Work

19 Single-cell biophysical profiling as an early predictor of tumor reactivity in tumor-infiltrating lymphocyte manufacturing

Year: 2026

Type: conference-abstract

Abstract: Background Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) is now an approved treatment for advanced melanoma, but manufacturing of cell product remains a resource bottleneck. TIL manufacturing requires the maintenance of parallel cultures for several weeks before reactivity can be assessed, significantly increasing the cost and complexity of the manufacturing process. A rapid, label-free measure of TIL quality applied early in manufacturing could allow prioritization of the cultures most likely to yield reactive product, reducing time and cost. Single-cell biophysical profiling has previously been shown to quantify immune cell activation, T cell dysfunction/exhaustion, and response to checkpoint blockade. We asked whether the same approach could serve as a biomarker of tumor reactivity in expanded melanoma TIL.Methods Expanded TIL cultures from 20 melanoma tumor fragment samples (8 unique patients) were cryopreserved after a 3-4 week expansion protocol. An aliquot of TIL from each fragment was co-cultured with autologous tumor cells and IFN-gamma was measured in supernatants. Of the 20 TIL cultures tested, 10 were tumor-reactive as indicated by the secretion of IFN-gamma, and 10 were non-reactive. The cryopreserved samples were later profiled at single-cell resolution using Travera’s integrated suspended microchannel resonator and imaging platform measuring single-cell mass, volume, density, and brightfield morphology. Each sample was measured at three points (post-thaw, 72 h without stimulation, and 72 h with aCD3 + aCD28 stimulation).Results Multiple single-cell biophysical features distinguished reactive from non-reactive TIL cultures. The strongest single feature was the mean mass of the presumed ‘activating’ subpopulation (highest mode cluster) measured at thaw, which differed significantly between groups (p = 0.015) and separated reactive from non-reactive samples with an area under the receiver operator curve of 0.82. The predictive signal was most pronounced when cells were measured directly at thaw, before any in vitro stimulation or culture, and class separation further improved with the addition of other biophysical features.Conclusions Single-cell biophysical profiling provided a rapid, label-free readout that distinguished tumor-reactive from non-reactive expanded melanoma TIL in this cohort, with the strongest signal detectable at thaw. Used as an in-process quality measure early in the TIL manufacturing process, this approach could rapidly prioritize cultures likely to contain tumor-reactive TIL leading to improved final product quality as well as reduced time and cost. Future work will validate these findings in larger cohorts and incorporate richer biophysical features, including imaging-based morphology, beyond the mass measurements reported here.Ethics Approval This study MCC20559 was approved by Moffitt’s Ethics Board Advarra; approval number CR00763140.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0019