Wolfgang Mueller-Klieser

Multicellular tumor spheroids: An underestimated tool is catching up again

The present article highlights the rationale, potential and flexibility of tumor spheroid mono- and cocultures for implementation into state of the art anti-cancer therapy test platforms. Unlike classical monolayer-based models, spheroids strikingly mirror the 3D cellular context and therapeutically relevant pathophysiological gradients of in vivo tumors. Some concepts for standardization and automation of spheroid culturing, monitoring and analysis are discussed, and the challenges to define the most convenient analytical endpoints for therapy testing are outlined. The potential of spheroids to contribute to either the elimination of poor drug candidates at the pre-animal and pre-clinical state or the identification of promising drugs that would fail in classical 2D cell assays is emphasised. Microtechnologies, in the form of micropatterning and microfluidics, are also discussed and offer the exciting prospect of standardized spheroid mass production to tackle high-throughput screening applications within the context of traditional laboratory settings. The extension towards more sophisticated spheroid coculture models which more closely reflect heterologous tumor tissues composed of tumor and various stromal cell types is also covered. Examples are given with particular emphasis on tumor-immune cell cocultures and their usefulness for testing novel immunotherapeutic treatment strategies. Finally, tumor cell heterogeneity and the extraordinary possibilities of putative cancer stem/tumor-initiating cell populations that can be maintained and expanded in sphere-forming assays are introduced. The relevance of the cancer stem cell hypothesis for cancer cure is highlighted, with the respective sphere cultures being envisioned as an integral tool for next generation drug development offensives.

Three-dimensional cell cultures: from molecular mechanisms to clinical applications

This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.

Elevated tumor lactate concentrations predict for an increased risk of metastases in head-and-neck cancer

PURPOSE Hypoxia shifts the balance of cellular energy production toward glycolysis with lactate generation as a by-product. Quantitative bioluminescence imaging allows for the quantitation of lactate concentrations in individual tumors. We assessed the relationship between pretreatment tumor lactate concentrations and subsequent development of metastatic disease in patients with newly diagnosed head-and-neck cancer. METHODS AND MATERIALS At the time of biopsy of the primary site, a separate specimen was taken and flash-frozen for subsequent quantitation of lactate concentration using a luciferase bioluminescence technique. The two-dimensional spatial distribution of the bioluminescence intensity within the tissue section was registered directly using a microscope and an imaging photon counting system. Photon intensity was converted to distributions of volume-related tissue concentrations (micromol per gram wet weight). Treatment consisted of either surgery and postoperative radiotherapy or primary radiotherapy, based on presenting disease stage and institutional treatment policies. The subsequent development of metastatic disease constituted the primary clinical endpoint. RESULTS Biopsies obtained from 40 patients were evaluable in 34. The larynx was the most frequent primary site (n = 25). Other sites included oropharynx (n = 5), hypopharynx (n = 3), and oral cavity (n = 1). Most patients (74%) presented with an advanced stage T3 or T4 primary tumor. Nodal involvement was present in 19 (54%) patients. The median tumor lactate concentration was 7.1 micromol/g. Tumors were classified as having either low or high lactate concentrations according to whether these values were below or above the median. The median follow-up time for surviving patients is 27 months. Two-year actuarial survival was 90% for patients with low-lactate-concentration tumor vs. 35% for patients with high-lactate-concentration primaries (<0.0001). Two-year metastasis-free survival was adversely influenced by high tumor lactate concentrations (90% vs. 25%, p < 0.0001). The median lactate concentration for tumors that subsequently metastasized was 12.9 micromol/g vs. 4.8 micromol/g for patients who remained continuously free of disease (p < 0.005). Lactate concentration was not correlated with presenting T stage or N stage. DISCUSSION Elevated tumor lactate concentrations are associated with the subsequent development of nodal or distant metastases in head-and-neck cancer patients. This more aggressive malignant phenotype is probably associated with hypoxia-mediated radioresistance and the upregulation of metastasis-associated genes.

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1– lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Tumor biology and experimental therapeutics.

Recent research using multicellular tumor spheroids has resulted in new insights in the regulation of invasion and metastasis, angiogenesis and cell cycle kinetics. The onset and expansion of central necrosis in tumor spheroids has been characterized to be a complex interaction of several mechanisms; in a number of cases, necrosis is not a consequence of hypoxia or anoxia, but emerges as secondary necrosis following an accumulation of apoptosis in spheroids. Recent therapeutically oriented studies have been directed towards novel hypoxic markers, targeted therapy, multicellular-mediated drug resistance, and heavy ion irradiation of spheroids. Research efforts should be enhanced mainly in the fields of tumor tissue modeling by heterotypic three-dimensional (3D) cultures and of apoptotic versus necrotic cell death. Based on the fundamental differences between monolayer and 3D cultures, spheroids should become mandatory test systems in therapeutic screening programs.

Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids.

The interrelationship among external O2 and glucose supply, oxygenation status, oxygen consumption rates and cellular viability in tumour microregions was studied using the multicellular spheroid model. For chronic exposure to various supply conditions multicellular EMT6/Ro spheroids were cultured in stirred media equilibrated either with 20% (v/v) or 5% (v/v) oxygen and containing four different glucose concentrations ranging from 0.8 mM to 16.5 mM. Spheroids were investigated using histology and O2-sensitive microelectrodes for measuring oxygen tension (PO2) values. A chronic decrease of the glucose concentration in the medium is associated with a substantial reduction in the thickness of the viable rim of cells and with a persistent increase in the cellular respiration rate. In general, both viable rim size and respiration are decreased through restriction of O2 supply during spheroid growth at a given external glucose concentration. The O2 consumption in spheroids appears to decrease with increasing spheroid size under most of the growth conditions investigated. These findings provide evidence for a large capacity of the spheroid cells to chronically adapt their metabolic rates to different supply situations. The experimental data and theoretical considerations indicate that necrosis may develop in the centre of these spheroids due to the lack of O2 and/or glucose under some of the growth conditions, but central necrosis can also occur despite sufficient O2 and glucose supply. Consequently, cellular metabolism and viability in tumour microregions may not be determined by the diffusion limitation of O2 or specific substrates alone, such as glucose, but may be influenced by a complex interaction of factors in the micromilieu the majority of which are still unknown.

Glycolytic metabolism and tumour response to fractionated irradiation

BACKGROUND AND PURPOSE To study whether pre-therapeutic lactate or pyruvate predict for tumour response to fractionated irradiation and to identify possible coherencies between intermediates of glycolysis and expression levels of selected proteins. MATERIALS AND METHODS Concentrations of lactate, pyruvate, glucose and ATP were quantified via bioluminescence imaging in tumour xenografts derived from 10 human head and neck squamous cell carcinoma (HNSCC) lines. Tumours were irradiated with 30 fractions within 6 weeks. Expression levels of the selected proteins in tumours were measured at the mRNA and protein level. Tumour-infiltrating leucocytes were quantified after staining for CD45. RESULTS Lactate but not pyruvate concentrations were significantly correlated with tumour response to fractionated irradiation. Lactate concentrations in vivo did not reflect lactate production rates in vitro. Metabolite concentrations did not correlate with GLUT1, PFK-L or LDH-A at the transcriptional or protein level. CD45-positive cell infiltration was low in the majority of tumours and did not correlate with lactate concentration. CONCLUSIONS Our data support the hypothesis that the antioxidative capacity of lactate may contribute to radioresistance in malignant tumours. Non-invasive imaging of lactate to monitor radiation response and testing inhibitors of glycolysis to improve outcome after fractionated radiotherapy warrant further investigations.

Intracapillary oxyhemoglobin saturation of malignant tumors in humans

Abstract The oxygenation of cancer tissue in humans has been assessed by measuring the intracapillary oxyhemoglobin saturations (i.c.HbO 2 ) in solid tumors of the oral cavity using a cryophotometric micrometbod. From histological investigations the tumors can be classified into three qualities of vascularization: (i) well vascularized tumors, (ii) tumors with medium quality of vascularization, and (iii) poorly vascularized tumors. In all three tumor types i.c.HbO 2 distribution curves are shifted to significantly lower values than saturation curves in normal oral mucosa. The extent of this shift can be related to the degree of the reduction in tumor vasculature. In addition, great regional inhomogeneities in i.c.HbO 2 values can be detected. Comparison of these results with earlier measurements in the DS-Carcinosarcoma of the rat reveals that i.c.HbO 2 values in implantation tumors of rats are considerably lower than saturations in tumors of the oral cavity in humans as involved in this study. The differences in tumor oxygenation can be explained by differences in growth stage, in the site of tumor growth, and in influences of the host on the tumor oxygenation.

Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release

In solid malignant tumors, lactate has been identified as a prognostic parameter for metastasis and overall survival of patients. To investigate the effects of lactate on tumor cell migration, Boyden chamber assays were applied. We could show here that lactate enhances tumor cell motility of head and neck carcinoma cell lines significantly in a dose-dependent manner. The changes in tumor cell migration could be attributed to L-lactate or a conversion of lactate to pyruvate, as only these two substances were able to increase migration. Addition of D-lactate or changes in osmolarity or intracellular pH did not alter the migratory potential of the cells investigated. Because lactate was shown earlier to impair the penetration of dendritic cells in a tumor spheroid model, which is contrary to the response of the malignant cell population in the present study, we included blood monocytes in our assay as a highly motile immune cell type and precursor of tumor-associated macrophages. Interestingly, high levels of L-lactate (20 mM) at a pH of 7.4 inhibited monocyte migration in the Boyden chamber system. In addition, cytokine release of TNF and IL-6 was inhibited. The obtained data suggest that high lactate content promotes tumor progression by contributing to the phenomenon of tumor immune escape and by enhancing the migratory potential of the malignant cell population which may directly be coupled to a higher incidence of metastasis.

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

BackgroundEven in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells.MethodsTo investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model.ResultsThe ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival.ConclusionIn summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.