumour‐suppressing signals (Akt/mTOR^low, aH3/aH4^high) are activated in parallel. Combined use of an HDAC‐ and mTOR inhibitor might then diminish pro‐tumour effects triggered by the HDAC‐ (Akt/mTOR^high) or mTOR inhibitor (aH3/aH4^low) alone.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.