Page 12 - EJMO-9-2

Eurasian Journal of
Medicine and Oncology Single-cell sequencing for lung cancer

lung cancer. As targeted therapy and immunotherapy can be achieved using methods such as limiting dilution,
6
have emerged as viable options, there has been improved micromanipulation, laser capture microdissection (LCM),
progression-free survival (PFS) and overall survival (OS) in fluorescence-activated cell sorting (FACS), and microfluidic
selected patients. Despite advances in treatment, the late- cell sorting. Capturing and isolating cells are the first and
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stage diagnosis, high aggressiveness, frequent metastasis, most critical step in single-cell sequencing. During this
and drug resistance continue to result in poor prognosis process, it is essential to maintain cell integrity to prevent
of lung cancer. 8 cell loss or rupture, as these events can result in nucleic
acid degradation, ultimately compromising the quality
Cancers are composed of complex multicellular
structures that include not only malignant cancer cells and accuracy of downstream genomic, transcriptomic, or
but also various epithelial, immune, and stromal cells. epigenomic analyses. Limiting dilution involves the serial
This complex and heterogeneous system is commonly dilution of a cell suspension to progressively reduce its
referred to as the tumor microenvironment (TME). concentration, aiming to achieve single-cell capture in each
9,10
The severity of cancer largely depends on factors such as well of a multiwell plate or sample chamber. This method
is simple and cost-effective; however, it is characterized by
the regulation of immune responses within the TME, the low throughput and a significant risk of rare cell depletion,
remodeling of the extracellular matrix, and the process of which restricts its utility in clinical and high-throughput
neovascularization. Traditional bulk sequencing methods research. Micromanipulation uses a micropipette under
typically rely on analyzing the average gene expression microscopic guidance to aspirate single cells. It is low-
levels within large cell populations, which can mask cellular
heterogeneity. In contrast, single-cell sequencing enables throughput, labor-intensive, and suited for isolating small
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the direct analysis of gene expression at the individual cell numbers. Similarly, LCM uses a focused laser beam
under a microscope to melt a thermoplastic ethylene-
cell level, facilitating the identification of distinct cellular vinyl acetate membrane, creating localized adhesion to
subpopulations. Single-cell sequencing technologies capture target cells, and while LCM offers exceptional
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have revolutionized lung cancer research, offering new spatial resolution that allows for the precise identification
hope for more precise and effective treatments. This and isolation of specific cell populations within complex
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review highlights the transformative impact of single- environments. However, like micromanipulation, it
cell technologies in advancing lung cancer research and remains inefficient and time-consuming. FACS primarily
therapy.
involves labeling the surface or interior of cells with specific
2. Advances of single-cell sequencing fluorescent dyes, and a flow cytometer emits a laser beam
technologies to excite these fluorescent labels, enabling the separation
of different cell types based on their distinct fluorescence
Single-cell sequencing is a high-resolution, high- signals. Moreover, FACS can sort target cells with very low
throughput technology that analyzes genetic material while levels of marker expression and rapidly process and isolate
eliminating batch effects and technical noise, enabling large numbers of single cells, making it an ideal method
accurate characterization of cellular heterogeneity. for preparing large-scale single-cell sequencing samples.
Furthermore, there are low expression abundance genes On the other hand, microfluidic cell sorting technology
that play an important role in tumor progression but are offers several advantages, including high throughput, high
often overlooked by traditional sequencing technologies. efficiency, and low reagent costs. However, it requires a high
Single-cell sequencing can detect these genes, which help degree of cell size uniformity, and its associated equipment
guide personalized therapy for cancer patients in clinical and consumables are relatively expensive, making it
practice, as well as for research into the mechanisms of particularly well-suited for applications demanding high-
disease onset. This technology has advanced rapidly in precision and high-throughput single-cell analysis.
recent years, with notable milestones including the first Single-cell sequencing encompasses a wide range of
single-cell transcriptomic analysis in 2009, the initial DNA analyses, including single-cell genome sequencing, which
sequencing of individual human cancer cells in 2011, investigates genetic variations and mutations; single-
and the first single-cell exome sequencing experiment in cell transcriptome sequencing, which examines RNA
2012. 12-14 expression profiles; single-cell epigenome sequencing,
The standard workflow for single-cell sequencing which explores DNA modifications and chromatin
comprises tissue preparation, single-cell isolation, library accessibility; and single-cell multi-omics sequencing,
preparation, sequencing, and data analysis. Among these which integrates multi-omics data across molecular layers.
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steps, single-cell isolation is a critical step, as it determines Other single-cell sequencing techniques also contribute to
the accuracy and amount of the amplified material. It advancing research, as shown in Figure 1.

Volume 9 Issue 2 (2025) 4 doi: 10.36922/ejmo.6883