A human mesenchymal spheroid prototype to replace moderate severity animal procedures in leukaemia drug testing

Abstract

Patient derived xenograft (PDX) models are regarded as gold standard preclinical models in leukaemia research, especially in testing new drug combinations where typically 45-50 mice are used per assay. 9000 animal experiments are performed annually in the UK in leukaemia research with these expensive procedures being classed as moderate severity, meaning they cause significant pain, suffering and visible distress to animal's state. Furthermore, not all clinical leukaemia samples engraft and when they do data turnaround time can be between 6-12 months. Heavy dependence on animal models is because clinical leukaemia samples do not proliferate in vitro. Alternative cell line models though popular for drug testing are not biomimetic - they are not dependent on the microenvironment for survival, growth and treatment response and being derived from relapse samples they do not capture the molecular complexity observed at disease presentation. Here we have developed an in vitro platform to rapidly establish co-cultures of patient-derived leukaemia cells with 3D bone marrow mesenchyme spheroids, BM-MSC-spheroids. We optimise protocols for developing MSC-spheroid leukaemia co-culture using clinical samples and deliver drug response data within a week. Using three patient samples representing distinct cytogenetics we show that patient-derived-leukaemia cells show enhanced proliferation when co-cultured with MSC-spheroids. In addition, MSC-spheroids provided improved protection against treatment. This makes our spheroids suitable to model treatment resistance - a major hurdle in current day cancer management Given this 3Rs approach is 12 months faster (in delivering clinical data), is a human cell-based biomimetic model and uses 45-50 fewer animals/drug-response assay the anticipated target end-users would include academia and pharmaceutical industry. This animal replacement prototype would facilitate clinically translatable research to be performed with greater ethical, social and financial sustainability.

Keywords: 3D models; Animal replacement; Preclinical models; cancer research; leukaemia.

Figure 1.. Comparison of existing in vivo preclinical leukaemia models with newly developed ex vivo animal replacement organoid models.

Key benefits of the animal replacement organoid models include species specificity and consequently higher biomimicry.

Figure 2.. Aggrewell plates generate uniform 3D spheroids from single cell suspension of the desired cell-type. SD from three independent experiments (using MSC from same patient) have been plotted as error bars. Phase contrast photographs showing (a) 2D MSC, scale bar = 100 μM and (b) 3D MSC, scale bar = 200 μM after 96 hours of growth. (c) Mesenchymal marker levels quantified through qPCR and normalised to GAPDH. (d,e) Phase contrast photograph of MSC spheroids generated through culture using Aggrewell plates. Scale bar = 1000 μM (f) Correlation between cell seeding densities and size of resultant spheroids. Data captured following 48 hours of seeding. (g) CD105, CD90 and CD73 expression in MSC. Black = unstained. Blue = 2D MSC. Red = 3D MSC. (h) Hoechst-33342-Calcein staining in 100 μM spheroids, taken at day five of 3D culture. Calcein (green) stains live cells, Hoechst-33342 (blue) stains live and dead cells.

Figure 3.. 3D MSC-spheroids confer higher proliferation and improved survival onto patient-derived ALL. Growth kinetics, measured via viable cell counts of ALL samples (a,d) L49120, (b,e) MS40 and (c,f) L707D across a 5 day period, with and without low dose dexamethasone (10nM) pressure, as co-cultures with 3D MSC spheres versus 2D MSC-co-culture. L49120, MS40 and L707D refer to three different patient samples, i.e., 3 biological repeats. (d,e,f) Dexamethasone treatment, data shown for 5nM treatment, is less effective in a 3D microenvironment. N= 3. (g) Flow cytometry data, representative plots, showing propidium iodide, PI, positive ALL cells (sample L707) following five days of 10nM dexamethasone treatment. The column graph shows % ALL cells stained positive for PI. N = 2.

Figure 4.. In vitro co-culture platform delivers drug response data with high in vivo predictivity to minimise in vitro to in vivo drug attrition rates. Leukaemia cells from leukaemia cytogenetic subgroup A is a responder to ABT199 (targets BCL2 in BCL2 positive ALL) and dexamethasone drug combination in vitro and in vivo. On the other hand, leukaemia cells from subgroup B show low sensitivity to this drug combination in vitro in both 2D and 3D formats, and subsequently poor response is also noted in vivo using PDX mice models. In vitro data readout includes viable cell counts; in vivo data (right hand graphs) is shown as bioluminescence imaging from luciferase expressing PDX cells in total flux(p/s). Total flux is a surrogate of blast number in vivo. Error bars shown refer to standard error (SE) using 4 mice per treatment group. Dexamethasone and ABT-199 are used in the clinics to treat ALL. b. ALL cell counts on day 0 versus day 5 of culture, with and without treatment with 25 nM dexamethasone. N = 3. Error bars = SD. c. Cell counts following dexamethasone and ABT-199 treatment of 3D MSC.

Figure 5.. A comparative costs analysis between 3D ex vivo and in vivo preclinical models show an approximate 30% costs benefit and consequently increased financial sustainability when using ex vivo organoid models.