(157a) Towards in Vitro Optimization of Chemotherapy for Leukaemia Under Environmental Stress: Moving from Two to Three Dimensional Cultures

Acute Myeloid Leukaemia (AML) is an aggressive type of leukaemia. According to the Cancer Research UK, approximately 8000 cases of AML occur annually in the UK. The most curative treatment for AML is chemotherapy, i.e., an intensive treatment currently depending on the physician’s experience. This treatment has life-threatening implications due to its high toxicity. It is, therefore, crucial to optimize chemotherapy treatment in order to balance the eradication of the cancerous population with the minimum possible toxicity to normal (healthy) tissues.

In order to optimize chemotherapy for AML treatment it is essential to conduct ex vivo studies in an appropriate platform that can effectively recapitulate the in vivo Bone Marrow (BM), which is the natural environment where normal and abnormal haematopoiesis take place in the body. Such a 3D BM scaffolding system has been previously developed in our group (Mortera et al., 2010;2011). Additionally to the BM architectural characteristics, i.e., porosity and presence of adhesion proteins, parameters such as oxygen and glucose concentration should be monitored. The value of these parameters could differ between patients as well as within the same patient at different stages of treatment, consequently affecting the resistance of the cancerous population to the anti-cancer agent (Guintoli et al., 2011).

The present work is a systematic comparative study of the growth kinetics and metabolic evolution of an AML model system in a classical two dimensional culturing platform as well as in a 3D scaffolding platform under the effect of oxidative, nutrient and cytotoxic stress.

K-562 cell line, which was originally derived from a patient with a blast crisis of chronic myeloid leukaemia, i.e., therefore resembling AML, was selected as a model system for AML. K-562 cells were seeded in 3D polyurethane porous scaffolds coated with collagen type I (Mortera et al., 2010) as well as in 2D suspension cultures in 5% and 20% oxygen, for three different glucose levels, i.e., 4.3 g/L (optimal level generally applied in laboratory growth media), 1.3 g/L (highest human physiological level in vivo) and 0.6 g/L (lowest human physiological level in vivo) for a time frame of 2 weeks. In vitro chemotherapy was conducted by exposing the cultures during 48 h (1 feeding cycle) to 0.1, 10 or 50 ug/mL of the cytotoxic drug cytarabine. The cellular proliferation and metabolic evolution was monitored approximately on a daily basis. Cellular proliferation was determined by cell counts using a hemocytometer, as well as with the MTS metabolic assay (MTS Promega ®). Nutrients (glucose, glutamine) and metabolites (lactate, glutamate) were measured with a bioprofiler (NOVA biomedicals).

Results indicate that the cellular growth kinetics and/or resistance to the cytotoxic agent –the latter defined as maintenance of cell viability and/or proliferation after exposure to the drug- significantly alter under oxygen and glucose stress as well as between 2D and 3D cultures. For example oxidative stress affects the leukaemic proliferation in both culturing systems but glucose stress is experienced more severely in the 3D system, reducing significantly the leukaemic proliferation and metabolic activity and, at a second stage, leading to an increased cell resistance to the chemotherapeutic drug. In general, the higher environmental stress experienced in the 3D system is attributed to concentration gradients (of oxygen or glucose) resulting from mass transfer limitations which are significantly higher at the centre as well as the bottom of the scaffold (in which diffusion of oxygen and/or glucose is extremely low). Mass balance analysis enables the quantification of thresholds amongst scaffold areas which are nutrient and/or oxygen depleted due to the architectural characteristics of the scaffolds (leading to low or no proliferation) and areas of controlled environmental stress (that alter the cell kinetics but do not minimize proliferation). Our findings point out the importance of recapitulating and further monitoring the BM micro-environmental conditions (independently and synergistically) when designing a chemotherapy protocol in vitro.  This effort belongs to the Imperial College Framework for the Design, Modelling and Optimization of Biomedical Systems (Velliou et al., 2014).

References

Giuntoli, S., Tanturli, M., Gesualdo, F.D., Barbetti, V., Rovida, E., Sbarba, P.D. (2011) Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate. Haematologica, 96(2), 204-212.

Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Panoskaltsis, N. (2011) Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds. Biomaterials, 32, 9263-9270.

Mortera-Blanco T., Mantalaris, A. Bismarck, A.,  Panoskaltsis, N. (2010) Development of a three-dimensional biomimicry of human acute myeloid leukemia ex vivo. Biomaterials, 31, 2243-2251.

Velliou E., Fuentes-Garí M., Misener R., Pefani E., Rende M., Panoskaltsis N., Pistikopoulos E. N., Mantalaris A. (2014) A framework for the design, modeling and optimization of biomedical systems. In M. Eden, J. D. Siirola and G. P. Towler (Ed.), Proceedings of the 8th International Conference on Foundations of Computer-Aided Process Design – FOCAPD. Cle Elum, Washington, USA; 2014.

Acknowledgements

This work is supported by ERC-BioBlood (no. 340719), ERC-Mobile Project (no. 226462), by the EU 7th Framework Programme [MULTIMOD Project FP7/2007-2013, no 238013] and by the Richard Thomas Leukaemia Research Fund. R.M. is further thankful for a Royal Academy of Engineering Research Fellowship.