3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B‑Cell Acute Lymphoblastic Leukemia

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B‑Cell Acute Lymphoblastic Leukemia

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B‑Cell Acute Lymphoblastic Leukemia

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling

Additional file 1 of PERK/NRF2 and autophagy form a resistance mechanism against G9a inhibition in leukemia stem cells

Additional file 1: Table S1. Synergistic effects of the G9a and PERK inhibitor on apoptosis of primary acute myeloid LSCs. Figure S1. Effects of treatment with the PERK inhibitor GKS2606414 for 48 h in the presence or absence of 10 μM BIX-01294, on apoptosis. Figure S2. Effect of PERK inhibition on BIX-01294- induced apoptosis in KG1a cells. (A) KG1a cells were treated with 10 μM BIX-01294 in the presence or absence of the PERK inhibitor GSK260641 at 5 μM. After incubation for 48 h, the apoptotic fraction was measured using flow-cytometric analysis. (B) KG1a cells were transfected with PERK siRNA or scrambled siRNA as described in the Materials and Methods and then treated with 10 μM BIX-01294 for 48 h. Figure S3. Effects of treatment for 48 h with the NRF2 inhibitor brusatol in the presence or absence of 10 μM BIX-01294 on apoptosis. Figure S4. Effects of PERK inhibition in the absence or presence of 2 nM bafilomycin A1 on autophagy induction

Supplementary Data from Adaptive Natural Killer Cells Facilitate Effector Functions of Daratumumab in Multiple Myeloma

Supplementary Figures 1-11</p

Supplementary Data from Adaptive Natural Killer Cells Facilitate Effector Functions of Daratumumab in Multiple Myeloma

Supplementary Tables 1-2</p