Frank Y. Huang

Chromosomal instability is a major driver of intratumoral heterogeneity (ITH), promoting tumor progression. In the present study, we combined structural variant discovery and nucleosome occupancy profiling with transcriptomic and immunophenotypic changes in single cells to study ITH in complex karyotype acute myeloid leukemia (CK-AML). We observed complex structural variant landscapes within individual cells of patients with CK-AML characterized by linear and circular breakage-fusion-bridge cycles and chromothripsis. We identified three clonal evolution patterns in diagnosis or salvage CK-AML (monoclonal, linear and branched polyclonal), with 75% harboring multiple subclones that frequently displayed ongoing karyotype remodeling. Using patient-derived xenografts, we demonstrated varied clonal evolution of leukemic stem cells (LSCs) and further dissected subclone-specific drug-response profiles to identify LSC-targeting therapies, including BCL-xL inhibition. In paired longitudinal patient samples, we further revealed genetic evolution and cell-type plasticity as mechanisms of disease progression. By dissecting dynamic genomic, phenotypic and functional complexity of CK-AML, our findings offer clinically relevant avenues for characterizing and targeting disease-driving LSCs.

Neutrophils transit through megakaryocytes in a process termed emperipolesis, but it is unknown whether this interaction is a single type of cell-in-cell interaction or a set of distinct processes. Using a murine in vitro model, we characterized emperipolesis by live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome to enter the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or the perinuclear endoplasmic reticulum. These findings reveal that megakaryocyte emperipolesis reflects at least 2 distinct processes differing in transit time and morphology, fast and slow emperipolesis, suggesting divergent physiologic functions.

Acute myeloid leukemia (AML) is an aggressive blood cancer in which disease initiation and relapse are driven by leukemic cells with stem-like properties, known as leukemic stem cells (LSCs). The LSC compartment is highly heterogenous and this contributes to differences in therapy response. This heterogeneity is determined by genetic and nongenetic factors including somatic mutations, the cell of origin, transcriptional and epigenetic states as well as phenotypic plasticity. While this complicates the identification and eradication of LSCs, it also presents an opportunity to tailor therapeutic strategies to the phenotypic and functional states of LSCs present in a patient, exploiting their specific vulnerabilities. The emergence of single-cell multiomics technologies has transformed our ability to dissect cellular heterogeneity in AML, enabling simultaneous interrogation of genomic, transcriptomic, epigenomic and proteomic layers and providing high-resolution molecular snapshots of individual cells. In this review, we discuss causes and consequences of LSC heterogeneity, highlight advances in single-cell multiomics technologies to resolve it and outline how they can address shortcomings in our understanding of LSC heterogeneity and plasticity to revolutionize diagnostics and disease monitoring of AML.

Abstract In emperipolesis, neutrophils transit through megakaryocytes, but it is unknown whether this interaction represents a single type of cell-in-cell interaction or a set of distinct processes. Using an in vitro model of murine emperipolesis, we characterized neutrophils entering megakaryocytes using live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome into the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or with the perinuclear endoplasmic reticulum, as confirmed by immunofluorescence microscopy. Together, these findings reveal that megakaryocyte emperipolesis reflects at least two processes, fast and slow emperipolesis, each with its own characteristic transit time, morphology, and intracellular localization, suggesting distinct functions. Key Points Neutrophil passage through megakaryocytes, termed emperipolesis, diverges into fast and slow forms that differ in transit time, morphology, and intracellular localization During emperipolesis, neutrophils can reside in vacuoles (emperisomes) or escape into the cell cytoplasm to assume positions near the megakaryocyte’s demarcation membrane system, endoplasmic reticulum, or nucleus.

Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) with limited biomarkers for early detection. While neutrophils contribute to SLE pathogenesis, their phenotypic heterogeneity in disease remains poorly characterized. Here, we used mass cytometry to profile blood neutrophils from patients with biopsy-confirmed proliferative LN and healthy controls. We identified a distinct population of activated neutrophils, marked by surface expression of lysosomal-associated membrane protein 1 (LAMP1/CD107a), that was virtually absent in healthy individuals. We demonstrate that LAMP1 resides intracellularly in resting neutrophils and translocates to the cell surface upon activation. Transcriptomic analysis revealed no difference in LAMP1 mRNA expression between patients with SLE and controls, confirming that surface LAMP1 reflects neutrophil activation rather than increased transcription. Soluble LAMP1 was significantly elevated in serum from patients with SLE compared with controls, with the highest levels in proliferative LN. In a large cohort of 225 patients with LN, urinary LAMP1 correlated with glomerular filtration rate, proteinuria, and histological activity indices. Together, our findings reveal LAMP1 as a marker of neutrophil activation in SLE and identify serum and urinary LAMP1 as potential noninvasive biomarkers for proliferative LN.