Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36 + LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

In the present study, we show that gonadal adipose tissue (GAT) serves as a reservoir for LSCs. We find that the interplay between resident leukemia cells and GAT results in lipolysis, which in turn fuels the fatty acid metabolism of leukemia cells. Further, we observe a distinct LSC subpopulation expressing the fatty acid transporter CD36. CD36 + cells demonstrate a relatively high level of FAO, have a quiescent and drug-resistant profile, and are protected by GAT from chemotherapy. Additionally, we find CD36 enhances leukemic colonization of GAT and also contributes to chemo-resistance of LSCs.

Stem cells have unique metabolic characteristics, which are accommodated by the microenvironment (). For example, a hypoxic microenvironment is required for HSCs to utilize glycolysis as their main energy pathway, and disruption of either the hypoxic niche or glycolysis in HSCs results in impaired stem cell functions (). Further, HSCs show an increased reliance on fatty acid metabolism. Inhibition of fatty acid oxidation (FAO) in HSCs leads to attenuation of stem cell capability (). The degree to which LSC biology is influenced by the microenvironment remains largely unknown. Interestingly, AT is capable of regulating metabolism of resident cells through several mechanisms to promote the survival and growth of cancer cells. For example, AT protects acute lymphoid leukemia (ALL) cells from L-asparaginase by regulating glutamine metabolism (). Additionally, AT produces several metabolism-regulating adipokines, such as leptin and adiponectin, whose receptors are commonly expressed by malignant cells to modulate cancer cell metabolism (). These findings suggest a metabolic regulatory role of AT and prompted us to examine a potential effect of AT on LSC metabolism.

A recently identified function of AT is its role as a reservoir for stem cells, including hematopoietic stem cells (HSCs) (), suggesting the presence of a distinct HSC niche. Interestingly, stromal components in AT are similar to those in the bone marrow (BM), which are essential regulators of HSC homeostasis (). Further, AT secretes multiple cytokines, such as CXCL12, to recruit and maintain the function of HSCs (). Leukemia stem cells (LSCs) co-opt the HSC marrow environment to gain survival benefits through hijacking the mechanisms utilized by HSCs to maintain homeostasis. For instance, disruption of interactions between LSCs and their stromal components, such as the extracellular matrix (ECM), is an effective means to eradicate LSCs (). Further, studies have shown that leukemia cells are protected from oxidative stress by stromal cells in the microenvironment via regulation of cysteine metabolism (). These studies led us to hypothesize that the AT HSC niche may be co-opted by LSCs to support their unique metabolic needs.

AT is multifunctional. It serves as a storage site for lipids and also functions as an endocrine organ (). Besides its canonical role in the regulation of systemic metabolism, AT is thought to play a role in the development and progression of solid tumors through various mechanisms (). For example, in obese patients, AT contributes to the chronic inflammatory state that induces DNA damage and leads to colon carcinogenesis by production of pro-inflammatory cytokines (adipokines) (). Further, AT has been reported to facilitate the metastasis of ovarian cancer through regulating cancer cell fatty acid metabolism as well as to promote the progression of breast cancer through modulating tumor microenvironment stromal components (). While these studies suggest a supportive role of AT in multiple steps of carcinogenesis, the biological behaviors of AT are, in turn, altered by cancer cells. For instance, cancer cell-derived cytokines cause lipolysis of AT, leading to tissue atrophy, which is a hallmark of cancer cachexia syndrome (). Together, these findings imply an interplay between solid tumors and AT. Whether there is a similar relationship between AT and leukemia cells is not yet clear.

Clinical studies have shown that survival for obese leukemia patients is poor relative to normal-weight patients (), suggesting that leukemia cells residing in adipose tissue (AT) may be more resistant to treatment and therefore contribute to disease persistence or relapse. Further, numerous studies have demonstrated the unique biology of the AT microenvironment (). However, the nature of interactions between leukemia cells and AT is poorly understood and was, therefore, the focus of studies described herein.

Together, these observations suggest that in at least some human bcCML and AML patients, CD36 also functions as a surface marker to segregate metabolically and functionally distinct primitive populations.

Lastly, to explore whether our findings from human bcCML are evident in other forms of disease, we examined the role of CD36 in eight primary samples derived from AML patients. To provide a broad analysis, we intentionally chose a range of AML specimen types with varying mutations and cytogenetic abnormalities ( Table S1 ). We first examined the expression of CD36 in the CD34leukemic compartment. Similar to bcCML, four of the eight specimens had readily detectable CD36cells in the CD34population (greater than 1%) (AML5, 6, 7, and 8) ( Figure 7 I). Further, CD36 functions as a fatty acid transporter in all four samples ( Figures 7 J and S7 I). Of note, other groups have shown that in AML patients, poor prognosis is associated with higher expression of CD36 (), indicating a potential role of CD36 in AML persistence or relapse.

To determine whether there is any functional difference between CD36and CD36cells, we examined the cell-cycle status of these two populations. Consistent with results from the murine model, CD36/CD34cells are more quiescent compared to CD36/CD34in all three samples ( Figure S7 F). We further compared their drug sensitivity in vitro and found that CD36/CD34cells are relatively drug resistant in all three samples ( Figure S7 G). Lastly, we asked whether CD36/CD34cells are also drug resistant in vivo. Animals engrafted with the two specimens were treated with Ara-C for 3 days, followed by analysis of marrow cells by flow cytometry. As shown in Figures 7 H and S7 H, the CD36/CD34cells from both samples were relatively drug resistant in vivo.

To investigate infiltration of GAT, we employed a xenograft model using transplantation of human specimens (bcCML1, 5, and 6) into immune-deficient NOD scid gamma (NSG) mice. Of the three specimens tested, we observed successful engraftment of bone marrow for two specimens (bcCML1 and bcCML5). The presence of human leukemia cells in GAT was observed for both engrafted specimens ( Figures 7 D and S7 C). Further, we found CD36/CD34cells are enriched in GAT ( Figures 7 E and S7 D). Additionally, CD36/CD34cells show greater homing to GAT than BM, and the CD36 inhibitor SSO impairs their homing to GAT ( Figures 7 F and S7 E). Notably, we found that serum FFA level is elevated in NSG mice engrafted with bcCML cells ( Figure 7 G).

Finally, we asked whether our findings in the murine model could be recapitulated in human bcCML cells. To this end, we first examined the expression of CD36 in the CD34leukemic compartment, which is typically enriched for primitive leukemia cells. Of the eight primary human specimens available for analysis, four had readily detectable CD36cells in the CD34population (bcCML1, 5, 6, and 8) ( Figure 7 A). Using the fluorescent fatty acid analog BODIPY-dodecanoic acid, we found in all four samples that the CD36/CD34population had a higher fatty acid uptake rate and was more sensitive to the treatment of SSO relative to CD36/CD34cells ( Figures 7 B and S7 A). These findings suggest that CD36 functions as a fatty acid transporter in human bcCML cells. Of these four samples, three had sufficient tissue for further analyses (bcCML1, 5, and 6). We examined the FAO rate and found that CD36/CD34cells have a higher FAO rate and are more sensitive to SSO in all three samples ( Figures 7 C and S7 B), indicating expression of CD36 is linked to increased FAO, as observed in the mouse model.

(J) Fatty acid uptake in AML cells. Leukemia cells were serum starved and pre-treated with or without SSO (50 μM) before incubation with BODIPY-dodecanoic acid (1μM). Error bars show means ± SD from triplicates.p < 0.005. See also Figure S7 I.

(H) NSG mice transplanted with bcCML cells were treated with Ara-C (100 mg/kg/day) or saline (control) for 3 days. Composition of BM-residual leukemia cells was examined to evaluate relative drug resistance of CD36/CD34versus CD36/CD34cells. Error bars show means ± SD. n = 4,p < 0.05,p < 0.005. See also Figure S7 H.

(F) Homing ability of CD36/CD34cells to BM and GAT. Leukemia cells were pre-treated with or without SSO (50 μM) for 1 hr before injection. Error bars show means ± SD. n = 5,p < 0.005. See also Figure S7 E.

(B) Fatty acid uptake in bcCML cells. Leukemia cells were serum-starved and pretreated with or without SSO (50 μM) before incubation with BODIPY-dodecanoic acid (1 μM). Error bars show means ± SD from triplicates.p < 0.005. See also Figure S7 A.

Lastly, we asked whether loss of CD36 would affect LSC drug sensitivity. We first examined viability of LSCs upon ex vivo treatment. CD36KO LSCs are more sensitive to chemotherapeutic drugs ( Figure S6 I). To test whether CD36KO LSCs are drug sensitive in vivo, we applied the same chemotherapeutic regimen shown in Figure 5 F to WT and KO leukemia mice. We found there are more residual leukemia cells in BM of WT leukemia mice compared to KO leukemia mice after chemotherapy ( Figure 6 H). Furthermore, LSCs are enriched in BM of WT leukemia mice, while similar results are not observed in KO leukemia mice ( Figure 6 I). Together, our data suggest that CD36 contributes to the survival of LSCs.

In terms of metabolic activity, we examined whether fatty acid uptake and FAO are impaired in CD36KO leukemia cells. As shown in Figure S6 H, fatty acid uptake is decreased in CD36KO Linleukemia cells compared to their WT counterparts. Further, FAO rate in CD36KO LSCs is significantly lower and less sensitive to the CD36 inhibitor SSO compared to WT LSCs ( Figure 6 G).

To determine whether the decreased leukemia burden in GAT from KO leukemia mice is due to compromised migration to GAT, we performed homing assays and found that CD36KO LSCs are less localized to GAT compared to WT LSCs ( Figure 6 F), while there are no differences in the homing ability to BM ( Figure S6 G).

We next investigated whether CD36 plays a role in leukemia migration to GAT. First, we examined LSCs in the MLL-AFP model, where migration to GAT was relatively low. Notably, while CD36 was expressed in some bulk tumor cells, there was no CD36 expression in the LSC populations ( Figure S6 A). Thus, lack of CD36 correlates with poor infiltration of GAT. To further explore this issue, we established the bcCML model using marrow cells derived from a CD36 knockout (CD36KO) mouse strain ( Figure S6 B). As shown in Figure 6 A, leukemic burden is significantly lower in GAT from animals transplanted with CD36KO leukemia cells compared to leukemia derived from wild-type (WT) cells. Importantly, the difference between CD36KO and WT leukemia cells was only evident in GAT, whereas all other tissues examined (BM, spleen, and PB) showed no difference as a function of CD36 ( Figures 6 A and S6 C). Additionally, no difference was found in the frequency of phenotypically defined LSCs between WT and CD36KO leukemia cells in different tissues ( Figures 6 B and S6 D). Consistent with the decreased leukemic burden in GAT, cachexia symptoms were reduced in knockout (KO) leukemia mice. We observed less body weight loss ( Figure S6 E) as well as less atrophy of both GAT and IAT in KO leukemia mice compared to WT leukemia mice ( Figures 6 C and S6 F). The serum FFA level is also lower in KO leukemia mice ( Figure 6 D). Interestingly, CD36KO leukemia cells produce lower amounts of IL-1α compared to WT leukemia cells in both unstimulated and OA-stimulated conditions ( Figure 6 E), which potentially explains the reduced cachexia in KO leukemia mice. Together, these findings indicate that the mobilization of fatty acid from AT in KO leukemia mice is reduced.

(F) Homing ability of WT and CD36KO LSCs to GAT. Homing ability of LSCs was determined by the ratio of the percentage of LSCs in the leukemia cells localized to GAT to the percentage of LSCs in leukemia cells before injection. Error bars show means ± SD. n = 4, ∗ p < 0.05.

(E) Secretion of IL-1α in WT and CD36KO leukemia cells. Leukemia cells (1 million/ml) were treated with BSA or OA (100 μM) for 24 hr, and ELISA was performed to determine the concentration of IL-1α in media. Error bars show means ± SD from triplicates. ∗∗ p < 0.005.

Since most conventional chemotherapy agents preferentially target cycling cells, we hypothesized that the more quiescent CD36LSCs would be relatively drug resistant. To test this hypothesis, we monitored the viability of CD36LSCs, CD36LSCs and bulk leukemia cells upon ex vivo treatment with different chemotherapeutic drugs, including cytarabine (Ara-C), doxorubicin, etoposide, SN-38, and irinotecan (CPT-11), as well as the kinase inhibitor dasatinib. We found that all LSCs, both CD36and CD36, were more drug resistant than bulk leukemia cells ( Figure S5 H). Furthermore, compared to CD36LSCs, CD36LSCs were preferentially drug resistant ( Figure S5 H). To determine whether drug resistance was also evident in vivo, we treated leukemic mice using a regimen that models the treatment for acute myeloid leukemia (AML) patients () and improves survival of leukemic mice ( Figure 5 F). We found that by the end of chemotherapy, CD36LSCs are enriched in BM residual leukemia cells while CD36LSCs are not ( Figure 5 G), suggesting that CD36LSCs are more drug resistant. Analysis of CD36LSCs in GAT also showed strong enrichment after chemotherapy ( Figure 5 G). Intriguingly, the percentage of CD36LSCs in GAT is even higher than BM while the percentage of CD36LSCs in GAT is reduced ( Figure 5 G), suggesting preferential survival of CD36LSCs in GAT. Together, these findings show that CD36LSCs are drug resistant and protected by GAT, implying that the heterogeneity found in LSCs is directly related to drug response in different leukemic sub-populations.

Collectively, these findings indicate that at least two metabolically distinct types of leukemia-initiating cells exist in the blast crisis model and that energy metabolism can differ as a function of anatomical location and expression of CD36.

The metabolic properties of CD36LSCs (high FAO rate, low ATP level, and sensitivity to inhibition of glycolysis) resemble the metabolic characteristics of quiescent HSCs (). Cell-cycle analyses showed that CD36LSCs are relatively quiescent in comparison to the CD36subpopulation ( Figure 5 E). In agreement with this result, CD36LSCs have higher expression of cell-cycle inhibitors and lower expression of cell-cycle promoters ( Figure S5 F). Interestingly, we do not detect any surface expression of CD36 in normal stem/progenitor populations ( Figure S5 G), suggesting CD36 is preferentially utilized by a subpopulation of LSCs.

Next, we examined relative metabolic status. As shown in Figure S5 C, CD36LSCs have a lower ATP content than CD36LSCs, even though their mitochondrial mass is comparable ( Figure S5 D). We further examined the response of each population to metabolic stress. CD36LSCs are more sensitive to 2-deoxy-D-glucose (2-DG) and more resistant to the complex I inhibitor rotenone compared to CD36LSCs ( Figure S5 E), suggesting the CD36cells are more dependent on glycolysis. These observations indicate that CD36and CD36LSCs are metabolically distinct.

The presence of phenotypically primitive CD36and CD36subsets raised the question of whether one or both populations contain true functionally defined LSCs. To investigate this issue, CD36versus CD36populations were isolated by flow cytometry and used in transplantation assays. As shown in Figure 5 C, both populations were able to recapitulate bulk leukemic disease in secondary recipients. Similarly, when cultured in vitro, these two populations were also able to recreate each other ( Figure S5 B). Additionally, limiting-dilution transplantation assays indicated a comparable LSC frequency between CD36and CD36populations ( Figure 5 D). These findings demonstrate that there are at least two phenotypically distinct types of LSCs in the blast crisis model employed for these studies. Notably, phenotypically distinct LSCs have recently been reported within primary human leukemia specimens ().

Since CD36LSCs have a high requirement for fatty acid and there is abundant FFAs in the GAT microenvironment, we hypothesized that CD36LSCs might preferentially localize to GAT. Indeed, we found that CD36LSCs are strikingly enriched in GAT ( Figure 5 A). Further, CD36LSCs have a higher tendency to migrate to GAT than to BM ( Figure 5 B), while homing to BM is comparable between CD36and CD36LSCs ( Figure S5 A).

(G) Preferential survival of CD36 + LSCs in GAT after chemotherapy. BM and GAT from control (vehicle-treated) and chemotherapy-treated leukemic mice were harvested, and the composition of residual leukemia cells was examined. Error bars show means ± SD. n = 5, ∗ p < 0.05, ∗∗ p < 0.005.

(C) Functional analysis of LSC potential in CD36 + versus CD36 − LSCs. Equal numbers of CD36 + and CD36 − LSCs were injected into recipient mice (1,000 cells/mouse). Two weeks later, BM cells from recipients were collected to examine the composition of leukemia cells.

Taken together, these results indicate that the fatty acid transporter CD36 differentially regulates FAO in LSCs. Furthermore, FAO in different LSC sub-fractions seems to be regulated by different mechanisms.

To gain insights into the mechanism underlying the high FAO rate in LSCs, we compared the expression of FAO-related genes in LSCs, Linleukemia cells, and their non-leukemic counterparts. LSCs show a distinct expression pattern of the FAO-related genes ( Figure S4 B). Specifically, we found one of the fatty acid transporters, CD36, is highly expressed by LSCs ( Figures S4 B, S4C, and 4 C), suggesting that CD36 may contribute to the increased FAO in LSCs. Notably, CD36 is not expressed uniformly in the LSC population, indicating that there may be heterogeneity in the stem cell compartment. To investigate a possible role for CD36 in LSCs, we first asked whether CD36-dependent fatty acid transport was evident in both leukemic and normal hematopoietic cells. To this end, we showed that purified CD36/Linleukemia cells demonstrate a higher fatty acid uptake rate and are preferentially sensitive to the CD36 inhibitor sulfosuccinimidyl oleate (SSO) compared to CD36/Linleukemia cells ( Figure S4 C). Additionally, using a fluorescent long-chain fatty acid analog, BODIPY-dodecanoic acid (), we showed that CD36/Linnormal cells have a higher fatty acid uptake rate and are more sensitive to SSO compared to CD36/Lincells ( Figure S4 E). These results suggest that CD36 functions as a fatty acid transporter in both leukemic and normal hematopoietic cells. Next, we asked whether CD36 regulates FAO in LSCs. We found that the FAO rate in total LSCs is selectively reduced by SSO, while FAO rates in Linleukemia cells and their normal counterparts are not ( Figure 4 D). Subsequent isolation of CD36versus CD36subpopulations showed that CD36LSCs have a much higher FAO rate and are more sensitive to SSO than CD36LSCs ( Figures S4 F and 4 E).

Next, we asked how leukemia cells might benefit from lipolysis of AT. We hypothesized that increased FFAs from lipolysis could serve as an energy source for leukemia cells both in GAT and possibly in other tissues. To investigate this hypothesis, we compared the ability of leukemic versus non-leukemic cells to utilize fatty acid. As shown in Figure 4 A, leukemia cells have a higher FAO rate compared to control non-leukemia cells. Furthermore, in examining various cell subpopulations, our data show that LSCs have a higher FAO rate than either more differentiated leukemia cells (Linleukemia cells) or their counterparts from normal hematopoietic cells ( Figure 4 B). Interestingly, treatment of leukemic and normal cells with adipocyte conditioned medium (CM) selectively increases FAO rate in LSCs ( Figure 4 B). Etomoxir, a CPT1 inhibitor, inhibits FAO in both leukemia cells and normal cells ( Figure 4 B). Of note, etomoxir only reduces FAO in LSCs by ∼60%, suggesting that peroxisomal FAO contributes to a substantial part of the overall FAO in LSCs. In addition, we noticed that LSCs exhibit the highest FAO rate when fatty acid serves as the only energy substrate ( Figure S4 A).

(D) FAO rates of LSCs, Lin + leukemia cells, and their counterparts from normal BM were determined with or without the presence of the CD36 inhibitor SSO (50 μM). Error bars show means ± SD from triplicates. ∗∗ p < 0.005.

(B) FAO rates of LSCs, Lin + leukemia cells, and their counterparts from normal BM were determined with or without the presence of adipocytes conditioned medium (CM) or the CPT1 inhibitor etomoxir (Ex) (100 μM). Error bars show means ± SD from triplicates. ∗ p < 0.05, ∗∗ p < 0.005.

(A) Leukemia cells and non-leukemia cells (GFP − /YFP − ) from the same sample were sorted and FAO assays monitoring the release of tritiated water ( 3 H 2 O) from tritium labeled palmitate were performed to determine their FAO rates. Error bars show means ± SD from triplicates. ∗∗ p < 0.005.

Together, these observations indicate that resident leukemia cells contribute to the lipolytic status of GAT by disrupting lipid metabolism in GAT through paracrine signaling of pro-inflammatory cytokines.

In addition to the pro-inflammatory cytokines outlined above, we note that fatty acid itself can be a potent pro-inflammatory agent (). Therefore, we hypothesized that FFAs from lipolysis would further exacerbate inflammation-induced lipolysis. Indeed, we found that leukemia cells treated with palmitate (PA) have increased expression of IL-1α and IL-1β, while normal BM hematopoietic cells do not ( Figure 3 H). Conversely, unsaturated fatty acid such as oleic acid (OA) has been reported to have anti-inflammatory effect (). We observed that OA represses mRNA expression of pro-inflammatory cytokines in normal cells ( Figure S3 E); however, this repression is not seen in leukemia cells ( Figure S3 E). Additionally, one recent study reported the induction of IL-1α by OA through an inflammasome-independent pathway (). Interestingly, we found that OA, but not PA, is able to induce IL-1α protein production in leukemia cells ( Figure 3 I).

To determine if inflammatory factors directly mediate lipolysis, we treated 3T3-L1 adipocytes as well as normal GAT explants with each of the pro-inflammatory cytokines/chemokines elevated in GAT. As shown in Figures 3 E and S3 B, tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), IL-1β, and CSF2 are capable of inducing lipolysis. Furthermore, these four cytokines result in reduced expression of both LPL and CIDEA in GAT explants and 3T3-L1 adipocytes ( Figures 3 F and S3 C). Additionally, analysis of conditioned medium (CM) from control and leukemic GAT explants as well as serum from control and leukemic mice showed increased secretion of IL-1α, IL-1β, TNF-α, and CSF2 ( Figures 3 G and S3 D). These findings suggest that TNF-α, IL-1α, IL-1β, and CSF2 secreted by leukemia cells mediates lipolysis.

To determine the molecular basis of lipolysis in leukemic GAT, we examined the expression of lipolysis-related genes. Comparison of control and leukemic GAT showed increased expression of adipose triglyceride lipase (ATGL), the gene that encodes the rate-limiting enzyme in lipolysis () ( Figures 3 C and 3D). Further, in leukemic GAT, we observed reduced expression of the negative regulators of lipolysis lipoprotein lipase (LPL) and cell death activator CIDE-A (CIDEA) ( Figures 3 C and 3D). LPL controls the influx of FFAs into adipocytes (). CIDEA is a lipid droplet (LD)-associated protein that shields LDs from lipases and thus inhibits lipolysis (). These results suggest that leukemic GAT triggers a lipolytic program that induces release of FFAs and mobilization of lipids that leads to atrophy of GAT.

Next, we focused on possible mechanisms controlling atrophy of GAT. Previous studies have indicated that lipolysis is mainly responsible for atrophy of AT in the setting of cancer (). To test whether leukemic GAT is lipolytic, we cultured GAT explants from control and leukemic mice and monitored lipolysis rates. As shown in Figure 3 A, leukemic GAT releases much more FFAs than control GAT. In addition, fatty acid binding protein 4 (FABP4), which facilitates the transportation of FFAs, is also abundantly secreted by leukemic GAT ( Figure 3 A). These data were further substantiated by detection of an elevated serum FFA level in leukemic mice ( Figure 3 B). We also found that leukemic serum is a potent inducer of lipolysis in 3T3-L1 adipocytes as well as in normal GAT explants ( Figure S3 A). Together, these findings strongly support the conclusion that leukemic GAT is lipolytic.

(I) Leukemia cells (1 million/ml) were treated with BSA, PA (100 μm), or oleic acid (OA) (100 μm) for 24 hr, and ELISA was performed to determine the concentration of IL-1α in media. Error bars show means ± SD from triplicates. ∗∗ p < 0.005.

(G) Conditioned medium (CM) from control and leukemic GAT was subjected to cytokine arrays. Array results as well as quantification for IL-1α, IL-1β, TNF-α, and CSF2 were shown. Error bars show means ± SD from the duplicate dots for each cytokines. ∗∗ p < 0.005.

(E and F) Normal GAT explants were cultured in DMEM containing 1% BSA and indicated cytokines (5 ng/ml for TNF-α and 10 ng/ml for others) for 24 hr. FFA level in culture medium (E) and expression of lipolysis related genes (F) were determined. Error bars show means ± SD from triplicates. ∗∗ p < 0.005.

(A) GAT explants (300 mg) from control (transplanted with normal BM hematopoietic cells) and leukemic mice were cultured in DMEM containing 1% BSA for 24 hr, and FFA and FABP4 levels in culture medium were determined. Error bars show means ± SD from triplicates. ∗∗ p < 0.005.

To examine whether similar symptoms are observed in other leukemic models, we utilized an MLL-AF9-induced leukemia model in which LSCs have been shown to arise in the granulocyte-macrophage progenitor compartment (GMP) (). This model differs from the bcCML model, where previous studies have shown that the HSC/multipotent progenitor (MPP) compartment represents the cell of origin (). Similar to what we found in the blast crisis model, loss of body weight as well as atrophy of GAT was observed in leukemic mice ( Figures S2 H and S2I). However, leukemic burden in GAT for the MLL-AF9 model is considerably lower than that in BM ( Figure S2 J), similar to our findings for IAT in the bcCML model. These findings suggest that localization to GAT may be dependent on both the leukemia cell of origin (i.e., HSC versus GMP) and the specific oncogenes that drive the malignant transformation process.

One possible physiological consequence of an altered inflammatory state could be increased mobilization of free fatty acids (FFAs) from AT. Notably, we observed severe atrophy of GAT ( Figure 2 D) as well as loss of body weight ( Figure S2 C) in leukemic mice. Additionally, despite only a low level of leukemia cells, atrophy of IAT was observed as well ( Figure S2 D). Further analyses indicated that in contrast to GAT, the non-leukemic population from IAT SVF in leukemic mice is more pro-inflammatory than IAT SVF in normal mice ( Figure S2 E). Together, these findings indicate cachexia in leukemic mice, where systemic factors lead to weight loss in organs that do not display large leukemia cell burden. Interestingly, we also observed that atrophy of GAT precedes body weight loss during the early stage of leukemia development ( Figures S2 F and S2G).

To gain insight into the characteristics of LSCs in AT, we compared transcriptomes of sorted LSCs from GAT, BM, PB, spleen, and their non-leukemic counterparts from BM (NBM) by RNA sequencing (RNA-seq). The data indicate that LSCs in GAT display a distinct gene expression pattern ( Figure 2 A) characterized by strong upregulation of pro-inflammatory cytokines/chemokines ( Figure S2 A). The most strongly upregulated pro-inflammatory genes are shown in Figure 2 B. The leukemic GAT SVF also shows strong upregulation of pro-inflammatory genes in comparison to controls ( Figure S2 B). To ensure that non-leukemic cells were not contributing to the pro-inflammatory phenotype, we also examined expression in the GFP/YFPpopulation derived from GAT SVF in leukemic mice ( Figure 2 C). In comparison to naive GAT SVF, cytokine/chemokine levels are actually somewhat lower, further indicating that the inflammatory state of GAT is due to resident leukemia cells in SVF ( Figure 2 C).

(A) Heatmap of genes differentially expressed by LSCs and their non-leukemic BM counterparts (NBM). RNA from LSCs in each tissue and NBM was isolated and subjected to RNA-seq. Three cohorts for each type of LSCs were analyzed. Each cohort consisted of pooled LSCs from ten mice. Genes that are only significantly differentially expressed in all three cohorts were chosen to make the heatmap (red, upregulated; blue, downregulated). See also Figure S2 A.

We next asked whether LSCs were also found in GAT. As reported in previous studies a Sca-1/Linphenotype encompasses all detectable LSCs in the bcCML model (). We observed enrichment of phenotypically defined LSCs in GAT relative to the hematopoietic tissues ( Figure 1 C). To investigate the level of functionally defined LSCs, the stromal vascular fraction (SVF) was isolated from leukemic GAT, which represents an adipocyte-depleted tissue suspension. Transplantation of SVF into recipient mice showed robust development of leukemia as evidenced by GFP/YFPcells in BM, GAT, and spleen ( Figure S1 C). Limiting-dilution transplantation assays indicate that the frequency of LSCs is slightly higher in GAT-derived leukemia cells compared to BM-resident leukemia cells ( Figure 1 D). Finally, we investigated the composition of leukemic populations that migrate to GAT. Homing assays demonstrated that LSCs are significantly enriched in leukemia cells that localize to GAT ( Figure 1 E). Taken together, these findings indicate that GAT functions as a reservoir for leukemia cells.

To investigate the presence of leukemia cells in AT, animals were established using the bcCML model. At advanced stages of disease, cells were isolated from both GAT and inguinal adipose tissue (IAT). GAT represents the largest visceral fat depot in mice, and IAT is the most prevalent reservoir of subcutaneous fat. In addition, several hematopoietic tissues were isolated for comparison, including BM, spleen, and peripheral blood (PB). Interestingly, the data show that leukemia cells are readily evident in GAT but only present at very low levels in IAT ( Figure 1 A). These data suggest that AT microenvironments may differ substantially with regard to their ability to attract and/or support leukemia cells, a finding supported by previous studies in which biological properties of IAT and GAT have been shown to differ substantially (). To further characterize GAT as a reservoir for leukemia cells, histological analyses were performed ( Figure 1 B). Leukemia cells are observed in direct contact with adipocytes and are prevalent throughout the AT.

(B) Histological analyses of leukemia infiltration of adipose tissue (AT) with H&E labeling of tissue morphology of GAT (20× magnification). Orange arrow indicates a blood vessel identified by CD31 labeling and morphology. Immunofluorescent labeling of GFP (indicated in red) shows extensive infiltration of leukemia cells throughout the AT. Labeling with CD31 and Plin indicate endothelial cells and adipocytes, respectively. DAPI indicates nuclei of cells. Overlay of all fields (bottom row) shows the position of numerous leukemia cells in direct contact with adipocytes. Scale bar, 50 μm.

A murine model of blast crisis chronic myeloid leukemia (bcCML) was used in this study to investigate the interplay between leukemia cells and AT. The system employs co-expression of the BCR-ABL and Nup98-HOXA9 translocations, each independently monitored using GFP and yellow fluorescent protein (YFP) expression, respectively ( Figures S1 A and S1B). These translocations are detected in human bcCML, and the model has previously been described in detail for studies of leukemia genetics and stem cell biology ().

Discussion

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Vipparala V.

Mayakesavan B. Characterization of human adipose tissue derived hematopoietic stem cell, mesenchymal stem cell and side population cells. Despite intensive research efforts, survival for patients with aggressive forms of myeloid leukemia remains poor. While many studies have focused on leukemia cell-intrinsic properties as an important challenge, an increasing number of reports have demonstrated a key role for microenvironmental signals in the biology and drug response of leukemia cells (). Because an almost infinite number of potential microenvironments may exist in mammalian organisms, the challenge in defining how various extrinsic factors influence anti-leukemia therapies is formidable. In the present study, we focused on AT for two reasons: (1) prognosis for obese patients is worse, and (2) AT has previously been shown to be a reservoir for normal HSCs (). These observations suggest that LSCs may reside in AT and that the unique microenvironment found in such tissue may protect leukemic cells from drug challenge.

To investigate the role of AT in leukemia pathogenesis, we employed a mouse model of bcCML in which LSCs have previously been described. We also examined primary human leukemia specimens. Collectively, these studies provide evidence supporting several previously unknown aspects of leukemia biology. First, GAT provides a unique microenvironmental niche for primitive leukemia cells. Residence in the adipose niche induces a strongly pro-inflammatory phenotype for leukemic cells, resulting in secretion of cytokines that elevate lipolysis and the release of FFAs. We propose that increased FFA levels act to positively reinforce the inflammatory condition of leukemia cells in GAT and to fuel metabolic processes of leukemic cells in both adipose and hematopoietic tissues, as evidenced by increased fatty acid oxidation for LSCs in both AT and marrow. Second, we observe that functionally defined LSCs exist in at least two distinct forms, differing in metabolism and cell-cycle status. The different types of LSC are characterized by expression of CD36, where expression serves to increase uptake of fatty acids. Intriguingly, CD36+ LSCs preferentially home to GAT, indicating a tropism for a microenvironment in which fatty acids are most readily available. Third, LSCs resident in GAT are preferentially resistant to challenge with conventional chemotherapy drugs, particularly the CD36+ LSCs, which reside in a more quiescent cell-cycle state. Taken together, these findings support a model in which leukemic cells co-opt AT to (1) create a microenvironment that supports systemic growth of leukemic disease and (2) provide a niche that confers a relatively drug-resistant phenotype.

+ drug-resistant LSCs have a higher FAO rate, indicating interplay between FAO and drug resistance; and (2) AT selectively regulates LSC FAO. Additionally, adipokines produced by AT have shown protective effects that could also benefit the survival of resident LSCs ( Khandekar et al., 2011 Khandekar M.J.

Cohen P.

Spiegelman B.M. Molecular mechanisms of cancer development in obesity. Pike et al., 2011 Pike L.S.

Smift A.L.

Croteau N.J.

Ferrick D.A.

Wu M. Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells. Jaswal et al., 2011 Jaswal J.S.

Keung W.

Wang W.

Ussher J.R.

Lopaschuk G.D. Targeting fatty acid and carbohydrate oxidation--a novel therapeutic intervention in the ischemic and failing heart. Lagadinou et al., 2013 Lagadinou E.D.

Sach A.

Callahan K.

Rossi R.M.

Neering S.J.

Minhajuddin M.

Ashton J.M.

Pei S.

Grose V.

O’Dwyer K.M.

et al. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. + LSCs ( Giordano et al., 2005 Giordano A.

Calvani M.

Petillo O.

Grippo P.

Tuccillo F.

Melone M.A.

Bonelli P.

Calarco A.

Peluso G. tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1. Paumen et al., 1997 Paumen M.B.

Ishida Y.

Han H.

Muramatsu M.

Eguchi Y.

Tsujimoto Y.

Honjo T. Direct interaction of the mitochondrial membrane protein carnitine palmitoyltransferase I with Bcl-2. An intriguing line of future investigation will be further analysis to determine the exact molecular details that underlie AT protection of LSCs. We speculate that regulation of resident LSC fatty acid metabolism is one of the mechanisms, because (1) our study shows that CD36drug-resistant LSCs have a higher FAO rate, indicating interplay between FAO and drug resistance; and (2) AT selectively regulates LSC FAO. Additionally, adipokines produced by AT have shown protective effects that could also benefit the survival of resident LSCs (). A related question is how fatty acid metabolism confers LSC drug resistance. We propose that regulation of cellular redox status is one possible mechanism. FAO contributes to the cellular NADPH pool (), which is critical for redox balance and survival. High rates of FAO produce large amounts of NADH and acetyl-coenzyme A, which in turn can inhibit mitochondrial oxidative activities () and thereby further reduce cellular oxidative stress. Lowered oxidative state favors quiescence as well as “stemness” for leukemia cells, as demonstrated by our previous studies in human AML (). Indeed, the present studies showed a more quiescent state for the CD36LSCs ( Figure 5 E), consistent with their improved resistance to chemotherapy. We also note that enzymes involved in FAO directly interact with anti-apoptotic proteins (), suggesting a potential interplay between FAO and cell survival.

Cui et al., 2015 Cui G.

Staron M.M.

Gray S.M.

Ho P.C.

Amezquita R.A.

Wu J.

Kaech S.M. IL-7-induced glycerol transport and TAG synthesis promotes memory CD8+ T cell longevity. Ito et al., 2012 Ito K.

Carracedo A.

Weiss D.

Arai F.

Ala U.

Avigan D.E.

Schafer Z.T.

Evans R.M.

Suda T.

Lee C.H.

Pandolfi P.P. A PML–PPAR-δ pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance. van der Windt et al., 2012 van der Windt G.J.

Everts B.

Chang C.H.

Curtis J.D.

Freitas T.C.

Amiel E.

Pearce E.J.

Pearce E.L. Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. Samudio et al., 2010 Samudio I.

Harmancey R.

Fiegl M.

Kantarjian H.

Konopleva M.

Korchin B.

Kaluarachchi K.

Bornmann W.

Duvvuri S.

Taegtmeyer H.

Andreeff M. Pharmacologic inhibition of fatty acid oxidation sensitizes human leukemia cells to apoptosis induction. An unexpected finding of our studies was the presence of two metabolically distinct LSC subpopulations, suggesting that metabolic heterogeneity may be an intrinsic property of leukemia biology or pathogenesis. This finding has practical ramifications, because we also observe differential drug response as a function of metabolic state. If these data are corroborated with additional studies in humans, it may be important to incorporate such knowledge into the design of anti-leukemia therapies. In principle, to achieve complete eradication of the LSC population, it will be necessary to employ therapies that can target metabolically distinct subpopulations. Notably, several studies have suggested an interaction between metabolic and functional heterogeneity. For example, long-lived and quiescent cells, including HSCs and memory T cells, have high FAO rates, and disruption of FAO results in dysfunction of these cells (). These observations lead us to postulate that selective modulation of LSC metabolism could be one component of regimens designed to more effectively eradicate LSC populations. Indeed, previous studies have shown that inhibition of FAO sensitizes leukemia cells to apoptosis stimulators ().

In summary, our findings demonstrate that GAT in leukemia mice functions as a reservoir for LSCs and confers chemo-resistance to resident leukemia cells, implying a potential role of GAT in the pathogenesis of leukemia and relative efficacy of therapeutic challenge. Furthermore, our data indicate metabolic heterogeneity within LSC populations, where pathways controlling energy consumption can differ. We propose that metabolic heterogeneity in LSCs may contribute to the challenge in effectively eradicating such cells and that modulation of fatty acid metabolism may be a promising way to eradicate LSCs.