Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.

Microfluidics for the detection of minimal residual disease in acute myeloid leukemia patients using circulating leukemic cells selected from blood.

We report a highly sensitive microfluidic assay to detect minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) that samples peripheral blood to search for circulating leukemic cells (CLCs). Antibodies immobilized within three separate microfluidic devices affinity-selected CLC subpopulations directly from peripheral blood without requiring pre-processing. The microfluidic devices targeted CD33, CD34, and CD117 cell surface antigens commonly expressed by AML leukemic cells so that each subpopulation's CLC numbers could be tracked to determine the onset of relapse. Staining against aberrant markers (e.g. CD7, CD56) identified low levels (11-2684 mL(-1)) of CLCs. The commonly used platforms for the detection of MRD for AML patients are multi-parameter flow cytometry (MFC), typically from highly invasive bone marrow biopsies, or PCR from blood samples, which is limited to <50% of AML patients. In contrast, the microfluidic assay is a highly sensitive blood test that permits frequent sampling for >90% of all AML patients using the markers selected for this study (selection markers CD33, CD34, CD117 and aberrant markers such as CD7 and CD56). We present data from AML patients after stem cell transplant (SCT) therapy using our assay. We observed high agreement of the microfluidic assay with therapeutic treatment and overall outcome. We could detect MRD at an earlier stage compared to both MFC and PCR directly from peripheral blood, obviating the need for a painful bone marrow biopsy. Using the microfluidic assay, we detected MRD 28 days following one patient's SCT and the onset of relapse at day 57, while PCR from a bone marrow biopsy did not detect MRD until day 85 for the same patient. Earlier detection of MRD in AML post-SCT enabled by peripheral blood sampling using the microfluidic assay we report herein can influence curative clinical decisions for AML patients.

Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor ß (TCRß) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRß clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRß repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRß clonotypes represented >17% of the entire TCRß repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRß clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRß clonotype sequences onto TCRß repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.