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[56]
biomarker of CTC-based cancer diagnostics . In pancreatic cancer, pancreas epithelial cells can be pres-
ent in the blood at pre-cancerous stages in pancreatic ductal adenocarcinoma patients [114] . In another study,
single-cell sequencing analysis on CTCs obtained from pancreatic ductal adenocarcinoma patients identi-
fied a macrophage-pancreatic tumor cell fusion product with high proliferative and metastatic potential [115] .
These studies suggest that early detection of these pancreatic epithelial cells in the blood stream can serve as
an important diagnostic tool for pancreatic cancer detection [114,115] .

The treatment of glioblastomas, an aggressive type of brain tumor has benefited from single-cell sequenc-
ing because of a high degree of tumor heterogeneity harboring a diverse population of cells with a large
[43]
spectrum of stemness, differentiation states, and variable proliferative capacity . By applying single-cell
sequencing to EGFR-amplified glioblastomas, novel EGFR truncation variants were identified [116] . In vitro
and in vivo functional studies revealed that a specific EGFR variant (EGFRvII, deletion of exons 14 and 15)
was sensitive to EGFR inhibitors, which are currently in clinical trials [116] . In chromosomally unstable B cell
leukemia patients, different degrees of karyotypic abnormalities were detected by single-cell whole genome
sequencing, which bulk sequencing failed to detect. Because karyotypic abnormalities associate with poor
clinical outcome in multiple cancers [102] , the degree of karyotypic anomalies assessed by single-cell sequenc-
ing can be utilized as an important readout for stratifying patient risk [117] . Single-cell analysis has identified
novel mutations in JAK2-negative myeloproliferative neoplasm such as SESN2 and NTRK1, chronic lympho-
cytic leukemia such as LCP1 and WNK1 and chromosomal abnormalities in melanoma such as chromo-
somal 12 amplification [78,113,118] , opening up opportunities to target these neoplasms. For example, NTRK1
encodes a tyrosine kinase receptor and inhibitors are available to target its NTRK1 gene fusions that results
in constitutive activation of the kinase [119] . For patients who are JAK2 mutation negative but harbor NTRK1
mutation, it is tempting to speculate that NTRK1 can be a target for the treatment of myeloproliferative neo-
plasm.

Disease monitoring and prognostic biomarkers
Cancer heterogeneity in part is driven by selection pressure that arises during drug treatment. Capturing
this dynamic heterogeneity at the genetic and cellular composition level prior to, during, or post-treatment is
crucial in assessing drug efficacy and predicting patient survival. Single-cell analysis is an extremely power-
ful tool to capture the dynamic events at a molecular level for disease monitoring and in predicting prognos-
tic biomarkers. Below are few examples of the application of single-cell sequencing in developing prognostic
and predictive biomarkers.

CTC analysis
Single-cell analysis of CTCs can provide prognostic markers in several cancers. Microfluidics-based RNA
sequencing has aided identification of CTC clusters held together by the cell junction component plakoglo-
bin that mediate intercellular adhesion. Presence of high levels of CTC clusters over single CTCs correlated
with poor prognosis indicating their role in the metastatic spread of cancer [120] . Indeed, heterogonous expres-
sion of plakoglobin in the primary tumor supports the evidence that tightly adhered groups of cells from
the primary tumors serve as the precursors to CTC clusters in circulation. Thus, single-cell identification of
plakoglobin-positive clonal cell populations of tumor cells in conjunction with the presence of CTC clusters
in the patient blood is a potent prognostic marker of breast cancer metastasis [120] .

TCR repertoire analysis
Anti-tumor immunity is largely driven by antigen-specific CD8 T cells, which recognize tumor-derived
neoantigenic peptides complexed with human leukocyte antigen also referred to as major histocompatibil-
ity complex (MHC) in mouse, to mount an anti-tumor immune response [121] . Adoptive cell therapy using
autologous tumor infiltrating lymphocytes (TILs) has been shown to be effective for the treatment of mul-
tiple cancers [122,123] . The anti-tumor effects observed post T cell therapy are associated with the activation of