Efficacy of catumaxomab in tumor spheroid killing is mediated by its trifunctional mode of action.

Catumaxomab is an intact trifunctional bispecific antibody targeting human EpCAM (epithelial cell adhesion molecule) and CD3 with further binding to Fcgamma receptor type I, IIa and III. We choose multicellular tumor spheroids (MCTS) of human EpCAM-positive FaDu tumor cells in co-culture with human peripheral blood mononuclear cells as an adequate three-dimensional in vitro model for pharmacological testing of catumaxomab. We found a strong dose-dependent antitumor response mediated by catumaxomab, with volume-decreased or completely destroyed tumor spheroids together with a massive immune cell infiltration and decreased signals for cancer cell viability and clonogenicity. In control experiments with F(ab')2 fragments of catumaxomab and the parental antibodies alone or in combination the effects in spheroid volume reduction were less than that of catumaxomab. All binding partners of the postulated tricell complex have to be present to exert catumaxomab's full mode of action. These distinct effects of catumaxomab are based on the unique composition of the trifunctional bispecific antibody. Since, in general, many cancers are treated by chemotherapy in combination with immunological tumor therapy, we additionally analyzed the effects of cisplatin alone and in combination with catumaxomab. For cisplatin alone we detected a dose-dependent response relating to decrease of spheroid volume. The combined approach resulted in a synergistic spheroid volume decrease and the colony formation was reduced to non-detectable levels.

Lactate: a metabolic key player in cancer.

Increased glucose uptake and accumulation of lactate, even under normoxic conditions (i.e., aerobic glycolysis or the Warburg Effect), is a common feature of cancer cells. This phenomenon clearly indicates that lactate is not a surrogate of tumor hypoxia. Tumor lactate can predict for metastases and overall survival of patients, as shown by several studies of different entities. Metastasis of tumors is promoted by lactate-induced secretion of hyaluronan by tumor-associated fibroblasts that create a milieu favorable for migration. Lactate itself has been found to induce the migration of cells and cell clusters. Furthermore, radioresistance has been positively correlated with lactate concentrations, suggesting an antioxidative capacity of lactate. Findings on interactions of tumor metabolites with immune cells indicate a contribution of lactate to the immune escape. Furthermore, lactate bridges the gap between high lactate levels in wound healing, chronic inflammation, and cancer development. Tumor cells ensure sufficient oxygen and nutrient supply for proliferation through lactate-induced secretion of VEGF, resulting in the formation of new vessels. In summary, accumulation of lactate in solid tumors is a pivotal and early event in the development of malignancies. The determination of lactate should enter further clinical trials to confirm its relevance in cancer biology.

Semiautomatic growth analysis of multicellular tumor spheroids.

Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCTS sediments are digitally scanned and spheroid volumes are calculated by computerized image analysis. The equipment includes regular office hardware (personal computer, flatbed scanner) and software (Adobe Photoshop, Microsoft Excel, ImageJ). The accuracy and precision of the method were tested using industrial precision steel beads with known diameter. In summary, in comparison with other methods, this approach provides benefits in terms of semiautomation, noninvasiveness, and low costs.

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.

Test system for trifunctional antibodies in 3D MCTS culture.

The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the trifunctional, bispecific antibody catumaxomab (anti-EpCAM x anti-CD3) or similar therapeutic molecules. The test system is accessible to various analytical methods and thus allows for characterizing multiple parameters, which can help elucidate the mode of action of immunotherapeutic anticancer treatment. For example, the novel approach enables precise, reproducible volume growth analysis of MCTSs under immunotherapeutic treatments. For evaluation of changes within individual spheroids, cryosections can be stained (e.g., for proliferating or apoptotic cells as well as infiltrating PBMCs). Molecular PCR-based assays or flow cytometric analyses allow for discrimination between different cell types, particularly leukocyte subtypes. Furthermore, MCTSs can be disaggregated to form standard monolayers for cell viability or plating efficiency experiments. For these reasons, the MCTS model is a powerful tool to analyze drug efficacy with various endpoints under highly reproducible, standardized conditions.