Isolation of Lipid Droplets from Acute Myeloid Leukemia Cells for Lipidomic Analysis

Lipids are ubiquitous components of cellular structures and processes, playing crucial roles in metabolism, energy reserve, signaling transduction, and cell stress homeostasis. Dysregulation of lipid metabolism has been identified in various cancer cell types as a key transformation to sustain proliferation, often accompanied by increased lipid uptake and de novo fatty acid synthesis. Excess lipids and fatty acids are primarily stored in lipid droplets (LDs), a cellular organelle comprised of mostly neutral triglycerides (TAGs), surrounded by a phospholipid monolayer and lipid droplet adjacent proteins. In recent studies, excessive cellular LDs have become a distinctive feature of cancer cells and are a possible hallmark of cancer cells. LDs closely interact with other cellular organelles such as mitochondria, providing substantial amounts of energy and components in mitochondrial biosynthesis. LDs are also shown to buffer lipo-toxicity and oxidative stress-induced cell death, suggesting their roles in cell death regulation. A greater understanding of LDs composition and dynamics may provide novel anti-cancer therapeutic concepts.

LDs can be isolated from cells due to their low specific density (<1g/ml) under high-speed centrifugation and used for downstream lipidomic studies. However, isolation and studies of LDs are mostly performed in solid tumors, hepatocytes, muscle, or adipose tissues with high LD abundance. While the topic of lipid-metabolism-targeting therapeutics is emerging in hematologic malignancies, LDs in the context of acute myeloid leukemia (AML) are still not yet well explored. Here we aim to establish an optimized protocol for isolating LDs in AML cells to aid future investigation of the roles of LDs in hematologic malignancies.

Methods:

Thirty million or more AML cells were collected by low-speed centrifugation. A snap-freeze step is employed to promote the release of intracellular contents. Homogenization was performed using a Dounce homogenizer in an organelle protective sucrose buffer. Firstly, the nuclear fraction was removed from cell homogenization by low-speed centrifugation (2000 x g). Floatation of LDs from the post-nuclear supernatant (PNS) were achieved by high-speed centrifugation (16000 x g). The resulting pellet contains crude mitochondria, the supernatant contains the cytosol, and the floating opaque-white layer contains LDs. Mitochondria and LDs fractions were separated and washed for downstream verification and analyses by SDS-PAGE immunoblotting and microscopy. Thus, this method enables the purification of not only LDs but also other organelles including mitochondria.

Results:

LDs and mitochondria were isolated from OCI-AML3 and MOLM13 cell lines. Immunoblotting of the LD surface protein perilipin-2 (PLIN2) and mitochondrial protein TOM20 was performed for molecular validation of isolated LD and mitochondrial fractions. High PLIN2 protein expression was found only in LDs samples, indicating the high purity of isolated LDs. The highest TOM20 protein expression was found in isolated mitochondria, with a lower signal in whole cell lysates and PNS.

The structure of isolated LDs was verified by confocal imaging using the neutral lipid dye BODIPY 493/503, yielding high fluorescence signal of ~1um-sized spherical structures. Additionally, PLIN2 was co-stained with BODIPY 493/503, suggesting the isolated lipid droplets retain intact phospholipid monolayer with surface proteins. Mass spectrometry-based lipidomic analysis of isolated LDs and mitochondria from AML is currently ongoing to demonstrate sufficient quality and purity of samples for high resolution lipidomic applications.

In summary, we have established an optimized protocol for isolating LDs in AML that avoids the usage of specialized instruments. Uniquely, this protocol enables collection of intact mitochondria, which are highly associated with LDs. The co-isolation of mitochondria allows investigation of the interplay between LDs and mitochondria, an established therapeutic target in AML. Our protocol provides a reliable solution for isolating LDs to explore potential uniqueness in lipid droplet biogenesis and its roles in AML.

Andreeff:Boehringer-Ingelheim: Honoraria; SentiBio: Current holder of stock options in a privately-held company, Honoraria, Research Funding; Ellipses: Research Funding; Chimerix: Current holder of stock options in a privately-held company; Oncolyze: Current holder of stock options in a privately-held company; Sellas: Honoraria, Research Funding; Glycomimetics: Honoraria; Aptose: Honoraria; Paraza: Honoraria; Daiichi-Sankyo: Research Funding; Syndax: Honoraria, Research Funding; Ona: Honoraria; Oxford Biomedical: Research Funding; Kintor Pharmaceutical: Research Funding; Roivant: Honoraria; Eterna: Current holder of stock options in a privately-held company, Honoraria, Research Funding.