Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

Measuring trogocytosis between ovarian tumor and natural killer cells.

Trogocytosis is an active transport mechanism by which one cell extracts a plasma membrane fragment with embedded molecules from an adjacent cell in a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting key experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. For complete details on the use and execution of this protocol, please refer to Gonzalez et al. (2021).

High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment.

Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy.

High-resolution myogenic lineage mapping by single-cell mass cytometry.

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.

Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.

The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38α and p38ß mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38α and p38ß in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.

Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.

Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a "knocked-in" EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and graded modalities combine to generate high-fidelity Stat5 signaling over the entire basal and stress Epo range. They suggest that dynamic behavior may encode information during STAT signal transduction.

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways.

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and ß-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-x(L) is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-x(L) pathway "resets," allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using "knock-in" mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-x(L) response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-x(L) induction provides a "stop-gap" in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-x(L) may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.

Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

Negative autoregulation by FAS mediates robust fetal erythropoiesis.

Tissue development is regulated by signaling networks that control developmental rate and determine ultimate tissue mass. Here we present a novel computational algorithm used to identify regulatory feedback and feedforward interactions between progenitors in developing erythroid tissue. The algorithm makes use of dynamic measurements of red cell progenitors between embryonic days 12 and 15 in the mouse. It selects for intercellular interactions that reproduce the erythroid developmental process and endow it with robustness to external perturbations. This analysis predicts that negative autoregulatory interactions arise between early erythroblasts of similar maturation stage. By studying embryos mutant for the death receptor FAS, or for its ligand, FASL, and by measuring the rate of FAS-mediated apoptosis in vivo, we show that FAS and FASL are pivotal negative regulators of fetal erythropoiesis, in the manner predicted by the computational model. We suggest that apoptosis in erythroid development mediates robust homeostasis regulating the number of red blood cells reaching maturity.