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random priming (SMART-Seq Stranded Kit, Takara Inc.) have been beneficial in extracting reliable gene
expression information from poor quality RNA from FFPE samples.

Single-cell isolation by mechanical or enzymatic dissociation
Conventionally, tumor tissues are dissociated into single cells by mechanical dissociation (e.g., meshing,
trituration with a pipette/tip) [40-42] or by enzymatic dissociation [43-45] or a combination of both. Enzymes such
[47]
[46]
[41]
as collagenase , DNase , trypsin are commonly used for dissociating the cell-cell contacts and the ex-
tracellular matrix to generate single cell suspensions. The various dissociation methods may largely differ in
their yield of viable cells [48,49] , limiting their downstream applications. Therefore, tumor dissociation proto-
cols optimized for different tumor types is a key gap that needs to be addressed for high-throughput single-
cell analysis.

Single-cell isolation by LCM
To preserve the native properties of tumor cells shaped by the complex tumor microenvironment, LCM can
be used to isolate tumor cells directly from sectioned tissues. It is a method to procure subpopulations of
tissue cells under direct microscopic visualization by cutting away unwanted cells and obtain histologically
[50]
pure cell population [Figure 4A] . A variety of downstream applications exist for microdissected cells such
as DNA genotyping, RNA transcript profiling or cDNA library generation. Even though the majority of the
studies take advantage of approximately 100-1000 dissected cells, LCM can also be used for single-cell isola-
tion directly [51-53] .

Isolation of rare CTCs
Currently, tumor biopsies are obtained to establish the diagnosis and determine whether the predictive bio-
markers are consistent between the primary and the metastatic tumors. However, getting biopsies is invasive,
expensive and not always feasible. Additionally, it is difficult to get biopsies of metastatic lesions or get repeat
biopsies for difficult to access tumors. Analysis of disseminated tumor cells (DTCs) is a useful alternative to
tumor biopsy in clinical setting for patient stratification, therapy selection and monitoring drug resistance
[54]
during the course of treatment . DTCs originate from the primary or metastatic tumors, extravasate into
the bloodstream or lymphatics and carry genomic profiles of tumors from which they originate [55,56] . Dis-
[54]
seminated cancer cells are usually detectable as CTCs in the circulation . A small fraction of them that
[54]
have reached to a secondary organ such as the bone marrow and lymph nodes is termed as DTCs . Though
for certain cancers, the presence of DTCs in distant organs is a strong predictive marker for cancer metasta-
sis, the challenge with DTC isolation due to the invasive procedure is a deterrent in studying this population
by single-cell sequencing. On the contrary, CTCs circulating in patient blood has proved to be a valuable
[57]
resource for diagnostic and prognostic biomarker discovery , although distinguishing a DTC from a pool
of CTCs is challenging.

CTCs contain signatures of tumor heterogeneity and carry the spectrum of somatic mutations present in
both the primary and metastatic lesions in different cancers [55,56,58] . Because conventional molecular analysis
of whole tumors provides genotype/phenotype information of the dominant clones or aggregated informa-
tion of all clones, single-cell analysis of the CTCs is a potential solution to investigate heterogeneity. By iso-
lating and sequencing single CTCs in the blood, it is possible to measure somatic mutations that are present
at both the primary and metastatic tumor sites without performing an invasive core biopsy [59,60] . Two types
of isolation methods - microfluidic-based and immunoaffinity-based are used for capturing CTCs.

Microfluidic-based cell isolation
The microfluidic platform can be used for single-step isolation of CTCs from unprocessed blood speci-
mens [61,62] . As whole blood flows through the CTC-chip, individual CTCs are captured onto the microposts
[63]
coated with anti-EpCAM antibody. This type of microfluidic processing enables high yield of pure CTCs .
Subsequent studies demonstrated the ability and reliability to isolate CTCs from patients with metastatic