The bone marrow adipose tissue (BMAT) (1) constitutes more than two-thirds of the bone marrow (BM) volume in adult life (2, 3). It is comprised of cells, BM adipocytes (BMAds), that bear a single, large cytoplasmic lipid droplet and express adipose-lineage markers such as fatty acid binding proteins, leptin, adiponectin and others (4). BMAds appear at birth in the extravascular marrow space to contribute to the three-dimensional stromal cell network that holds hematopoietic cells, and progressively expand during skeletal growth in parallel with the centripetal retraction of the hematopoiesis (Neumann’s law) (5). Based on this inverse relationship with the hematopoietic tissue, BMAds have long been thought as white fat cells with a simple supporting/filling function. However, it is now widely recognized that BMAds represent a distinct fat depot with metabolic and functional properties that are different from those of the other human fat tissues (6). In addition, it is now well known that BMAds participate in the regulation of skeletal homeostasis, in systemic metabolism and in the pathogenesis of different bone and BM diseases (4, 7).

Over the last years, the potential pathogenetic role of BMAds and its molecular bases have been intensively investigated in marrow neoplastic conditions such as hematological malignancies and cancer metastasis. Thus, it has been shown that BMAds are able to modulate the migration and aggressiveness of neoplastic cells (8), to sustain their growth (9) and, in some cases, to promote their survival during therapy (10, 11). These effects are achieved by multiple mechanisms such as the generation and transfer of fatty acids (9) and the secretion of different types of adipokines (4). On the other side, it has also been reported that tumor cells are able to harness the metabolic and biological functions of BMAds to generate a microenvironment that promotes their own growth. The tumor-dependent modifications of BMAds reported so far include the stimulation of a lipolytic state (9), the increased expression of lipid transporter genes (9) and the induction of a senescence-associated secretory phenotype (12). However, although these studies have significantly expanded the knowledge on the molecular crosstalk between BMAds and neoplastic cells, they have been performed predominantly in ex-vivo experimental systems or in animal models thus providing little, if any, information on the in situ features of BMAds in the human neoplastic BM.

In this study, we have analyzed the number, morphology and phenotype of BMAds in archival bone biopsies obtained from patients with marrow metastasis, either from epithelial and non-epithelial tumors, and myeloproliferative neoplasms featuring grade-3 myelofibrosis. We selected BM metastasis since in situ features of BMAds in this condition have never been investigated. The samples of chronic myeloproliferative neoplasms were chosen based on the absence of data on the in situ immunophenotype of BMAds in these haematological malignancies. In order to generate a homogenous experimental group, the latter samples were selected based on the presence of myelofibrosis, which is frequently found in patients with chronic myeloproliferative neoplasms. We report that BMAds in the neoplastic marrow samples were characterized by altered histomorphometric and immunophenotypic features that might reflect their tumor-promoting activity.

The human BM is the site of origin of most haematological malignancies and a frequent site of metastasis of non-haematological tumors. In marrow neoplasia, the BMAT undergoes metabolic and molecular changes that help cancer cells to thrive at the expense of the other BM cell types. BMAds are known to secrete adipokines (e.g., adiponectin and leptin) and inflammatory factors (e.g., interleukin-6 and tumor necrosis factor-α) and are thought to be the energy source of tumor cells (e.g., through the release of free fatty acids), which, in turn, modulate their activities by releasing different types of molecules (e.g., cytokines and chemokines) (4, 8, 11, 19).

Previous work showed that in peripheral white adipose tissue (WAT), adipocytes located in close proximity of invasive cancer cells undergo morphological changes [i.e., loss of lipid content (delipidation) and acquisition of a fibroblast-like/pre-adipocyte phenotype (dedifferentiation)] and functional modifications (e.g., decreased expression of adipocyte-related genes and increased production of pro-inflammatory cytokines) (20). However, only a few studies investigated the in situ morphology and phenotype of BMAT in the human neoplastic BM.

The analysis of BMAds neighboring marrow tumors may provide information of relevance for the pathogenesis and possibly for the diagnosis of marrow neoplastic diseases. For example, it may reveal changes of BMAds that occur at a specific time (e.g., early vs late stage of marrow cancer infiltration) and/or anatomical space (e.g., in subcortical vs perisinusoidal BMAT) during the progression of the disease. On the other side, it may assist in the diagnostic identification of neoplastic BM samples when cancer cells are very few and barely detectable. Currently available work on the in situ features of BMAds in the neoplastic BM consists essentially in a few histomorphometry studies performed exclusively on hematological tumors (21).

Here, we report the results of the quantitative and qualitative analyses of BMAds in bone biopsies from patients with different types of marrow cancer metastasis and with myeloproliferative neoplasia with grade-3 myelofibrosis. We provide the first data on the in situ immunophenotype of BMAds in haematological malignancies and the first analysis ever of their in situ quantitative and immunophenotypic features in BM cancer metastasis.

Our study revealed a significant decrease in the number of BMAds in affected bone biopsies, regardless of the type of neoplasia. The quantitative reduction of BMAds in the presence of tumors seems to be a recurrent finding and was previously observed in Multiple Myeloma (MM) (12), Acute Lymphoblastic Leukaemia (ALL) (22), Myelodysplasia (21, 23), Acute Myeloid Leukaemia (AML) (24) and MPN (21). The reason for the lowering of BMAds in marrow neoplastic conditions is currently under investigation. Some studies showed that it is unrelated to the amount of marrow cellularity and may not be explained solely by the physical pressure caused by tumor infiltration (12, 22). Other studies reported a compromised growth and differentiation of BMAd progenitor cells (22, 24, 25). BMAds derive from skeletal stem/progenitor cells that appear after birth in the marrow cavity (26) and are included within the fibroblast-like cell subset of the BM stroma (Bone marrow stromal cells, BMSCs) (27). Reduced adipogenic differentiation was shown in BMSCs isolated from AML patients, in which the defective generation of mature adipocytes was not reproduced in the extra-skeletal WAT depots (24), and from patients with MM (25). In addition, in ALL patients, Heydt et al. observed an altered growth of BMSCs with accumulation of adipocyte-primed cells and suggested that the differentiation of adipocyte progenitors was stalled by blast-dependent mechanisms (22). Interestingly, we previously observed that BM stromal progenitor cells isolated from patients with AML were characterized by an enhanced terminal adipogenic differentiation compared to controls when transplanted in vivo in the absence of leukemic blasts (28). The negative modulation of BM adipogenesis seems to be in contrast with the positive effect of BMAds on cancer growth. A plausible explanation for our results is that this positive effect is an early event no longer required in the established disease. However, it must be reminded that BMAds also support the maturation of the normal myeloid-erythroid lineages (24), which compete with the neoplastic cells for the same microenvironment. Thus, it is possible that the modulation of BMAd number in the neoplastic marrow reflects, at least in part, the need for the tumor to generate a supporting microenvironment while suppressing the normal haematopoiesis.

We also observed a reduced size of BMAds in all our samples, consistent with previous report on MM (29) and other lympho- and myelo-proliferative conditions (21). The change in the dimension of individual BMAds was previously related to the enhanced lipolysis induced by neoplastic cells (9, 30), although subsequent studies did not detect a lipolytic activity in BMAds (6). Regardless of the mechanism(s), the shrinking of BMAds may have multiple potential consequences on the neoplastic microenvironment that should not be overlooked. For example, since BMAds are in close contact with the marrow sinusoidal network (31), a pronounced modification of their dimension may affect the blood flow and, as a consequence, the amount of oxygen and nutrients for neoplastic cells. In addition, as previously reported in WAT, variations in the cell size within BMAT could be correlated with different chemokine and lipid secretory profiles and therefore with different modulatory effects on the tumor growth (32).

As major changes in the overall phenotype of BMAT, we detected a reduced number of ADIPOQ positive and an increased number of p-HSL positive BMAds in the neoplastic marrow compared to control biopsies. ADIPOQ is the most abundant molecule produced by the adipose tissue and in humans it is highly expressed in BMAT compared to WAT (33). Reduced levels of ADIPOQ, in parallel with an increase in the level of leptin, are associated with an enhanced risk of development of different tumors, including hematological neoplasia (34, 35). Indeed, ADIPOQ inhibits proliferation and promotes apoptosis in different types of cancer cells (36). Recently, it has been reported that the content of this adipokine in the marrow plasma is lower in patients with ALL compared to healthy subjects (22). In agreement with these data, we demonstrate here for the first time a reduction in the number of ADIPOQ positive BMAds in the presence of cancer metastasis and myeloproliferative neoplasia with grade-3 myelofibrosis, thus confirming that the modulation of the secretory activity of BMAT contributes to the changes in the marrow/serum levels of this adipokine in neoplastic conditions.

HSL is a lipase that is activated upon phosphorylation and is involved in the breakdown of triglycerides (37). Cancer cells may upregulate its expression and phosphorylation in WAT (38, 39). Although BMAds seem to have a specific lipid metabolism compared to WAT (6), HSL phosphorylation was reported to be induced in BMAds co-cultured with ALL blasts (9). In this study we have confirmed this effect in situ showing that, in the presence of neoplastic cells, the fraction of p-HSL positive BMAds was expanded. The metabolic implication of the increase of p-HSL and its significance in the context of BM cancer involvement remains to be clarified. However, it is interesting to note that in the marrow metastasis of glioma, we observed BMAds bearing a large lipid vacuole and a polarized cluster of small droplets beneath the plasma membrane. This finding is reminiscent of the remodeled BMAds previously detected in the mouse femur upon stimulation with a β3 adrenergic receptor agonist (40). Thus, it is possible that a remodeling phenomenon occurs in human BMAds too, at least in some specific rare conditions as the exposure to glioma cells. Of note, it is known in the literature that astrocytomas may cause lipodystrophy (41) and that lipolysis is increased in the glioma microenvironment (42).

In conclusion, although this study has important limitations (such as the reduced number of bone biopsies, the absence of correlation with patients’ clinic and anthropometric data), it demonstrates for the first time that, in different types of marrow cancers, BMAds undergo significant quantitative and morpho-phenotypical changes, which need to be further investigated in future studies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Institutional Review Board, Department of Molecular Medicine, Sapienza University of Rome, Italy, 17/02/2022. Written informed consent was not deemed necessary owing to the retrospective nature of the study.

MDSV, BP, SD, GF, FA and IC performed morphometric analysis and immunohistochemical stains. MDSV, BP, MS, AC and MR analyzed the data. MDSV and BP performed statistical analysis and wrote the draft of the manuscript. MDSV, BP, MS, AC and MR edited the manuscript. All authors approved the final version of the manuscript.

Sapienza University to AC (RM118164289636F0) and MR (RM11916B839074A8 and RM120172B8BF5C15).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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