The Limits of In Vitro and Ex Vivo Immuno-Oncology Assays
Studying the immune system in vivo can be challenging, and in vitro and ex vivo assays are often used to reduce system complexity. This post looks at the types of I/O assays available, the data they provide, and the caveats to their use in immuno-oncology drug discovery.
Immuno-Oncology In Vivo Models Provide Complex Living Assessment Systems
Research in immuno-oncology is typically conducted in the realm of the animal model, which should come as no surprise. The immune system is a complex set of interacting cell types, each potentially in different states of activation or inhibition. With such systems, however, a deeper understanding of the biology at play requires a reduction in complexity – and therefore the use of ex vivo and in vitro tools.
At this point we should clarify some vocabulary:
- In vitro refers to things living in culture. For instance, cancer cell lines live in an in vitro world.
- Ex vivo refers to things that were once living in vivo, but are removed from that context for study. Lymphocytes live in an in vivo world, for example, but are being measured or manipulated outside of this context.
These are functional or practical designations, rather than dictionary definitions.
Ex Vivo Immuno-Oncology Assays
In general, ex vivo assays are typically used to count things and/or to determine their activation state. Flow cytometry, for example, is widely used to evaluate the number of immune cells infiltrating into a tumor (i.e. tumor infiltrating lymphocytes or TILs). However, flow cytometry is not the only technique to evaluate TILs. Histology and immunohistochemistry are also used.
Although slower with lower throughput, histological analysis preserves spatial information that is lost upon preparing single cell suspensions for flow cytometry. The activation state of cells can be measured by technologies such as ELISPOT or staining cells for intracellular cytokine production by flow cytometry. Tetramer staining can be used with flow cytometry to assess the presence and frequency of epitope specific T cell populations.
In Vitro Immuno-Oncology Assays
In vitro assays are intended to asses function, probe mechanism of action, and monitor temporal changes. Cells are grown or manipulated in culture, and the unifying characteristic of in vitro assays are that changes to those cells are measured over time.
The assay endpoints typically measured in in vitro experiments are numerous. These include:
- activation (or inhibition) of cytokine expression through engagement (or blocking) of signaling receptors
- assessment of cell dependent killing of target cells
- assessment of events following cell-cell interactions.
Limitations in Immuno-Oncology
In “traditional” drug discovery, small molecules that interfere with specific biochemical reactions influence cellular function. In this setting, there is generally strong correlation between the nature of the interaction between the small molecule and its target, and the subsequent effect on cellular function. This correlation can be measured and is generally called Potency.
What I think is missing in the realm of immuno-oncology is an appreciation of Potency in this traditional sense. I believe this stems from the fact that in immuno-oncology applications the proximal effector on the cancer cell is not the drug itself, but the cell(s) of the immune system that have been acted upon by the drug. This complicates assessment of potency, compared to the relatively simple correlations of in vitro or ex vivo assays.
In “traditional” drug discovery, where the drug is the proximal effector, we are confident that when the agent works in vitro, it will very likely work in vivo. Despite revealing exquisite mechanism of action insights, I feel there is much less confidence that an I/O agent will work in vivo given demonstration that is does work in vitro – the context of action is simply too different.
This would seem to me a natural consequence of the reduction of a very complex system. The forest is lost as we look closer at the individual trees; in immuno-oncology, this forest is dense, dark, and deep.
Improved Technologies will Allow More Complex Model/Platform Integration
As technologies improve, so will our ability to model and integrate systems with ever increasing complexity. With these continued advances it is hoped that in vitro and ex vivo measurements will continue to provide clarity in molecular and cell biology, but extend into intricate systems.