Stromal Cell Supported High-Throughput Drug Testing Of Primary Leukemia Cells For Comprehensive Assessment Of Sensitivity To Novel Therapies

Ex vivo drug sensitivity testing of cancer cells taken directly from patients would significantly facilitate optimization of clinical therapies. However, in the past, such testing has been performed in suboptimal conditions, where patient cells gradually stop proliferating and undergo apoptosis, with poor translation of Results. More reliable prediction of drug sensitivity is needed and recent focus has been directed towards Methods that take into account the supporting impact of the surrounding tumor microenvironment. Primary leukemia cell viability and long-term survival ex vivo can be promoted with co-culture Methods using stromal cells (McMillin et al. 2013). While high throughput (HT) drug testing enables rapid assessment of sensitivity to 100s of drugs or drug combinations, application of co-culture Methods is challenging considering the mixed readouts from multiple cell types. In this study we describe a HT platform based on stroma-conditioned medium for assessing the anti-leukemic activity of compounds against fresh and vital biobanked primary leukemia samples ex vivo.

Stroma-conditioned medium (CM) was collected from the HS-5 human bone marrow (BM) cell line and combined with RPMI medium for drug sensitivity testing. Mononuclear cell medium (Promocell) was used as the standard medium comparison. Sensitivity of primary leukemia or healthy cells to 306 approved and investigational drugs was measured at 5 different concentrations covering a 10,000-fold concentration range. Cell viability was measured after 72 h with the CellTiter-Glo assay and dose response curves generated for each tested drug. Drug sensitivity scores (DSS) were calculated based on the area under the dose response curve. Here, we compared comprehensive drug sensitivity ex vivo responses between stroma-conditioned medium and standard medium using mononuclear cells from 8 acute myeloid leukemia (AML) patients and 4 healthy donors.

HS-5 CM supported fresh and biobanked primary AML cells, promoting proliferation and overall survival. Freshly isolated AML cells had a mean viability of 123% after 3 days in CM compared to 59% in the absence of CM. The viability of biobanked cells was 85% with CM vs. 20% in conventional medium. Improved ex vivo cell survival increased the therapeutic window of drug sensitivity testing and more drugs could be assessed with CM compared to conventional medium. Results from different healthy samples tested with the same type of medium were highly similar, but sensitivities differed significantly when comparing CM to standard medium Results. In contrast, drug sensitivity Results of AML cells from different patients were more diverse, reflecting the heterogeneity of the disease. However, comparison of CM and standard medium drug sensitivities of cells from individual AML patients showed modest differences that were primarily indicative of the increased proliferation of cells incubated with CM. Overall, both AML and healthy cells showed greater sensitivity to anti-mitotic drugs when incubated with CM. For example, the average DSS of vinblastine for healthy controls was 17 in CM vs. 9 in standard medium. In addition, AML cells often exhibited increased sensitivity to JAK inhibitors such as ruxolitinib when tested with CM compared to standard medium (DSS 14 vs. 9). In contrast, stress-related protein-targeting drugs (e.g. HSP90 inhibitors) and certain tyrosine kinase inhibitors (e.g. dasatinib, quizartinib) exhibited reduced efficacy when AML cells were incubated with CM compared to conventional media. This may be due to soluble factors present in CM that mimic the protection provided by the BM niche.

Our data support the concept that conditioned medium from stromal cells improves application of drug sensitivity testing to AML patient samples ex vivo. Stromal medium supports both fresh and biobanked AML cells, likely providing environmental cues present in the BM niche and necessary for AML cell growth and survival. This may lead to more reliable ex vivo assessment of the anti-leukemic activity of compounds for cells from leukemia patients. Importantly, stromal cell based conditions support the growth of vital biobanked leukemia samples and enable use of retrospective samples for a multitude of assays including HT drug testing.

Porkka:Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Kallioniemi:Medisapiens: Membership on an entity’s Board of Directors or advisory committees; Roche: Research Funding.