Normal Mouse Marrow Research Articles

Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice is visualized by μPET-CT with 64Cu-labeled anti-CD11b and prevented by anti-CSF-1.

To investigate the utility of noninvasive µPET-CT with 64Cu-DOTA-anti-CD11b (64Cu-αCD11b) in assessing bone marrow status after anticancer therapies, and the protective role of anti-CSF-1 (αCSF-1) against bone marrow suppression induced by Abraxane.Methods: MDA-MB-435 tumor-bearing mice were treated with Abraxane, αCSF-1, or αCSF-1 plus Abraxane. µPET-CT and biodistribution of 64Cu-αCD11b were performed after intravenous injection of the radiotracer. Cells from mouse bone marrow and MDA-MB-435 tumor were analyzed by flow cytometry. A humanized αCSF-1 was investigated for its role in protecting bone marrow cells, using a transgenic mouse model that expresses functional human CSF-1.Results: μPET-CT showed that 64Cu-αCD11b had high uptake in the bone marrow and spleen of both normal and tumor-bearing mice. Abraxane significantly reduced 64Cu-αCD11b uptake in the bone marrow and spleen of treated mice compared to untreated mice. Interestingly, 64Cu-αCD11b μPET-CT revealed that αCSF-1 alleviated the depletion of bone marrow cells by Abraxane. These changes in the bone marrow population of CD11b+ myeloid cells were confirmed by flow cytometry. Moreover, αCSF-1 potently enhanced tolerance of bone marrow granulocytic myeloid cells to Abraxane, decreased cell migration, and suppressed recruitment of myeloid cells to the tumor microenvironment. The humanized αCSF-1 also alleviated the effects of Abraxane on bone marrow cells in transgenic mice expressing human CSF-1, suggesting clinical relevance of αCSF-1 in prevention of bone marrow suppression in addition to its role in reducing tumor-infiltrating myeloid cells.Conclusions: Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice could be noninvasively assessed by μPET-CT with 64Cu-αCD11b and prevented by αCSF-1.

Ganoderma spore lipid protects mouse bone marrow mesenchymal stem cells and hematopoiesis from the cytotoxicity of the chemotherapeutic agent.

Cancer chemotherapeutic agents are frequently toxic to bone marrow and impair bone marrow functions. It is unclear whether ganoderma spore lipid (GSL) can protect bone marrow cells from the cytotoxicity of chemotherapy. To investigate the protective effects of GSL on bone marrow mesenchymal stem cells (MSCs) and hematopoiesis, we examined the effects of GSL on MSCs in vitro and hematopoiesis in vivo after treatment with the chemotherapeutic agent cyclophosphamide. MSCs and peripheral blood cells were isolated and counted from the bone marrow of normal mice were pre-treated with GSL before CTX treatment or co-treated with GSL and CTX, followed by examining the changes in phenotype, morphology, proliferation, apoptosis, and differentiation potentials. The results showed that GSL could reduce the CTX-induced changes in the phenotype of MSCs and maintain the elongated fibroblast-like morphology. MTT and annexin V/propidium iodide (PI) analyses found that GSL pre-treatment and co-treatment increased the proliferation and decreased the apoptosis in CTX-treated MSCs. Furthermore, GSL improved the osteogenic and adipogenic differentiation potentials of CTX-treated MSCs. In vivo, GSL treatment increased the number of peripheral blood cells including white blood cells (WBC) and platelets (PLT) in the CTX-treated mice and enhanced the in vitro formation of hematopoietic lineage colonies (erythrocyte colony forming unit, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte macrophage colony-forming units, CFU-GM) from bone marrow cells in these mice. These findings suggest GSL could protect MSCs and hematopoiesis from the cytotoxicity of CTX and might become an effective adjuvant to attenuate side effects of chemotherapy during cancer treatment.

Abstract LB-332: Evidence for cutaneous squamous tumors derived from carcinogen-exposed bone marrow

Abstract Cytokeratins are frequently found in the blood and bone marrow of patients with epithelial cancers and are attributed to metastasis. We wondered whether we could find keratin expression in blood and bone marrow in untreated adult mice. To address this problem, we have used classical immunoreactivity, Krt1-14;mTmG transgenic mice together with fluorescence activated cell sorting, and quantitative reverse transcriptase polymerase chain reaction. We have made several novel findings. First, we discovered, rare but distinctive, keratin-14 and keratin-15 immunoreactive cells the size of small lymphocytes in blood and bone marrow of untreated mice. Second, using Krt1-14;mTmG transgenic mice, we found low (8.6 GFP positive cells per 10^6 cells analyzed), but significant numbers (p&lt0.0005) of GFP positive cells in bone marrow of normal adult mice when compared with negative controls. Third, qRT-PCR demonstrated very low but reproducibly detectable expression of keratin-14 mRNA in blood and bone marrow when compared with epidermal keratinocytes: with blood expressing one thousand times, and bone marrow, one hundred thousand times, less than epidermal keratinocytes. Moreover, FACS analysis of fresh bone marrow disclosed a subpopulation of keratin-14 immunoreactive cells that was negative for hematopoietic lineage markers. Furthermore, and even more curious, recipients of bone marrow transplants from DMBA-treated UBC/GFP mice (“toxic bone marrow”) developed GFP-immunoreactive skin tumors upon TPA tumor promotion: four of fifteen mice developed GFP-immunoreactive skin tumors. Of these tumors, four were squamous papillomas, and one was a squamous cell carcinoma. None of the ten control mice receiving normal whole bone marrow developed any skin tumors upon tumor promotion. We conclude from these observations that keratin-14 protein and mRNA are expressed at low, but detectable levels in the blood and bone marrow of mice, and that a subset of “toxic bone marrow” derived cells can be recruited to form epithelial benign and malignant squamous tumors. Building upon these observations should open new avenues of understanding for cutaneous biology, non-melanoma skin cancer, and other epithelia and their cancers. Citation Format: Sonali Lad, Kelly Johnson, Kelsey Boland, Nyssa Readio, Todd Schuster, Yong Li, Derek Gordon, Rebecca J. Morris. Evidence for cutaneous squamous tumors derived from carcinogen-exposed bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-332.

Abstract 126: MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells

Abstract The MYC oncogene is commonly overexpressed in hematopoietic cancers. In a transgenic mouse model of MYC-induced T-cell lymphoma, we found that there was a systemic reduction in NK cell (CD3- NKp46High) numbers and activation in lymphoid organs such as spleen and bone marrow. MYC inactivation in lymphoma-bearing MYCON mice partially restored NK cell numbers and activation. Hence, the MYC oncogene appears to suppress NK cell-mediated immune surveillance of lymphomas. We evaluated whether reduction in mature and activated NK cells (CD3- NKp46High) in MYCON mice was due to increased death of NK cells in these mice when compared to normal and MYCOFF mice. Using flow cytometry, NK cells were measured in age matched spleens and bone marrows of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. Surprisingly, we observed a significant reduction in proportions of dead NK cells (NKp46+ PI+) in spleen and bone marrow of MYCON mice, in comparison to normal and MYCOFF cohorts. Hence, the reduction in activated NK cell numbers during MYC-driven lymphomagenesis does not occur because of increased death of NK cells. Next, we investigated whether MYC arrests early NK cell development in the bone marrow of lymphoma mice, leading to systemic NK suppression. NK cell lineage specification begins with CD122 expression, that is maintained throughout NK development starting at the precursor stage. NK precursors (NKP) transition to immature NK (iNK) cells by expressing the cytotoxicity marker NKp46. iNK cells leave the bone marrow to populate peripheral lymphoid organs. We measured NKP (CD122+ NKP46-), and iNK (CD122+ NKp46+) cells in bone marrow of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. We observed no significant changes in the percentages of NKP. However, the proportions of iNK cells were significantly reduced in MYCON mice, in comparison to MYCOFF and normal mice. Our results suggest that MYC may block transition from NKP to iNK stage during early NK cell development. The reduction of iNK cells in spleens of MYCON mice was concordant with the reduction of iNK cells in bone marrow from the same mice. We conclude that MYC-induced lymphomagenesis blocks early NK cell development, thereby suppressing NK-mediated immune surveillance. Citation Format: Line D. Heftdal, Srividya Swaminathan, Dean W. Felsher. MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 126.

Sitagliptin-mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes.

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.

Hematopoietic stem and multipotent progenitor cells produce IL-17, IL-21 and other cytokines in response to TLR signals associated with late apoptotic products and augment memory Th17 and Tc17 cells in the bone marrow of normal and lupus mice

We studied effects of early and late apoptotic (necroptotic) cell products, related damage associated alarmins and TLR agonists, on hematopoietic stem and progenitor cells (HSPC). Surprisingly, normal HSPC themselves produced IL-17 and IL-21 after 1½days of stimulation, and the best stimulators were TLR 7/8 agonist; HMGB1–DNA; TLR 9 agonist, and necroptotic B cells. The stimulated HSPC expressed additional cytokines/mediators, directly causing rapid expansion of IL-17+ memory CD4 T (Th17), and CD8 T (Tc17) cells, and antigen-experienced IL-17+ T cells with “naïve” phenotype. In lupus marrow, HSPC were spontaneously pre-stimulated by endogenous signals to produce IL-17 and IL-21. In contrast to HSPC, megakaryocyte progenitors (MKP) did not produce IL-17, and unlike HSPC, they could process and present particulate apoptotic autoantigens to augment autoimmune memory Th17 response. Thus abnormally stimulated primitive hematopoietic progenitors augment expansion of IL-17 producing immune and autoimmune memory T cells in the bone marrow, which may affect central tolerance.

Abstract 5096: ALIBI: a novel, truncated tubulin isotype in AML and stem cells

Abstract Tubulin isotype expression is tissue restricted, with β-1 tubulin (TUBB1) restricted to hematopoietic tissues. Using the TCGA database, we found that β-1 is highly expressed in acute myeloid leukemia (AML) among all hematologic malignancies. β-1 may therefore be an attractive target for the development of novel therapeutics for AML. Analysis of a panel of AML cell lines using a polyclonal antibody against the C-terminus of β-1 revealed the presence of a band at 35kD, distinct from the canonical tubulin molecular weight of 50kD. This 35kD band, which we call Acute Leukemia Isoform β1 (ALIBI), was present in 10 of the 19 AML cell lines tested of various molecular and morphologic subtypes. In addition, we analyzed 12 AML primary AML patient blast samples and found ALIBI expression in six patient samples, suggesting that ALIBI is clinically relevant and prevalent Immunofluorescent analysis of ALIBI's subcellular localization revealed that ALIBI is present in distinct cytoplasmic aggregates not co-localizing with native microtubules as well as in the nucleus. Interestingly, ALIBI was also enriched in AML patient CD34+/CD38- cells containing leukemia stem cells. Using a murine β-1 antibody in a mouse model of acute leukemia (HOXA9-NUP98/BCR-ABL) we found that ALIBI expression was significantly higher in the leukemic marrow as compared to normal mouse marrow. As leukemia stem cells share features with embryonic stem cells (ESCs) we next investigated ALIBI expression in mouse ESCs after differentiation along megakaryocytic and erythroid lineages. ALIBI, but not canonical β-1, was expressed in ESCs, while hematopoietic differentiation led to loss of ALIBI and expression of only canonical β-1 tubulin, suggesting that expression of canonical β-1 and ALIBI are differentially regulated and that ALIBI expression is associated with stemness. RNASeq and 5′RACE of cell lines and patient samples revealed that ALIBI mRNA is missing exons 1 and 2 of canonical β-1 RNA, while it retains part of exon 3, exon 4 and the intervening intron. These results suggest that ALIBI mRNA is alternatively spliced, leading to the use of a cryptic translational start site in exon 4 flanked by a strong Kozak sequence. Exogenous expression of the putative coding region of ALIBI in ALIBI-negative cell lines produced a protein product with similar molecular weight and subcellular localization as endogenous ALIBI. Studies of ALIBI function using genome editing are currently underway. In conclusion, ALIBI is a novel truncated isoform of tubulin that has unique biologic features never previously described for tubulins. While the exact function of ALIBI within leukemic cells is still being uncovered, it nevertheless is a highly attractive target of novel therapeutic development based on its high expression in AML and its unique biologic properties. Citation Format: Paul Basciano, Xi Li, Jason Matakas, Susanna Liu, Silvana Di Giandomenico, Siddhartha Sen, Todd Evans, Joseph Scandura, Monica Guzman, Paraskevi Giannakakou. ALIBI: a novel, truncated tubulin isotype in AML and stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5096. doi:10.1158/1538-7445.AM2015-5096

Renal and Hematological Effects of CLCF-1, a B-Cell-Stimulating Cytokine of the IL-6 Family.

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

In Vivo Quantification of Active Decitabine-Triphosphate Metabolite: A Novel Pharmacoanalytical Endpoint for Optimization of Hypomethylating Therapy in Acute Myeloid Leukemia

Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 μM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.

Cyclin Dependent Kinase Inhibitor P21 Negatively Regulates PBX-1 Via Internal Tandem Duplication of Flt3 Dependent Manner, a Novel Mechanism That Does Not Exist in Normal Hematopoietic Progenitor Cells

Abstract 1310The cyclin dependent kinase inhibitor p21 CDKN1 (p21) regulates proliferation of diverse cell types by modulating cell cycle and apoptosis in variety of ways, however, its molecular mechanism underlying cell fate decisions remains to be explored. We previously reported that p21 expression is up-regulated in mouse Ba/F3 cells by Internal Tandem Duplication mutations on the Flt3 gene (ITD-Flt3), which induce constitutive activation of Flt3 signaling and associate with poor prognosis of patients with acute myeloid leukemia (AML). However, despite up-regulation of p21 by ITD-Flt3, deletion of p21 gene accentuated growth factor independent proliferation of primary mouse hematopoietic progenitor cells (HPC) transformed by ITD-Flt3 (Abe et al. ASH 2010). The data suggests that the paradoxical increase of p21 induced by ITD-Flt3 negatively regulates oncogenic proliferation of ITD-Flt3+ HPC. Although p21 is generally considered to reduce cell proliferation by inhibiting G1/S transition, p21 can enhance proliferation of normal HPC stimulated by hematopoietic growth factors (Mantel et al. 1995, Fukuda et al. 2004). These data suggest that differential mechanism is involved in regulating p21 function between normal versus transformed HPC. Herein in order to determine the mechanism responsible for growth factor independent proliferation of ITD-Flt3+HPC regulated by p21, we identified selective molecules regulated by p21 exclusively in ITD-Flt3+ HPC that are distinct from normal HPC.Deletion of p21 gene modulated expression of 247 genes in c-kit+, Sca-1+, lineage− (KSL) cells over-expressing ITD-Flt3. Out of 247 genes modulated by p21 deletion in ITD-Flt3+ KSL cells, 112 genes were either not modulated or inversely regulated by p21 in normal mouse marrow KSL cells in vivo. The results implicate that alteration of 112 mRNA expression by p21 may be specific for transformed KSL cells by ITD-Flt3, which is distinct from normal KSL cells. Out of 112 genes, 13 genes are listed in the known genes deregulated in human AML stem cells (LSC) (Majeti et al. 2009). Among the 13 molecules that are also deregulated in AML LSC, pbx-1, a transcriptional factor, which is profoundly associated with hematopoietic stem cell function but down-regulated in AML LSC (Ficara et al. 2008), was up-regulated by 1.6±0.2 fold in ITD-Flt3+ KSL cells lacking p21 (P<0.05, N=3) compared to p21+/+ counterparts. Whereas, loss of p21 did not affect pbx-1 expression in normal KSL cells (1.1±0.1 fold compared to p21+/+ KSL cells, NS). Pbx-1 mRNA expression was significantly lower in ITD-Flt3 transduced KSL cells compared to freshly isolated KSL cells (P<0.05, N=3), regardless of p21 status, a finding consistent with down-regulation of pbx-1 in AML LSC compared to normal CD34+CD38− cells and presence of ITD-Flt3 in AML LSC (Levis et al. 2005). In contrast, p21 mRNA expression was coincidently elevated in ITD-Flt3+KSL cells (P<0.05) compared to normal KSL cells, indicating that p21 and pbx-1 are concomitantly but inversely modulated by ITD-Flt3 in ITD-Flt3+ KSL cells. Consistently, p21 expression was negatively correlated with pbx-1 expression in 293 AML subjects obtained from a public database Gene Expression Omnibus (GEO: GSE1159). However, the association between p21 and pbx-1 was not observed in the gene expression profile of normal human CD34+CD38− cells. In addition, pbx-1 expression was significantly lower in ITD-Flt3+ AML cells (N=78) compared to the ITD-Flt3 negative AML cells (N=215, P<0.05) listed in GSE1159, providing additional evidence for the negative regulation of pbx-1 by ITD-Flt3 in AML cells.These results suggest that p21 and ITD-Flt3 down-regulate pbx-1 expression, but the effect of p21 on pbx-1 is ITD-Flt3 dependent. As opposed to p21/pbx1 axis identified in transformed HPC and AML, pbx-1 and p21 is not associated in normal KSL cells and CD34+CD38− cells. The data suggests that p21/pbx-1 axis selectively exist in AML cells, a mechanism that is not present in normal HPC. Enhancement of growth factor independent proliferation of ITD-Flt3+ KSL cells resulting from p21 deletion may be partly due to up-regulation of pbx-1induced by p21 deletion, rather than merely a consequence of modulation of G1/S transition by p21. In this regard, manipulation of selective p21/pbx-1 axis that is dependent on ITD-Flt3 may represent a novel therapeutic strategy for ITD-Flt3+AML that are refractory to ITD-Flt3 inhibitors. Disclosures:No relevant conflicts of interest to declare.

Abstract 470: Role of Fli1 in hematopoietic stem cell-derived fibroblast promotion of tumor cell migration and invasion

Abstract Cells and paracrine factors of the tumor microenvironment play a central role in tumor angiogenesis, invasion, migration and proliferation, making the tumor microenvironment an exciting therapeutic target. Among the most prominent cell types in the tumor stroma are fibroblasts, termed carcinoma-associated fibroblasts (CAFs). We have identified a novel population of CAFs and CAF precursors (circulating fibroblast precursors, CFPs) that are of hematopoietic stem cell (HSC) origin. These cells preferentially migrate and differentiate in response to tumor and their inhibition results in decreased tumor size. While these studies have identified a novel HSC-derived CAF population, the mechanisms by which these cells promote tumorigenesis are unknown. Previous studies have indicated a critical role for Fli1, an Ets family transcription factor, in regulation of HSCs and differentiation/maturation of hematopoietic lineages. Additionally, loss of Fli1 has been associated with cancer progression. Based on these findings, we hypothesize that HSC-derived CFPs/CAFs directly affect tumor cell migration and invasion and that loss of Fli1 in CFPs/CAFs enhances these effects. To address this hypothesis, we examined the ability of HSC-derived fibroblast populations to affect tumor cell migration and invasion in vitro. Fibroblasts were established from peripheral blood and bone marrow of normal mice and mutant Fli1 mice expressing a truncated Fli1 protein (Fli1ΔCTA) lacking the carboxy-terminal regulatory (CTA) domain. The effects of conditioned media from these fibroblast populations on tumor cell migration and invasion were examined in chemotactic transwell migration assays and matrigel-based invasion assays, respectively. Data show that conditioned media from all HSC-derived fibroblast populations promoted migration and invasion of tumor cells versus control, and that this effect was significantly enhanced using conditioned media from fibroblasts derived from Fli1ΔCTA/Fli1ΔCTA mice. Ongoing studies are directed at identifying the mechanisms by which Fli1 target genes affect HSC-derived fibroblast modulation of tumor cell migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2011-470

Chemoprotective effect of the tetrahydrofuran lignan grandisin in the in-vivo rodent micronucleus assay

The chemoprotective effect of the tetrahydrofuran lignan grandisin against DNA damage induced by cyclophosphamide (200 mg/kg) has been evaluated using the in vitro rodent micronucleus assay. The effects of a daily oral administration of grandisin (2, 4, or 8 mg/kg) for five days before exposure to cyclophosphamide on the frequency of micronucleus in the bone marrow of normal mice exposed and unexposed to cyclophosphamide were investigated (n = 5 per group). Electrochemical measurements were applied to investigate whether the antimutagenic effects of grandisin could be, at least in part, a consequence of its or its metabolite's antioxidant properties. Grandisin did not show mutagenic effects on the bone marrow cells of exposed mice. On the other hand, the oral administration of grandisin (2, 4, or 8 mg/kg) per day reduced dose-dependently the frequency of micronucleus, induced by cyclophosphamide, in all groups studied. Cyclic voltammograms showed two peaks for a grandisin metabolite, which were absent for grandisin. Under the conditions tested herein, this study has shown that mice treated with grandisin presented, in a dose-dependent manner, a protective effect against cyclophosphamide-induced mutagenicity. This effect could be, at least in part, associated to grandisin bioactivation. These data open new perspectives for further investigation into the toxicology and applied pharmacology of grandisin.

Hematopoiesis in 3 dimensions: human and murine bone marrow architecture visualized by confocal microscopy

AbstractIn many animals, blood cell production occurs in the bone marrow. Hematopoiesis is complex, requiring self-renewing and pluripotent stem cells, differentiated progenitor and precursor cells, and supportive stroma, adipose tissue, vascular structures, and extracellular matrix. Although imaging is a vital tool in hematology research, the 3-dimensional architecture of the bone marrow tissue in situ remains largely uncharacterized. The major hindrance to imaging the intact marrow is the surrounding bone structures are almost impossible to cut/image through. We have overcome these obstacles and describe a method whereby whole-mounts of bone marrow tissue were immunostained and imaged in 3 dimensions by confocal fluorescence and reflection microscopy. We have successfully mapped by multicolor immunofluorescence the localization pattern of as many as 4 cell features simultaneously over large tiled views and to depths of approximately 150 µm. Three-dimensional images can be assessed qualitatively and quantitatively to appreciate the distribution of cell types and their interrelationships, with minimal perturbations of the tissue. We demonstrate its application to normal mouse and human marrow, to murine models of marrow failure, and to patients with aplastic anemia, myeloid, and lymphoid cell malignancies. The technique should be generally adaptable for basic laboratory investigation and for clinical diagnosis of hematologic diseases.

Prolonged effects of short‐term anti‐CD20 B cell depletion therapy in murine systemic lupus erythematosus

Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes. (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each). Tissue samples were harvested and analyzed by flow cytometry. The development and extent of nephritis were assessed by monitoring proteinuria (using a urine dipstick) and by immunohistochemical analysis of the kidneys. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay. After a single injection of anti-mCD20, BCD was more efficient in the peripheral blood, lymph nodes, and spleen compared with the bone marrow and peritoneum of normal mice as well as younger mice with lupus. Since depletion of the marginal zone and peritoneal B cells was incomplete and variable, particularly in older mice with established nephritis, a strategy of sequential weekly dosing was subsequently used, which improved the extent of depletion. BAFF blockade further enhanced depletion in the spleen and lymph nodes. Early BCD therapy delayed disease onset, whereas BCD therapy in mice with advanced disease reduced the progression of nephritis. These effects were long-lasting, even after B cell reconstitution occurred, and were associated with a reduction in T cell activation but no significant change in autoantibody production. The lasting benefit of a short course of BCD therapy in lupus-prone mice with an intact immune system and established disease highlights the validity of this treatment approach.

Counts of Stromal Precursor Cells in the Bone Marrow and Heterotopic Bone Marrow Transplants from Mice Immunized with Group A Streptococcus Antigens during Different Periods after Immunization

The counts of stromal precursor cells in bone marrow transplants obtained from animals 2 months after their immunization with killed type 5 group A streptococcus vaccine drop almost 3-fold in comparison with transplants from normal donors. Six months after donor immunization, the count of stromal precursor cells in the transplants reaches the normal level. The count of stromal precursor cells in bone marrow transplants from normal mice transplanted to recipients 6 months after their immunization with killed streptococcus vaccine also virtually did not change in comparison with the counts in bone marrow transplants from normal donors transplanted to normal recipients. The weight and size of bone capsules of 6.5-month bone marrow transplants in intact recipients after transplantation from donors immunized 2 months before with killed type 5 group A streptococcus vaccine was 3-fold lower than in bone marrow transplants collected from intact donors. The content of stromal precursor cells in the femoral bone marrow of animals immunized with killed streptococcus vaccine was 2.5 time higher in comparison with the parameter in the femoral bone marrow of normal mice even 8 months after immunization. The results indicate a significant long-acting effect of streptococcal antigens on the bone marrow stromal tissue, specifically, on its osteogenesis potential.

Retroviral Insertion and Temozolomide Selection Promote T-Cell Lymphomas in Tumor-Prone Mice.

In vivo selection is a key strategy for increasing contribution of gene-modified cells in hematopoietic stem cell gene therapy. The combination of DNA-alkylating agents such as temozolomide (TMZ) with O6-benzylguanine (BG) allows selection for cells expressing a drug-resistant O6-methylguanine-DNA-methyltransferase (MGMT) gene, resulting in hematopoietic repopulation with gene-modified cells in numerous mouse studies. TMZ is used clinically for treatment of glioma and other malignancies, with dose-limiting myelotoxicity, and has been used with BG in a phase I trial of drug-resistant MGMT gene transfer for chemoprotection in treatment of advanced solid malignancies. We report here that in transplants of lymphoma-prone LMO1 transgenic or retrovirus-transduced normal mouse marrow to study the effect of in vivo selection on insertional mutagenesis by retrovirus vectors in gene therapy, administration of BG+TMZ post-transplant increased the incidence of donor-derived T-cell lymphomas. In transplants of LMO1 transgenic mouse lineage-negative (Lin−) marrow progenitor cells, BG+TMZ significantly increased (p=0.0026) the incidence of lymphoma (87.5% incidence in mice surviving drug-induced hematologic toxicity; n=8) compared to mice without drug (41% incidence by day 315 post-transplant; n=17). Increased tumor incidence after BG+TMZ was observed both in transplants of untransduced LMO1 Lin− cells and in LMO1 cells transduced with an MFG gamma-retroviral vector encoding a drug-resistant P140K MGMT gene and a GFP reporter gene. Additionally, in transplants of normal C57BL/6 mouse Lin− cells transduced with the P140K-GFP retroviral vector, BG+TMZ significantly increased (p=0.0043) the tumor incidence (100% incidence by day 169 post-transplant in mice surviving drug toxicity; n=6) compared to mice without drug (0% incidence by day 315 post-transplant; n=9). This is a higher frequency of lymphomas than we have previous observed in numerous selection studies with MGMT transduction of normal mouse whole marrow. Administration of BG+TMZ following transplant of transduced C57BL/6 mouse cells resulted in an approximately 2-fold increase in vector-expressing cells in peripheral blood, compared to an approximately 57% decline in untreated mice, demonstrating in vivo selection of MGMT-expressing cells. However, in all cases of LMO1 or C57BL/6-derived lymphomas, the tumor cells were found to lack expression of the P140K-GFP vector, suggesting that cells expressing drug-resistant MGMT were protected from the mutagenesis caused by BG+TMZ, as well as from its hematologic toxicity. Despite the lack of transgene expression, retroviral vector DNA was detected by LAM-PCR in some of the LMO1-derived and all of the C57BL/6-derived lymphomas, suggesting that insertional mutagenesis by an integrated but non-expressing (or silenced) retroviral vector may have contributed to TMZ-associated tumorigenesis in some instances. Two cases of treatment-related secondary myelodysplastic syndrome and acute myeloid leukemia have been reported in the literature for glioma patients treated with TMZ, indicating a clinical risk of TMZ tumorigenesis in some cancer patients. Here we present evidence that TMZ can cooperate with a tumor-promoting transgene (in LMO1 marrow) or potentially with insertional mutagenesis (in gamma-retrovirus transduced C57BL/6 or LMO1 marrow) for hematopoietic transformation, and that hematopoietic cells not protected by drug-resistant MGMT expression can give rise to lymphomas in a majority of mice following TMZ treatment.

The Hypomorphic Gata1low Mutation Alters the Proliferation/Differentiation Potential of the Common Megakaryocytic-Erythroid Progenitor.

Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. We had previously described that hematopoietic tissues from mice carrying the hypomorphic Gata1low mutation contain high numbers (~10%) of “unique” progenitor cells that generate colonies composed by erythroblasts, megakaryocytes and mast cells (Migliaccio et al, J Exp Med 197:281, 2003). Predicted by the stochastic model of hematopoietic commitment, such a tri-lineage progenitor cell has not been isolated prospectively as yet from the marrow of normal mice. The aim of this study was to clarify the role of Gata1 in hematopoietic commitment by identifying the antigenic profile and proliferation potential of the Gata1low progenitors giving rise to tri-lineage colonies. First, we compared the frequency of granulo-monocytic (GMP), megakaryocytic-erythroid (MEP), common myeloid (CMP) and mast-cell restricted (MCP) progenitors in marrow, and spleen, from wild-type and Gata1low littermates. Next, the different populations were isolated, and their differentiation and proliferation potential characterized under conditions of limiting dilution followed by single cell re-cloning. In Gata1low mice, the frequency of cells with the antigenic profile of CMP, MEP, and GMP was normal in marrow and markedly increased in spleen, while cells with the MCP profile were not detectable. However, mutant cells isolated according to the MEP phenotype, had the “unique” property to generate mast cells and their precursors in 7 days of culture, in addition to erythroblasts and megakaryocytes. Furthermore, the progeny of ~11% of mutant MEP could be propagated in culture, as single cells, with 95% efficiency, for up of 4 months. In comparison, the progeny of wild-type MEP became extinguished in 7–14 days. In agreement with these results, tri-lineage (erythroid, megakaryocytic and mastocytic) cell lines were consistently isolated from bone marrow and spleen cells of Gata1low mice. These results indicate that cells with the phenotype corresponding to myeloid, but not those corresponding to mastocytic, progenitors are detectable in tissues from Gata1low mice. However, in these mice, the mast cell generating activity is abnormally acquired by MEP, that, therefore, are similar in phenotype, but not in function, to wild-type MEP. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which CMP became either MEP or MCP.

Survivin Regulates Proliferation of Normal Hematopoietic Stem and Progenitor Cells In Vivo.

The inhibitor of apoptosis protein Survivin is barely detectable in most normal adult tissues but is over-expressed in almost all cancers. Survivin regulates apoptosis, cell division and cell cycle, making anti-Survivin therapy an attractive cancer treatment strategy. We reported that Survivin is expressed and regulated by hematopoietic growth factors in normal human CD34+ cells and that over-expression of wild-type Survivin in bone marrow cells enhances in vitro proliferation and survival of normal hematopoietic progenitor cells, whereas disrupting Survivin reduced their proliferation and survival. These results suggest that Survivin regulates normal hematopoietic progenitor cell function. Although targeted anti-Survivin therapies for cancers demonstrate efficacy without overt toxicity in animal models, the consequences of in vivo Survivin disruption in normal hematopoietic stem and progenitor cells (HSPC) has not been determined. In order to understand the physiological roles of Survivin in normal HSPC function in vivo, we created Cre-ER™/Survivin flox/flox mice, in which the Survivin gene can be excised by Tamoxifen treatment and characterized HSPC growth following Survivin gene deletion. RT-PCR analysis showed that Survivin mRNA is expressed in freshly isolated normal mouse marrow Sca-1+, c-kit+, lin− (SKL) cells and more primitive CD34−SKL cells, which contain long term repopulating hematopoietic stem cells (HSC). Administration of 5mg of Tamoxifen for 6 days (3 days injection, 3 days off, 3 additional days and analyzed 14 days after final injection) in Cre-ER™/Survivin flox/flox mice induced Survivin gene deletion in marrow cells, but had little effect on peripheral blood cell count, marrow cellularity (3.5+/−7.1%, NS) or the proportion or total number of lineage committed cells (Gr-1+, Mac-1+, B220+, CD4+ and/or CD8+) in marrow and in peripheral blood. In contrast, short term Survivin deletion significantly decreased the frequency and the absolute number of undifferentiated linneg cells (37+/−6% reduction), c-kit+, lin− cells (35.2+/−8.4% reduction,), CFU-GM (31+/−9 % reduction), Lin−, IL7Ra−, Sca-1−, c-kit+, CD34+, Fcglow common myeloid progenitor cells (52+/−13% reduction), SKL cells (56.8+/−5.4% reduction) and CD34−SKL cells (60.6+/−5.5% reduction) in bone marrow compared to control mice. The effect of Survivin gene deletion was more dramatic on primitive hematopoietic populations compared to mature cells, which is consistent with down-regulation of Survivin in hematopoietic cells with terminal differentiation. Similarly, treatment of bone marrow cells from Cre-ER™/Survivin flox/flox mice with 1uM of Tamoxifen in vitro significantly reduced the number of CFU-GM, (c-kit+, lin−) KL, SKL and CD34−SKL cells cultured with hematopoietic cytokines and increased apoptosis measured by Annexin-V staining. These results suggest that Survivin is required and regulates normal hematopoietic stem and progenitor function in vivo and that Survivin function may be selectively essential for growth and differentiation of primitive hematopoietic cells. In addition, acute ablation of Survivin may cause adverse toxicity on HSPC that provide long term hematopoiesis in the patients receiving anti-Survivin target therapies.

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH) 2D 3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-κB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10 − 10 M 1,25-(OH) 2D 3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH) 2D 3. ECF-L-Fc by itself only modestly increased NF-κB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1α and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10 − 10 M 1,25-(OH) 2D 3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH) 2D 3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.

Interference with nuclear factor kappa B and c-Jun NH2-terminal kinase signaling by TRAF6C small interfering RNA inhibits myeloma cell proliferation and enhances apoptosis

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of six adaptor proteins (TRAF1-6) links the TNFR superfamily to the nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) transcriptional activators. Unlike other TRAFs, TRAF6 is also involved in Toll-like/interleukin (IL)-1 receptor (TIR) signal transduction. Thus, inhibition of TRAF6 function could interrupt both CD40 (TNFR family) and IL-1 growth signals, pathways critical to myeloma proliferation. To block TRAF6-mediated IL-1 signaling, we constructed small interfering RNA (siRNA) against TRAF6. We found that siRNA targeting the TRAF6 C-terminal (siTRAF6C) receptor interaction domain specifically reduced only TRAF6 protein expression, without affecting TRAF2 or 5 levels, and substantially interfered with IL-1-induced NF-kappaB and c-Jun/AP-1 activation. Inhibition by siTRAF6C was concentration-dependent. SiTRAF6C also significantly reduced myeloma proliferation and enhanced apoptosis in a similar dose-dependent fashion in vitro. More importantly, marked siTRAF6C growth inhibition was detected in vivo when these cells were implanted into the bone marrow of irradiated normal mice. In contrast, introduction of siRNA derived from the TRAF6 Zn-finger domain or an irrelevant siRNA construct failed to alter cell growth or cell death. These studies suggest that TRAF6 may be a new molecular target to block cell signal transduction important for the survival and proliferation of multiple myeloma cells.