HIGH-THROUGHPUT IMAGING ANALYSIS FOR DRUG COMBINATION EFFICACY AND SPECIFICITY IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKAEMIA
Author(s): ,
Edward Law
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Lynsey McKenzie
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Helen Blair
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Deepali Pal
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Katarzyna Szoltysek
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Hesta McNeill
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Simon Bomken
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
John Luec
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Julie Irving
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom
,
Josef Vormoor
Affiliations:
Prinses Maxima Centrum,Utrecht,Netherlands
Olaf Heidenreich
Affiliations:
Northern Institute for Cancer Research,Newcastle University,Newcastle upon Tyne,United Kingdom;Prinses Maxima Centrum,Utrecht,Netherlands
EHA Library. Law E. Jun 15, 2019; 267216; PS915
Dr. Edward Law
Dr. Edward Law
Contributions
Abstract

Abstract: PS915

Type: Poster Pitch

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Childhood acute lymphoblastic leukaemia (ALL) responds well to conventional treatment, however there is a need to find more targeted therapies without the associated toxicities. There is great inconsistency of drug responses in cell lines, and often these do not represent the outcomes observed in the clinic. Patient-derived xenografts (PDXs) have been shown to more closely represent patient cancers, and increasingly better demonstrate the drug response clinically observed. However, in vivo assessment of multiple drug combinations is costly and time consuming. The development of an ex vivo ALL patient-derived xenograft/mesenchymal stromal cell (MSC) co-culture platform has allowed for screening of novel drug combinations in multiple childhood ALL cytogenetic subtypes. 

Aims
The aim of this study was to develop a high-throughput whole-well image-based analysis of PDX and MSC cell numbers after exposure to double combinations of 4 drugs. A panel of PDXs were assessed to take into account heterogeneity in pharmacological response.

Methods
A previously reported PDX/MSC platform was utilised to assess double drug combinations of dexamethasone, palbociclib, idasanutlin and ABT-199 in a panel of childhood ALL PDXs. Thawed PDXs were seeded on MSCs and treated with drug combinations in a 7x7 matrix format for 96h. These were subsequently stained with a fluorescent DNA dye, and high-throughput microscopy was conducted to obtain whole-well images of PDX and MSC nuclei.  Images were segmented and measurements of nuclear intensity, size and shape were produced with CellProfiler software. Segmented images were used to train a random forest classifier, within CellProfiler Analyst software, to distinguish PDX and MSC nuclei, utilising a multitude of image features. Combination treatments were assessed for synergy with the zero interaction potency model (ZIP).

Results
Whole-well cell numbers were achieved for PDXs and MSCs in the context of drug combinations across a range of ALL cytogenetic subgroups. Comparing fluorescence intensity readings with imaging and cell scoring revealed a background fluorescence level that previously could not be removed, leading to the underestimation of drug response. Combination of dexamethasone and ABT-199, previously shown to be additive or synergistic was also confirmed across our panel. Cell numbers in control wells were consistently equal to or higher than the original seeding densities, demonstrating the capacity of the platform to support PDX proliferation. Interestingly, the CDK4/6 inhibitor palbociclib scored strongly across many of the PDXs, and even more so in combination with ABT-199: something which has not been previously observed. Particularly strong synergy of ABT-199 and idasanutlin was observed in a PDX with a t(9;22) translocation (max ZIP score 69). None of the double combinations tested had an effect on MSC cell number.

Conclusion
This platform allows accurate quantification of absolute PDX cell number and relative MSC cell number. DNA stains have previously been shown to be superior to many standard viability assays such as fluorescent metabolic readouts. This methodology goes further to enable stratification of different cell types. The effective combination of dexamethasone and ABT-199 in childhood ALL, previously seen in mice, was confirmed with this platform, and several novel combinations were identified which appeared to show synergy, and will be taken forward to in vivo testing. This image-based platform is now being expanded to increase the complexity of the co-culture model with other haematopoietic niche cells.

Session topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research

Keyword(s): Acute lymphoblastic leukemia, Childhood, Drug sensitivity, Synergy

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