Inducible CXCL12/CXCR4-dependent extramedullary hematopoietic niches in the adrenal gland.

ABSTRACT: Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. Although the complex BM niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic-supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here, we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α/leptin receptor (PDGFRα+/LEPR+/-)-expressing stromal nodules. We further show in CXCL12-green fluorescent protein reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-abundant reticular cells, which compose the BM HSPC niche. Mechanistically, HSPC homing to hormonally induced adrenal glands was found dependent on the CXCR4-CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells coexpressing master niche regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches, which may pave the way to therapeutic applications.

Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition.

Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues.

MarrowQuant 2.0: A Digital Pathology Workflow Assisting Bone Marrow Evaluation in Experimental and Clinical Hematology.

Bone marrow (BM) cellularity assessment is a crucial step in the evaluation of BM trephine biopsies for hematologic and nonhematologic disorders. Clinical assessment is based on a semiquantitative visual estimation of the hematopoietic and adipocytic components by hematopathologists, which does not provide quantitative information on other stromal compartments. In this study, we developed and validated MarrowQuant 2.0, an efficient, user-friendly digital hematopathology workflow integrated within QuPath software, which serves as BM quantifier for 5 mutually exclusive compartments (bone, hematopoietic, adipocytic, and interstitial/microvasculature areas and other) and derives the cellularity of human BM trephine biopsies. Instance segmentation of individual adipocytes is realized through the adaptation of the machine-learning-based algorithm StarDist. We calculated BM compartments and adipocyte size distributions of hematoxylin and eosin images obtained from 250 bone specimens, from control subjects and patients with acute myeloid leukemia or myelodysplastic syndrome, at diagnosis and follow-up, and measured the agreement of cellularity estimates by MarrowQuant 2.0 against visual scores from 4 hematopathologists. The algorithm was capable of robust BM compartment segmentation with an average mask accuracy of 86%, maximal for bone (99%), hematopoietic (92%), and adipocyte (98%) areas. MarrowQuant 2.0 cellularity score and hematopathologist estimations were highly correlated (R2 = 0.92-0.98, intraclass correlation coefficient [ICC] = 0.98; interobserver ICC = 0.96). BM compartment segmentation quantitatively confirmed the reciprocity of the hematopoietic and adipocytic compartments. MarrowQuant 2.0 performance was additionally tested for cellularity assessment of specimens prospectively collected from clinical routine diagnosis. After special consideration for the choice of the cellularity equation in specimens with expanded stroma, performance was similar in this setting (R2 = 0.86, n = 42). Thus, we conclude that these validation experiments establish MarrowQuant 2.0 as a reliable tool for BM cellularity assessment. We expect this workflow will serve as a clinical research tool to explore novel biomarkers related to BM stromal components and may contribute to further validation of future digitalized diagnostic hematopathology workstreams.

The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines.

BACKGROUND: The edible fruit Annona cherimola has previously shown many nutritional and medicinal properties. The current study evaluates the anti-cancer and anti-proliferative properties of Annona cherimola ethanolic leaf extract (AELE) on Acute Myeloid Leukemia (AML) cell lines cultured in vitro (Monomac-1 and KG-1). METHODS: The anti-proliferative effect of A. cherimola ethanolic leaf extract was evaluated via cell viability assay. Its pro-apoptotic effect was assessed through Cell Death ELISA and dual Annexin V/PI staining. To further investigate the molecular mechanism by which the extract promoted apoptosis and inhibited the proliferation of the AML cells used, apoptotic protein expression was determined through western blots. Extract composition was elucidated by Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: Our results showed that the treatment with A. cherimola ethanolic leaf extract exhibited an inhibitory effect on the proliferation of both cancer cell lines used in a dose- and time-dependent manner, with no toxic effects on normal mononuclear cells (MNCs) isolated from human bone marrow. This effect was mediated by DNA fragmentation and apoptosis, as revealed by Cell Death ELISA and dual Annexin V/PI staining. Western blot analysis revealed a Bax/Bcl2 dependent mechanism of apoptosis, as well as PARP cleavage, confirming the apoptotic results observed previously. These effects may be attributed to the presence of terpenes which constitute a large component of the leafy extract, as revealed via GC-MS. CONCLUSION: All the data presented in our study show that the terpene-rich A. cherimola ethanolic leaf extract exhibits an anti-proliferative and pro-apoptotic effect on the AML cell lines used.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

MarrowCellDLD: a microfluidic method for label-free retrieval of fragile bone marrow-derived cells.

Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.

Urtica dioica Leaf Infusion Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to Cisplatin Treatment.

Urtica dioica (UD) has been widely used in traditional medicine due to its therapeutic benefits, including its anticancer effects. Natural compounds have a promising potential when used in combination with chemotherapeutic drugs. The present study explores the anticancer and anti-proliferative properties of UD tea in combination with cisplatin on MDA-MB-231 breast cancer cells in vitro. To elucidate the effect of this combination, a cell viability assay, Annexin V/PI dual staining, cell death ELISA, and Western blots were performed. The results showed that the combination of UD and cisplatin significantly decreased the proliferation of MDA-MB-231 cells in a dose- and time-dependent manner compared to each treatment alone. This was accompanied by an increase in two major hallmarks of apoptosis, the flipping of phosphatidylserine to the outer membrane leaflet and DNA fragmentation, as revealed by Annexin V/PI staining and cell death ELISA, respectively. DNA damage was also validated by the upregulation of the cleaved PARP protein as revealed by Western blot analysis. Finally, the increase in the Bax/Bcl-2 ratio further supported the apoptotic mechanism of death induced by this combination. Thus, a leaf infusion of Urtica dioica enhanced the sensitivity of an aggressive breast cancer cell line to cisplatin via the activation of apoptosis.

Next Generation Bone Marrow Adiposity Researchers: Report From the 1st BMAS Summer School 2021.

The first International Summer School on Bone Marrow Adiposity was organized by members of Bone Marrow Adiposity Society and held virtually on September 6-8 2021. The goal of this meeting was to bring together young scientists interested in learning about bone marrow adipose tissue biology and pathology. Fifty-two researchers from different backgrounds and fields, ranging from bone physiopathology to adipose tissue biology and hematology, participated in the summer school. The meeting featured three keynote lectures on the fundamentals of bone marrow adiposity, three scientific workshops on technical considerations in studying bone marrow adiposity, and six motivational and career development lectures, spanning from scientific writing to academic career progression. Moreover, twenty-one participants presented their work in the form of posters. In this report we highlight key moments and lessons learned from the event.

The pro-apoptotic properties of a phytonutrient rich infusion of A. cherimola leaf extract on AML cells.

Annonaceae family has broad uses in herbal medicine for treatment of several diseases, whether through seeds' or leaves' extracts. The present study investigates the antiproliferative and antitumor activity of Annona cherimola aqueous leaf (AAL) extract/infusion in acute myeloid leukemia (AML) cell lines in vitro. High-resolution LC-MS was first used to analyze the composition of the aqueous extract. Cell proliferation assay, Annexin V staining, cell cycle analysis, dual Annexin V/PI staining, cell death quantification by ELISA, ROS level detection and Western Blotting were then performed to elucidate the therapeutic effects of AAL extract. The results obtained revealed a potent antioxidant activity of AAL extract. Moreover, the extract exhibited dose- and time-dependent antiproliferative effects on AML cell lines by decreasing cell viability with an IC50 of 5.03% (v/v) at 24 h of treatment of KG-1 cells. This decrease in viability was accompanied with a significant increase in apoptotic cell death with cell cycle arrest and flipping of the phosphatidylserine from the inner to the outer leaflet of the cell membrane. The respective overexpression and downregulation of proapoptotic proteins like cleaved caspase-8, cleaved PARP-1 and Bax and antiapoptotic proteins like Bcl-2 further validated the apoptotic pathway induced by AAL on AML cells. Finally, LC-MS revealed the presence of several compounds like fatty acids, terpenes, phenolics, cinnamic acids and flavonoids that could contribute to the antioxidant and anti-cancer effects of this herbal infusion. In addition to the generally known nutritional effects of the Annona cherimola fruit and leaves, the presented data validates the antioxidant and anti-cancerous effects of the leaf infusion on AML cell lines, proposing its potential therapeutic use against acute myeloid leukemia with future in vivo and clinical trials.

The selective anti-proliferative and pro-apoptotic effect of A. cherimola on MDA-MB-231 breast cancer cell line.

BACKGROUND: Herbal medicines have been a major target for numerous studies through the past years as an alternative treatment for cancer, mainly due to their minimal effects on normal healthy cells. Annona cherimola, popularly known as Cherimoya, is an edible natural fruit rich in phytochemical components and known to possess various biological activities. Previous studies have reported the anti-cancerous effect of A. cherimola ethanolic leaf extract (AELE) on leukemia. This study aims at studying the potential anti-cancer activity of this extract in vitro in two different breast cancer cell lines, namely MDA-MB-231 and MCF-7, in addition to investigating its toxicity on normal mesenchymal stem cells. METHODS: The anti-proliferative effect of AELE was evaluated via cell viability assay. Propidium iodide staining, Cell Death Detection ELISA and flow cytometry analysis of Annexin V binding were used to assess cell cycle progression, DNA fragmentation and apoptosis induction, respectively. Protein expression was determined via Western Blot analysis to decipher the underlying apoptotic molecular mechanism induced upon AELE treatment. RESULTS: The anti-proliferative effect of the extract was found to be selective on the triple-negative breast cancer cell line (MDA-MB-231) in a time- and dose-dependent manner with an IC50 of 390.2 µg/mL at 48 h, with no cytotoxic effects on normal murine mesenchymal stem cells. The pro-apoptotic effect was confirmed by the increase in cellular and DNA fragmentation, flipping of the phosphatidylserine moiety to the outer leaflet, and the increase in Annexin V binding. The underlying molecular mechanism revealed the involvement of the mitochondrial pathway, as shown by alterations in mitochondrial permeability and the upregulation of cytochrome c expression. CONCLUSION: All the data presented in our study suggest that AELE exhibits a selective anti-proliferative and pro-apoptotic effect on the chemo-resistant MDA-MB-231 breast cancer cells, providing evidence for the anti-tumor effects of A. cherimola.

Malva pseudolavatera Leaf Extract Promotes ROS Induction Leading to Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Malva pseudolavatera Webb & Berthel. is a plant from the Malvaceae family that has long been included in the human diet due to its various curative effects. Many plant leaf extracts from the various species of Malva genus have been reported to possess anti-cancer properties, however, studies on M. pseudolavatera Webb & Berthel. leaves have documented anti-inflammatory and anti-oxidant effects with no emphasis on their possible anti-cancer potential. The present study explores the anti-cancer properties of Malva pseudolavatera Webb & Berthel. leaf extract on acute myeloid leukemia (AML) cell lines in vitro and deciphers the underlying molecular mechanism. Treatment of AML cell lines with M. pseudolavatera methanolic leaf extract showed a dose- and time-dependent inhibition of proliferation and a dose-dependent increase in apoptotic hallmarks such as an increase in phosphatidylserine on the outer membrane leaflet and membrane leakage in addition to DNA fragmentation. The pro-apoptotic effect was induced by reactive oxygen species (ROS) as well as an upregulation of cleaved poly(ADP-ribose) polymerase (PARP), increase in Bax/Bcl-2 ratio, andrelease of cytochrome-c from the mitochondria. Major compounds of the extract included methyl linolenate, phytol, γ-sitosterol, and stigmasterol as revealed by gas chromatography coupled with mass spectrometry, and amino acids, amino acid derivatives, tiliroside, 13-hydroxyperoxyoctadecadienoic, and quercitrin as detected by liquid chromatography coupled to mass spectrometry.

MarrowQuant Across Aging and Aplasia: A Digital Pathology Workflow for Quantification of Bone Marrow Compartments in Histological Sections.

The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-µCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in, MarrowQuant, which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner. MarrowQuant discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature, AdipoQuant, fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with MarrowQuant lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of in vivo recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies MarrowQuant successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.

Annona cherimola Seed Extract Activates Extrinsic and Intrinsic Apoptotic Pathways in Leukemic Cells.

Annona cherimola Mill is a large green fruit with black seeds widely known to possess toxic properties due to the presence of Annonaceous acetogenins. The present study investigates the anti-cancer properties of an Annona cherimola Mill ethanolic seed extract on Acute Myeloid Leukemia (AML) cell lines in vitro and elucidates the underlying cellular mechanism. The anti-proliferative effects of the extract on various AML cell lines and normal mesenchymal cells (MSCs) were assessed using WST-1 viability reagent. The pro-apoptotic effect of the extract was evaluated using Annexin V/PI staining and Cell Death ELISA. The underlying mechanism was deciphered by analyzing the expression of various proteins using western blots. Treatment with an A. cherimola seed ethanolic extract promotes a dose- and time-dependent inhibition of the proliferation of various AML cell lines, but not MSCs. Positive Annexin V staining, as well as DNA fragmentation, confirm an increase in apoptotic cell death by upregulating the expression of pro-apoptotic proteins which control both intrinsic and extrinsic pathways of apoptosis. GC/MS analysis revealed the presence of phytosterols, in addition to other bioactive compounds. In conclusion, Annona cherimola Mill seed extract, previously known to possess a potent toxic activity, induces apoptosis in AML cell lines by the activation of both the extrinsic and the intrinsic pathways.