Real-time cellular metabolic phenotyping in immuno-oncology research Evolving Science For The Future | Articles

Real-time cellular metabolic phenotyping in immuno-oncology research

Central cell metabolism – also known as energy metabolism – describes the interconnected network of biochemical processes that occur within living cells to maintain energy homeostasis and build biomass.1 By studying cell metabolic pathways, researchers can gain functional insights into specific tissue characteristics, develop novel therapeutics and better understand the critical drivers of cell therapy applications.

Metabolic flux is flexible and variable across tissues, cell types and differentiation states.2,3,4
Hence, careful metabolic analyses are essential in supporting basic research and the development of cell-based therapeutics. While all metabolic pathways contribute to cellular function, glycolysis in particular is recognized for its dichotomous role in immune cell health and tumorigenic disease.1,5,6
As it's involved in generating the key intermediates required for cell growth and differentiation, glycolysis often holds a critical role in determining cell fate and function.7,8,9

Typically, glycolysis is responsible for the efficient yet controlled catabolism of glucose into pyruvate, generating adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) in the process.10,11 Mitochondrial metabolism, notably, oxidative phosphorylation (OXPHOS), is then responsible for meeting the remaining ATP deficit.
In a sufficiently oxygenated environment, glycolysis also yields a small but quantifiable concentration of lactate which is rapidly metabolized by lactate dehydrogenase (LDH). In cancer, aerobic glycolysis is upregulated fueling rapid cell proliferation and increased lactate production.1,6 This phenomenon is broadly termed the Warburg effect and contributes to the unique metabolic profile of tumorigenic cells.

Metabolic intermediates can also function as important intercellular signaling molecules.12,13,14 Therefore, studying the cellular microenvironment also holds relevance in biotherapeutic research. In responsive immune cells, glycolysis drives the effector functions of activated T lymphocytes, yet, their activity may also be suppressed by a lactic acid environment.8,13,14
Live-cell glycolytic analyses can provide functional insights into both the specific tumor microenvironment (TME) and the efficacy of anti-tumordirected cell therapies.15

This article outlines the critical role of live-cell metabolic phenotyping in immuno-oncology research. It also explores the pitfalls of traditional sampling-based methods in contrast to the benefits of continuous monitoring for cell therapy research applications.

Metabolism in immunology and
immuno-oncology research

Metabolism research lies at the core of immune cell therapy development; in fact,
its role in immunology and immuno-oncology research has gained increasing attention in recent years.16,17,18 Immuno-oncology focuses on the application of adaptive immune components as anti-cancer therapies.19 In particular, chimeric antigen receptor (CAR)-T cell technology is a key research focus,having shown great promise both in vitro and in the clinic.

CAR-T cells are designed to express specific receptors on their extracellular surface. These receptors target cancer cell antigens, initiating the recognition and destruction of tumorigenic cells without causing damage to healthy tissue. Metabolic activity is a key indicator of CAR-T cell fitness and function.5,18 In fact, all T lymphocytes undergo significant metabolic reprogramming as they differentiate and mature, shifting from a quiescent to a highly energetic state in support of their effector functions (Figure 1).

Figure 1: Lymphocyte metabolism during activation and quiescence.20

Naive T cells (TN) circulate in the blood in a steady metabolic state.20 Upon contact with a complementary cancer cell antigen, PI3K-AKT-mTOR signaling pathways are activated, resulting in the expression of transcription factors that upregulate glycolysis.20,21 If TN can meet the metabolic requirements, they differentiate into effector T cells (TEFF) that can execute immune functions. Once the antigen-presenting cells are cleared, the glycolytic rate of TEFF is reduced. This stimulates a small yet significant number of TEFF to persist as fully differentiated memory T cells (TM). TM exhibit a similar glycolytic profile to TN, although higher rates of mitochondrial respiration enable them to re-activate quickly upon a resurgence of the same threat. If metabolic parameters are not met during the maturation process, T cells may lose their ability to differentiate or to target tumor cells effectively.22,23,24

Optimizing adoptive cell therapies

The real-time characterization of CAR-T cell metabolic activity can support the understanding of TEFF activation and guide the engineering of optimal therapeutic characteristics.14,21,25,26
These may include increased efficacy and persistence against specific tumor types or effectivity in combination therapy approaches.27,28 For example, CAR-T cells expressing specific costimulatory receptor pairings have been found to possess metabolic characteristics that may render them more effective against acute lymphoblastic leukemia (ALL) than other cell types.27 In addition, certain receptor and coreceptor subdomain pairings can influence the metabolic characteristics of CAR-T cells contributing to a less differentiated TEFF phenotype that has shown to be highperforming in in vivo studies.29

Through a detailed understanding of metabolism,researchers have elucidated the effects of specific transmembrane costimulatory combinations, allowing them to optimize CAR-T cell design for the highest chance of therapeutic success. In addition, the engineered overexpression of specific glycolytic transcription factors can support CAR-T cell persistence in the TME, reducing the impact of lactate-mediated activity suppression.15
Meanwhile, the upregulation of non-glycolytic pathways reduces CAR-T cell exhaustion and increases their therapeutic persistence.30
Without accurate metabolic analyses, all such research efforts could be limited in their applications.

Immuno-oncology therapeutics are often administered as part of a combination therapy or multi-drug approach.31,32 Combination therapies are an exciting area of research surrounding the treatment of solid tumors.33,34 Programmed Cell Death Protein 1 (PD-1) is an immune response inhibitor that has been widely targeted in anti-tumor therapies.35 PD-1 exists on the surface of many immune cell types; it can interact with its complementary ligand on the tumor cell surface and offer a mechanism for tumor immune escape.28,35 PD-1 stimulation can also abrogate CAR-T cell glycolytic rates and result in a metabolic shift toward mitochondrial respiration.35
Hence, metabolic analyses can guide CAR-T cell engineering and, in the face of potential immune escape, support the design of therapeutic persistence alongside small molecule PD-1 inhibitors.

Challenges and opportunities in cell glycolytic phenotyping

Several metabolic phenotyping approaches currently exist to support immuno-oncology research. The latest technologies utilize probe-based methods to measure key metabolic parameters in cell-adjacent media (oxygen consumption, extracellular acidification or changes in metabolite concentrations). While such methods claim to provide dynamic, real-time insights into cellular energy homeostasis, traditional sampling-based methods can fail to capture critical glycolytic data.

Figure 2: The latest live-cell metabolic phenotyping technology for continuous sampling of glucose and lactate production.38

Lactate production rate is one parameter by which researchers can measure glycolytic activity (Figure 2).36 It can also be used to evaluate changes in the balance of other metabolic processes. However, the rapid clearance rate of this critical metabolite means that samplingbased methods can easily underestimate glycolytic function.37 Additionally, the highly flexible nature of CAR-T cell metabolism means rapid changes can easily be missed.4,5
Continuous measurements, taken without undue cell or media disturbance, can therefore offer superior accuracy to their sampling-based counterparts. By measuring glucose consumption and lactate production, researchers can take a more direct approach to measuring metabolic flux with a direct inference of ATP production. Hence, continuous measurements and considered experimental design can enable researchers to easily and clearly analyze TEFF activation without the risk of data misinterpretation from anomalous or inaccurate results.

While the advent of instruments for live-cell metabolic analysis has significantly enhanced the scope for in vitro research, many methods include complex cell culture requirements such as specialist media and/ or re-plating and de-gassing protocols. Not only can such requirements be costly in time and resources, but they may also induce unwanted treatment effects and confound important controls. Therefore, analyzer protocols that take real-time measurements from within standard culture vessels without undue processing can be considered a more robust experimental approach. Additionally, they can support research on hard-toculture or non-adherent cell populations while boasting affordability and compatibility with existing lab systems and infrastructure.

The latest technology in continuous live-cell metabolic phenotyping is capable of offering real-time observations of cultured cells in the standard environment (Figure 2). Thus, it offers greater consistency, flexibility and reproducibility than other methods, and can provide data that is directly comparable to other supporting experiments.

It is essential that real-time metabolism data is both accurate and robust if technological advancements are to be made. Continuous monitoring not only supports the exclusion of anomalous results, but it also provides unrivaled insights into CAR-T cell metabolic reprogramming.38
During experiments, it may be pertinent to measure the metabolic effects of coreceptor engineering over time; thus uncovering differences in cell differentiation or persistence as they occur.15,31,39,40 This helps researchers select appropriate cell populations for further analysis or clinical applications. With continuous monitoring, it is possible for researchers to observe realtime metabolic changes as they occur, providing the best understanding of how novel therapy designs may differentiate and mature in vitro.38,40

Conclusion and future perspectives

Real-time metabolic phenotyping is a crucial aspect of immuno-oncology research and can pave the way for the development of more advanced adaptive cell therapies. Understanding the metabolic dynamics of immune cells is vital for optimizing their efficacy. Meanwhile, metabolic analyses can also provide insights into the TME and the potential challenges it poses for in vivo applications.

Metabolic phenotyping could be used to provide insights into the activation and function of CAR-T cells, guiding their engineering and optimizing therapeutic outcomes. However, the future of such technologies will likely focus on the application of continuous monitoring approaches to increase the reliability, robustness and publication potential of cell metabolic data. For this reason, continuous monitoring is likely to become the gold standard approach in metabolic research with significant impacts on the field of immuno-oncology.


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