Circulating tumor cells (CTCs) are malignant cells released into the bloodstream 
with the potential to form metastases in secondary sites. These cells, acquired 
non-invasively, represent a sample of highly relevant tumor tissue that is an 
alternative to difficult and low-yield tumor biopsies. In recent years, there 
has been growing interest in genomic profiling of CTCs to enable longitudinal 
monitoring of the tumor's adaptive response to therapy. However, due to their 
extreme rarity, genotyping CTCs has proved challenging. Relevant mutations can 
be masked by leukocyte contamination in isolates. Heterogeneity between 
subpopulations of tumor cells poses an additional obstacle. Recent advances in 
single-cell sequencing can overcome these limitations but isolation of single 
CTCs is prone to cell loss and is prohibitively difficult and time consuming. To 
address these limitations, we developed a single cell sample preparation and 
genome sequencing pipeline that combines biophysical enrichment and single cell 
isolation using laser capture microdissection (LCM). A key component of this 
process is the encapsulation of enriched CTC sample in a hydrogel matrix, which 
enhances the efficiency of single-cell isolation by LCM, and is compatible with 
downstream sequencing. We validated this process by sequencing of single CTCs 
and cell free DNA (cfDNA) from a single patient with castration resistant 
prostate cancer. Identical mutations were observed in prostate cancer driver 
genes (TP53, PTEN, FOXA1) in both single CTCs and cfDNA. However, two 
independently isolated CTCs also had identical missense mutations in the genes 
for ATR serine/threonine kinase, KMT2C histone methyltransferase, and FANCC DNA 
damage repair gene. These mutations may be missed by bulk sequencing libraries, 
whereas single cell sequencing could potentially enable the characterization of 
key CTC subpopulations that arise during metastasis.