Marina Kreutz

Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology

The growth of tumour cells as three‐dimensional multicellular spheroids in vitro has led to important insights in tumour biology, since properties of the in vivo‐tumour such as proliferation or nutrient gradients, can be studied under controlled conditions. While this review starts with an update of recent data on spheroid monocultures, especially concerning tumour microenvironment and therapeutic modalities, the main emphasis is put on the spectrum of heterologous cultures which have evolved in previous years. This type of culture includes tumour cell interaction with endothelial, fibroblast or immunocompetent cells. The relation of the spheroid culture model to other types of three‐dimensional culture and our critical evaluation and presentation of the technical aspects of growing and analysing spheroids are included in the text. These topics are chosed to help the experimental pathologist design experiments with tumour spheroids and to stimulate discussion.

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

Differential screening identifies genetic markers of monocyte to macrophage maturation.

Maturation of cells of the mononuclear phagocyte lineage from bone marrow precursors to tissue macrophages (MAC) via circulating blood monocytes (MO) is a multistep process only partially understood. Similarly, MAC differentiation can be observed if MO are cultured in vitro. In an attempt to further characterize molecular changes occurring during this process we carried out differential screening of a MO‐derived MAC cDNA library using MO and MAC cDNA. After subcloning and confirmation by a second round of screening, partial sequencing of 41 cDNA clones was performed. In 33 clones the sequences of 7 different previously identified cDNAs were found. The mRNA expression of two of the corresponding genes (apolipoprotein E, ferritin light chain) is already known to be up‐regulated during MAC maturation. For one gene (cathepsin B), a specific up‐regulation of mRNA expression could be shown corresponding to previous protein data. For four genes [human cartilage glycoprotein (HC‐gp39), osteopontin, type IV collagenase, and tryptophanyl‐tRNA synthetase] the specific expression in MAC versus MO was previously unknown but could be confirmed by the use of Northern blot analysis. Of these genes, HC‐gp39 is especially interesting because it is only expressed during the late stages of MAC differentiation. J. Leukoc. Biol. 60: 540–545; 1996.

The fibroblast: Sentinel cell and local immune modulator in tumor tissue

Development and progression of epithelial malignancies are frequently accompanied by complex phenotypic alterations of resident tissue fibroblasts. Some of these changes, such as myofibroblastic differentiation and an oncofetal extracellular matrix (ECM) expression profile, are also implicated in inflammation and tissue repair. Studies over the past decade revealed the relevance of reciprocal interactions between tumor cells and tumor‐associated host fibroblasts (TAF) in the malignant process. In many tumors, a considerable fraction of the inflammatory infiltrate is located within the fibroblast‐ and ECM‐rich stromal compartment. However, while fibroblasts are known as “sentinel cells” in various nonneoplastic diseases, where they often regulate the composition and function of recruited leucocytes, they are hardly considered active participants in the inflammatory host response in tumors. This article focuses on the functional impact of TAF on immune cells. The complex network of immune‐modulating effects transduced by TAF and TAF‐derived factors is highlighted, and recent reports that support the hypothesis that TAF are involved in the inflammatory response and immune suppression in tumors are reviewed. The role of TAF‐dependent ECM remodeling and TAF‐derived peptide growth factors, cytokines, and chemokines in the immune modulation is stressed and the idea of TAF as an important therapeutic target is emphasized.

GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis.

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

Lactic Acid and Acidification Inhibit TNF Secretion and Glycolysis of Human Monocytes

High concentrations of lactic acid (LA) are found under various pathophysiological conditions and are accompanied by an acidification of the environment. To study the impact of LA on TNF secretion, human LPS-stimulated monocytes were cultured with or without LA or the corresponding pH control. TNF secretion was significantly suppressed by low concentrations of LA (≤10 mM), whereas only strong acidification had a similar effect. This result was confirmed in a coculture model of human monocytes with multicellular tumor spheroids. Blocking synthesis of tumor-derived lactate by oxamic acid, an inhibitor of lactate dehydrogenase, reversed the suppression of TNF secretion in this coculture model. We then investigated possible mechanisms underlying the suppression. Uptake of [3-13C]lactate by monocytes was shown by hyphenated mass spectrometry. As lactate might interfere with glycolysis, the glycolytic flux of monocytes was determined. We added [1,2-13C2]glucose to the culture medium and measured glucose uptake and conversion into [2,3-13C2]lactate. Activation of monocytes increased the glycolytic flux and the secretion of lactate, whereas oxygen consumption was decreased. Addition of unlabeled LA resulted in a highly significant decrease in [2,3-13C2]lactate secretion, whereas a mere corresponding decrease in pH exerted a less pronounced effect. Both treatments increased intracellular [2,3-13C2]lactate levels. Blocking of glycolysis by 2-deoxyglucose strongly inhibited TNF secretion, whereas suppression of oxidative phosphorylation by rotenone had little effect. These results support the hypothesis that TNF secretion by human monocytes depends on glycolysis and suggest that LA and acidification may be involved in the suppression of TNF secretion in the tumor environment.

Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes.

Macrophages in lymphoid organs are in close contact to nerve terminals of the sympathetic nervous system. Hence, these cells could be targets of neuronal modulation. We studied sympathetic neurotransmitters as chemoattractants enabling the aggregation of macrophages and nerve terminals. Norepinephrine (NE), neuropeptide Y (NPY), isoproterenol (β‐adrenergic), p‐aminoclonidine (α2‐adrenergic), methoxamine (α1‐adrenergic), and adenosine triphosphate (ATP) were used to study human monocyte and macrophage migration in 48‐well Boyden chambers. NE stimulated chemotaxis of monocytes and macrophages at an optimal concentration of 10−10 M (P < 0.025). Isoproterenol, but not p‐aminoclonidine or methoxamine, induced chemotaxis of monocytes (10−10 M, P < 0.05). In these studies, elevation of cAMP is a critical step in NE‐induced chemotaxis of monocytes. NPY (10−11 M, P < 0.05) stimulated monocyte chemotaxis as well. ATP at 10−4 and 10−5 M stimulated undirected cell mobility (P < 0.05). All tested neurotransmitters of the sympathetic nerve terminal were potent chemoattractants. These findings may explain the close association of nerves and macrophages in tissue and lymphoid organs and may thus be of functional relevance in neuroimmunomodulation. J. Leukoc. Biol. 67: 553–558; 2000.

Lactate promotes glioma migration by TGF-β2–dependent regulation of matrix metalloproteinase-2

Lactate dehydrogenase type A (LDH-A) is a key metabolic enzyme catalyzing pyruvate into lactate and is excessively expressed by tumor cells. Transforming growth factor-beta2 (TGF-beta2) is a key regulator of invasion in high-grade gliomas, partially by inducing a mesenchymal phenotype and by remodeling the extracellular matrix. In this study, we tested the hypothesis that lactate metabolism regulates TGF-beta2-mediated migration of glioma cells. Small interfering RNA directed against LDH-A (siLDH-A) suppresses, and lactate induces, TGF-beta2 expression, suggesting that lactate metabolism is strongly associated with TGF-beta2 in glioma cells. Here we demonstrate that TGF-beta2 enhances expression, secretion, and activation of matrix metalloproteinase-2 (MMP-2) and induces the cell surface expression of integrin alpha(v)beta(3) receptors. In spheroid and Boyden chamber migration assays, inhibition of MMP-2 activity using a specific MMP-2 inhibitor and blocking of integrin alpha(v)beta(3) abrogated glioma cell migration stimulated by TGF-beta2. Furthermore, siLDH-A inhibited MMP2 activity, leading to inhibition of glioma migration. Taken together, we define an LDH-A-induced and TGF-beta2-coordinated regulatory cascade of transcriptional regulation of MMP-2 and integrin alpha(v)beta(3). This novel interaction between lactate metabolism and TGF-beta2 might constitute a crucial mechanism for glioma migration.

Comparative analysis of integrin expression on monocyte‐derived macrophages and monocyte‐derived dendritic cells

Both macrophages (MAC) and dendritic cells (DC) are members of the mononuclear phagocyte system (MPS) with monocytes (MO) as common precursor cells. Cells of the MPS are able to take up, process and present antigens to T lymphocytes, thereby inducing a primary or secondary immune response. Adhesion molecules are of crucial importance for the interaction of antigen‐presenting cells with immune cells, especially T lymphocytes. By representational difference analysis, we identified CD49c (VLA‐3), a member of the β1‐integrin family of adhesion receptors, as differentiation‐associated antigen in MO‐derived MAC. In contrast, MO‐derived DC did not express CD49c mRNA. These data prompted us to compare the integrin expression pattern of MAC and DC. Both cell types showed a low expression of the α‐chains of the β1‐integrins CD49a, CD49b, CD49d and CD49e, whereas a marked difference was observed for CD49c and CD49f. Expression of both integrins increased during MO to MAC differentiation, but was not detectable on DC. In parallel the β1‐chain (CD29) was clearly up‐regulated during MO to MAC differentiation but was only weakly expressed on DC. On the other hand, the β2‐integrins CD11a, CD11b, CD11c and CD18 were all expressed on MAC and DC. Beside their role in cell–cell interaction and adhesion, β2‐integrins are also known as possible binding molecules for bacteria and lipopolysaccharide (LPS), especially for high LPS concentrations. Therefore we investigated the LPS response of MAC versus DC in terms of tumour necrosis factor‐α (TNF‐α) release. DC were less responsive to low doses of LPS, which can easily be explained by the very low CD14 expression on DC compared for MAC. In contrast, the TNF‐α response was comparable to MAC when DC were stimulated with high LPS concentrations. Our results show a specific, differentiation‐dependent pattern of β1‐ and β2‐integrin expression on in vitro‐generated MAC and DC. We suggest that the high expression of CD11/CD18 on DC could be involved in the LPS binding of DC. As LPS is not only an activation but also a differentiation stimulus for DC, the expression of CD11/CD18 on DC may be important for the successful maturation of DC and thereby the initiation of a primary immune response.

Expression of CD68 in non-myeloid cell types.

CD68, the human homologue of macrosialin, is commonly regarded as a selective marker for human monocytes and macrophages. Its expression is thought to be regulated by a macrophage‐specific promoter. However, several immunohistochemical studies have indicated that CD68 antibodies also react with other haematopoietic and non‐haematopoietic cell types. We investigated the expression of CD68 in various primary cells and carcinoma cell lines using immunohistochemistry, flow cytometry, Western blot analysis and qRT‐PCR. Weak but significant immunoreactivity was detected in lymphocytes and several tumour cell lines whereas staining of primary fibroblasts and endothelial cells was comparable to macrophages. The intensity of CD68 staining in individual cell types depended on the antibody clone and the fixation technique. Anti‐CD68 mAb KP1 should be used with great caution for frozen tissue sections due to its reactivity with a wide variety of cell types. Also, care should be taken when distinguishing macrophages from fibroblasts/stromal cells in paraffin sections after formalin fixation since both cell types are stained highly positive for CD68. In accordance, mRNA expression of CD68 was not only detected in macrophages and monocytes but also in fibroblasts as well as endothelial cells and tumour cells, although with a varying intensity. Cloning of full length 5′‐sequences and determination of transcription start sites shows that macrophages and fibroblasts initiate transcription within the known promoter region; however, from different start sites, indicating alternative promoter architecture in myeloid versus non‐myeloid cells. We suggest that CD68 is not a selective macrophage marker but rather a lysosomal protein that is enriched in macrophages.