Arc-well: a new method for single-cell DNA-seq analysis of formalin-fixed paraffin-embedded samples

Clinical research is vital for developing new cancer therapies, clinical trials, and monitoring treatment outcomes. These studies rely on patient samples such as biopsies taken at the time of diagnosis and during treatment. The value of comparing the actual tumor with the tissue obtained in the first biopsy for a patient undergoing a lengthy treatment with a lack of response is enormous. This comparison allows clinicians to understand how the tumor evolved, how different it is from the primary tumor, and even which treatment is better for the modified tumor. However, the availability, storage mode, and previous sample processing can limit their use for genomic analysis.

There is a technical challenge to performing the genomic profiling of tumor tissue as formalin-fixed paraffin-embedded (FFPE) blocks at single-cell resolution. The available technologies can analyze only a few samples each time or a number of samples for specific targets. These limitations were recently overcome by researchers at the MD Anderson Cancer Center working with the ICELL8 cx Single-Cell System from Takara Bio.

Wang, K. et al. (2023) developed a novel high-throughput method, called Arc-well, that enabled the analysis of formalin-fixed paraffin-embedded (FFPE) samples. Arc-well is an automated and cost-effective method based on whole-genome amplification for sequencing single-cell DNA of samples archived for more than 30 years.

The researchers profiled over 40,000 single cells from two cell lines, frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors to validate their method and compare the quality control (QC) metrics. An essential step of Arc-well is the single-cell DNA-seq library preparation, for which they used the ICELL8 cx system for dispensing, integrated imaging, and single-cell selection. After nuclei separation, the samples were automatically dispensed into ICELL8 350v chips using ICELL8 cx system. Then, the nanowells were imaged with the CellSelect Software (Takara Bio) to identify the wells containing single nuclei. These steps are vital for method optimization and reducing the cost of expensive reagents by lowering reaction volume from microliter-scale to nanoliter-scale. The method showed reduced variance of read count distributions, increased genome coverage performance, and better metrics than data generated from non-fixed samples. Moreover, their comparisons showed that older samples were not correlated with poor QC metrics.

The researchers also performed longitudinal analysis on matching initial ductal carcinoma in situ (DCIS) and recurrent diseases collected more than 10 years apart. They identified persistent subclones between the initial DCIS and recurrent diseases, which helped to understand and propose evolutionary models of tumor progression. They showed that the primary tumors shared genomic lineages with persistent subclones in the recurrences.

The ICELL8 cx system enabled an easy setup of nanoliter-scale, high-throughput, automated, and optimized single-cell analysis of genomic profiles. The ICELL8 cx system was vital to select the nanowells with high-quality single-cells to ensure the high quality of the final scDNA-seq libraries. Single-cell DNA-seq gave them detailed information on clonal substructure in tumor-paired samples and confirmed a common genetic lineage between primary and recurrent cancers.

Being able to use these samples represents a significant impact on clinical research. Arc-well is helpful for researchers who study tumor progression and heterogeneity and need to perform subclone analysis on FFPE samples collected from tumor tissues. The method is highly valuable for scientists who do not want to waste archived FFPE samples that can hide the answers to therapeutic questions in cancer research. 

References

Wang, K. et al. Archival single-cell genomics reveals persistent subclones during DCIS progression. Cell 186, 3968-3982.e15 (2023).