Publications
The CellChorus platform is the only approach that can associate motility, contact, killing, cytokine secretion and other dynamic readouts over time for individual cells and cell-cell interactions at high throughput. This approach is called dynamic single-cell analysis. The platform has broad application across antibodies, cell therapies, vaccines and other technologies, as shown in the publications below from leading journals such as Blood, Clinical Cancer Research, Nature Immunology, the JITC, Journal of Immunology, and Science Advances.
Authors from the University of Texas MD Anderson Cancer Center, the University of Houston, the Technical University of Munich, Baylor College of Medicine, the University of Nebraska Medical Center at Omaha, and CellChorus published data in Cancer Discovery describing the design of a novel CAR27 NK therapy with CD28 costimulation for patients with CD70+ hematologic malignancies and solid cancers. The TIMING™ platform quantified the difference in serial killing between the CARs harboring varying intracellular domains. Approximately three times more NK cells were able to kill more than one target cancer cell when the best intracellular domain was used, as shown below.
The authors present hydroporation (Indee Labs, Berkeley, CA) as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7 to 2x higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion, in addition to improved CAR-T yield by day 5 post-transfection. The authors demonstrated scaled-up hydroporation can process 5 x 108 cells in less than 10 seconds, showcasing the platform as a viable solution for high-yield, precise CAR-T cell manufacturing with the potential for improved therapeutic outcomes. TIMING data showed that CAR-T cells manufactured with hydroporation have superior motility and resistance to activation-induced cell death (AICD).
Researchers published data in Nature Cancer identifying a subset of CAR T-cells within axicabtagene ciloleucel (axi-cel) products that result in patient responses. Key results—including single-cell migration, killing, and serial killing—were generated with the company’s Time-lapse Imaging In Nanowell Grids (TIMING™) platform, which applies artificial intelligence (AI) to evaluate cells over time.
Mechanistic studies using TIMING based single-cell reporter assays demonstrated that redundancy is important for efficacious T cells. Leukemic targets are sensitive to multiple granzymes whereas solid tumors are sensitive to granzyme and Fas ligand.
Hydroporation is an alternative way to deliver the CRISPR/Cas9 gene-editing machinery into the T cells. Hydroporation uses a gentle fluid force to permeabilize the cell membrane, allowing the gene-editing components to enter the T cells. Using a Time-lapse Imaging Microscopy in Nanowell Grids (TIMING) assay, scientists compared CAR-T cells from five donors after processing them with either hydroporation or nucleofection.
To improve the detection of apoptosis, researchers directly detected apoptotic bodies in a label-free manner. The trained network identified nanowells containing apoptotic bodies with 92% accuracy and predicted the onset of apoptosis with an error of one frame (5 min/frame). The apoptotic body segmentation yielded an IoU accuracy of 75%, allowing associative identification of apoptotic cells.
The paper presents an integrated methodology to identify single cancer cells with differences in extracellular vesicle (EV) secretion and further link EV secretion to their transcriptome. The authors elucidated a four gene signature that is associated with EV secretion and correlated with severity of cancer progression. In the publication, the researchers applied a version of TIMING to profile single-cell EV secretion and retrieve individual cancer cells for further molecular and functional analysis.
In less than 18 months, the FuseBio team developed a TCE lead candidate that targets ROR1 (FUSE394). The data presented at SITC 2022 demonstrated that FUSE394 is potent and successfully decouples anti-tumor activity from cytokine release and T cell exhaustion. TIMING data from the CellChorus early access program demonstrated that FUSE394 preserves T cell motility and healthy morphology and promotes 50% more synapse formation than a non-decoupled control.
Profiling of TILs used for human ACT and their autologous tumor cells included function-based single-cell profiling by timelapse imaging microscopy in nanowell grids (TIMING); multi-omics using RNA-sequencing and proteomics; metabolite inference using genome-scale metabolic modeling, and pulse-chase assays based on confocal microscopy to profile the uptake and fate of fatty acids (FA). Phenotypically, the ACT TILs from both responders (Rs) and nonresponders (NRs) were comprised of predominantly effector memory T cells (TEM cells) and did not express a high frequency of programmed death ligand-1 (PD-L1) and showed no differences in TCR diversity. The results demonstrate that while tumor cells from both Rs and NRs are efficient at uptaking FAs, R TILs are significantly more efficient at utilizing FA through fatty acid oxidation (FAO) than NR TILs under nutrient starvation conditions.
Researchers applied the TIMING platform to test NK-mediated cytotoxicity at single-cell resolution as part of research showing that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness.
Researchers applied the CellChorus TIMING platform to evaluate interactions between thousands of tumor cells and CAR-T cells that were to be infused into patients to preview how the CAR-T cells being given to patients can eradicate tumor cells after infusion. The researchers robotically selected super-killer T cells for transcriptional profiling to characterize the molecular properties of the CAR-T cells with the best features for eradicating tumors.
Time-lapse imaging microscopy in nanowell grids (TIMING™) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering.
By simultaneously evaluating thousands of individual interactions between T cells and target cells bearing virally derived peptides, the company’s artificial intelligence-powered TIMING™ platform reveals individual T cells that are capable of polyfunctionality based on killing, serial killing, and secretion of the cytokine interferon gamma (IFNγ).
Time-lapse imaging in nanoliter wells was applied to understand the kinetics of translocation at the single‐cell level and to enable tracking of the same individual cells. The micromesh array contains nanoliter wells that enabled imaging protein translocation dynamically.
Understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. These 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.
Using time-lapse imaging microscopy to monitor T cell-mediated tumor killing at the single cell level to gain more complete understanding of the kinetics of killing in studies that reveal two distinct types of immune resistance regulators and demonstrate their potential as therapeutic targets to improve the efficacy of immunotherapy.
Immune cells such as T cells and natural killer cells, and target cells such as NALM6, K562 and EL4, were incubated in PDMS nanowell arrays and imaged using time-lapse fluorescent microscopy. The proposed cell segmentation and tracking algorithms allowed automated quantification of cell pairs, cell location, morphology, interactions and movement as well as cell viability, without the need for manual processing. The result revealed that cytotoxic T cells have higher motility than noncytotoxic T cells, both before and during synapse formation.
We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.
CD19/20/22CAR T-cells killed CD19(−) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.
By utilizing both traditional blob detection to generate binary mask labels from the stained channel images and the deep learning Mask RCNN model to train a detection and segmentation model, we managed to segment nuclei based only on phase images. The detection average precision is 0.82 when the IoU threshold is to be set 0.5. And the mean IoU for masks generated from phase images and ground truth masks from experts is 0.735. Without any ground truth mask labels during the training time, this is good enough to prove our hypothesis. This result enables the ability to detect nuclei without the need for exogenous labeling.
This paper proposes an efficient variant of capsule networks (CapsNets) as an alternative to CNNs. Extensive experimental results demonstrate that the proposed CapsNets achieve competitive performances in target cell apoptosis classification, while significantly outperforming CNNs when the number of training samples is small. To utilize temporal information within microscopy videos, we propose a recurrent CapsNet constructed by stacking a CapsNet and a bi-directional long short-term recurrent structure. Our experiments show that when considering temporal constraints, the recurrent CapsNet achieves 93.8% accuracy and makes significantly more consistent prediction than NNs.
Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that the master metabolic regulator AMP kinase (AMPK) is required for CAR+ T cells with high motility. We used a xenograft leukemia mouse model (CD19+ NALM-6) and validated that the motile cells have enhanced persistence and superior anti-cancer effect in vivo compared to the parental un-sorted population. Collectively, our multi-dimensional results demonstrated that persistent motility is a selectable biomarker of expanded CAR+ T cell bioactivity.
On a desktop computer, TIMING 2.0 takes 5 s/block/image frame, four times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number = 0.821). A graphical user interface provides the ability to inspect the video analysis results, make corrective edits efficiently (one-click editing of an entire nanowell video sequence in 5–10 s) and display a summary of the cell killing efficacy measurements.
Genetically engineered T cells that express chimeric antigen receptors (CAR+) are heterogeneous and thus, understanding the immunotherapeutic efficacy remains a challenge in adoptive cell therapy. We developed a high-throughput single-cell methodology, Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to monitor interactions between immune cells and tumor cells in vitro. Using TIMING we demonstrated that CD4+ CAR+ T cells participate in multi-killing and benefit from improved resistance to activation induced cell death in comparison to CD8+ CAR+ T cells. For both subsets of cells, effector cell fate at the single-cell level was dependent on functional activation through multiple tumor cells.
A comprehensive understanding of the polyfunctionality of T lymphocytes in ICI or adoptive cell transfer (ACT), at single-cell resolution, will quantify T-cell properties that are essential for therapeutic benefit. We briefly highlight several emerging integrated single-cell technologies focusing on the profiling of multiple properties/functionalities of T cells. We envision that these tools have the potential to provide valuable experimental and clinical insights on T-cell biology, and eventually pave the road for the discovery of surrogate T-cell biomarkers for immunotherapy.
CD8+BTLA- TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA- T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened "serial killing" capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation. Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151-64. ©2017 AACR.
We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.
In aggregate, these results demonstrate the utility of our TIMING single cell methodology in uncovering not only the dynamic profile of T-cell behavior but also the ability to identify subpopulations of T-cell with enhanced polyfunctionality. Our studies support the use of motility as a surrogate and selective marker of higher CAR+ T cell bioactivity. These results also open up avenues to molecularly engineer T cells for an increased motility that could translate to better in vivo outcomes.
Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact.