P1333: chemotherapy-activated hscs are defined by highly increased mitochondrial mass and potential independent of cell cycle status

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: The most common therapy for different cancers consists of multiple cycles of chemotherapy and irradiation to eradicate malignant cells. However, these treatments also damage the rapidly proliferating healthy bone marrow (BM) cells, which can result in myelosuppression, anaemia and thrombocytopenia in patients. Hematopoietic stem cells (HSCs) are responsible for hematopoietic regeneration after myeloablative chemotherapy, yet their activation is poorly understood. HSCs are defined and isolated from murine BM at steady state based on cell surface phenotype (Lin-, c-kit+, Sca-1+, CD48-, CD150+). However, this profile is unreliable during hematopoietic or inflammatory stress, since expression of multiple surface markers changes (e.g. diminished surface expression of c-kit). This has hampered the identification and characterisation of HSCs under chemotherapy-induced stress as well as the pathways and genes governing early HSC activation. Aims: We aimed to reliably identify activated HSCs after chemotherapy, to characterise the pathways and processes that are critical for mouse and human HSC activation and to study how activated HSCs can be supported to enhance or accelerate BM recovery. Methods: To achieve HSC activation through chemotherapy, we injected 5-Fluorouracil (5-FU) into C57Bl/6 mice or human xenografted NRG mice. We optimised methods to identify HSCs after chemotherapy which allowed us to perform time-course analyses of activated HSCs, measuring metabolic parameters (transcription rate, translation rate, mitochondrial potential and mass), cell cycle status and transcriptome. Function of activated HSCs was assayed through (serial) transplantation to test engraftment capacity and lineage kinetics. Results: First we identified a cell surface phenotype to capture pure mouse HSCs from steady state and chemotherapy-treated mice that does not rely on c-kit (Lin-, CD244-, CD48-, CD150+, EPCR+). Functional testing of these cells by transplantation confirmed that this marker combination captures highly pure HSCs both at steady state and early after chemotherapy. Our time course analysis revealed a striking state of HSC activation two days after chemotherapy, in which all tested metabolic parameters are significantly upregulated, in absence of cell cycle entry (90% in G0). This allows us to separately test the consequences of metabolic stem cell activation versus stem cell cycling, the latter of which is known to negatively impact long-term HSC function. Our transcriptomic data revealed that nearly 3000 genes are significantly differentially expressed between activated HSCs two days after 5-FU and steady state equivalents. This transcriptional shift relates to changes in lineage priming and upregulation of inflammatory genes, HSC self-renewal genes and mitochondrial genes. In transplant assays, these activated HSCs (day 2) show equally high engraftment levels as controls, but characterised by a B-cell bias. Importantly, we could observe a similar state in activated human HSCs treated with 5-FU in xenografted NRG mice: highly increased mitochondrial mass and protein translation rate independent of cell cycle status. Summary/Conclusion: Mouse and human HSC activation is characterised by increased protein translation and mitochondrial activity prior to cell cycling. We have herein captured novel aspects of HSC activation that may lead to additional approaches, such as mitochondrial-targeted treatments (Mansell, Sigurdsson et al, Cell Stem Cell, 2021), to support regenerating HSCs after cancer treatment or transplantation. Keywords: Chemotherapy toxicity, Hematopoietic stem cell, Mitochondria, Myelosuppression