Page 66 - JCTR-11-5

Journal of Clinical and
Translational Research Metabolism of healthy and leukemic stem cells

can provide a comprehensive metabolic map to better escape mechanisms. Incorporating these technologies into
understand the heterogeneity within HSCs and LSCs in HSC and LSC research helps bridge mechanistic insight
the BM niche microenvironment, discern key metabolites with significant translational potential.
and metabolic pathways, and develop novel therapeutic
interventions. 136-138 4.4. Problems and opportunities in therapeutic
translation
4.3. Technologies for live cells in mitochondria and Despite the advancements in metabolomics toward
metabolism defining the metabolic profiles of HSCs or identifying
Live-cell technologies provide real-time functional LSC-specific vulnerabilities, translating these findings into
insights into cellular processes and mitochondrial activity, effective therapies remains a challenge. The wide range and
making them powerful tools for studying metabolically complexity of the metabolome can hide low-abundance
dynamic populations like LSCs. Instruments such as but functionally critical metabolites. Furthermore, flux-
120
the Oroboros Oxygraph-2k (O2k) and the Seahorse XF based metabolic insights usually depend on stable isotope
Analyzer are widely used to assess bioenergetic function tracing, which is technically difficult to perform on limited
in intact or permeabilized cells, tissues, and isolated clinical patient samples. Reproducibility also depends
mitochondria under physiologically relevant conditions heavily on the standardization of sample preparation,
120
(Figure 3). These platforms allow for the determination of instrumentation, and data analysis across platforms.
oxygen consumption rate, enabling detailed profiling of Nevertheless, recent technological advances are driving
ATP-linked respiration, proton leak, and spare respiratory basic research toward clinical translation. An example of
capacity. The Seahorse technology is also capable of this innovation is the use of in vivo C tracing to reveal
13
measuring the extracellular acidification rate to determine metabolic circuits that are essential to LSC survival and
glycolytic activity. to directly demonstrate the value of metabolomics in
144
The Agilent Seahorse XFe96 Analyzer was recently identifying targetable metabolic flux in vivo. Additional
employed in CML LSCs, unveiling a deeply quiescent research demonstrates the efficacy of co-targeting
subset leukemia initiators (LI) characterized by suppressed mitochondrial sirtuin 3 and cholesterol homeostasis to
145
Complex I activity yet enhanced FAO dependency. selectively disrupt mitochondrial function in AML LSCs,
139
Single-cell metabolomic profiling in parallel confirmed that while other studies demonstrate the efficacy of metformin
this LI subset maintained low ROS levels despite high FAO for activating the AMPK pathway in AML to enhance
chemosensitivity. Together, these studies show a growing
105
flux, suggesting that mitochondrial complex I suppression capacity to move from metabolomic discovery toward
is a protective adaptation within a functionally discrete mechanistically driven interventions. As metabolomic
LSC subpopulation. Previous research has shown that technologies become more sensitive, especially in single-
139
cytarabine-resistant AML cells maintain a high oxygen cell and live-cell contexts, they hold potential to define
consumption rate, indicating that persistent mitochondrial new biomarkers and therapies tailored specifically to LSCs,
respiration is a hallmark of chemoresistant LSCs. 107
thereby advancing translational leukemia research.
Alternative to the Seahorse, the Oroboros O2k high-
resolution respirometry provides a further depth of 5. Conclusion
understanding by directly measuring Complex I–IV The study of stem cell metabolism offers critical insight into
activity and coupling efficiency in primary AML samples. the networks that govern HSCs and the precise regulation
140
In contrast, the Seahorse provides high throughput, of quiescence and activation. Disruption of these tightly
dynamic stress testing. The Oroboros O2k offers extensive regulated metabolic programs leads HSCs to acquire
control over substrate and inhibitor addition, enabling abnormal metabolic profiles that contribute to oncogenic
precise dissection of respiratory chain function. 120,141 transformation. While LSCs maintain features of normal
This makes Oroboros particularly valuable for evaluating stemness, such as quiescence and drug resistance, they
subtle mitochondrial defects or drug-induced changes in exhibit distinct metabolic characteristics that enable them
electron transport that may underlie LSC persistence. 142,143 to survive and resist therapeutic intervention. Recent
In the context of HSCs and LSCs, Oroboros O2k live-cell advances in metabolic profiling, including single-cell
approaches not only reveal vulnerabilities in oxidative analytics and live-cell functional assays, have enabled
metabolism but also allow investigators to assess dynamic high-resolution assessment of these divergent metabolic
responses to metabolic inhibitors in real time. Today, the states. Novel and emerging technologies supporting
use of live-cell functional assays offers a critical readout for metabolic profiling not only reveal alterations in the
evaluating therapeutic efficacy and identifying bioenergetic biochemical pathways that contribute to HSC quiescence

Volume 11 Issue 5 (2025) 60 doi: 10.36922/JCTR025320053