Mesenchymal stem cell-derived S100A8 facilitates leukemia stem cell maintenance via TLR4/PI3K/Akt signaling

Mesenchymal stem cell-derived S100A8 facilitates leukemia stem cell maintenance via TLR4/PI3K/Akt signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mesenchymal stem cell-derived S100A8 facilitates leukemia stem cell maintenance via TLR4/PI3K/Akt signaling Fuling Zhou, Xiaoyan Liu, Jinxian Wu, Xinqi Li, Ruiyang Pan, Li Liu, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4374015/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The role of microenvironmental inflammation in the regulation of acute myeloid leukemia (AML) and stressed hematopoiesis is significant, though the molecular mechanisms are not fully understood. Here, we found that MSCs in a leukemic microenvironment had dysregulated expression of the inflammatory cytokine S100A8. Upregulating S100A8 in MSCs increased the proliferation and chemoresistance of AML cells in vitro. In contrast, removing S100A8 from MSCs in the murine MLL-AF9 AML model resulted in longer survival and less infiltration of leukemia cells and leukemic stem cells (LSCs). S100A8 binds to the TLR4 receptor on leukemia cells, which activates the PI3K/Akt pathway. In addition, removing S100A8 from MSCs causes a temporary increase in their quantity, followed by a decline in hematopoietic stem cells (HSCs) in mice exposed to stressful environments. Furthermore, the absence of S100A8 alters the properties of MSCs, impairing their ability to differentiate into osteoblasts and decreasing the expression of osteopontin, which is required to support HSCs. Our findings highlight the importance of MSC-derived S100A8 in promoting the maintenance of LSCs while impeding the maintenance of HSCs, providing new insights into the potential for the management of AML and hematopoietic regeneration. Biological sciences/Stem cells/Haematopoietic stem cells Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Points Niche S100A8 accelerates AML development by regulating TLR4/PI3K/Akt pathway. Niche S100A8 promoted short-term hematopoiesis recovery but impaired long-term hematopoiesis regeneration under stress. Introduction Hematopoietic stem cells (HSCs) establish themselves in a safe bone marrow (BM) environment composed of a complex interconnected network of hematopoietic and nonhematopoietic cells that play critical roles in regulating HSC dormancy, proliferation, and migration( 1 ). Under normal circumstances, hematopoiesis strikes a delicate balance between expansion and quiescence to achieve maximum production output( 2 ) ( 3 ). Both extrinsic( 1 ) and intrinsic factors( 4 ) work hard to maintain this homeostasis while also allowing for adaptive responses( 5 ) and recovery( 6 ) in the face of emergencies like chemotherapy and radiotherapy. Acute myeloid leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of myeloid blasts evolved from hematopoietic stem and progenitor cells (HSPCs)( 7 ). Adequate hematopoiesis regeneration under stress determines the outcome of AML and is governed by BM HSC niche( 8 , 9 ). Mesenchymal stromal cells (MSCs) have a pivotal niche component( 10 – 15 ), which forms various BM components, such as adipogenic, osteogenic, and chondrogenic lineages( 10 , 16 – 19 ). Inflammation influences HSC activity and AML development through niche-dependent interactions, as demonstrated by a growing body of research( 20 – 23 ). In response to external stimulation, BM niche cells release factors that promote myelopoiesis( 24 – 26 ). However, the precise mechanisms and roles of proinflammatory factors in the mesenchymal niche are poorly understood. At present, the proinflammatory factors that play a key role in the mesenchymal niche are not well studied. S100A8, also known as "calprotectin" or "myeloid-related protein 8 (MRP8)," is a member of the S100 family that regulates various cellular processes, including Ca 2+ balance, apoptosis, migration, proliferation, differentiation, energy metabolism, and inflammation( 27 , 28 ). Extracellular S100A8 and S100A9, as well as the heterodimer S100A8/A9, are potent inflammatory factors( 29 ). The heterodimer S100A8/A9 indicates the predominant form, which accounts for up to 45% and ∼5% of all cytosolic proteins in neutrophils and monocytes, respectively( 27 , 28 ). Recent research has found elevated levels of MSC-derived S100A8 in patients with myeloid neoplasms such as myelodysplastic syndrome (MDS) and (yeloproliferative neoplasm(MPN)( 30 ). Escalating S100A8 expression in MSCs is associated with increased MSC proliferation during the early osteogenic differentiation phase, whereas mature osteogenesis is inhibited, resulting in the disruption of trabecular bone structure(31–33). The effects of S100A8 on stromal cells are similar to the remodeling of the BM microenvironment observed in AML. Notably, MSC-derived S100A8 has been linked to MDS disease progression( 34 ) by causing mitochondrial dysfunction, eliciting oxidative stress responses, and impairing DNA damage repair in hematopoietic stem progenitors. In this study, we investigated the effects of aberrant expression of MSC-derived S100A8 on HSCs regeneration and AML development. Methods Mice S100A8 flox/flox mice were obtained from GemPharmatech LLC.. Prx1-Cre mice were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. C57BL/6 and B6.SJL mice were bought from GemPharmatech LLC.. S100A8 flox/flox mice were crossed with Prx1-Cre mice to produce Prx1-Cre; S100A8 flox/flox and S100A8 flox/flox mice. Mice ranging from eight to twelve weeks old were used. All animal procedures followed the animal care guidelines approved by the Institutional Animal Care and Use Committees at Zhongnan Hospital Wuhan University. AML patient and healthy donor samples All BM specimens from AML patients and healthy donors were with informed consent obtained from Zhongnan Hospital and Wuhan University respectively. All studies involving human samples were approved by the Ethics Committee of Zhongnan Hospital and Wuhan University. Flow cytometry analysis and cell sorting Mice BM and peripheral blood (PB) samples were prepared as described previously. Phenotypic analysis of lineage cells, HSPCs, leukemia stem cells (LSCs), and leukemic GMP populations (L-GMPs), MSCs, and osteoblasts (OBCs) was performed according to previous studies. LSK cells (Lin − Sca-1 + c-Kit + ), LT-HSCs (Lin − Sca-1 + c-Kit + CD34 − Flt3 − ), ST-HSCs (Lin − Sca-1 + c-Kit + CD34 + Flt3 − ), MPPs (Lin − Sca-1 + c-Kit + CD34 + Flt3 + ), GMPs (Lin − Sca-1 − c-Kit + CD16/32 hi CD34 hi ), CMPs (Lin − Sca-1 − c-Kit + CD16/32 Med CD34 hi ), MEPs (Lin − Sca-1 − c-Kit + CD16/32 − CD34 − ), CLPs (Lin − IL-7R + Sca-1 Med c-Kit Med ), LSCs (YFP + CD117 + Gr-1 − ), L-GMPs (IL-7R/Lin − YFP + c-Kit hi CD34 + CD16/32 hi ), MSCs (CD45 − TER119 − CD31 − LepR + ), and OBCs (CD45 − TER119 − CD31 − CD166 + Sca-1 − ) were analyzed with FACSCanto™ Ⅱ (BD Biosciences, San Jose, CA, USA). Immature cells were obtained by sorting mouse HSPC and human LSCs with CD117 MicroBeads and CD34 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The antibody details are provided in Supplementary Table S1 . Measurement of ROS The intracellular (reactive oxygen species) ROS were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Briefly, cells were incubated with HSC or LSC marker antibodies for 30–45 min before being treated with 10 µM DCFH-DA (Beyotime, S0033S) for 10 min at 37°C in the dark. Following incubation, the cells were washed twice with PBS and analyzed by flow cytometry (CantoII; BD). A shift to the right indicates higher ROS levels. TMRM staining Mitochondrial membrane potential was identified by tetramethylrhodamine, ethyl ester (TMRE, Beyotime, C2001S) following the manufacturer’s protocol. Briefly, cells were incubated with HSC or LSC marker antibodies for 30–45 min, then stained with 50 nM TMRE for 10 min and analyzed using flow cytometry (CantoII; BD). Statistics The significance of differences between the two groups was assessed using unpaired two-tailed Student ’s t- tests. The results in the bar graphs are mean value ± SEM. Overall survival curves were plotted using the Kaplan–Meier method, with log-rank tests used for comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001 Results Expression of MSC-derived S100A8 increased in cases of AML In our previous study, we found a significant increase in the expression of BM MSC-derived S100A8 in a cohort of rapidly advancing murine models of AML (Fig. 1 A–B), indicating that MSC-derived S100A8 plays a role in the regulation of leukemic disease progression. Here, we validated the expression of S100A8 in MSCs derived from BM with AML status. Volunteer-derived BM mononuclear cells were obtained and in vitro cultured to produce human BM MSCs (Fig. 1 C). MSC expression of the marker (Fig. 1 D–K)and differentiation functions were validated (Fig. 1 L–M). After 48 hours of cocultivation with leukemic cells, MSCs showed an increase in S100A8 expression (Fig. 1 N–O). Furthermore, MSC-derived S100A8 expression in MLL-AF9 AML mice increased similarly to that in normal mice (Fig. 1 P). Furthermore, our analysis of human BM sections showed very little expression of S100A8 in healthy human MSCs, whereas MSCs derived from patients with AML expressed a lot of S100A8 (Fig. 1 Q). The data presented above support the aberrant expression of S100A8 in the presence of AML, implying that S100A8 may be involved in the progression of AML. MSC-derived S100A8 is essential for the growth of human AML cells To further validate the role of MSC-derived S100A8 in AML development, we performed an experiment in which S100A8 was overexpressed in human BM MSCs (Fig. 2 A–C) and then cocultured with the human AML cell line Kasumi-1. Overexpression of S100A8 in MSCs increased the growth of primary human AML cells (Fig. 2 D) and facilitated their progression through the cell cycle (Fig. 2 E). Furthermore, overexpression of S100A8 did not significantly alter the rate of apoptosis (Fig. 2 F), but it increased the resistance of Kasumi-1 cells to etoposide (Fig. 2 G). Then, leukemia cells from AML patients were used, and CD34 + leukemia cells were enriched using magnetic-activated cell sorting (MACS). CD34 + leukemia cells were cocultured with S100A8-overexpressing and control MSCs for 48 h. Then, leukemia cells were transplanted into immunodeficient mice, along with the MSC suspension (Fig. 2 H). After 30 days, the WBC count in S100A8-overexpressing mice was higher than in the control group, as were the levels in the BM and SP (Fig. 2 I–K). Finally, these findings indicate that S100A8 plays a role in the development and drug resistance of human AML cells. MSC-derived S100A8 drives murine AML progression and maintains LSC function To fully understand the role of S100A8 in leukemogenesis, MSC-specific S100A8 knockout (Prx1-Cre +/− ; S100A8 flox/flo x ) mice and control mice was created (Prx1-Cre −/− ; S100A8 flox/flox ) by crossing S100A8 flox/flox mice with Prx1-Cre mice (Figure S1 ), then MLL-AF9-induced murine AML model was constructed with S100A8 knockout and control mice as recipients (Fig. 3 A–B). S100A8 deletion significantly improved the survival rate of AML mice (Fig. 3 C). Not surprisingly, S100A8 knockout recipients had lower spleen (SP) weights than controls (Fig. 3 D). Furthermore, we found that S100A8 knockout recipients had a significantly lower ratio of YFP + leukemic cells in the PB, SP, and BM, as well as lower malignant cell and total WBC counts in the PB (Fig. 3 E–H). Furthermore, the percentages and numbers of LSCs (YFP + c-Kit + Gr-1 − cells) in PB, SP, and BM were significantly reduced after S100A8 deletion (Fig. 3 I–M), as were L-GMPs (IL-7R/Lin − YFP + c-Kit hi CD34 + CD16/32 hi ) in SP and BM (Fig. 3 N–P). Finally, we examined the characteristics of LSCs in a murine AML model, and we discovered that S100A8 knockout recipients had more G0 phase cells and higher apoptosis rates than control mice (Fig. 3 Q–R). Collectively, we can reasonably conclude that S100A8 plays an important role in promoting the maintenance of LSCs. MSC-derived S100A8 regulates AML development via the TLR4/PI3K/Akt pathway RAGE and TLR4 are the most common S100A8 receptors, and to investigate the mechanism by which S100A8 acts on leukemic cells, we added a RAGEE inhibitor (FPS-ZM1) or a TLR44 inhibitor (Resatorvid) to MSC and Kasumi-1 coculture systems, and the addition of TLR4 inhibitors resulted in the most significant decrease in leukemic cell proliferation (Fig. 4 A–B), indicating that MSC-derived S100A8 acts primarily by binding leukemic cell TLR4. To delve deeper into the molecular mechanism, we conducted transcriptomic analyses. It revealed notable differences in gene expression in Kasumi-1 cells between the group cocultured with overexpressed S100A8 and the control group (Fig. 4 C), as well as significant alterations in the PI3K/Akt signaling pathway (Fig. 4 D–E). WB experiments revealed that phosphorylated PI3K and Akt protein levels were significantly higher in the S100A8 overexpression group but lower in the S100A8-deficient group, and PI3K inhibitor (LY294002) significantly reduced proliferation of leukemic cells (Fig. 4 H). The most recent article reported that PI3K/Akt regulates oxidative stress to promote drug resistance in leukemia progression, and changes in the HIF-1 pathway were also revealed in our sequencing results (Fig. 4 D). We then examined ROS levels in LSCs. Flow cytometry analysis revealed a lower intracellular level of ROS in LSCs from S100A8 knockout mice compared to the control group (Fig. 4 I). TMRE staining was used to determine mitochondrial membrane potential. Flow cytometry analysis revealed a decrease in TMRE fluorescence in LSCs from S100A8 knockout mice compared to the control group (Fig. 4 J). Furthermore, DNA damage was found to be lower in BM cells from S100A8 null AML mice compared to control cells (Fig. 4 K). Overall, these findings showed that S100A8 promoted leukemia progression through TLR4/PI3K/Akt signaling. MSC-derived S100A8 deletion is dispensable for normal hematopoiesis but inhibits HSC expansion under short-term stress Impairment of normal hematopoiesis and leukemia progression are 2 closely related processes during leukemia development and are regulated by the BM niche. We discovered that removing S100A8 had no discernible effect on the frequencies and numbers of HSCs, hematopoietic progenitors (HPCs) (except for multipotent progenitors, MPPs) (Figure S2 A–E), and mature cells were comparable in the BM and PB of control and knockout mice (Figure S2 F–I). Thus, MSC-derived S100A8 was not required for homeostatic hematopoiesis in mice. However, in the MLL-AF9 mouse model, we discovered that S100A8 knockout recipients had more HPCs and mature myeloid cells than control group mice (Figure S2 J–R), implying that S100A8 may regulate hematopoiesis under stress conditions. Interestingly, we found that S100A8 was significantly upregulated in MSCs after 5-fluorouracil (5-FU) and irradiation (IR) exposure (Figure S3). As expected, the frequencies and numbers of LSKs, long-term HSCs (LT-HSCs), and short-term HSCs (ST-HSCs) were nearly all lower in S100A8 -/- BM at day 14 after 5-FU treatment and IR exposure than in controls (Fig. 5 A–D). Notably, 5-FU treatment and IR exposure resulted in lower percentages and numbers of granulocyte/macrophage progenitors (GMPs), while decreased megakaryocyte/erythroid progenitors (MEPs) was observed in S100A8 -/- mice after IR exposure, whereas common myeloid progenitors (CMPs) showed no significant difference between the two groups after 5-FU treatment and IR exposure (Fig. 5 E–F). Consistent with these findings, S100A8 -/- mice had significantly fewer mature myeloid cells following 5-FU and IR treatment in both BM and PB (Fig. 5 G–J). Overall, our findings indicate that S100A8 ablation promotes short-term HSC expansion under hematopoietic stresses. Deletion of MSC-derived S100A8 increases HSC quiescence under hematopoietic stress Given the observation that S100A8 causes short-term expansion of HSCs during hematopoietic stress, we hypothesized that S100A8 may modulate HSC quiescence. As expected, HSCs from S100A8 knockout mice had a higher percentage in the G0 phase but a decreasing trend in the G1 and S/G2/M phases after 5-FU treatment and IR exposure (Fig. 6 A–C). Furthermore, an in vivo BrdU incorporation assay revealed that deletion of S100A8 reduced HSC proliferation of HSCs in mice following 5-FU and IR challenge, while apoptosis rates were comparable between S100A8 knockout and control HSCs (Fig. 6 D–H). These findings suggest that S100A8 promotes hyperactivation of HSCs in response to stress stimuli. To see how S100A8 affected HSCs functionality, we used a colony formation assay. The results showed that the number of colonies formed in the group with S100A8 deletion was significantly higher after treatment with 5-FU and IR (Fig. 6 I–J). These findings suggest that S100A8 promotes the entry of HSCs into the proliferation cycle as a means of short-term hematopoietic recovery under stressful conditions, albeit at the expense of impairing the general function of HSCs. To see if S100A8 affects long-term HSC maintenance, we gave S100A8 knockout and control mice four injections of 5-FU (50mg/kg) once per week. The hematopoietic phenotypes of S100A8 knockout and control mice were then assessed at 4, 8, 12, and 16 weeks (Fig. 6 K). The results revealed a great increase in white blood cell (WBC) count following the deletion of S100A8 after 4 weeks (Fig. 6 L). Surprisingly, the S100A8 knockout mice had lower percentages of mature PB and BM myeloid cells than control mice (Fig. 6 M–N), but higher percentages of GMPs (Fig. 6 O). However, it is worth noting that the proportion and quantity of LT-HSCs in the S100A8 knockout group were lower than in the control group (Fig. 6 P). This finding suggests that S100A8 reduces HSCs and myeloid differentiation bias in a prolonged stressful environment, causing HSCs to exhibit a senescent phenotype. S100A8 remodeled the BM microenvironment We investigated whether dysregulated expression of S100A8 causes microenvironment remodeling, which affects hematopoietic homeostasis and leukemia progression. After overexpressing S100A8, MSCs were cultured for 72 h. Then, colony-forming unit-fibroblast (CFU-F) experiments were performed, which revealed that S100A8 overexpression significantly increases CFU-F formation (Fig. 7 A–B). Flow cytometry analysis revealed that the group with S100A8 overexpression had a higher proliferation rate by flow cytometry analysis (Fig. 7 C). In addition, the S100A8 overexpression group showed a decrease in G0 and G1 phase cells while increasing S/G2/M phase cells (Fig. 7 D). Furthermore, the S100A8 overexpression group had a lower apoptosis ratio than the control group (Fig. 7 E). Subsequent investigations into MSCs in the mouse model revealed a lower percentage of MSCs in S100A8 knockout mice than in control mice following 5-FU and IR exposure, as well as in AML mice (Fig. 7 F–I). Furthermore, our findings showed that S100A8 overexpression in vitro inhibited MSCs differentiation into OBCs and reduced osteopontin (OPN) and osteocalcin (OCN) expression (Fig. 7 J–L). In contrast, S100A8-deficient mice showed an increase in osteoblasts following 5-FU and IR exposure in AML mice, leading to increased secretion of OPN (Fig. 7 M–S). This suggests that remodeling of the BM microenvironment by S100A8 may have an indirect effect on stress hematopoiesis and leukemia progression. Discussion Myeloid cells exhibit the baseline alarming protein S100A8 expression as a marker, whereas MSCs typically do not express S100A8 under steady-state conditions. In this study, we found that the presence of AML 5-FU treatment and IR, all led to an increase in S100A8 expression. These findings highlight the critical role of S100A8, derived from MSCs, as a potent proinflammatory factor in AML and hematopoietic regeneration during stress. Using an in vitro coculture system of MSCs with leukemia cells and an MLL-AF9 leukemia mouse model, we discovered that MSC-derived S100A8 promotes leukemia cell proliferation and drug resistance while reducing the survival of leukemic mice. These findings indicate that S100A8 in the microenvironment greatly contributes to the progression of leukemia. According to reports, the cell membrane receptors RAGE and TLR4 are the primary binding receptors for S100A8 homodimers and S100A8/A9 heterodimers( 35 ). Our findings further show that TLR4 is the primary binding receptor for S100A8 in leukemia cells, as evidenced by the inhibition of RAGE and TLR4 receptors in the coculture system. S100A8 activates the downstream PI3K/Akt signaling pathway, which regulates leukemia cell growth and proliferation. Dysregulation of the PI3K pathway is common in various human cancers, accounting for approximately 50% of de novo AML cases exhibiting this aberration( 36 – 40 ). The PI3K inhibitor was introduced into the coculture system, and subsequent findings confirmed the role of S100A8 in modulating leukemia cell proliferation via the PI3K pathway. A recent study discovered that DNA damage is inextricably linked to the PI3K/Akt pathway, which controls the senescence of hematopoietic stem cells( 41 ). In the current study, we discovered that leukemic stem cells in the S100A8-deficient group had lower levels of ROS and DNA damage, indicating that S100A8 may play a regulatory role in the PI3K/Akt pathway, which is associated with DNA damage and subsequent promotion of leukemia cell proliferation. Previous research has shown that the development of AML and the aging of HSCs are linked to the accumulation of DNA damage and increased ROS levels. Modifications in the levels of antioxidant enzymes in the plasma of individuals with AML, the decreased antioxidant status found in the plasma of patients with AML is most likely due to elevated levels of ROS, as evidenced by the decline in antioxidant activity. This decrease in antioxidant activity indicates that oxidative stress is widely recognized as a significant factor in AML progression and recurrence( 42 – 47 ). In contrast, BM cells from transcription factor Meis1-deficient mice had reduced colony formation and significantly fewer long-term HSCs, which lost quiescence due to ROS accumulation in HSCs. ATF4 deficiency causes severe defects in HSCs, leading to a complex aging-like phenotype, the HSC defects exhibited in ATF4 −/− mice are linked to elevated production of mitochondrial ROS. In our study, we found that MSC-derived S100A8 increased ROS levels not only in LSCs but aslo in HSCs (Figure S4). This study demonstrates that increased S100A8 expression in MSCs contributes to short-term hematopoietic recovery following IR and 5-FU-induced stress, which is consistent with previous research that has highlighted the role of an inflammatory microenvironment in promoting the swift recovery of hematopoiesis. Nonetheless, this phenomenon has been shown to have a negative impact on the long-term preservation of hematopoietic stem cells, eventually reducing the stem cell reservoir. Our findings also indicate that the long-term maintenance of HSCs is compromised by sustained 5-FU stress. According to our findings, S100A8 promotes the entry of G0-phase hematopoietic stem cells into the cell cycle. HSC quiescence and activation are highly complex interactions governed by a variety of cell-intrinsic and cell-extrinsic factors( 48 – 50 ). Our findings identify S100A8 as a critical regulator in determining the fate of HSCs, particularly in "awakening" dormant HSCs in response to hematological insults caused by IR and 5-FU. S100A8 clearly directs HSC differentiation toward the myeloid lineage, but the sustained inflammatory response mediated by S100A8 may eventually lead to HSC exhaustion. Increased levels of ROS may explain the contradictory effect of S100A8 expression, which inhibits the preservation of healthy HSCs while concurrently facilitating the progression of leukemia. The decline of HSCs and hematopoietic impairment associated with S100A8 may be linked to MSC senescence. Under stress conditions such as IR, 5-FU treatment, and AML, abnormal S100A8 causes MSCs to undergo cell cycle progression, while concurrently impairing their ability for osteogenic differentiation, which are signs of MSC senescence. Consequently, diminished osteogenic differentiation reduces the expression and secretion of OPN, a critical bridging protein primarily from MSCs and osteoblasts. Previous research has shown that a decrease in OPN causes a phenotype associated with hematopoietic senescence. Specifically, when young HSCs are exposed to an OPN-deficient niche, their ability to engraft is reduced, while the frequency of long-term HSCs increases, and stem cell polarity is lost. Conversely, when aged HSCs are exposed to thrombin-cleaved OPN, the aging process is reversed, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity, and the restoration of the balance between lymphoid and myeloid cells in PB( 51 ). In summary, our findings reveal the dual role of the mesenchymal niche inflammatory factor S100A8 in facilitating AML progression via TLR/PI3K/Akt pathway and suppressing stress hematopoiesis. In additin, S100A8 stimulates the production of ROS in both HSCs and LSCs, causing HSC senescence and promoting leukemia cell self-renewal. This novel finding sheds new light on the relationship between niche inflammation, hematopoiesis, and malignant transformation. Declarations Authorship Contributions Conceptualization: XYL, JXW, and FLZ; Methodology: XYL, JXW, and XQL; Validation: FLZ; Formal analysis: XYL, and JXW; Investigation: XYL, JXW, XQL, LLM, GPC, QW, NZ, XQT, YXT, HQJ, YXL, RHL, and WYY; Resources: LL, TTH, JXW, and XQL; Data Curation: XYL, JXW, and RYP; Writing - Original Draft: XYL; Writing - Review & Editing: FLZ; Visualization: XYL, JXW and RYP; Supervision: FLZ; Project administration: XYL and FLZ; Funding acquisition: XYL, XZ and FLZ. All authors reviewed and authorized the final manuscript. Disclosure of Conflicts of Interest All authors have read and approved the submission of the final manuscript. No conflicts of interest were declared. Acknowledgments We thank all of the patients who took part in this study, as well as their families. This study was funded by the Natural Science Foundation of China program [grant numbers 81900116, 82370176, 82000127, 82200254], and the Zhongnan Hospital of Wuhan University discipline construction platform project [grant numbers 202021, PDJH202217]. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Wei Q, Frenette PS. Niches for Hematopoietic Stem Cells and Their Progeny. Immunity. 2018;48(4):632–48. Doulatov S, Notta F, Laurenti E, Dick JE. Hematopoiesis: a human perspective. Cell Stem Cell. 2012;10(2):120–36. Pietras EM, Warr MR, Passegue E. Cell cycle regulation in hematopoietic stem cells. J Cell Biol. 2011;195(5):709–20. Yu VWC, Yusuf RZ, Oki T, Wu J, Saez B, Wang X, et al. Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells. Cell. 2017;168(5):944–5. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4374015","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":299797945,"identity":"d029a508-769e-419f-b6f6-1c2077ba7fbc","order_by":0,"name":"Fuling 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Park","correspondingAuthor":false,"prefix":"","firstName":"Junyi","middleName":"","lastName":"Liu","suffix":""},{"id":299797977,"identity":"6b8122e6-a49e-4487-8112-92eb10e39d2b","order_by":17,"name":"Ruihang Li","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Ruihang","middleName":"","lastName":"Li","suffix":""},{"id":299797980,"identity":"ef6b109d-25ef-4970-b5c9-d08a7934d490","order_by":18,"name":"Wanyue Yin","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Wanyue","middleName":"","lastName":"Yin","suffix":""},{"id":299797983,"identity":"726b4cef-f2e9-4f49-82de-008bd2def5e1","order_by":19,"name":"Xian Zhang","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-05-06 04:40:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4374015/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4374015/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56442021,"identity":"13ae159f-4f73-478b-9e3e-e1e758d89b09","added_by":"auto","created_at":"2024-05-14 08:44:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1318289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of MSC-derived S100A8 under leukemic state. \u003c/strong\u003e(A) Transcriptome sequencing data compares the expression of cytokines in MSC-derived fast and slow AML. (B) S100A8 expression in fast and slow AML (n = 3). (C) Flow chart of coculture of patient-derived BM MSC with leukemic cells Kasumi-1. (D-K) Flow cytometry analyzed surface markers of MSC. (L) Identify the adipose differentiation capacity of MSC by Oil red O staining. (M) Identify the osteogenic differentiation capacity of MSC by Azane Red staining. (N) Flow plots of representative fluorescence intensity of S100A8 in MSC coculture with Kasumi-1 and control MSC. (O) MFI of S100A8 in MSCs following coculture with Kasumi-1 cells (n = 3). (P) Expression of S100A8 in BM MSC from MLL-AF9 mice and normal mice (n = 3). (Q) Immunofluorescent images of human BM sections from a healthy donor and an AML patient are shown after staining for CD271 (red), S100A8 (green), and 4′,6-diamidi no-2-phenylindole (DAPI, blue). Scale bars, 20 μm. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/519c71dd1cc717a039087a40.png"},{"id":56442019,"identity":"8744e9cd-4774-4846-87cd-6dc842c5240d","added_by":"auto","created_at":"2024-05-14 08:44:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":802899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of S100A8 overexpression in MSCs on proliferation and resistance of human AML cells in vitro. \u003c/strong\u003e(A) Fluorescence plot of lentivirus transfected MSC from the Control and S100A8 overexpression group. (B) The transcriptional level of S100A8 expression following lentivirus transfected (n = 3). (C) S100A8 MFI of MSCs in S100A8 overexpression and control group (n = 3). (D) The effect of S100A8 overexpression on Kasumi-1 cell proliferation, flow representation profiles on the left, and statistical map on the right (n = 3). (E) Effects of S100A8 overexpression on the cell cycle of Kasumi-1, flow diagram on the left, and statistical plots on the right (n = 3). (F) S100A8 overexpression affects apoptosis in Kasumi-1 cells, with flow representative plots on the left and statistical plots on the right (n = 3). (G) Effect of S100A8 overexpression on etoposide resistance in Kasumi-1 cells, with flow diagram on the left and statistical plots on the right (n = 3). (H) Flow of PDX modeling following coculture of patient-derived AML cells with MSC. (I) PB WBC counts in PDX mice with S100A8 overexpression versus the control group. (J) Percentage of BM leukemia cells of PDX mice in the S100A8 overexpression and control group. (K) The percentage of SP leukemia cells in PDX mice with S100A8 overexpression versus the control group. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/bb45d7f34d7d08be7d00cd6b.png"},{"id":56442023,"identity":"2e56a86a-9e81-4d90-89ae-05b457037359","added_by":"auto","created_at":"2024-05-14 08:44:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1333117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of S100A8 deletion in MSC on disease progression and leukemic stem cells in MLL-AF9 leukemic mice. \u003c/strong\u003e(A) A flow diagram of the MLL-AF9 leukemia model, with S100A8-deficient and control mice as recipients. (B) Leukemic cell infiltration revealed by BM Wright–Giemsa staining in S100A8-deficient and control MLL-AF9 mice. (C) Survival of MLL-AF9 mice in the S100A8-deficient and control groups (n = 8–9). (D) The spleen size of MLL-AF9 mice in the S100A8-deficient and control groups. (E–F) The proportion (E) and count (F) of PB leukemia cells in S100A8-deficient and control MLL-AF9 mice (n = 5–6). (G–H) The proportion (G) and count (H) of SP and BM leukemia cells in S100A8-deficient and control MLL-AF9 mice (n = 5–7). (I) A flow representation of BM LSCs (YFP\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e−\u003c/sup\u003e) in MLL-AF9 mice. (J–K) The proportion (K) and count (L) of PB LSCs in S100A8-deficient and control MLL-AF9 mice (n = 5–6). (L–M) Proportion (L) and count (M) of SP and BM LSCs in S100A8-deficient and control MLL-AF9 mice (n = 5–7). (N) Flow representation of BM L-GMPs (IL-7R/Lin\u003csup\u003e−\u003c/sup\u003eYFP\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003ehi\u003c/sup\u003eCD34\u003csup\u003e+\u003c/sup\u003eCD16/32\u003csup\u003ehi\u003c/sup\u003e) in MLL-AF9 mice. (O–P) Proportion (O) and count (P) of L-GMPs of SP and BM of S100A8-deficient versus control MLL-AF9 mice (n = 5–7). (Q) Cell cycle of BM LSCs in S100A8-deficient and control MLL-AF9 mice (n = 5–6). (R) Apoptosis of BM LSCs in S100A8-deficient and control MLL-AF9 mice (n = 6). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 (\u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/ee6f4d4727903ccb09a4c806.png"},{"id":56442024,"identity":"385941ed-fd46-466f-99ea-b24d5fcf7a5c","added_by":"auto","created_at":"2024-05-14 08:44:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":868943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular mechanisms by which MSC-derived S100A8 promotes AML progression. \u003c/strong\u003e(A) Flow chart of rescue experiment with RAGE inhibitor or TLR4 inhibitor added to the cocultivation system. (B) Effect of RAGE inhibitor or TLR4 inhibitor treatment on leukemic cell proliferation in the S100A8 overexpression group (n = 4). (C) The cocultivation of MSCs with KASUMI-1 cells after the overexpression of S100A8 was followed by transcriptome sequencing of the Kasumi-1 cells. A volcano plot was created to compare the upregulated and downregulated genes in the overexpression group to those in the control group. \u0026nbsp;(D) Gene Ontology analysis of the signaling pathway of Kasumi-1 in the S100A8 overexpression group versus the control group. (E) Gene set enrichment analyses evaluating changes in Kasumi-1 of the S100A8 overexpression group compared to control group. NES, normalized nrichment score; FDR q-val, false-discovery rate q-value. (F) PI3K, Akt expression and its phosphorylation level in Kasumi-1 of the S100A8 overexpression group compared to control group. (G) PI3K, Akt expression and its phosphorylation level in leukemic cells of S100A8-deficient and control AML mice. (H) Effect of PI3K inhibitor treatment on leukemic cells proliferation in the S100A8 overexpression group (n = 4). (I) MFI of DCFDA in leukemic cells and LSCs in S100A8-deficient and control AML mice (n = 5. (J) MFI of TMRE in leukemic cells and LSCs in S100A8-deficient and control AML mice (n = 5). (K) Representative γ-H2AX images \u0026nbsp;of BM cells of S100A8-deficient and control AML mice. (n = 3). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, \u003cem\u003et\u003c/em\u003e test (I, J) and 1-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (B, H) .\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/798613f0889d271bd266e37b.png"},{"id":56442020,"identity":"5f361114-41a2-4e6c-a564-c98b046c6950","added_by":"auto","created_at":"2024-05-14 08:44:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":577873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MSC-derived S100A8 deletion on 5-FU and IR stress hematopoiesis. \u003c/strong\u003e(A) Construction of the 5-FU and IR stress model. (B) A representative flow pattern of HSPC in BM. (C–D) Proportion (C) and absolute number (D) of BM HSC in the S100A8-deficient and control groups 14 days after 5-FU and IR treatment (n = 6-7). (E–F) The proportion (E) and absolute number (F) of BM hematopoietic progenitors in the S100A8-deficient and control groups 14 days after 5-FU and IR treatment (n = 6-7). (G–H) The percentage (G) and absolute number (H) of BM mature lineage cells in the S100A8-deficient and control groups 14 days after 5-FU and IR (n = 6-7). (I–J) Proportion (J) and absolute number (K) of PB mature lineage cells in the S100A8-deficient and control groups 14 days after 5-FU and IR treatment (n = 6-7). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/c6193d800d42281b97755bb0.png"},{"id":56442022,"identity":"5ad29b7c-722a-413a-b746-b347687bed70","added_by":"auto","created_at":"2024-05-14 08:44:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":427489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of S100A8 deficiency on the cell cycle and function of HSCs under stress. \u003c/strong\u003e\u0026nbsp;(A) Flow representation of the LSK cell cycle. (B–C) Cell cycle of LSK cells in the S100A8-deficient and control groups after 14 days of 5-FU treatment (B) and IR exposure (C) (n =4–5). (D) A flow representation of proliferation and apoptosis of LSK cells. (E–F) Proliferation of LSK cells in the S100A8-deficient and control group after 14 days of 5-FU treatment (E) and IR exposure (F) (n =4–5). (G–H) Apoptosis of LSK cells in the S100A8-deficient and control group after 14 days of 5-FU treatment (G) and IR exposure (H) (n = 4–5). (I–J) The total number of BM colonies in the S100A8-deficient and control groups after 14 days of 5-FU treatment (I) and IR exposure (J) (n =4).\u003cstrong\u003e \u003c/strong\u003e(K) Flow of 5-FU treatment for long-term hematopoietic model development. (L) Dynamic changes in PB WBC in S100A8 deletion and control groups treated with 5-FU (n = 7–8). (M–N) The proportion of PB (M) and BM (N) mature myeloid cells and lymphocytes cells 16 weeks after the first 5-FU treatment (n = 6–7). (O) Proportion of BM HPCs at 16 weeks after 5-FU treatment (n = 6–7). (P) Proportion of BM HSCs 16 weeks after 5-FU treatment (n = 6–7). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/03d0365348950eb566e58fb8.png"},{"id":56442028,"identity":"045133b6-6fd6-49e2-8b5e-59d638028ae8","added_by":"auto","created_at":"2024-05-14 08:44:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":782614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of S100A8 on MSC proliferation and differentiation.\u003c/strong\u003e (A) Representative plots of CFU-F for the S100A8 overexpression group and control group. (B) CFU-F counts in the S100A8 overexpression group and control group (n = 3). (C) The proliferation of MSCs in the S100A8 overexpression and control groups (n = 4). (D) Cell cycle status of MSCs in the S100A8 overexpression and control groups (n = 3). (E) Apoptosis of MSCs in the S100A8 overexpression and control groups (n = 3). (F) Flow diagram of BM MSCs of S100A8-deficient and control mice. (G–I) MSC ratio in BM of S100A8-deficient and control mice after 5-FU treatment (G), IR exposure (H), and AML (I) (n = 5–7). (J) Representative images of Azane Red staining (osteogenic differentiation) of MSC in the S100A8 overexpression and control groups. (K–L) qRT-PCR analysis of osteoblastic differentiation relative genes (\u003cem\u003eOPN\u003c/em\u003e, and \u003cem\u003eOCN\u003c/em\u003e) in MSCs from the S100A8 overexpression group and control group (n = 3). (M) A flow diagram of BM OBCs from S100A8-deficient and control mice. (N–O) OBC ratio in the BM of S100A8-deficient and control mice after 5-FU treatment (N) and IR exposure (O) (n = 5–6). (P–R) Microcomputed tomography analysis of the trabecular bone in S100A8-deficient and control AML mice. Representative images are displayed in (P). Scale bars measure 1 mm. Trabecular bone volume/total volume (BT/BV) and trabecular number (Tb. N) in the femoral metaphysis are illustrated in (Q–R) (n = 4). (S) Enzyme-linked immunosorbent assay analysis of BM protein concentrations of OPN in S100A8-deficient and control AML mice (n = 3). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 (\u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/6d37ed5861914c4f8769c8c3.png"},{"id":58046250,"identity":"c879d5ae-b40d-4fd9-882e-4ebcd153dbab","added_by":"auto","created_at":"2024-06-10 11:43:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7314176,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/0e835dc2-19b2-426f-9021-5e611a9392db.pdf"},{"id":56442026,"identity":"b6d710b0-5192-4a5e-a643-182f0b5f72b2","added_by":"auto","created_at":"2024-05-14 08:44:39","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1061417,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4374015/v1/7d5f55193261be5137355c13.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Mesenchymal stem cell-derived S100A8 facilitates leukemia stem cell maintenance via TLR4/PI3K/Akt signaling","fulltext":[{"header":"Key Points","content":"\u003cp\u003eNiche S100A8\u0026nbsp;accelerates AML development by regulating TLR4/PI3K/Akt pathway.\u003c/p\u003e\n\u003cp\u003eNiche S100A8 promoted short-term hematopoiesis recovery but impaired long-term hematopoiesis regeneration under stress.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eHematopoietic stem cells (HSCs) establish themselves in a safe bone marrow (BM) environment composed of a complex interconnected network of hematopoietic and nonhematopoietic cells that play critical roles in regulating HSC dormancy, proliferation, and migration(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Under normal circumstances, hematopoiesis strikes a delicate balance between expansion and quiescence to achieve maximum production output(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Both extrinsic(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and intrinsic factors(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) work hard to maintain this homeostasis while also allowing for adaptive responses(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) and recovery(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) in the face of emergencies like chemotherapy and radiotherapy. Acute myeloid leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of myeloid blasts evolved from hematopoietic stem and progenitor cells (HSPCs)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Adequate hematopoiesis regeneration under stress determines the outcome of AML and is governed by BM HSC niche(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMesenchymal stromal cells (MSCs) have a pivotal niche component(\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), which forms various BM components, such as adipogenic, osteogenic, and chondrogenic lineages(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Inflammation influences HSC activity and AML development through niche-dependent interactions, as demonstrated by a growing body of research(\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In response to external stimulation, BM niche cells release factors that promote myelopoiesis(\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, the precise mechanisms and roles of proinflammatory factors in the mesenchymal niche are poorly understood. At present, the proinflammatory factors that play a key role in the mesenchymal niche are not well studied.\u003c/p\u003e \u003cp\u003eS100A8, also known as \"calprotectin\" or \"myeloid-related protein 8 (MRP8),\" is a member of the S100 family that regulates various cellular processes, including Ca\u003csup\u003e2+\u003c/sup\u003e balance, apoptosis, migration, proliferation, differentiation, energy metabolism, and inflammation(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Extracellular S100A8 and S100A9, as well as the heterodimer S100A8/A9, are potent inflammatory factors(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The heterodimer S100A8/A9 indicates the predominant form, which accounts for up to 45% and \u0026sim;5% of all cytosolic proteins in neutrophils and monocytes, respectively(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Recent research has found elevated levels of MSC-derived S100A8 in patients with myeloid neoplasms such as myelodysplastic syndrome (MDS) and (yeloproliferative neoplasm(MPN)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEscalating S100A8 expression in MSCs is associated with increased MSC proliferation during the early osteogenic differentiation phase, whereas mature osteogenesis is inhibited, resulting in the disruption of trabecular bone structure(31\u0026ndash;33). The effects of S100A8 on stromal cells are similar to the remodeling of the BM microenvironment observed in AML. Notably, MSC-derived S100A8 has been linked to MDS disease progression(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) by causing mitochondrial dysfunction, eliciting oxidative stress responses, and impairing DNA damage repair in hematopoietic stem progenitors. In this study, we investigated the effects of aberrant expression of MSC-derived S100A8 on HSCs regeneration and AML development.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003e\u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice were obtained from GemPharmatech LLC.. Prx1-Cre mice were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. C57BL/6 and B6.SJL mice were bought from GemPharmatech LLC.. \u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice were crossed with Prx1-Cre mice to produce Prx1-Cre; \u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice. Mice ranging from eight to twelve weeks old were used. All animal procedures followed the animal care guidelines approved by the Institutional Animal Care and Use Committees at Zhongnan Hospital Wuhan University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAML patient and healthy donor samples\u003c/h2\u003e \u003cp\u003eAll BM specimens from AML patients and healthy donors were with informed consent obtained from Zhongnan Hospital and Wuhan University respectively. All studies involving human samples were approved by the Ethics Committee of Zhongnan Hospital and Wuhan University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis and cell sorting\u003c/h2\u003e \u003cp\u003eMice BM and peripheral blood (PB) samples were prepared as described previously. Phenotypic analysis of lineage cells, HSPCs, leukemia stem cells (LSCs), and leukemic GMP populations (L-GMPs), MSCs, and osteoblasts (OBCs) was performed according to previous studies. LSK cells (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003e), LT-HSCs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eCD34\u003csup\u003e\u0026minus;\u003c/sup\u003eFlt3\u003csup\u003e\u0026minus;\u003c/sup\u003e), ST-HSCs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eCD34\u003csup\u003e+\u003c/sup\u003eFlt3\u003csup\u003e\u0026minus;\u003c/sup\u003e), MPPs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eCD34\u003csup\u003e+\u003c/sup\u003eFlt3\u003csup\u003e+\u003c/sup\u003e), GMPs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e\u0026minus;\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eCD16/32\u003csup\u003ehi\u003c/sup\u003eCD34\u003csup\u003ehi\u003c/sup\u003e), CMPs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e\u0026minus;\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eCD16/32\u003csup\u003eMed\u003c/sup\u003eCD34\u003csup\u003ehi\u003c/sup\u003e), MEPs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e\u0026minus;\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eCD16/32\u003csup\u003e\u0026minus;\u003c/sup\u003eCD34\u003csup\u003e\u0026minus;\u003c/sup\u003e), CLPs (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eIL-7R\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003eMed\u003c/sup\u003ec-Kit\u003csup\u003eMed\u003c/sup\u003e), LSCs (YFP\u003csup\u003e+\u003c/sup\u003eCD117\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e\u0026minus;\u003c/sup\u003e), L-GMPs (IL-7R/Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eYFP\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003ehi\u003c/sup\u003eCD34\u003csup\u003e+\u003c/sup\u003eCD16/32\u003csup\u003ehi\u003c/sup\u003e), MSCs (CD45\u003csup\u003e\u0026minus;\u003c/sup\u003eTER119\u003csup\u003e\u0026minus;\u003c/sup\u003eCD31\u003csup\u003e\u0026minus;\u003c/sup\u003eLepR\u003csup\u003e+\u003c/sup\u003e), and OBCs (CD45\u003csup\u003e\u0026minus;\u003c/sup\u003eTER119\u003csup\u003e\u0026minus;\u003c/sup\u003eCD31\u003csup\u003e\u0026minus;\u003c/sup\u003eCD166\u003csup\u003e+\u003c/sup\u003eSca-1\u003csup\u003e\u0026minus;\u003c/sup\u003e) were analyzed with FACSCanto\u0026trade; Ⅱ (BD Biosciences, San Jose, CA, USA). Immature cells were obtained by sorting mouse HSPC and human LSCs with CD117 MicroBeads and CD34 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The antibody details are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of ROS\u003c/h2\u003e \u003cp\u003eThe intracellular (reactive oxygen species) ROS were measured using 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Briefly, cells were incubated with HSC or LSC marker antibodies for 30\u0026ndash;45 min before being treated with 10 \u0026micro;M DCFH-DA (Beyotime, S0033S) for 10 min at 37\u0026deg;C in the dark. Following incubation, the cells were washed twice with PBS and analyzed by flow cytometry (CantoII; BD). A shift to the right indicates higher ROS levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTMRM staining\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential was identified by tetramethylrhodamine, ethyl ester (TMRE, Beyotime, C2001S) following the manufacturer\u0026rsquo;s protocol. Briefly, cells were incubated with HSC or LSC marker antibodies for 30\u0026ndash;45 min, then stained with 50 nM TMRE for 10 min and analyzed using flow cytometry (CantoII; BD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eThe significance of differences between the two groups was assessed using unpaired two-tailed Student\u003cem\u003e\u0026rsquo;s t-\u003c/em\u003etests. The results in the bar graphs are mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Overall survival curves were plotted using the Kaplan\u0026ndash;Meier method, with log-rank tests used for comparisons. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExpression of MSC-derived S100A8 increased in cases of AML\u003c/h2\u003e \u003cp\u003eIn our previous study, we found a significant increase in the expression of BM MSC-derived S100A8 in a cohort of rapidly advancing murine models of AML (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;B), indicating that MSC-derived S100A8 plays a role in the regulation of leukemic disease progression. Here, we validated the expression of S100A8 in MSCs derived from BM with AML status. Volunteer-derived BM mononuclear cells were obtained and in vitro cultured to produce human BM MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). MSC expression of the marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;K)and differentiation functions were validated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u0026ndash;M). After 48 hours of cocultivation with leukemic cells, MSCs showed an increase in S100A8 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN\u0026ndash;O). Furthermore, MSC-derived S100A8 expression in MLL-AF9 AML mice increased similarly to that in normal mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP). Furthermore, our analysis of human BM sections showed very little expression of S100A8 in healthy human MSCs, whereas MSCs derived from patients with AML expressed a lot of S100A8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ). The data presented above support the aberrant expression of S100A8 in the presence of AML, implying that S100A8 may be involved in the progression of AML.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC-derived S100A8 is essential for the growth of human AML cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further validate the role of MSC-derived S100A8 in AML development, we performed an experiment in which S100A8 was overexpressed in human BM MSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C) and then cocultured with the human AML cell line Kasumi-1. Overexpression of S100A8 in MSCs increased the growth of primary human AML cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and facilitated their progression through the cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, overexpression of S100A8 did not significantly alter the rate of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), but it increased the resistance of Kasumi-1 cells to etoposide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Then, leukemia cells from AML patients were used, and CD34\u003csup\u003e+\u003c/sup\u003e leukemia cells were enriched using magnetic-activated cell sorting (MACS). CD34\u003csup\u003e+\u003c/sup\u003e leukemia cells were cocultured with S100A8-overexpressing and control MSCs for 48 h. Then, leukemia cells were transplanted into immunodeficient mice, along with the MSC suspension (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). After 30 days, the WBC count in S100A8-overexpressing mice was higher than in the control group, as were the levels in the BM and SP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI\u0026ndash;K). Finally, these findings indicate that S100A8 plays a role in the development and drug resistance of human AML cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC-derived S100A8 drives murine AML progression and maintains LSC function\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo fully understand the role of S100A8 in leukemogenesis, MSC-specific S100A8 knockout (Prx1-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e;\u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flo\u003cem\u003ex\u003c/em\u003e\u003c/sup\u003e) mice and control mice was created (Prx1-Cre\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e;\u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e) by crossing \u003cem\u003eS100A8\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice with Prx1-Cre mice (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), then MLL-AF9-induced murine AML model was constructed with S100A8 knockout and control mice as recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B). S100A8 deletion significantly improved the survival rate of AML mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Not surprisingly, S100A8 knockout recipients had lower spleen (SP) weights than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, we found that S100A8 knockout recipients had a significantly lower ratio of YFP\u003csup\u003e+\u003c/sup\u003e leukemic cells in the PB, SP, and BM, as well as lower malignant cell and total WBC counts in the PB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026ndash;H). Furthermore, the percentages and numbers of LSCs (YFP\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e\u0026minus;\u003c/sup\u003e cells) in PB, SP, and BM were significantly reduced after S100A8 deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI\u0026ndash;M), as were L-GMPs (IL-7R/Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eYFP\u003csup\u003e+\u003c/sup\u003ec-Kit\u003csup\u003ehi\u003c/sup\u003eCD34\u003csup\u003e+\u003c/sup\u003eCD16/32\u003csup\u003ehi\u003c/sup\u003e) in SP and BM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN\u0026ndash;P). Finally, we examined the characteristics of LSCs in a murine AML model, and we discovered that S100A8 knockout recipients had more G0 phase cells and higher apoptosis rates than control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eQ\u0026ndash;R). Collectively, we can reasonably conclude that S100A8 plays an important role in promoting the maintenance of LSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC-derived S100A8 regulates AML development via the TLR4/PI3K/Akt pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRAGE and TLR4 are the most common S100A8 receptors, and to investigate the mechanism by which S100A8 acts on leukemic cells, we added a RAGEE inhibitor (FPS-ZM1) or a TLR44 inhibitor (Resatorvid) to MSC and Kasumi-1 coculture systems, and the addition of TLR4 inhibitors resulted in the most significant decrease in leukemic cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B), indicating that MSC-derived S100A8 acts primarily by binding leukemic cell TLR4. To delve deeper into the molecular mechanism, we conducted transcriptomic analyses. It revealed notable differences in gene expression in Kasumi-1 cells between the group cocultured with overexpressed S100A8 and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), as well as significant alterations in the PI3K/Akt signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;E). WB experiments revealed that phosphorylated PI3K and Akt protein levels were significantly higher in the S100A8 overexpression group but lower in the S100A8-deficient group, and PI3K inhibitor (LY294002) significantly reduced proliferation of leukemic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The most recent article reported that PI3K/Akt regulates oxidative stress to promote drug resistance in leukemia progression, and changes in the HIF-1 pathway were also revealed in our sequencing results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We then examined ROS levels in LSCs. Flow cytometry analysis revealed a lower intracellular level of ROS in LSCs from S100A8 knockout mice compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). TMRE staining was used to determine mitochondrial membrane potential. Flow cytometry analysis revealed a decrease in TMRE fluorescence in LSCs from S100A8 knockout mice compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Furthermore, DNA damage was found to be lower in BM cells from S100A8 null AML mice compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Overall, these findings showed that S100A8 promoted leukemia progression through TLR4/PI3K/Akt signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMSC-derived S100A8 deletion is dispensable for normal hematopoiesis but inhibits HSC expansion under short-term stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImpairment of normal hematopoiesis and leukemia progression are 2 closely related processes during leukemia development and are regulated by the BM niche. We discovered that removing S100A8 had no discernible effect on the frequencies and numbers of HSCs, hematopoietic progenitors (HPCs) (except for multipotent progenitors, MPPs) (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u0026ndash;E), and mature cells were comparable in the BM and PB of control and knockout mice (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF\u0026ndash;I). Thus, MSC-derived S100A8 was not required for homeostatic hematopoiesis in mice. However, in the MLL-AF9 mouse model, we discovered that S100A8 knockout recipients had more HPCs and mature myeloid cells than control group mice (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eJ\u0026ndash;R), implying that S100A8 may regulate hematopoiesis under stress conditions.\u003c/p\u003e \u003cp\u003eInterestingly, we found that S100A8 was significantly upregulated in MSCs after 5-fluorouracil (5-FU) and irradiation (IR) exposure (Figure S3). As expected, the frequencies and numbers of LSKs, long-term HSCs (LT-HSCs), and short-term HSCs (ST-HSCs) were nearly all lower in S100A8\u003csup\u003e-/-\u003c/sup\u003e BM at day 14 after 5-FU treatment and IR exposure than in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;D). Notably, 5-FU treatment and IR exposure resulted in lower percentages and numbers of granulocyte/macrophage progenitors (GMPs), while decreased megakaryocyte/erythroid progenitors (MEPs) was observed in S100A8\u003csup\u003e-/-\u003c/sup\u003e mice after IR exposure, whereas common myeloid progenitors (CMPs) showed no significant difference between the two groups after 5-FU treatment and IR exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE\u0026ndash;F). Consistent with these findings, S100A8\u003csup\u003e-/-\u003c/sup\u003e mice had significantly fewer mature myeloid cells following 5-FU and IR treatment in both BM and PB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026ndash;J). Overall, our findings indicate that S100A8 ablation promotes short-term HSC expansion under hematopoietic stresses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDeletion of MSC-derived S100A8 increases HSC quiescence under hematopoietic stress\u003c/h2\u003e \u003cp\u003eGiven the observation that S100A8 causes short-term expansion of HSCs during hematopoietic stress, we hypothesized that S100A8 may modulate HSC quiescence. As expected, HSCs from S100A8 knockout mice had a higher percentage in the G0 phase but a decreasing trend in the G1 and S/G2/M phases after 5-FU treatment and IR exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C). Furthermore, an in vivo BrdU incorporation assay revealed that deletion of S100A8 reduced HSC proliferation of HSCs in mice following 5-FU and IR challenge, while apoptosis rates were comparable between S100A8 knockout and control HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;H). These findings suggest that S100A8 promotes hyperactivation of HSCs in response to stress stimuli. To see how S100A8 affected HSCs functionality, we used a colony formation assay. The results showed that the number of colonies formed in the group with S100A8 deletion was significantly higher after treatment with 5-FU and IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u0026ndash;J). These findings suggest that S100A8 promotes the entry of HSCs into the proliferation cycle as a means of short-term hematopoietic recovery under stressful conditions, albeit at the expense of impairing the general function of HSCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo see if S100A8 affects long-term HSC maintenance, we gave S100A8 knockout and control mice four injections of 5-FU (50mg/kg) once per week. The hematopoietic phenotypes of S100A8 knockout and control mice were then assessed at 4, 8, 12, and 16 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). The results revealed a great increase in white blood cell (WBC) count following the deletion of S100A8 after 4 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Surprisingly, the S100A8 knockout mice had lower percentages of mature PB and BM myeloid cells than control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM\u0026ndash;N), but higher percentages of GMPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO). However, it is worth noting that the proportion and quantity of LT-HSCs in the S100A8 knockout group were lower than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eP). This finding suggests that S100A8 reduces HSCs and myeloid differentiation bias in a prolonged stressful environment, causing HSCs to exhibit a senescent phenotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eS100A8 remodeled the BM microenvironment\u003c/h2\u003e \u003cp\u003eWe investigated whether dysregulated expression of S100A8 causes microenvironment remodeling, which affects hematopoietic homeostasis and leukemia progression. After overexpressing S100A8, MSCs were cultured for 72 h. Then, colony-forming unit-fibroblast (CFU-F) experiments were performed, which revealed that S100A8 overexpression significantly increases CFU-F formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B). Flow cytometry analysis revealed that the group with S100A8 overexpression had a higher proliferation rate by flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In addition, the S100A8 overexpression group showed a decrease in G0 and G1 phase cells while increasing S/G2/M phase cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Furthermore, the S100A8 overexpression group had a lower apoptosis ratio than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Subsequent investigations into MSCs in the mouse model revealed a lower percentage of MSCs in S100A8 knockout mice than in control mice following 5-FU and IR exposure, as well as in AML mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF\u0026ndash;I). Furthermore, our findings showed that S100A8 overexpression in vitro inhibited MSCs differentiation into OBCs and reduced osteopontin (OPN) and osteocalcin (OCN) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ\u0026ndash;L). In contrast, S100A8-deficient mice showed an increase in osteoblasts following 5-FU and IR exposure in AML mice, leading to increased secretion of OPN (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM\u0026ndash;S). This suggests that remodeling of the BM microenvironment by S100A8 may have an indirect effect on stress hematopoiesis and leukemia progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMyeloid cells exhibit the baseline alarming protein S100A8 expression as a marker, whereas MSCs typically do not express S100A8 under steady-state conditions. In this study, we found that the presence of AML 5-FU treatment and IR, all led to an increase in S100A8 expression. These findings highlight the critical role of S100A8, derived from MSCs, as a potent proinflammatory factor in AML and hematopoietic regeneration during stress.\u003c/p\u003e \u003cp\u003eUsing an in vitro coculture system of MSCs with leukemia cells and an MLL-AF9 leukemia mouse model, we discovered that MSC-derived S100A8 promotes leukemia cell proliferation and drug resistance while reducing the survival of leukemic mice. These findings indicate that S100A8 in the microenvironment greatly contributes to the progression of leukemia. According to reports, the cell membrane receptors RAGE and TLR4 are the primary binding receptors for S100A8 homodimers and S100A8/A9 heterodimers(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Our findings further show that TLR4 is the primary binding receptor for S100A8 in leukemia cells, as evidenced by the inhibition of RAGE and TLR4 receptors in the coculture system. S100A8 activates the downstream PI3K/Akt signaling pathway, which regulates leukemia cell growth and proliferation. Dysregulation of the PI3K pathway is common in various human cancers, accounting for approximately 50% of de novo AML cases exhibiting this aberration(\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The PI3K inhibitor was introduced into the coculture system, and subsequent findings confirmed the role of S100A8 in modulating leukemia cell proliferation via the PI3K pathway. A recent study discovered that DNA damage is inextricably linked to the PI3K/Akt pathway, which controls the senescence of hematopoietic stem cells(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In the current study, we discovered that leukemic stem cells in the S100A8-deficient group had lower levels of ROS and DNA damage, indicating that S100A8 may play a regulatory role in the PI3K/Akt pathway, which is associated with DNA damage and subsequent promotion of leukemia cell proliferation.\u003c/p\u003e \u003cp\u003ePrevious research has shown that the development of AML and the aging of HSCs are linked to the accumulation of DNA damage and increased ROS levels. Modifications in the levels of antioxidant enzymes in the plasma of individuals with AML, the decreased antioxidant status found in the plasma of patients with AML is most likely due to elevated levels of ROS, as evidenced by the decline in antioxidant activity. This decrease in antioxidant activity indicates that oxidative stress is widely recognized as a significant factor in AML progression and recurrence(\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In contrast, BM cells from transcription factor Meis1-deficient mice had reduced colony formation and significantly fewer long-term HSCs, which lost quiescence due to ROS accumulation in HSCs. ATF4 deficiency causes severe defects in HSCs, leading to a complex aging-like phenotype, the HSC defects exhibited in ATF4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice are linked to elevated production of mitochondrial ROS.\u003c/p\u003e \u003cp\u003eIn our study, we found that MSC-derived S100A8 increased ROS levels not only in LSCs but aslo in HSCs (Figure S4). This study demonstrates that increased S100A8 expression in MSCs contributes to short-term hematopoietic recovery following IR and 5-FU-induced stress, which is consistent with previous research that has highlighted the role of an inflammatory microenvironment in promoting the swift recovery of hematopoiesis. Nonetheless, this phenomenon has been shown to have a negative impact on the long-term preservation of hematopoietic stem cells, eventually reducing the stem cell reservoir. Our findings also indicate that the long-term maintenance of HSCs is compromised by sustained 5-FU stress. According to our findings, S100A8 promotes the entry of G0-phase hematopoietic stem cells into the cell cycle. HSC quiescence and activation are highly complex interactions governed by a variety of cell-intrinsic and cell-extrinsic factors(\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Our findings identify S100A8 as a critical regulator in determining the fate of HSCs, particularly in \"awakening\" dormant HSCs in response to hematological insults caused by IR and 5-FU. S100A8 clearly directs HSC differentiation toward the myeloid lineage, but the sustained inflammatory response mediated by S100A8 may eventually lead to HSC exhaustion. Increased levels of ROS may explain the contradictory effect of S100A8 expression, which inhibits the preservation of healthy HSCs while concurrently facilitating the progression of leukemia.\u003c/p\u003e \u003cp\u003eThe decline of HSCs and hematopoietic impairment associated with S100A8 may be linked to MSC senescence. Under stress conditions such as IR, 5-FU treatment, and AML, abnormal S100A8 causes MSCs to undergo cell cycle progression, while concurrently impairing their ability for osteogenic differentiation, which are signs of MSC senescence. Consequently, diminished osteogenic differentiation reduces the expression and secretion of OPN, a critical bridging protein primarily from MSCs and osteoblasts. Previous research has shown that a decrease in OPN causes a phenotype associated with hematopoietic senescence. Specifically, when young HSCs are exposed to an OPN-deficient niche, their ability to engraft is reduced, while the frequency of long-term HSCs increases, and stem cell polarity is lost. Conversely, when aged HSCs are exposed to thrombin-cleaved OPN, the aging process is reversed, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity, and the restoration of the balance between lymphoid and myeloid cells in PB(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, our findings reveal the dual role of the mesenchymal niche inflammatory factor S100A8 in facilitating AML progression via TLR/PI3K/Akt pathway and suppressing stress hematopoiesis. In additin, S100A8 stimulates the production of ROS in both HSCs and LSCs, causing HSC senescence and promoting leukemia cell self-renewal. This novel finding sheds new light on the relationship between niche inflammation, hematopoiesis, and malignant transformation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthorship Contributions\u003c/h2\u003e \u003cp\u003eConceptualization: XYL, JXW, and FLZ; Methodology: XYL, JXW, and XQL; Validation: FLZ; Formal analysis: XYL, and JXW; Investigation: XYL, JXW, XQL, LLM, GPC, QW, NZ, XQT, YXT, HQJ, YXL, RHL, and WYY; Resources: LL, TTH, JXW, and XQL; Data Curation: XYL, JXW, and RYP; Writing - Original Draft: XYL; Writing - Review \u0026amp; Editing: FLZ; Visualization: XYL, JXW and RYP; Supervision: FLZ; Project administration: XYL and FLZ; Funding acquisition: XYL, XZ and FLZ. All authors reviewed and authorized the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eDisclosure of Conflicts of Interest\u003c/h2\u003e \u003cp\u003e All authors have read and approved the submission of the final manuscript. No conflicts of interest were declared.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank all of the patients who took part in this study, as well as their families. This study was funded by the Natural Science Foundation of China program [grant numbers 81900116, 82370176, 82000127, 82200254], and the Zhongnan Hospital of Wuhan University discipline construction platform project [grant numbers 202021, PDJH202217].\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWei Q, Frenette PS. Niches for Hematopoietic Stem Cells and Their Progeny. Immunity. 2018;48(4):632\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoulatov S, Notta F, Laurenti E, Dick JE. 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EMBO J. 2017;36(7):840\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4374015/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4374015/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of microenvironmental inflammation in the regulation of acute myeloid leukemia (AML) and stressed hematopoiesis is significant, though the molecular mechanisms are not fully understood. Here, we found that MSCs in a leukemic microenvironment had dysregulated expression of the inflammatory cytokine S100A8. Upregulating S100A8 in MSCs increased the proliferation and chemoresistance of AML cells in vitro. In contrast, removing S100A8 from MSCs in the murine MLL-AF9 AML model resulted in longer survival and less infiltration of leukemia cells and leukemic stem cells (LSCs). S100A8 binds to the TLR4 receptor on leukemia cells, which activates the PI3K/Akt pathway. In addition, removing S100A8 from MSCs causes a temporary increase in their quantity, followed by a decline in hematopoietic stem cells (HSCs) in mice exposed to stressful environments. Furthermore, the absence of S100A8 alters the properties of MSCs, impairing their ability to differentiate into osteoblasts and decreasing the expression of osteopontin, which is required to support HSCs. Our findings highlight the importance of MSC-derived S100A8 in promoting the maintenance of LSCs while impeding the maintenance of HSCs, providing new insights into the potential for the management of AML and hematopoietic regeneration.\u003c/p\u003e","manuscriptTitle":"Mesenchymal stem cell-derived S100A8 facilitates leukemia stem cell maintenance via TLR4/PI3K/Akt signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-14 08:44:24","doi":"10.21203/rs.3.rs-4374015/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b65c7bf7-9e40-46e3-ad35-a89cdcbce6c0","owner":[],"postedDate":"May 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31630963,"name":"Biological sciences/Stem cells/Haematopoietic stem cells"},{"id":31630964,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia"}],"tags":[],"updatedAt":"2024-06-10T11:35:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-14 08:44:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4374015","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4374015","identity":"rs-4374015","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}