Baseline single-cell mass distributions correlate with clinical response to acalabrutinib, venetoclax, and obinutuzumab in mantle cell lymphoma

Background: Mantle cell lymphoma (MCL) exhibits heterogeneous responses to targeted therapy, which are incompletely explained by genomic and immunophenotypic markers. Building on our prior work linking single-cell mass to BCR signaling activity and Bruton's tyrosine kinase inhibitors (BTKi) sensitivity, we hypothesized that baseline biophysical mass distributions may integrate proliferative, metabolic, and signaling states and predict clinical responses to BTKi-based therapy. Methods: Baseline tumor cells from treatment-naïve MCL patients in a phase 1/2 trial of Acalabrutinib, Venetoclax, and Obinutuzumab (NCT04855695) were enriched by fluorescence-activated cell sorting and analyzed via suspended microchannel resonators (SMR) without drug exposure to measure distributions of single-cell buoyant mass. Durable response (DR) was defined as achieving complete metabolic remission ≥12 months; non-durable response (NDR) as refractory disease or relapse within 12 months. Of the nine cases analyzed by SMR, seven also underwent parallel CyTOF profiling. Results: Baseline median mass of MCL cells was higher in NDR (n = 3; mean of patient medians 13.1 pg) than in DR (n = 6; mean of patient medians 13.1 pg; p = 0.02). The fraction of cells >15 pg-a threshold previously linked to BTK inhibitor response-was elevated in NDR ( p =0.02) and correlated with clinical Ki-67 index (available in 6/9, r²=0.87). Eight of nine cases exhibited classic MCL morphology; the single blastoid case (NDR) had a median mass of 14.6 pg. In CyTOF-profiled samples, the >15 pg tumor cell fraction correlated with B-cell expression of Ki-67 (r² = 0.99), the glucose transporter GLUT1 (r² = 0.55), and the immune checkpoint ligand PD-L1 (r² = 0.99), but not with the amino acid transporter CD98 or fatty acid transporter CD36. These associations are consistent with sequelae of heightened BCR. Across the cohort, an increased proportion of tumor cells >15 pg correlated with increased PD-1 expression on CD4⁺ T cells (r² = 0.66) and expansion of T follicular helper (Tfh) cells (r² = 0.74). Increased tumor cell mass distributions also positively correlated with Tfh activation markers, including ICOS, Ki-67, and checkpoint molecules TIGIT (r² = 0.56-0.69). Taken together, these features raise the possibility of chronic B-cell antigen stimulation driving both Tfh activation and immune-regulatory feedback. Conclusions: Baseline MCL cell biophysical mass distributions correlate with tumor-intrinsic proliferation and metabolic activity as well as systemic Tfh activation and immune checkpoint expression. These results suggest that tumor biophysical properties ex vivo may capture in vivo BTKi sensitivity through tumor-intrinsic and immunologic mechanisms. Evaluation in larger cohorts and deeper mechanistic analyses are required to clarify its potential as a predictive biomarker.