Live-cell Pick-Seq (LiP-Seq): Interrogating ultra-rare mantle cell lymphoma persistent cells after CART19 therapy.

Many cancer therapies induce high response rates, with some resulting in undetectable disease as assessed using standard clinical assays. This is particularly true in leukemia and lymphoma, in which patients often achieve deep remission yet ultimately experience relapse. These outcomes highlight a critical need to better understand ultrarare persistent cells that survive therapy but remain inaccessible to current techniques. Here, we developed Live-cell Pick-Seq (LiP-Seq), an advanced platform leveraging multiplexed live-cell imaging to identify and retrieve individual target cells for downstream analysis. LiP-Seq enables high-resolution transcriptomic profiling of single, viable lymphoma cells present at frequencies as low as 10-6, providing a technological window into the biology of these elusive reservoirs. Applying this method to patients with mantle cell lymphoma after treatment with CD19 chimeric antigen receptor T-cell (CAR-T) therapy, we identified recurrent upregulation of the immune modulator IFITM2 in the persistent cell fraction. Functional validation demonstrated that IFITM2 overexpression conferred protection against CAR-T cytotoxicity in vitro, implicating it as a potential survival mechanism under therapeutic pressure. Our results provide, to our knowledge, the first transcriptomic characterization of viable, ultrarare persistent cells using LiP-Seq, establishing a new paradigm for identifying and targeting features that may enable treatment evasion.