Profiling of 24 Standard of Care Drugs in a Panel of 20 Human Hematological Cell Lines Using Xenograft-Derived Three-Dimensional (3D) Cultures Ex Vivo and In Vivo
Authors: Maier Armin , Engelhardt Monika , Fiebig Heinz-Herbert , Schüler Julia 
Published: October 2019

Abstract
Abstract 4995

Introduction:
Leukemia and lymphoma account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of hematological malignancies induce unique therapeutic challenges. It is well known that pluripotent as compared to differentiated cells possess the potential for anchorage independent growth in semisolid medium. This can be monitored via clonogenic or colony formation assays, in which cells grow in vitro in a three-dimensional (3D) manner without adherence to plastic culture material support. These assays can be utilized to evaluate growth and drug sensitivity of tumor stem and progenitor cells (Fiebig HH et al. Eur J Cancer 40:802, 2004). In addition, these 3D cell culture assays often mimic the in vivo scenario better than 2D cell culture assays with adherent tumor cells.

Material and methods:
For our ex vivo anti-tumor efficacy profiling using clonogenic assays, we established a panel of 20 hematological cell lines comprising different entities like acute lymphoblastic leukemia (ALL, 4 cell lines), acute myeloid leukemia (AML, 6 cell lines), chronic myeloid leukemia (CML, 5 cell lines), Hodgkin- (1 cell line) and non-Hodgkin-lymphoma (NHL, 3 cell lines), as well as multiple myeloma (MM, 3 cell lines). Tumor cells were injected into the flanks of NOD/SCID mice in order to obtain subcutaneous tumor xenografts, which were kept at low passages (n <3). These xenografts served as starting material either to prepare single cell suspensions for ex vivo analysis, or to carry out in vivo efficacy tests using either subcutaneous or disseminated growing tumor xenografts.

Results:
Twenty-four standard of care agents were tested in terms of their ex vivo chemosensitivity (e.g. cytarabine, cyclophosphamide, dexamethasone, doxorubicin, etoposide, melphalan, prednisolone, vincristine), including selected targeted drugs also (e.g. bortezomib, imatinib, nilotinib, sorafenib). The drugs showed diverse patterns of selectivity and potency: vincristine, doxorubicin and cytarabine, but also the proteasome inhibitor bortezomib exhibited pronounced activity with IC50 values in the nanomolar range (mean IC50 = 1 – 100nM), not only in their respective clinical application, but also in various other tumor entities, such as in ALL and AML with use of bortezomib. Differential activity was determined e.g. for prednisolone and dexamethasone, which were active in a micromolar range (mean IC50 = 22 – 58μM) in the ALL cell lines CCRF-CEM and MOLT-4, AML cell lines NOMO-1, NHL DAUDI and U-937, as well as the MM cell line IM-9. All-trans-retinoic acid (mean IC50 = 1.3μM) as well as interferon-gamma-1b (mean IC50 = 0.43 μM) showed specific activity patterns with pronounced growth inhibition in AML (3/6 tested AML cell lines: KG-1, NOMO-1, OCI-AML2), but also in CML (1/5 tested CML cell lines: EM-2) and MM (1/3 tested MM cell lines: L-363). The strong correlation of both tyrosine kinase inhibitors imatinib and nilotinib (spearman coefficient: 0.73, p <0.001) and their differential activity restricted to bcr-abl-positive cells served as a positive control for the implemented test system. In vivo follow-up testing in defined tumor xenografts confirmed the results obtained ex vivo. For example, cyclophosphamide that showed strong antitumor activity with use of the NHL cell line DAUDI via clonogenic assay (IC50 = 0.3μM), also induced tumor remissions of 80% in xenografts with subcutaneously growing DAUDI cells as compared to untreated control animals. Moreover, an exceedingly promising antitumor activity of sorafenib in AML cells assessed via clonogenic assay (mean IC50 0.84μM in AML cells vs. mean IC50 4.0μM over all tested entities) could be confirmed in the disseminated in vivo model using HL-60 cells (reduction of 99% vs. untreated control; Schueler J. et al. Blood 116 (21):2141, 2010).

Conclusions:
The presented panel screen using clonogenic assays is of great value for time and cost effective profiling of traditional cytotoxic as well as new targeted anti-cancer agents which can be confirmed in tumor models of hematological malignancies and can thereby guide to more effectively designed in vivo experiments. Diverse activity and resistance patterns ex vivo and in vivo also contribute to create clinical development strategies of standard and novel compounds.

Disclosures:
No relevant conflicts of interest to declare.