Application of LDH assay for therapeutic efficacy evaluation of ex vivo tumor models

Megan C Cox,Inês A Isidro,Ana Félix,Julie J Purkal,Kathleen Halwachs,Erwin R Boghaert,Adelyn Zelaya-Lazo,Fernanda Silva,Catarina Brito,Rita Mendes,Teresa F Mendes

doi:10.1038/s41598-021-97894-0

Abstract

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.

Highlights

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials
We demonstrate that the lactate dehydrogenase (LDH) assay can assess the therapeutic efficacy of chemotherapeutics in ovarian cancer patient-derived explants (PDE) (OvC-PDE) cultures
Other criteria considered in the selection of readouts to directly compare the LDH assay include methods that are (1) compatible with low cell numbers due to the labor-intensive process of xenosphere generation and limited availability of patient material making methods requiring high cell numbers unfeasible in these culture formats; (2) do not require generation of single cell suspensions as the cell line and cell status may present distinct sensitivity to dissociation affecting assay results; (3) do not require sectioning of the sample to capture the signal over the larger area of the xenospheres (~ 500–600 μm diameter) and PDE (1–2 mm diameter) which would involve a more complex experimental procedure and data processing

Summary

Introduction

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. It is crucial to validate readout methods in complex culture formats, considering the advantages and limitations of each technique, and to create protocols for preclinical drug screening studies employing in vitro and ex vivo tumor models that are standardized, reproducible, and applicable to the culture system of choice[12]. We demonstrate that the LDH assay can assess the therapeutic efficacy of chemotherapeutics in ovarian cancer PDE (OvC-PDE) cultures

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