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

Bone marrow adiposity and the hematopoietic niche: A historical perspective of reciprocity, heterogeneity, and lineage commitment.

Here we review the current knowledge on bone marrow adipocytes (BMAds) as active contributors to the regulation of the hematopoietic niche, and as potentially pivotal players in the progression of hematological malignancies. We highlight the hierarchical and functional heterogeneity of the adipocyte lineage within the bone marrow, and how potentially different contexts dictate their interactions with hematopoietic populations. Growing evidence associates the adipocyte lineage with important functions in hematopoietic regulation within the BM niche. Initially proposed to serve as negative regulators of the hematopoietic microenvironment, studies have also demonstrated that BMAds positively influence the survival and maintenance of hematopoietic stem cells (HSCs). These seemingly incongruous findings may at least be partially explained by stage-specificity across the adipocytic differentiation axis and by BMAds subtypes, suggesting that the heterogeneity of these populations allows for differential context-based interactions. One such distinction relies on the location of adipocytes. Constitutive bone marrow adipose tissue (cBMAT) historically associates to the "yellow" marrow containing so-called "stable" BMAs larger in size, less responsive to stimuli, and linked to HSC quiescence. On the other hand, regulated bone marrow adipose tissue (rBMAT)-associated adipocytes, also referred to as "labile" are smaller, more responsive to hematopoietic demand and strategically situated in hematopoietically active regions of the skeleton. Here we propose a model where the effect of distinct BM stromal cell populations (BMSC) in hematopoiesis is structured along the BMSC-BMAd differentiation axis, and where the effects on HSC maintenance versus hematopoietic proliferation are segregated. In doing so, it is possible to explain how recently identified, adipocyte-primed leptin receptor-expressing, CXCL12-high adventitial reticular cells (AdipoCARs) and marrow adipose lineage precursor cells (MALPs) best support active hematopoietic cell proliferation, while adipose progenitor cells (APCs) and maturing BMAd gradually lose the capacity to support active hematopoiesis, favoring HSC quiescence. Implicated soluble mediators include MCP-1, PAI-1, NRP1, possibly DPP4 and limiting availability of CXCL12 and SCF. How remodeling occurs within the BMSC-BMAd differentiation axis is yet to be elucidated and will likely unravel a three-way regulation of the hematopoietic, bone, and adipocytic compartments orchestrated by vascular elements. The interaction of malignant hematopoietic cells with BMAds is precisely contributing to unravel specific mechanisms of remodeling. BMAds are important operative components of the hematopoietic microenvironment. Their heterogeneity directs their ability to exert a range of regulatory capacities in a manner dependent on their hierarchical, spatial, and biological context. This complexity highlights the importance of (i) developing experimental tools and nomenclature adapted to address stage-specificity and heterogeneity across the BMSC-BMAd differentiation axis when reporting effects in hematopoiesis, (ii) interpreting gene reporter studies within this framework, and (iii) quantifying changes in all three compartments (hematopoiesis, adiposity and bone) when addressing interdependency

<i>SCL</i>, <i>LMO1</i> and <i>Notch1</i> Reprogram Thymocytes into Self-Renewing Cells

<div><p>The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive <i>NOTCH1</i> allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive <i>NOTCH1</i> cannot reprogram thymocytes on its own, despite the fact that <i>NOTCH1</i> is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating <i>NOTCH1</i> triggers a parallel pathway involving <i>Hes1</i> and <i>Myc</i> that dramatically enhances the activity of <i>SCL-LMO1</i> We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, <i>LYL1</i> and <i>LMO1</i> or <i>LMO2</i> are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.</p></div

Model of the collaboration between the SCL, LMO1 and <i>Notch1</i> oncogenes.

<p>(<b>A</b>) <i>SCL</i> and <i>LMO1</i> interact to upregulate <i>Lyl1</i> gene expression and create a feed forward loop that activates self-renewal in DN3 thymocytes. DN3 cells are prone to <i>SCL-LMO1</i> self-renewal activity due to higher physiological NOTCH levels. (<b>B</b>) The <i>Notch1</i> oncogene drastically enhances <i>SCL-LMO1</i>-induced self-renewal activity to expand the pool of target cells to DN1-4 and ISP8 in a parallel pathway via <i>Hes1</i> and <i>c-Myc</i>. <i>SCL-LMO1</i> initiated cells (A) subsequently acquire gain of function <i>Notch1</i> mutations (B), causing target cell expansion and escape from thymic environmental control.</p

Transcription activation driven by SCL-LMO1 interaction is critical for thymocyte reprogramming and T-ALL induction.

<p>(<b>A</b>) Generation of transgenic mice expressing the LMO1-binding defective mutant SCLm13. The sequence coding for wild type human SCL or human SCLm13 HLH domain mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004768#pgen.1004768-Lecuyer1" target="_blank">[37]</a> were cloned into the VA h<i>CD2</i> cassette to generate transgenic mice. Shown are amino acids of the HLH region of SCL or SCLm13. (<b>B</b>) Immunofluorescence of human SCL (wt or m13) by flow cytometry. Thymocytes were stained with the monoclonal antibody against human SCL (BTL73). Control cells were stained with the second antibody only. (<b>C</b>) Expression of E protein target genes is inhibited both by <i>SCL-LMO1</i> and <i>SCLm13-LMO1</i> transgenes in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to <i>β-Actin</i> (Mean +/- SD, n = 3). (<b>D</b>) Kaplan-Meier curves of the time to leukemia for <i>LMO1^tg</i>, <i>E2a^+/-LMO1^tg</i>, <i>SCL^tgLMO1^tg</i> and <i>SCLm13^tgLMO1^tg</i> mice. (<b>E</b>) The interaction between SCL and LMO1 is required to activate the transcription of the self-renewal genes <i>Lyl1</i>, <i>Hhex</i> and <i>Nfe2</i> in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to <i>β-Actin</i> (Mean +/- SD, n = 3). (<b>F–G</b>) SCL but not the LMO1-binding defective SCL-m13 mutant collaborates with LMO1 to induce abnormal thymic reconstitution potential to thymocytes. Pre-leukemic thymocytes (1.5×10⁷ cells) from 3-week-old mice were transplanted. Recipient mice were analysed for thymic reconstitution (CD45.2⁺Thy1⁺) after 6 weeks (F) and the proportion of DP cells in engrafted CD45.2⁺Thy1⁺ thymocytes was assessed by FACS (G).</p

Notch1 collaborates with SCL-LMO1 to increase the pool of pre-LSCs and their competitiveness independently of a functional pre-TCR.

<p>(<b>A</b>) The engraftment of <i>SCL-LMO1</i> DN3 thymocytes is abrogated by γ-secretase inhibitor (GSI) treatment prior to transplantation. DN3 thymocytes were purified from pre-leukemic <i>SCL</i>^tg<i>LMO1</i>^tg mice and co-cultured on OP9-DL1 stromal cells in the presence or absence (vehicle) of 2.5 µM DAPT (GSI) for 4 days. The total numbers of viable cells recovered per culture are shown (<i>right panel</i>). Following drug treatment, equal numbers of viable cells were transplanted (5×10⁴ per mouse, n = 5). Engrafted mice: number of positive mice showing thymic reconstitution per group. (<b>B</b>) A hyperactive <i>Notch1</i> allele is insufficient to induce aberrant self-renewal in thymocytes but significantly enhances the engraftment of <i>SCL</i>^tg<i>LMO1</i>^tg thymocytes. Total thymocytes (1.5×10⁷) from 1-week-old mice of the indicated genotype were transplanted; recipient mice were analyzed for thymic engraftment 3 weeks later. (<b>C</b>) Oncogenic <i>Notch1</i> increases the frequencies of <i>SCL-LMO1</i> pre-LSCs independently of a functional pre-TCR. Purified DN3 thymocytes from <i>SCL</i>^tg<i>LMO1</i>^tg and <i>Notch1</i>^tg<i>SCL</i>^tg<i>LMO1</i>^tg mice with (<i>Cd3ε</i>^+/+) or without (<i>Cd3ε</i>^-/-) a functional pre-TCR were transplanted in limiting dilution assays (<i>upper panel</i>). Mice were scored positive when T-cell lineage reconstitution was more than 1%; pre-LSC frequencies and confidence intervals (<i>lower panel</i>) were calculated by applying Poisson statistics using the Limiting Dilution Analysis software (StemCell Technologies). (<b>D</b>) <i>Cd3ε</i>^-/-<i>Notch1</i>^tg<i>SCL</i>^tg<i>LMO1</i>^tg pre-leukemic thymocytes outcompete <i>Cd3ε</i>^-/-<i>SCL</i>^tg<i>LMO1</i>^tg thymocytes. Reconstitution by <i>Cd3ε</i>^-/-<i>Notch1</i>^tg<i>SCL</i>^tg<i>LMO1</i>^tg (CD45.2⁺ GFP^-, closed circles) and <i>GFP</i>^tg<i>Cd3ε</i>^-/-<i>SCL</i>^tg<i>LMO1</i>^tg (CD45.2⁺ GFP⁺, open circles) thymocytes transplanted with the indicated cell numbers at 1∶1 or 1∶20 ratio. (<b>E</b>) <i>Notch1</i> expands the cellular targets of <i>SCL-LMO1</i> to DN1-4 and ISP8 cells. Pre-leukemic thymocyte subsets (DN1-4, ISP8 and DP) were purified from <i>Notch1^tgSCL^tgLMO1</i>^tg mice and transplanted at 5×10⁴ cells per recipient mouse. The absolute numbers of donor-derived DN1-4 and ISP8 cells was calculated for each transplantation.</p

Functional importance of <i>Hes1</i> and <i>c-Myc</i> downstream of <i>Notch1</i> in thymocyte reprogramming induced by SCL-LMO1.

<p>(<b>A</b>) Expression of GSI-responsive NOTCH1 target genes during thymocyte differentiation. Global gene expression data of thymocyte subpopulations were obtained from the Immunological Genome Project (<a href="http://www.immgen.org/" target="_blank">http://www.immgen.org/</a>). The percentage of GSI-responsive NOTCH1 target genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004768#pgen.1004768-Wang1" target="_blank">[68]</a> or MYC target genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004768#pgen.1004768-Lachmann1" target="_blank">[120]</a> that are up-regulated at each transitional stage during thymocyte differentiation (>1.3-fold change) are shown. (<b>B</b>) Filtering of GSI-responsive NOTCH1 target genes that increase at the DN2 to DN3a transition and are present in HSC self-renewal resources (<a href="http://www.bioinfo.iric.ca/self-renewal/Main" target="_blank">www.bioinfo.iric.ca/self-renewal/Main</a> and <a href="http://www.bonemarrowhsc.com" target="_blank">www.bonemarrowhsc.com</a>). (<b>C</b>) Gene expression profiles of <i>Notch1</i> and their target genes (<i>Notch3</i>, <i>Hes1</i>, <i>IL7r</i>, <i>Myc</i> and <i>Bcl6</i>) during thymocyte differentiation were collected from the Immunological Genome Project and represented as a heat map. (<b>D</b>) Schematic strategy to study the role of <i>Hes1</i> and <i>c-Myc</i> in self-renewal activity induced by <i>SCL-LMO1</i>. Lineage negative (LIN^-) cells from <i>SCL</i>^tg<i>LMO1</i>^tg mice (CD45.2⁺) were transduced with either MSCV-<i>Hes1</i> and MSCV- <i>Myc</i> retroviral vectors or with control MSCV-GFP. Equal number (5×10⁴ cells) of purified GFP⁺LIN^- cells were then transplanted in primary mice (CD45.2^-). Donor-derived GFP⁺CD45.2⁺ thymocytes were transplanted at the limiting dose of ∼1 CRU (10⁵ cells) per mouse into secondary recipients. (<b>E</b>) Immunophenotype of donor-derived GFP⁺CD45.2⁺ thymocytes in primary mice was analyzed by FACS (<i>left panel</i>) and the absolute number of DN3 cells was calculated (<i>right panel</i>). (<b>F</b>) The fold expansion of donor-derived GFP⁺CD45.2⁺ DN3 thymocytes was calculated in secondary mice.</p