Precision DNA Mixture Interpretation with Single-Cell Profiling

Wet-lab based studies have exploited emerging single-cell technologies to address the challenges of interpreting forensic mixture evidence. However, little effort has been dedicated to developing a systematic approach to interpreting the single-cell profiles derived from the mixtures. This study is the first attempt to develop a comprehensive interpretation workflow in which single-cell profiles from mixtures are interpreted individually and holistically. In this approach, the genotypes from each cell are assessed, the number of contributors (NOC) of the single-cell profiles is estimated, followed by developing a consensus profile of each contributor, and finally the consensus profile(s) can be used for a DNA database search or comparing with known profiles to determine their potential sources. The potential of this single-cell interpretation workflow was assessed by simulation with various mixture scenarios and empirical allele drop-out and drop-in rates, the accuracies of estimating the NOC, the accuracies of recovering the true alleles by consensus, and the capabilities of deconvolving mixtures with related contributors. The results support that the single-cell based mixture interpretation can provide a precision that cannot beachieved with current standard CE-STR analyses. A new paradigm for mixture interpretation is available to enhance the interpretation of forensic genetic casework.

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Faculty Opinions recommendation of Single cell profiling of potentiated phospho-protein networks in cancer cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020412.323536 ◽

2005 ◽

Author(s):

Alfonso Martinez Arias

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Protein Networks ◽

Single Cell Profiling

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Faculty Opinions recommendation of Single cell profiling of potentiated phospho-protein networks in cancer cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020412.232570 ◽

2004 ◽

Author(s):

James Woodgett

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Protein Networks ◽

Single Cell Profiling

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Faculty Opinions recommendation of Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732844079.793548900 ◽

2018 ◽

Author(s):

Yann Herault

Keyword(s):

Spinal Cord ◽

Single Cell ◽

Mouse Brain ◽

Single Cell Profiling ◽

Developing Mouse Brain ◽

Brain And Spinal Cord

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Faculty Opinions recommendation of Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732909626.793544340 ◽

2018 ◽

Author(s):

Bruce Appel

Keyword(s):

Single Cell ◽

Cell Types ◽

Vertebrate Brain ◽

Single Cell Profiling

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Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization

Nature Neuroscience ◽

10.1038/s41593-020-00789-y ◽

2021 ◽

Author(s):

Ana Rita Pombo Antunes ◽

Isabelle Scheyltjens ◽

Francesca Lodi ◽

Julie Messiaen ◽

Asier Antoranz ◽

...

Keyword(s):

Single Cell ◽

Myeloid Cells ◽

Disease Stage ◽

Single Cell Profiling

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Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001877 ◽

2021 ◽

Vol 9 (3) ◽

pp. e001877

Author(s):

Irfan N Bandey ◽

Jay R T Adolacion ◽

Gabrielle Romain ◽

Melisa Martinez Paniagua ◽

Xingyue An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Adoptive Cell Therapy ◽

Population Heterogeneity ◽

Serial Killer ◽

Car T Cells ◽

Car T ◽

Single Cell Profiling ◽

Killer T Cells

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

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Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Nature Communications ◽

10.1038/s41467-021-21168-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Tracy Rabilloud ◽

Delphine Potier ◽

Saran Pankaew ◽

Mathis Nozais ◽

Marie Loosveld ◽

...

Keyword(s):

Targeted Therapy ◽

T Cell ◽

Single Cell ◽

Remission Rate ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

T Cell Therapy ◽

Car T Cell Therapy ◽

Car T ◽

Single Cell Profiling

AbstractChimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

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