Explore
Dive into TROVO's various applications and see how labs are using TROVO to bypass the marker-dependency of Flow and the noise of bulk readouts.
TROVO: Bridging the gap between in viTRO and in viVO
Preprint now available!
From Yale University – the Katz Lab identifies high-performing CAR-T cells with efficacy in solid tumors, using TROVO in their screening and validation workflow.
See below for more info:
Nanobody MET CAR T cells show efficacy in solid tumors
Chen PH, Li Q, Deveraux S, Sohai D, Cha PC, Raghunandan R, Chen N, Li Y, Nguyen M, Stankewich MC, Morrow JS, Augert A, Yan QC, Katz SG. Nanobody MET CAR T cells show efficacy in solid tumors. bioRxiv [Preprint]. 2026 Jan 30:2026.01.27.702111. doi: 10.64898/2026.01.27.702111. PMID: 41659518; PMCID: PMC12873946.
Background
MET overexpression is associated with poor prognosis in many solid tumors due to its central role in tumor survival, invasion, metastasis, and chemoresistance. While targeting MET with antibody-drug conjugates has shown promising results, engineered cellular immunotherapeutic approaches have not been extensively explored. Compared to conventional single-chain variable fragments (scFv), naturally occurring single-domain antibodies consisting of variable heavy chains only (VHH or nanobodies) are smaller, retain high specificity, and exhibit remarkable biochemical stability. In this study, we tested the efficacy of MET-targeting VHH-CART (chimeric antigen receptor T cells).
Methods
We generated a panel of VHH-CARTs using mRNA electroporation. VHH-CART cells were evaluated in functional assays including cell binding avidity, cytokine production profiles, hydrogel microwell-based cellular kinetics, and in vitro cytotoxicity. We also assessed the therapeutic effectiveness of VHH-CART in an in vivo mouse model of metastatic triple negative breast cancer.
Results
Among the tested VHH, we identified those with intermediate avidity as most effective for in vitro tumor killing. Using the CD28 costimulatory domain with the VHH augmented cytotoxicity, yet the CARTs maintained selectivity as activation required a minimum antigen density threshold. Mechanistically, VHH-CARTs demonstrated low tonic signaling, high avidity, potent cytokine production, and rapid tumor killing kinetics. When administered in an mRNA format, VHH-CARTs exhibited potent and prolonged tumor growth control in an in vivo metastatic model of triple negative breast cancer.
Conclusion
Taken together, these results demonstrate that VHH-CARTs exhibit robust tumor selectivity and potent therapeutic efficacy both in vitro and in vivo. Given their demonstrated potency and specificity, VHH-CART represent a promising immunotherapeutic strategy for targeting MET-overexpressing solid tumors.
Key Takeaways:
High performance discovery with precision: Traditional scFv-based CAR-Ts often struggle with stability and tonic signaling in solid tumors. This study demonstrates the efficacy of a new class of Nanobody (VHH) MET-targeting CAR-Ts, using TROVO’s unique screening platform to identify the most effective candidates.
Functional Kinetics at Scale: The researchers utilized hydrogel microwell-based cellular kinetics to evaluate cytotoxicity and T-cell behavior in real-time.
The “Best” Clones: By tracking rapid tumor-killing kinetics, the study identified that intermediate avidity clones were actually the most effective for in vitro killing—a finding that requires the precise functional ranking TROVO provides.
Low Tonic Signaling: The VHH-incorporated CAR-Ts showed superior biochemical stability and favorable cytokine profiles compared to traditional designs.
Proven in TNBC Models: Demonstrated potent and prolonged growth control in metastatic triple-negative breast cancer (TNBC) models both in vitro and in vivo.
This research highlights how TROVO’s ability to monitor individual cell-killing kinetics is essential for optimizing CAR-T design against difficult-to-target solid tumor antigens.
Case Studies
T cell clone selection based on tumor co-cultures by TROVO System
Stop Betting on Short-Term Killing Bursts.
Standard activation markers don’t always correlate with real-world clinical performance. Most assays stop too early, missing the “persistence wall” where clones drop off. Distinguish between true persistence clones and short-term killers in as little as 3 days.
Massive Clonal Search: Simultaneously monitor and pick from up to 36,000 clones in parallel on a single six-well plate.
Long-term assays: Use near-vivo tumor feeding strategies to track cytotoxicity and persistence kinetics live for up to 12 days.
Retrieve the Best Cells: Use fluidics-free, light-induced capture to pull specific, viable clones directly for downstream sequencing or expansion.
Why it matters: The TROVO system provides a clear ranking of functional avidity, ensuring you only move forward with the clones that have actual staying power.
Capturing and retrieval workflow
- Microwells of interest are captured by selecting a grouping of microwells from a long-term T cell vs cancer cell assay.
- Microwells are encapsulated in a biocompatible gel, and the unwanted cells can be washed away.
- The capture gel can be enzymatically (or non-enzymatically) degraded to release the cells for downstream processing.
- Retrieved cells are viable and ready for downstream sequencing or expansion
Capturing and expanding T cells
T cells of interest were captured using TROVO and then expanded in culture. The cells maintained normal phenotypic expression and functionality.
High-throughput B Cell Screening
Stop Chasing False Positives.
Single-readout screenings often get stuck on sticky targets or non-specific IgGs. TROVO enables a two-round, multi-signal screening process to identify “double-positive” clones before you ever commit to sequencing.
Native Conformation Matters: Identify antibodies that bind to both small target peptides and full-length proteins in their native conformation.
Speed to Discovery: Go from seeding PBMCs to identifying and retrieving secretor clones in under 2 hours.
High-Throughput Mapping: Screen thousands of hydrogel microwells on a single 6-well plate to link individual cell behavior to secretion.
Cost-Effective Precision: Achieve the single-cell resolution of complex microfluidic platforms without the “nanopen” price tag or complexity.
Why it matters: Rapidly identify, capture, and retrieve high-affinity B cells ready for RT-PCR and sequencing.
Approach and Methods
- Immunize animals and obtain PBMCs
- Coat streptavidin beads with biotinylated target peptide
- Seed PBMCs and beads with target peptide into microwells. PBMCs are pre-stained with green fluorescence. Incubate for 2 hours. Positive antibody secreted by B cells will bind to the target peptide on beads.
- Remove media with excess antibody. Add anti-rabbit IgG antibody with red fluorescence for staining. Image the plate with TROVO. Beads that bind to positive antibody will be stained red along with B cells.
- Identify microwells with positive antibody production in the TROVO software by selecting microwells with high red fluorescent signal intensity on a distribution chart and/or looking at the images.
- Capture cells in positive microwells with TROVO: a liquid capture gel (gelatin-based hydrogel) is added onto cells. In selected microwells, the capture gel is solidified locally by photo-crosslinking to immobilize cells of interest. Uncaptured cells and beads are then washed away.
- Captured cells are retrieved by dissolving solid capture gels in a tube and are ready for downstream analysis such as sequencing.
Precision scRNA-seq for Rare & Fragile Samples
When Bulk Isn’t Good Enough. If you have millions of cells, use a magnet. But when you are working with rare clinical biopsies or fragile edited lines where every single cell counts, you can’t afford the sample loss or “blind” nature of bulk enrichment.
Image-Verified Selection: Unlike MACS, you aren’t just pulling everything with a certain surface marker; you are visually confirming viability and morphology before capture.
High-Stakes Low Input: Specifically designed for samples as small as $10^4$ cells where traditional sorters or bulk kits result in too much “dead volume” or sample loss.
No Fluidic Stress: Avoid the high-pressure environment of FACS that can alter the transcriptome of fragile cells.
Zero Clog Risk: A fluidics-free process that handles tissue debris and clumps that would immediately shut down a traditional flow sorter.
Proven Data Quality: Achieves higher % fraction reads in cells compared to unenriched controls across breast, lung, and colon tissues.
Fast Workflow: Go from raw tissue to a single live cell suspension in under 4 hours for 3 samples.
Webinars
WEBINAR: Integrated High-Throughput Platform for Function Profiling of Myeloid Cells in Models of Pancreatic Cancer
Dr. Won Jin Ho of Johns Hopkins discusses how to utilize TROVO to link real-time functional killing kinetics with high-plex Imaging Mass Cytometry (IMC) by performing in situ proteomic profiling of rare clones directly within hydrogel microwells.
Click the Thumbnail to watch the full length webinar:
WEBINAR: Functional Selection of CAR-T Clones from 2D and 3D Brain Tumor Cocultures
See how scientists at City of Hope use TROVO to monitor T cell behavior in 2D and 3D brain tumor co-cultures, then directly retrieve selected clones for downstream analysis.
Click the Tumbnail to watch the full length webinar:
