Culturing Patient Derived Explants (PDE)

An Overview of Culturing Patient Derived Explants

Patient Derived Explants or PDEs are ex vivo models where fresh tumors from biopsy or surgical resections are directly used for drug studies. Typically, tumor pieces are sectioned or minced and placed in culture prior to drug treatments. Once the drug exposure is complete, the tissues are fixed or homogenized for endpoint analysis. PDEs are generated using little to no tissue disruption and include tumor cells, stroma, immune cells and vasculature, so they are an accurate microcosm of the native tumor environment (Powley et al. 2020). PDEs facilitate the interrogation of molecular and histological tumor characteristics in a single sample to construct a more complete picture of the tumor. PDEs have been developed from various tumor indications including prostate (Tieu et al, 2021) and endometrial (Collins et al. 2020) cancers. However, PDEs can be extremely fragile and are liable to disintegrate rapidly and degrade over time. Researchers have optimized culture conditions to increase PDE viability. For example, Majumder et al. developed a novel ex vivo system that included autologous patient serum and immune cells as well as tumor indication and grade matched matrix proteins. This autologous PDE system supported culture for about 7 days allowing a window of opportunity to test several drug regimens to identify the optimal therapy for the patient (Majumder et al. 2015).


Endpoint Analysis in PDEs

Endpoint analysis in PDEs post drug treatment can follow one of two paths – the explants can be formalin fixed and paraffin embedded (FFPE) for histological and immunohistochemical analyses or homogenized for DNA, RNA and total protein analyses. FFPE sections can be stained using H&E (hematoxylin and eosin) staining to study changes in tumor and stroma compartments as well as understand the impact of culture conditions on tissue integrity. Sections can also be stained with specific antibodies to measure important parameters such as proliferation using Ki-67, apoptosis using cleaved caspase-3 or PARP and immune cell markers such as PD-L1 and CD3. DNA and RNA isolated from homogenized explants are largely used to identify tumor mutational burden or transcriptome profiling while protein analysis is used to understand overall changes in protein expression and post translational modification.

In addition to endpoint analysis of the explant tissues, secreted proteins such as cytokines can be measured in the culture media at various timepoints to evaluate early and delayed changes in cytokine expression after drug treatment.


PDEs have several advantages and limitations compared to other 3D cell models (reviewed in Powley et al. 2020). Explants maintain the individual patient specific tumor architecture and microenvironment including immune cells such as tumor infiltrating lymphocytes (TILs) and macrophages as well as stromal cells. The retention of the native environment facilitates studies on tumor stroma interactions and paracrine signaling and also permits the comparison of tumor cells to matched normal cells. Since explants are generated from fresh tissue, they are more predictive of patient response and the data generated from the explants can be correlated with the individual patient response. PDEs are a very useful model to study changes in immune cells in response to checkpoint inhibitors that are the primary drug targets for most tumor indications.

PDEs have limitations primarily in terms of source tissue availability and the culture time frame. Since fresh tissues are needed in sufficient quantities, PDEs can be generated primarily from surgical resections which requires access to hospital networks, surgeons and pathologists. Building a network to reliably source fresh tumors is a major undertaking. PDEs are not suited for longitudinal studies as they tend to start degrading in about 3 days so there is a tight timeline to generate as much data as possible. Due to the short culture time, it is difficult to measure direct tumor killing effects of immunotherapies that can take several weeks to induce cytotoxicity. The amount of fresh tissue that is available varies between indications but the finite amount of tissue does limit the potential to screen multiple therapies. This is compounded by the fact that each timepoint and treatment data point should include multiple replicates to account for intra- and inter-explant variation of tumor tissues and immune cells.

Despite these limitations, PDEs have a unique role in preclinical drug development of novel cancer therapies as they are the only model that truly represents the native tumor state.


References:
  1. Tieu, T., Irani, S., Bremert, K. L., Ryan, N. K., Wojnilowicz, M., Helm, M., Thissen, H., Voelcker, N. H., Butler, L. M., Cifuentes‐Rius, A., Patient‐Derived Prostate Cancer Explants: A Clinically Relevant Model to Assess siRNA‐Based Nanomedicines. Adv. Healthcare Mater. 2021, 10, 2001594.
  2. Collins, Anna & Miles, Gareth & Wood, Joanna & MacFarlane, Marion & Pritchard, Catrin & Moss, Esther. (2019). Patient-derived explants, xenografts and organoids: 3-dimensional patient-relevant pre-clinical models in endometrial cancer. Gynecologic Oncology. 156.
  3. Majumder, B., Baraneedharan, U., Thiyagarajan, S. et al. Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity. Nat Commun 6, 6169 (2015).
  4. Powley, I.R., Patel, M., Miles, G. et al. Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery. Br J Cancer 122, 735–744 (2020).