Amouzgar, M.; Favaro, P. C.; Ho, D.; Bruce, T.; Bendall, S. C.

The cell cycle (CC) is involved in diverse cell processes like cell differentiation, immune cell expansion, and tumorigenesis but current single-cell (sc) strategies study CC as: (i) coarse phases, (ii) are based on transcriptomic data, (iii) leverage imaging modalities that are not extensible outside adherent cell culture, or (iv) do not enable high-throughput, single-cell multiplexing. To solve this, we developed an expanded, metal-tagged antibody approach for Mass Cytometry (MC) with 48 CC-related molecules that more deeply phenotypes the diversity of scCC states. Using cytometry by time of flight (CyTOF), we quantified CC states across various suspension and adherent cell lines and stimulated primary human T cells. Our approach captures the diversity of scCC states, including atypical molecular signatures that do not meet the canonical definitions of CC phases in the literature. Multiplexing with pharmacologically-induced CC arrest reveals that drug-induced CC perturbations can exacerbate reported noncanonical states and induce previously unobserved states. Notably, cells escaping CC inhibitor action in primary cells demonstrated more aberrant CC states compared to their untreated counterparts. Overall, our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems and can simultaneously multiplex experimental perturbation & disease systems, or integrate with other MC measurement platforms.