(584x) The Effect of Oxygen and Glucose Stress On the Evolution of a Leukaemia Model System in An in Vitro Bone Marrow Biomimicry

The effect of oxygen and glucose stress on the evolution of a leukaemia model system in an in vitro bone marrow biomimicry

E. G. Velliou^1,2, M. Fuentes Garí^1,2, S. Brito Dos Santos², R. Misener^1,2,

N. Panoskaltsis³, A. Mantalaris², E.N. Pistikopoulos¹

¹Centre for Process Systems Engineering (CPSE), Department of Chemical Engineering, Imperial Imperial College London, South Kensington Campus, London SW7 2AZ, UK

²Biological Systems Engineering Laboratory (BSEL), Department of Chemical Engineering, Imperial Imperial College London, South Kensington Campus, London SW7 2AZ, UK

³Department of Hematology, Imperial College London, Northwick Park & St. Mark’s Campus, London HA1 3UJ, UK

Acute Myeloid Leukaemia (AML) is one of the most common types of leukaemia in adults. AML is a malignant disease of the bone marrow (BM) and blood. Immature white blood cells which are not able to develop into normal functioning blood cells are overproduced and build up in the bone marrow and blood. This inhibits the development of healthy blood and immune cells due to space restrictions as well as inhibitory and clonal factors specific to the disease. Another type of Leukaemia is the Chronic Lymphocytic Leukaemia (CLL) which is also characterized by an accumulation of abnormal white blood cells in the bone marrow, but in a slower rate compared to AML. The most common treatment for AML and CLL is chemotherapy. It is well known that this therapy can result in several life-threatening complications as only few patient-specific factors are taken into consideration in current protocols and the choice of treatment often depends on the physician’s experience. Furthermore, inter-patient and intra-leukaemia significantly increase the complexity in determining these treatment protocols. As a consequence, no widely accepted ‘optimal’ chemotherapy treatment protocol exists, as the evolution of the cancer as well as the chemotherapy (drug) metabolism is highly dependent on patient characteristics such as age, weight, height, medical and family history as well as on a variety of unknown factors/parameters.

Moreover, the absorption/ metabolism of a chemotherapy drug can highly vary within the same patient, depending on the condition of the patient during chemotherapy. For example when the body temperature is higher, i.e., 40^oC, than the optimal/normal one, i.e., 37^oC, the liquid tumor will experience a temperature stress and both the leukaemia metabolism and the chemotherapy absorption may alter. Another important issue is the variability of parameters such as oxygen or glucose concentration among different body compartments. For example the oxygen concentration in the bone marrow is much lower than the one in peripheral blood. Glucose blood concentration is another crucial factor that may fluctuate within a patient’s body. It differs among organs as well as among patients or even within the same patient at different time points (hypoglycemia-hyperglycemia).

The alteration of these environmental factors, i.e., glucose concentration, oxygen concentration, temperature, can be experienced as a severe environmental stress by the cancer population. Several studies have shown that the latter impacts both the cancer growth and/or inactivation kinetics as well as the efficiency of the chemotherapy drug (see as examples Fecteau et al., 2013; Giuntoli et al., 2011; Lodi et al., 2011).

Therefore, understanding the influence of environmental stress such as oxygen and glucose stress is crucial for the optimization of AML chemotherapy treatment. A first step towards that direction is the efficient in vitro biomimicry of the human BM. Haematopoiesis, both normal and abnormal, is regulated by spatially organised cellular microenvironments (niches) within the BM which is a three dimensional (3D) tissue. The deregulation of the niches is implicated in the pathogenesis of cancers of the BM, i.e., leukaemia. Without the provision of growth factors from the niche, the cultivation of leukaemic cells is extremely difficult and requires co-culture with allogeneic/xenogeneic stroma or fibroblasts, or the addition of abnormally high concentrations of exogenous cytokines to the culture. This results in the introduction of artifacts to cell culture kinetics and biasing in outgrowth of cells which are dependent on these signals for survival (Panoskaltsis et al., 2005).  An “in vitro” 3D BM biomimicry for the successful cultivation of  leukaemic cells without the use of exogenous factors or allogeneic cell lines would be a powerful tool for the study of different types of treatments in leukaemia, as incorporating molecular, cellular and microenvironmental factors is required for a more efficient and accurate reproduction of the in vivo environment ex vivo.

The overall aim of the current research is the experimental kinetic study of a leukaemia model system, i.e., leukaemic cell lines, in a 3D polyurethane based collagen coated scaffolding system under oxygen, i.e., ex vivo mimicry of hypoxia, and glucose stress, i.e., ex vivo mimicry of hypo and hyperglycemia.

K-562 and MEC-1 cell lines are selected as a model system for AML and CLL, respectively. The leukaemic cells are seeded in the 3D system that has been developed in our team (Mortera et al., 2010;2011), which is made up of a highly porous polyurethane based scaffold coated with collagen. Hypoxia is imposed by decreasing the oxygen level of the culturing incubator to 5%, which represents the oxygen level of the human BM, as opposed to normoxia at which oxygen is set at 20%. Glucose stress is imposed by alteration of the glucose concentration of the nutrient medium from the normal one, i.e., 4.3 g/L used in most laboratory culture media to 0.6 g/L, i.e., lower level in human peripheral blood or 1.3 g/L, i.e., higher level in human peripheral blood.

In order to evaluate the cellular growth within the 3D system, several tests take place. More specifically, the MTS (tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium) metabolic assay is applied for the estimation of the cellular proliferation within the scaffolds. The MTS cell proliferation is a colorimetric assay that measures the reduction of the tetrazolium compound by the cells into a coloured formazan product that is soluble in tissue culture media. The amount of formazan produced, as  measured by the absorbance (490 nm), is directly proportional to the number of living cells in culture. Metabolites present in the growth medium such as lactate, glutamine, glutamate, NH₄⁺ are measured with a bioprofiler on a daily basis. Cells are extracted manually with a syringe, from the scaffolds at regular time points in order to determine the percentage of viable cells which is estimated by performing a cell count with a hemocytometer. Moreover, extracted cells are analyzed by Flow Cytometry in order to determine the cell cycle distribution. The latter is of great importance, on the one hand as it is an indication of the level of the environmental stress response and adaptation of the cells (higher stress resulting in more cells shifting to the dormant state G₀) and, on the other hand, because knowing the cell cycle distribution is crucial for chemotherapy treatment, as most chemotherapy drugs target cells which are at a specific phase of the cell cycle.

Results indicate that the cellular growth kinetics significantly alter under oxygen and glucose stress. For example extreme glucose stress leads to a higher cellular proliferation the first days of culturing –showing a possible induction of the autophagic mechanism- or in normoxia there is a higher level of glutamine consumption and glutamate synthesis compared to hypoxia. Therefore, our results are pointing out the importance of taking into consideration fluctuations of the micro-environmental conditions (independently and synergistically) of the leukaemic population when designing a chemotherapy protocol and/or when constructing pharmacokinetic and pharmacodynamic mathematical predictive models.

References

Fecteau, J.-F., Messmer, D., Zhang, S., Cui,  B., Chen,, L., and Kipps, T.J. (2013) Impact of oxygen concentration on growth of mesenchymal stromal cells from the marrow of patients with chronic lymphocytic leukemia, Blood, 121, 971-974.

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.

Lodi, A., Tiziani, S., Khanim, F.L., Drayson, M.T., Gunther, U.L., Bunce, C.M., Viant, M.R. (2011). Hypoxia Triggers Major Metabolic Changes in AML Cells without Altering Indomethacin-Induced TCA Cycle Deregulation, ACS Chemical Biology, 6, 169-175.

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

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

Panoskaltsis, N., Mantalaris, S., Wu, D. (2005). Engineering a mimicry of bone marrow tissue Ex vivo. Journal of Bioscience and Bioengineering, 100: 28-35.

Acknowledgements

This work is supported by ERC-Mobile Project (n^o 226462), by the European Union Seventh Framework Programme [MULTIMOD Project FP7/2007-2013, n^o 238013] and by Richard Thomas Leukaemia Research Fund. Ruth Misener is further thankful for a Royal Academy of Engineering Research Fellowship (Award number: RAEng 10216/118).