HS27a Cells Research Articles

P1253: MAVORIXAFOR DISRUPTS THE CROSS TALK BETWEEN WALDENSTRÖM’S MACROGLOBULINEMIA CELLS AND THE BONE MARROW MICROENVIRONMENT RESULTING IN ENHANCED SENSITIVITY TO B-CELL TARGETED THERAPIES

Background: Waldenström’s macroglobulinemia (WM) is a rare B-cell malignancy characterized by infiltration of bone marrow (BM) and lymphoid tissues with lymphoplasmacytic cells along with hypersecretion of monoclonal immunoglobulin M (IgM) by progenitor B cells. Studies have shown that more than 90% of patients with WM have an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), and 30% to 40% of those patients show mutations in the carboxyl terminus of C-X-C receptor 4 (CXCR4). The CXCR4/C-X-C motif ligand 12 (CXCL12) axis plays an important role in the homing and retention of WM cells in the BM. The interaction of WM cells and the BM microenvironment, via cell-to-cell contacts or indirectly through secreted factors (e.g. interleukin 6 [IL-6], C-C motif ligand 5 [CCL5]), is thought to be essential for the pathogenesis of WM and to confer drug resistance. A clinical trial evaluating the efficacy of the CXCR4 antagonist, mavorixafor, in combination with the BTK inhibitor, ibrutinib in patients with MYD88L265P and CXCR4WHIM WM is currently ongoing (NCT04274738). However, the effects of mavorixafor with ibrutinib and other B-cell targeted therapies on WM cells harboring only the single mutation in MYD88L265P and without a CXCR4 mutation have not been evaluated. Aims: This study was designed to elucidate the crosstalk between WM cells and the BM stromal cell (BMSC)-based microenvironment and to explore the effect of mavorixafor in combination with different B-cell targeted therapies in a WM/BMSC co-culture model. Methods: WM cells (MWCL-1 cell line, MYD88L265PCXCR4WT) pretreated with mavorixafor and B-cell targeted therapies (BTK antagonists: ibrutinib, zanubrutinib, evobrutinib, pirtobrutinib, nemtabrutinib; or BCL2 inhibitor venetoclax) were co-cultured with established BMSCs (HS27a cells). Apoptosis, IL-6 production, and IgM release were measured after 48 or 72 hours. The effects of mavorixafor on calcium mobilization, cell migration, and adhesion to BMSCs were also measured. Results: Co-culture of WM cells with established BMSCs led to increased IgM secretion and enhanced surface expression of CXCR4 in WM cells by activation of the IL-6 receptor (IL-6R)/ Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Co-culture with established BMSCs protected WM cells from the effects of B-cell targeted therapies, reducing apoptosis of tumor cells and decreasing the IgM-reducing efficacy of the targeted therapies. Mavorixafor alone reduced IgM secretion, blocked CXCL12-induced cell migration and Ca2+ mobilization and disrupted the adhesion of WM cells to established BMSCs. Mavorixafor in combination with B-cell targeted therapies synergistically enhanced antitumor activity in WM cells, inhibited BMSC-induced IgM hypersecretion, and overcame the protective effects of BMSCs on tumor cells. Summary/Conclusion: Our studies provide preliminary evidence for the potential use of mavorixafor in disrupting the interaction of WM cells with the BMSC-based microenvironment, enhancing the efficacy of B-cell targeted therapies in the treatment of WM and potentially other lymphomas. Further studies using additional WM cell lines and/or primary patient cells are warranted to support these findings.

Abstract 6093: Mavorixafor enhances efficacy of Bruton tyrosine kinase inhibitors by overcoming the protective effect of bone marrow stroma on tumor cells in Waldenström’s macroglobulinemia

Abstract Waldenström’s macroglobulinemia (WM) is a rare B-cell malignancy characterized by monoclonal immunoglobulin M (IgM) hypersecretion and invasion of B cells in the bone marrow (BM) and lymphoid tissues. >90% of WM cases show mutations in MYD88 and 30-40% show mutations in the carboxyl terminus of CXCR4.The CXCR4/CXCL12 axis is crucial for the homing/retention of WM cells in the BM. Emerging clinical trial data (NCT04274738; ongoing) suggest that mavorixafor, a CXCR4 antagonist, in combination with ibrutinib results in clinically meaningful changes in levels of IgM and hemoglobin in patients with MYD88L265P CXCR4WHIM WM. However, the effects of mavorixafor with ibrutinib and other Bruton tyrosine kinase (BTK) inhibitors on WM cells harboring only the single mutation (MYD88L265P without CXCR4 mutation) have not been evaluated. This study was designed to test the ability of mavorixafor to sensitize WM cells carrying MYD88L265P CXCR4WT to BTK inhibitors in a WM/BM stromal cell (BMSC) co-culture model. The effects of mavorixafor on Ca2+ mobilization, cell migration, and adhesion to BMSC were also measured. WM cells (MWCL-1 cell line, MYD88L265PCXCR4WT) pretreated with mavorixafor and BTK inhibitors (ibrutinib, zanubrutinib, evobrutinib, LOXO-305, ARQ 531) were co-cultured with established BMSCs (HS27a cells). Cell viability, apoptosis, and IgM release were measured after 72 hours. BTK inhibitors, but not mavorixafor, decreased tumor cell viability and increased apoptosis of WM cells in the absence of BMSCs. Co-culture with BMSCs enhanced CXCR4 expression and protected WM cells from the effects of BTK inhibitors. BMSCs also significantly increased IgM secretion 2- to 5-fold compared with WM cells grown in the absence of BMSCs. Mavorixafor alone inhibited CXCL12-stimulated Ca2+ mobilization and migration of WM cells and disrupted the adhesion of WM cells to BMSCs. Combination of mavorixafor with BTK inhibitors overcame the protective effect of BMSCs on tumor cells, decreasing cell viability and/or increasing apoptosis compared with BTK inhibitors alone. Mavorixafor also reduced IgM secretion, which was further decreased when combined with BTK inhibitors.This study is the first to show in vitro that the protection of WM cells against BTK inhibitors conferred by BMSCs can be overcome by inhibition of the CXCR4/CXCL12 axis. The observations and responses to mavorixafor suggest a contribution of CXCR4WT to the pathogenicity of WM cells carrying only the MYD88L265P mutation. Mavorixafor addition enhanced the efficacy of not only ibrutinib but the other BTK inhibitors tested, supporting the greater potential of this combination therapeutic strategy in WM patients with or without CXCR4WHIM mutations. Further studies using additional WM cell lines and/or primary patient cells are warranted to support these findings. Citation Format: Chi Nguyen, Halenya Monticelli, Tom Kruitwagen, Matteo Tardelli, Barbara Maierhofer, Thalia Martins Rebelo, Sabine Maier-Munsa, Katarina Zmajkovicova, Lukas Dillinger, Adriana Badarau, Arthur G. Taveras. Mavorixafor enhances efficacy of Bruton tyrosine kinase inhibitors by overcoming the protective effect of bone marrow stroma on tumor cells in Waldenström’s macroglobulinemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6093.

Acute Lymphoblastic Leukaemia (ALL) Cells and Commonly-Used Chemotherapy Drugs Can Induce Bone Marrow-Derived Mesenchymal Stromal Cells to Become Cancer Associated Fibroblasts (CAF), Leading to Enhanced ALL Proliferation and Treatment Resistance

To investigate the role of mesenchymal stromal cells (MSCs) in treatment resistance in acute lymphoblastic leukaemia (ALL) we systematically isolated and studied, ISCT-confirmed, bone-marrow-derived MSCs isolated from 151 adults (22-69 years) with ALL registered on the UKALL14 and ALL60+ trials both at diagnosis and during therapy.CD11b, CD68 and CD163-expressing tumour-associated macrophages were observed in 34/75 (45%) of MSC cultures from follow-up samples (termed M+MSC). By contrast, macrophages were absent (M-MSC) from all diagnostic (0/56) and healthy donor specimens (0/7) tested. ELISA on supernatant from expanded M+MSC showed significantly higher levels of pro-inflammatory cytokines IL8 (4961 pg/ml vs 1093 pg/ml, p = 0.01) and CXCL2 (106 pg/ml vs 36 pg/ml, p = 0.03) than that from expanded M-MSC. The B-ALL cell line Nalm6 proliferated more rapidly when co-cultured with primary M+MSCs than with primary M-MSC (1.93 fold, p = 0.03). Importantly, primary M+MSCs strongly expressed α-smooth muscle actin (αSMA) by immunostaining, consistent with a cancer associated fibroblast (CAF) phenotype.To establish the role of ALL cells and/or chemotherapy in the induction of CAFs, we used the MSC cell line HS27a to model CAF generation in-vitro . Co-culture of HS27a with B-ALL cell lines Nalm6, SD1 and 697 cells induced increased aSMA expression and cytoskeleton remodeling including increased actin stress fibers in the HS27a consistent with a CAF phenotype. These changes were not observed in HS27a in co-culture with healthy donor B-cells or the B-ALL cell line SEM. A targeted RQPCR gene panel showed up-regulation of CAF-defining genes (FAPA 2-4.4.6 fold, COX2 5.9 - 32.1 fold, IL6 11.2 - 495 fold, FN 2.2 - 3.0 fold, TGFβ1 2.0 - 4.6 fold, IL8 15.5 - 85.2 fold, CCL2 10.6 - 33.3 fold, VEGF 2.9 - 13.4 fold, FGF2 2.9 - 14.6 fold) and expected down-regulation of CAV1 (1.8 - 5.4 fold) when HS27a were cultured with SD1 and 697 cells compared to HS27a alone. HS27a exposed to healthy donor B-cells or SEM cells in contrast did not induce differential regulation of CAF defining genes. In a flow-based, cytokine bead assay, SD1 cells induced HS27a to secrete pro-inflammatory cytokines (IL8, IL6, CCL2, CXCL10 and CCL5) and growth factor bFGF at several fold higher levels than normal B-cells or SEM cells consistent with a CAF secretion profile. Similar morphological changes, gene expression profile and cytokine profile were observed when cells were co-cultured in a transwell system, suggesting a soluble mechanism of CAF induction. TGFβ1 and reactive oxygen species, known to induce CAFs in solid tumors, were both increased in all B-ALL cell lines able to induce CAFs compared to SEM cells, suggesting a probable mechanism behind CAF induction.Therapeutically-relevant concentrations of chemotherapy agents cytarabine and daunorubicin - but not vincristine and dexamethasone - induced gross morphological changes in HS27a cells with an increase in actin stress fibres and αSMA. RQPCR of cytarabine-treated HS27a showed upregulation of several genes consistent with a CAF phenotype and cytokine bead assay showed increased secretion of IL8 and CCL2 compared to untreated HS27a (973 pg/ml vs 430pg/ml, p < 0.0001 and 1089pg/ml vs 717.3pg/ml p = 0.02 respectively).To examine the functional significance of CAF on ALL cell proliferation and chemoresistance, we pre-treated HS27a with cytarabine to induce a CAF phenotype and then assessed the impact on SEM (selected for their lack of CAF induction) proliferation and chemosensitivity. Pretreatment with vincristine or dexamethasone and untreated HS27a comprised the controls. Cytarabine-generated, HS27a-derived CAF increased SEM cell proliferation compared to both the untreated and vincristine and dexamethasone pre-treated HS27a (1.2 fold p = 0.002, 1.3 fold p = 0.0004 and 1.2 fold p = 0.0002 respectively). Cytarabine-generated, HS27a-derived CAF also reduced the sensitivity of SEM cells to both dexamethasone and cytarabine compared to both the untreated and dexamethasone or vincristine pre-treated HS27a.We have demonstrated for the first time in both primary cells and cell-line models that bone-marrow derived MSCs can adopt a CAF phenotype in response to both ALL cells and ALL therapies. The functional impact is to enhance both ALL cell proliferation and chemotherapy resistance. Ongoing work will delineate the precise nature of the interaction between MSC and ALL cells. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Cellular senescence induced by S100A9 in mesenchymal stromal cells through NLRP3 inflammasome activation.

Bone marrow stromal cells from patients with myelodysplastic syndrome (MDS) display a senescence phenotype, but the underlying mechanism has not been elucidated. Pro-inflammatory signaling within the malignant clone and the bone marrow microenvironment has been identified as a key pathogenetic driver of MDS. Our study revealed that S100A9 is highly-expressed in lower-risk MDS. Moreover, normal primary mesenchymal stromal cells (MSCs) and the human stromal cell line HS-27a co-cultured with lower-risk MDS bone marrow mononuclear cells acquired a senescence phenotype. Exogenous supplemented S100A9 also induced cellular senescence in MSCs and HS-27a cells. Importantly, Toll-like receptor 4 (TLR4) inhibition or knockdown attenuated the cellular senescence induced by S100A9. Furthermore, we showed that S100A9 induces NLRP3 inflammasome formation, and IL-1β secretion; findings in samples from MDS patients further confirmed these thoughts. Moreover, ROS and IL-1β inhibition suppressed the cellular senescence induced by S100A9, whereas NLRP3 overexpression and exogenous IL-1β supplementation induces cellular senescence. Our study demonstrated that S100A9 promotes cellular senescence of bone marrow stromal cells via TLR4, NLRP3 inflammasome formation, and IL-1β secretion for its effects. Our findings deepen the understanding of the molecular mechanisms involved in MDS reprogramming of MSCs and indicated the essential role of S100A9 in tumor-environment interactions in bone marrow.

Reduced SLIT2 Are Associated with Increased Cell Proliferation and Arsenic Trioxide Resistance in APL Cells

Background: Recently, the SLIT/ROBO axis became a therapeutic target for a variety of non-cerebral solid tumors because of its relevance in the regulation of angiogenesis, inflammatory cell chemotaxis, tumor cell migration and metastasis. In acute myeloid leukemia (AML), lower levels of SLIT2 expression were associated with poorer prognosis in studies that excluded patients with acute promyelocytic leukemia (APL) of the analysis (Golos et al., Arch Immunol 2019). Our group previously reported that higher SLIT2 expression was associated with improved overall survival, disease-free survival and decreased cumulative incidence of relapse. Subsequently, our in vitro functional studies indicated anti-proliferative effects exercised by exogenous SLIT2 peptide treatment in APL (Weinhauser et al., Blood 2018). Although, the expression of SLIT/ROBO was detected on leukemic cells, the fact that in healthy subjects the main source of SLIT2 derives from bone marrow stromal cells (BMSC) (Smith-Berdan et al., Cell Cycle 2012) was so far neglected in the context of hematological malignancies. Aims: Here, we expanded our data on the role of SLIT2 using lentivirally knocked down APL cells, and addressed the issue of the importance of BMSC-derived SLIT2 on APL development. Methods: NB4 and NB4R2 (RA-resistant) and primary APL cells (age, 25-47y; n=4) were transduced with shRNA-SLIT2 or shCT (control). After synchronization using double-thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For the apoptosis analysis, cells were treated with ATO (1 µM) alone or in combination with RA (1 µM) for 24, 48 and 72 hours. The granulocytic differentiation in response to RA treatment alone (1 µM) or in combination with ATO (1 µM) for 48 and 72 hours, was evaluated based on the CD11b and CD11c levels. To assess the effect of SLIT2 silencing in BMSCs, HS5 and HS27A cell lines were transduced with shSLIT2 or shCT. Non-transduced NB4, NB4R2 and primary APL cells were co-cultured with engineered BMSCs and proliferation, apoptosis and granulocytic differentiation was performed in response to ATO alone or in combination with RA for 48 and 72 hours. In vivo assessment was performed using four 8-12 week-old C57/BL6PepBoy (CD45.1) lethally irradiated, transplanted with 5 x 106 hCG-PMLRARa blasts (CD45.2) transduced with shSlit2-murine and shCT. Results: Silencing SLIT2 in primary APL samples resulted in a significant increase in cell growth at the sixth day of culture compared to shCT samples (P&lt;.001). Accordingly, enhanced staining of Ki67 was detected in NB4 and NB4R2-shSLIT2 transduced compared to shCT cells. Moreover, NB4-shSLIT2 cells exhibited lower ATO-induced apoptosis either when ATO was used alone or in combination with RA. Nevertheless, SLIT2 knockdown had no effect on NB4R2 cells suggesting that RARA moiety of PML/RARA may be important to the observed effect. Concomitantly, NB4 cell differentiation rate, was greatly increased in NB4-shSLIT2 in the presence of RA after 48 and 72h (P&lt;.05) and 48h when RA was combined with ATO (P&lt;.05). In vivo transplantation of primary murine shSLIT2 hCG-PMLRARa blasts, resulted in significant increase of white blood cell count during the course of APL emergence and increased extramedullary hematopoiesis in the spleen, yet with no difference in the BM at the time of sacrifice. Next, we evaluated the number of transcripts and the function of SLIT2 in AML derived MSC cells. SLIT2 transcript levels were significantly reduced in primary AML-derived BMSC compared to BMSCs obtained from healthy donors (P&lt;.05). In vitro assays co-culturing NB4 and NB4R2 cells on engineered HS5 or HS27A BMSC cells showed no difference in drug-induced apoptosis or differentiation (P&gt;.05). In contrast to shSLIT2 APL induced cell growth, co-culture of primary APL blast cells with transduced HS27A cells had no impact on proliferation, while the expression of the neutrophilic differentiation gene ELANE2 was significantly decreased in APL cells co-cultured with HS27A-shSLIT2.ConclusionSLIT2 is expressed in APL cells and exerts an anti-proliferative effect. On the other hand, SLIT2 produced by BMSC seemed to promote APL cellular stemness. Our results reinforce that SLIT2 function is cell type dependent and may be involved in the cross-talk between microenvironment and leukemic cells. Disclosures Figueiredo-Pontes: Novartis: Honoraria.

Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress

AbstractWe investigated and modeled the mesenchymal stromal cell (MSC) niche in adult acute lymphoblastic leukemia (ALL). We used gene expression profiling, cytokine/chemokine quantification, flow cytometry, and a variety of imaging techniques to show that MSCs, directly isolated from the primary bone marrow specimens of patients with ALL, frequently adopted an activated, cancer-associated fibroblast phenotype. Normal, primary human MSCs and the MSC cell line HS27a both were activated de novo, when exposed to the reactive oxygen species (ROS)–inducing chemotherapy agents cytarabine (AraC) and daunorubicin (DNR), a phenomenon blocked by the antioxidant N-acetyl cysteine. Chemotherapy-activated HS27a cells were functionally evaluated in a coculture model with ALL targets. Activated MSCs prevented therapy-induced apoptosis and death in ALL targets, via mitochondrial transfer through tunneling nanotubes (TNTs). Reduction of mitochondrial transfer by selective mitochondrial depletion or interference with TNT formation by microtubule inhibitors, such as vincristine (VCR), prevented the “rescue” function of activated MSCs. Corticosteroids, also a mainstay of ALL therapy, prevented the activation of MSCs. We also demonstrated that AraC (but not VCR) induced activation of MSCs, mitochondrial transfer, and mitochondrial mass increase in a murine NSG model of disseminated SEM cell–derived ALL, wherein CD19+ cells closely associated with nestin+ MSCs after AraC, but not in the other conditions. Our data propose a readily clinically exploitable mechanism for improving treatment of ALL, in which traditional ROS-inducing chemotherapies are often ineffective at eradicating residual disease, despite efficiently killing the bulk population.

Analysis of the effect of DEK overexpression on the survival and proliferation of bone marrow stromal cells

Abstract Objective DEK is ubiquitously expressed and encodes a nuclear protein, which is also released from some cells. Overexpression of DEK suppresses proliferation of some blood cell progenitors whereas it increases proliferation of epithelial tumors. We showed that DEK is overexpressed in BM cells of 12% of multiple myeloma (MM) patients. Here, we aimed to test if DEK overexpression effects the proliferation and viability of BM stromal cells or MM cells co-cultured with DEK-overexpressing stromal cells, mimicking the BM microenvironment. Methods DEK is stably overexpressed in the BM stromal cell line HS27A. Periodic growth curve and fluorescent activated cell sorting (FACS) analysis was performed to determine the effect of DEK overexpression on HS27A cells and MM cell lines (RPMI-8226 and U266) that are co-cultured with these HS27A cells. Results We showed that, on the contrary to blood progenitors or ephitelial cells, DEK overexpression doesn’t alter the viability or proliferation of the HS27A cells, or the MM cell lines which are co-cultured with DEK-overexpressing HS27A cells. Conclusions Our results suggest that effect of DEK overexpression on the proliferation is cell type and context dependent and increased DEK expression is tolerable by the stromal cells and the co-cultured MM cell lines without effecting proliferation and viability.

Commonly Used Chemotherapy Drugs Differentially Determine Microenvironment-Mediated Protection, Via Mitochondrial Transfer, to B-Precursor Acute Lymphoblastic Leukaemia Cells

Murine models of Acute Lymphoblastic Leukaemia (ALL) suggest relapse arises not from intrinsic chemoresistance by genetically distinct cells, but from a subset of cells protected within a specific niche.To confirm the existence of such a niche in patients with ALL and elucidate the mechanism by which stromal cells (MSC) protect ALL cells we isolated MSC from bone marrow of 70 B-ALL patients enrolled on the UKALL14 trial. Immunostaining for f-actin, gene expression profiling and cytokine/chemokine quantification showed that a significant proportion of bone marrow specimens, especially after treatment with a cytarabine (AraC)-containing block, contained MSC with an activated phenotype, analagous to cancer associated fibroblasts (ALL-CAF).We demonstrated that primary ALL cells, ALL cell lines, AraC and daunorubicin (but not vincristine (VCR) or dexamethasone (dex)) generated ALL-CAF de novo from both healthy donor MSC and the MSC cell line HS27a. Notably, the chemotherapy drugs induced distinct morphological changes and differential alpha-smooth muscle expression (figure 1a).Control of oxidative stress via modification of reactive oxygen species (ROS) presented a potential unifying explanation for ALL-CAF generation by both ALL cells and chemotherapy. Using flow cytometry we demonstrated that AraC significantly increased ROS in the B-ALL cell line, SEM, in monoculture but in co-culture with HS27a, ROS was significantly lowered and was not impacted by AraC. The MSC co-culture-mediated reduction in ROS in co-culture corresponded to a significant reduction in cell death (10.5% vs 36%, p = 0.0001). By contrast, VCR did not impact ROS significantly and Dex reduced it. Both were significantly more effective than AraC at inducing SEM cell death in co-culture (VCR 20.2% vs AraC 10.5%, p = 0.0003; Dex 39.1% vs AraC 10.5%, p = 0.0007), despite inducing the same degree of cell death in monoculture.We hypothesised that mitochondrial transfer between ALL-CAF and B-ALL cells could provide a generalised mechanism to overcome the deleterious impact of cell-intrinsic and chemotherapy-driven ROS in B-ALL cells. A ‘mitotracker’ flow cytometry assay showed differential mitochondrial transfer from HS27a to B-ALL cells, in proportion to the baseline ROS levels. We confirmed that mitochondria could also be transferred from healthy donor MSC co-cultured with primary patient ALL cells. Furthermore, AraC, but not VCR or Dex, significantly enhanced mitochondrial transfer, and did so in a dose-dependent manner. To rule out passive transfer of dye, we used the murine stromal line MS5 as an alternative mitochondria donor to SEM cells. Murine mitochondrial, but not nuclear, DNA was clearly seen in flow-sorted SEM cells after co-culture with MS5, at baseline and at higher levels after AraC therapy. We also directly visualised the transfer of mitochondria along tunnelling nanotubes (TNT) by time-lapse confocal imaging (figure 1b). To confirm that mitochondrial transfer was essential in MSC ‘rescue’ of ALL cells, we generated HS27a cells deficient in mitochondria following prolonged culture with low dose ethidium bromide. The mitochondrially-deficient cells retained viability as well as the ability to become ALL-CAF but were clearly defective in their ability to rescue SEM ALL cells from AraC induced cell death.To confirm the functional impact of mitochondrial transfer via TNT, we used actin inhibitor latrunculin B (LatB) and the microtubule damaging agent nocodazole which both significantly blocked the phenomenon. Both LatB and nocodazole significantly restored AraC-related cytotoxicity. Colchicine, another microtubule damaging agent had a similar impact to nocodazole. VCR completely overcame the protective impact of HS27a on AraC cytotoxicity and was additive with AraC in the co-culture system.We have shown that CAF-like MSC provide support to ALL cells under oxidative stress by mitochondrial transfer via TNT. This is disrupted by microtubule damaging agents and conditions provoking their formation are mitigated by Dex, both mainstays of ALL therapy. Our data may explain the ineffectiveness of ROS-inducing chemotherapy at eradicating disease at the niche and provides an explanation of why low dose, microtubule damaging agents such as VCR used in maintenance therapy are effective in ALL. Our findings have immediate implications for the design and scheduling of current combination chemotherapies for ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PO-184 Role of ubiquitin-conjugating enzymes in chromosome instability and breast cancer metastasis

IntroductionLab-on-a-chip (LOC) systems can present cells multiple cues that cells are subjected to, including gradients of cytokines and secreted proteins from neighbouring cells, biochemical and mechanical interactions with the extracellular matrix, and direct cell-to-cell contacts in a controllable and reproducible fashion. Simulating microenvironment with LOC devices is crucial to understand metastasis preference of cancer cells especially for planning personalised therapies in hepatocellular carcinoma (HCC).Material and methodsPeristaltic-pump was used to circulate HCC cells, HuH-7 and SNU-449. Survival under shear stress was analysed by fluorometric and microscopic analysis. Adhesion and proliferation were measured by live cell analysis system. HUVEC cells were used to simulate extravasation of circulating cells. BEAS2B, HS-27A and THLE-2 cells were used as homing targets to simulate bone, lung and liver, respectively. Image analysis was performed with fluorescent and confocal microscopy.Results and discussionsFirstly, we determined the effect of shear stress in a time-dependent manner on adhesion and proliferation of HCC cells. Circulation under shear stress up to 30’ caused an increase in both adhesion and proliferation significantly, whereas longer exposure time has a negative effect on adhesion, proliferation and, survival. HuH-7 cells with epithelial phenotype found to gain increased survival capacity under shear stress than its mesenchymal counterpart SNU-449. Then, we investigated the homing preference of HCC cells by using a novel micro-circulation LOC device. Shear stress, microenvironmental conditions (medium, matrigel, coating, etc.), cell labels and numbers (homing, HCC) were optimised for LOC. Representation of endothelial barrier was succeeded by monolayer coating of ’flow-channel’ by HUVEC cells. HCC cells were circulated for 4 hours then LOCs were incubated for 3 days. The extravasation and homing preference of HCC cells were determined by confocal imaging. Consistent with the high frequency of intrahepatic metastasis in HCC, SNU-449 cells mostly preferred to home into the channel with normal liver cells.ConclusionOur study suggests designed LOC system has a potential to predict homing capacity and organ preference of circulating tumour cells in HCC. In addition to precision medicine applications such as drug screening, this device could be used as a three-dimensional tool for understanding tumor-microenvironment interactions with mechanistic explanations in metastasis.

Abstract 5910: Influence of 3D- cultured prostate cancer and stromal cells on drug response

Abstract Background: Response to chemotherapeutic drugs presents a significant challenge to effective treatment of advanced prostate cancer. Exploration of therapeutic response has focused largely on the tumor cell. However, evidence suggests mechanisms that involve the tumor microenvironment mediate response to therapeutics. Therefore, it is imperative to mimic the in vivo tumor microenvironment, to bridge the gap between in vitro studies and the development of therapeutics. The concept of three-dimensional (3D) cell culture and co-culture of multiple cell types is critical to predict therapeutic efficacy and outcome. Thus, these studies sought to determine the distinct responses to anti-cancer drugs in 3D cell spheroids. Methods: In this study, we utilized metastatic prostate cancer cells (PC3) and fluorescently labeled prostate bone stromal cell line (HS27a) to generate tumor spheroids. Tumor spheroid viability was measured at 72 and 120 h using the CellTiter-Glo 3D assay after treatment with three common anti-cancer drugs, Docetaxel (4ηM), Enzalutamide (30μM), and Bicalutamide (30μM).Treatments were compared with spheroids originated from HS27a and PC3 cells alone and in co-culture. The cell viability percentage was normalized using the untreated cells as 100% cell survival. Results: Spheroid formation increased resistance to Docetaxel, Enzalutamide, and Bicalutamide. However, PC3 spheroid cells was significantly higher compared to HS27a and co-culture spheroids. For all three spheroids, as the size of the spheroids increase, cellular resistance to anti-cancer and hormone drugs increased. Conclusion: The findings in this study demonstrated that the use for 3D-cell cultures influence many aspects of PCa behavior including, proliferation rates, and response to chemotherapeutics. Note: This abstract was not presented at the meeting. Citation Format: Dominique N. Gales, Sonni Miller, Temesgen Samuel, Clatyon Yates. Influence of 3D- cultured prostate cancer and stromal cells on drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5910. doi:10.1158/1538-7445.AM2017-5910

The Bone Marrow Environment Promotes Resistance to Mitoxantrone and Etoposide By Distinct Mechanisms in Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704).To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children’s Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45high, SSClow) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p<0.0001). Results were similar for mitoxantrone treatment. We also found increased DNA damage signaling (%cPARP-/gH2AX+) after mitoxantrone treatment for AML cells in stromal co-culture (34.5 ±3.0% in cells cultured alone, v. 58.6 ±3.1% in HS5 co-cultures, p<0.0001). However, DNA damage signaling was not significantly increased after etoposide treatment. For 5 samples treated with both chemotherapy agents, we also measured pDNA-PKcs and pATM by FACS. Treatment with mitoxantrone, but not etoposide, induced more pDNA-PKcs (MFI, 25.7 ±4.2 untreated, v. 61.7 ±5.1 mitoxantrone, p<0.001, v. 31.5 ±4.8 etoposide) in AML cells cultured alone. However, stroma did not further increase pDNA-PKcs. These results suggest that the NHEJ pathway is important for the repair of DNA damage caused by mitoxantrone, and that stromal cells increase DNA damage signaling by a mechanism not involving increased pDNA-PKcs.To better understand etoposide resistance, 29 of the 44 primary AML samples were cultured on stroma overnight, and activation of intracellular signaling pathways, including pY-STAT3, pY-STAT5, and pERK1/2, was measured by FACS. Responses were heterogeneous overall, but we found several patterns of stroma-induced signaling. For example, a sample that strongly activated a given pathway when co-cultured with HS5 also responded strongly to HS27A cells, and this was particularly true for pY-STAT3 (R=0.71, p<0.0001) and pY-STAT5 (R=0.76, p<0.0001). We also found a correlation between pY-STAT3 and pY-STAT5, such that samples that showed a strong activation of STAT3 with stromal co-culture had a similar activation of STAT5 (R=0.94, p<0.0001, on HS27A). As for signaling pathways in relationship to apoptosis, higher levels of pERK1/2 were associated with lower levels of apoptosis (R=-0.5182, p<0.01, on HS27A), suggesting that ERK1/2 activation may promote resistance to etoposide.In summary, we found that DNA damage signaling is induced in AML cells by mitoxantrone, and it is augmented in cells co-cultured with stroma, likely contributing to mitoxantrone resistance. This mechanism does not occur with etoposide. Instead, we found that stromal environment-induced ERK1/2 signaling may enhance etoposide resistance in pediatric AML patient samples. Further studies to confirm the role of pERK1/2 in stroma-induced etoposide resistance are underway. DisclosuresNo relevant conflicts of interest to declare.

Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method.

Two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells, have frequently been used in studies of cross talk between cells in the bone marrow microenvironment and hematopoietic cells. Altered expression of proteins is typically associated with cell-cell signal transduction and regulation of cellular functions. Many studies have focused on key proteins that contribute to functional differences in cell co-culture models, but global quantitative proteome analysis of HS5 and HS27a has not been performed. We employed the stable isotope labeling by amino acids in cell culture (SILAC) method using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. Labeled proteins were analyzed by 2-D ultrahigh-resolution liquid chromatography- LTQ/Orbitrap mass spectrometry. Among 4,213 unique identified and annotated proteins in the cell lines, 1,462 were detected in two independent experiments. Of these, 69 exhibited significant upregulation and 48 significant downregulation (>95% confidence) in HS27a relative to HS5 cells. Gene ontology term and pathway analysis indicated that the differentially regulated proteins were involved in cellular movement, cell-to-cell signaling and interaction, and hematologic system development and function. A total of 55 items were identified in both genomic and proteomic databases. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed on 7 proteins randomly selected from 28 differentially expressed proteins that were identified in both databases and were involved in the top networks/pathways. We observed a decrease in apoptosis in co-cultured KG1a cells when integrin αV was inhibited in HS27a cells, which suggested the functional role of integrin αV in the co-culture system. The integrated genomic/proteomic approach described here, and the identified proteins, will provide a useful basis for further elucidation of molecular mechanisms in the bone marrow microenvironment and for ongoing studies of cross talk among stromal cells and myeloma cells in co-culture systems.

Monocyte and Fibroblast Directed Generation of a Marrow Vascular Niche

Hematopoietic cells dynamically interact with their surrounding microenvironment during their residence, maturation and differentiation. Individual marrow components have been isolated and studied in 2D in vitro cultures, yet their functional contributions to a complete niche are not fully understood. In vivo studies in mouse models are complex, and the inaccessibility of marrow architecture has precluded systematic analysis of each component. Here, we employ an in vitro 3D microfluidic vascular system to study the effect of microenvironmental cues on cell trafficking in an engineered hematopoietic niche. Our system allows for control of 3D geometric cues, hydrodynamic flow, multi-cellular compositions, and cellular matrix remodeling by combining soft lithography and injection modeling in type I collagen gel (Zheng et al. PNAS 2012). Endothelial cells perfused through the embedded microfluidic network form a confluent, patent endothelium within the collagen. Incorporating hematopoietic cells and stromal cells into the extravascular collagen space allows us to visualize how cells interact with vasculature during culture.First, we developed a marrow stromal microenvironment to understand how stromal cells modify the marrow microvascular environment, by incorporating two different human marrow-derived stromal fibroblast cell lines (HS27a and HS5) in the extravascular space surrounding the vessels (Fig 1A). HS27a and HS5 cells created distinct vascular microenvironments by secreting divergent cytokine profiles: stem cell niche-associated and inflammatory cytokines, respectively. Both stromal lines modified the vascular phenotype by reducing endothelial expression of vWF and junctional proteins. In particular, co-culture with HS5 increased expression of inflammatory markers on the endothelium.Next, we evaluated the function of this modified vasculature on hematopoietic stem cell trafficking through the vessels. When CD34+ cells alone were perfused through the microvessels, they adhered on the vessel wall and migrated into the matrix. The HS27a and HS5-induced microenvironment did not significantly change trafficking behavior (Fig 1B, C). When monocytes were perfused through the microvessels, they also adhered on the vessel wall and migrated into the matrix. Monocytes are a critical part of the marrow space, and are known to modify the endothelium and stromal microenvironment. When CD34+ cells were perfused through the vessels 24 hours after monocytes, crosstalk shifted trafficking patterns of both cell types and led to increased adhesion and migration within the HS5- and HS27a-modified vessels (Fig 1D).To explore the competent marrow niche in vitro, we further examined hematopoietic cell trafficking from the extravascular space into the circulation by embedding cells from fresh human bone marrow screens within the collagen matrix. We found this cell fraction contained a mixed population of hematopoietic and stromal cells, and could be cultured in the microvascular niche for at least two weeks. Scanning electron microscopy showed various types of marrow cells residing in both the abluminal and luminal side of the endothelium (Fig 1E-G). Throughout culture with continuous vascular perfusion, we collected the media flow-through and identified different hematopoietic cell populations released from the matrix into the circulation over the course of two weeks. Specifically, we identified CD34+ hematopoietic progenitor cells along with megakaryocyte, erythroid, lymphoid, and myeloid lineage cells by flow cytometry analysis.In summary, we developed an in vitro 3D microvascular marrow niche and gained insight into hematopoietic cell trafficking between the stroma and the circulation. By guiding the interplay of heterogeneous cell populations, we have demonstrated the capacity to define distinct microenvironment spaces. This platform shows promise for long term culture of a whole marrow population and for the ex vivo generation of hematopoietic cells. Further development of this 3D marrow niche will allow us to better understand the complexities mediating stem cell trafficking, residence, proliferation, mobilization, and differentiation in both health and disease. [Display omitted] DisclosuresNo relevant conflicts of inteqerest to declare.

β-catenin signaling induces the osteoblastogenic differentiation of human pre-osteoblastic and bone marrow stromal cells mainly through the upregulation of osterix expression

Both β-catenin (β-cat) and osterix (OSX) are known to be essential for embryonic and postnatal osteoblast differentiation and bone growth. In the present study, we explored the crosstalk between β-cat signaling and OSX, and assessed its effect on the osteoblastogenic differentiation of human pre-osteoblastic cells (MG-63) and bone marrow stromal cells (HS-27A). In the HS-27A and MG-63 cells, the selective β-cat signaling inhibitor, CCT031374, and the stable overexpression of a constitutively active β-cat mutant respectively decreased and increased the cytoplasmic/soluble β-cat levels, and respectively decreased and increased TOPflash reporter activity, the mRNA levels of β-cat signaling target genes c-Myc and c-Jun, as well as the mRNA and protein expression levels of OSX. Mutational analyses and electrophoretic mobility shift assays revealed that the increased binding activity of c-Jun at a putative c-Jun binding site (−858/−852 relative to the translation start codon, which was designated as +1) in the human OSX gene promoter was required for teh β-cat signaling-induced expression of OSX in the HS-27A and MG-63 cells. During osteoblastogenic culture, stimulating β-cat signaling activity by the stable overexpression of the active β-cat mutant markedly increased alkaline phosphatase (ALP) activity and calcium deposition in the HS-27A and MG-63 cells, which was abolished by knocking down OSX using shRNA. On the other hand, the inhibition of β-cat signaling activity with CCT031374 decreased the ALP activity and calcium deposition, which was completely reversed by the overexpression of OSX. On the whole, the findings of our study suggest that β-cat signaling upregulates the expression of OSX in human pre-osteoblastic and bone marrow stromal cells by trans-activating the OSX gene promoter mainly through increased c-Jun binding at a putative c-Jun binding site; OSX largely mediates β-cat signaling-induced osteoblastogenic differentiation. The present study provides new insight into the molecular mechanisms underlying osteoblast differentiation.

Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy

Altered expression of glycans is associated with cell-cell signal transduction and regulation of cell functions in the bone marrow micro-environment. Studies of this micro-environment often use two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells. We hypothesized that differential protein glycosylation between these two cell lines may contribute to functional differences in in vitro co-culture models. In this study, we applied an integrated strategy using genomic, proteomic, and functional glycomic techniques for global expression profiling of N-glycans and their related genes and enzymes in HS5 cells versus HS27a cells. HS5 cells had significantly enhanced levels of bisecting N-glycans (catalyzed by MGAT3 [β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase]), whereas HS27a cells had enhanced levels of Galβ1,4GlcNAc (catalyzed by β4GalT1 [β4-galactosyltransferase I]). This integrated strategy provides useful information regarding the functional roles of glycans and their related glycogenes and glycosyltransferases in the bone marrow microenvironment, and a basis for future studies of crosstalk among stromal cells and myeloma cells in co-culture.

Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells.We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance.CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stromal Cells Enhance DNA Damage Signaling Independent Of Stat3 Activation In Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. It is clear that the bone marrow microenvironment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. We recently reported that patients whose AML blasts activated Stat3 in response to two bone marrow-derived ligands, G-CSF and IL-6, had a significantly superior survival rate, compared to patients whose blasts failed to activate Stat3 (Redell, et al, 2013, Blood121:1083). In this followup study of a subset of these patients, we further investigated the basis for the failure of the Stat3 response, as well as mechanisms of environment-mediated chemotherapy resistance. Our hypotheses were A) Stat3 responsiveness to G-CSF/IL-6 reflects the cell's general ability to respond to ligand stimulation, rather than a specific defect in a Stat3 pathway; and B) Stat3 responsiveness correlates with other functional responses to stromal exposure, including survival, proliferation, and chemotherapy resistance. Second samples from 27 patients were studied by FACS analysis of intracellular markers. To study stroma-induced changes, AML cells were co-cultured on stromal cells overnight, or briefly stimulated with stroma-conditioned medium (CM). We used HS5 cells, which secrete high levels of many soluble factors, and HS27A cells, which secrete very few factors. The stromal cell lines express mOrange for exclusion by FACS.First, primary AML cells were stimulated with G-CSF (10 ng/ml) or IL-6 + soluble IL-6R (5 + 10 ng/ml) for 15 min, then pY-Stat3 (PE) and pERK1/2 (AlexaFluor488) were measured by FACS. Data were acquired on the LSRII (BD) and analyzed with FCSExpress4 (DeNovo). Ligand responses were expressed as the fold change in mean fluorescence intensity over unstimulated cells (ΔMFI). We found a strong correlation between G-CSF-induced pY-Stat3 ΔMFI and pERK1/2 ΔMFI (R=0.858, p<0.001), suggesting that failure to activate Stat3 in unresponsive samples was not due to a Stat3-specific block. As expected, HS5 CM strongly activated Stat3 in samples that also responded to G-CSF and IL-6, while HS27A CM did not activate Stat3 in any samples. We compared responses to G-CSF v. HS5 CM, and found a strong correlation for pY-Stat3 ΔMFI (R=0.886, p<0.001) and for pERK1/2 ΔMFI (R=0.907, p<0.001). Samples that failed to respond to G-CSF also failed to respond to a combination of multiple soluble factors, supporting the idea that signaling function or dysfunction is a generalized property of AML cells.To further investigate functional changes induced in AML cells by stromal cells, we co-cultured blasts from 17 samples overnight with HS5 or HS27A cells. We labeled cleaved PARP (AlexaFluor647) to measure viable cells (%cPARP-) in each condition. Within the viable AML cell population, we measured the proliferative fraction (Ki67-FITC+ or Ki67-AlexaFluor700+) and the fraction with active DNA damage signaling (γH2AX-AlexaFluor488+). We found a small increase in the %viable for co-cultured cells v. cultured alone (p<0.05 for HS27A v. alone; Wilcoxon Signed Rank Test) and in the %Ki67+ (p<0.005 for HS27A v. alone). Moreover, increases were seen in γH2AX, with 5.6 ±6%+ in cells cultured alone, v. 7.9 ±6% in HS5 co-cultures (p<0.05) and v. 11.5 ±9% in HS27A co-cultures (p<0.001). For 10 samples, cell number was sufficient for additional overnight cultures with etoposide (up to 10 mM). Both stromal cell lines enhanced DNA damage signaling, reflected by an increase in γH2AX from 7 ±10% in etoposide-treated cells cultured alone, to 18 ±19% in HS5 co-cultures (p<0.05) and 19 ±16% in HS27A co-cultures (p<0.005). To test whether HS5 CM-induced pY-Stat3 or pERK1/2 were related to these parameters, we examined bivariate correlations. We found no correlation in most cases, but an inverse relationship between both ΔMFIs and %viable after etoposide (pY-Stat3: R=-0.733, p<0.05 and pERK1/2: R=-0.683, p<0.05). In other words, samples characterized by signaling failure tended to have better survival after etoposide treatment. This result is consistent with our prior study showing that patients whose blasts failed to activate Stat3 had poorer outcome than those whose blasts activated Stat3 normally. In summary, we found that environment-induced Stat3 signaling did not promote chemoresistance, and that stromal cells may instead confer chemoresistance by enhancing DNA damage signaling. Disclosures:No relevant conflicts of interest to declare.

Abstract 4962: Stroma-induced molecules and chemotherapy resistance in acute myeloid leukemia cells.

Abstract Overall objective: Acute myeloid leukemia (AML) is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the survival rate is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stroma protects a subset of leukemia cells from chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML. Methods, Results &amp; Conclusions: We used a human bone marrow stromal cell line, HS-27A and two human AML cell lines, THP-1 and NB-4. HS-27A cells provide important contact-dependent interactions with AML cells and secrete only a few soluble factors. Our HS-27A cells constitutively express mOrange, to allow discrimination from AML cells after co-culture. We performed co-culture experiments to determine the ability of stromal cells to confer resistance to chemotherapy. THP-1 and NB-4 cells were cultured alone or co-cultured with HS-27A cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours before labeling with annexin V for apoptosis assay by FACS. HS-27A cells significantly protected THP-1 and NB-4 cells from all three chemotherapy agents. To discover genes in AML cells that are induced by stromal cells and may contribute to chemotherapy resistance, oligonucleotide microarray analysis was done using total RNA extracted from THP-1 or NB-4 cells cultured alone or co-cultured with HS-27A cells. We identified over 1000 genes that were similarly differentially expressed in both AML cell lines by HS-27A co-culture. Among the significantly upregulated genes was cysteine-rich, angiogenic inducer 61 (CYR61). CYR61 is a secreted, extracellular matrix-associated signaling molecule, which has been related to chemotherapy resistance in breast cancer cells and malignant melanoma cells. To validate the microarray results, qRT-PCR was done with RNA from THP-1 cells and NB-4 cells, cultured alone or co-cultured with HS-27A cells. CYR61 expression was increased over 100-fold in those cell lines when co-cultured with HS-27A cells. Next, we examined CYR61 protein expression by immunocytochemistry, with co-staining for myeloperoxidase to confirm that CYR61-expressing cells were AML cells. This study confirmed that CYR61 was highly expressed in THP-1 cells co-cultured with HS-27A cells, but barely detectable in THP-1 cells cultured alone. Further studies are underway to determine if CYR61 contributes to stroma-induced chemotherapy resistance in AML. This study suggests that CYR61 induced by the microenvironment may promote chemotherapy resistance in AML cells. Elucidating the pathways involved in chemotherapy resistance is likely to result in promising combination therapies to reduce relapse, and thereby improve survival for children with AML. Citation Format: Xin Long, Laszlo Perlaky, Tsz-Kwong Man, Michele S. Redell. Stroma-induced molecules and chemotherapy resistance in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4962. doi:10.1158/1538-7445.AM2013-4962

Abstract 4950: Targeting a breast cancer xenograft by modulation of the microenvironment with zoledronic acid.

Abstract Introduction - Targeting breast cancer (BC) via its microenvironment is a way to avoid tumor plasticity and development of resistance towards therapy. Bisphosphonates (BPs) are used to treat BC bone metastases. Prior studies suggest that BPs also exert an anti tumor effect by interacting with the microenvironment. However, the precise nature of this interaction is unclear. The chorioallantoic membrane (CAM) assay allows assessment of both human BC and microenvironment cells, circumventing issues of species specificity. Aim - 1) evaluate whether an indirect anti-BC effect by BPs, can be determined using the CAM assay 2) Assess potential biomarkers for this intervention. Methods - MTT survival assays were performed to assess a differential effect of BP zoledronic acid (ZA) on survival of human BC cell lines (MCF-7, SUM149 and MDA-MB-231 progeny cell line SCP2) and human stromal cell line Hs27a. For biomarker analysis, excretion of vascular endothelial growth factor (VEGF)-A and Transforming Growth Factor (TGF) β was measured in the supernatant of Hs27a and MCF-7 cultures and co-cultures treated with ZA by Enzyme-Linked Immuno Sorbent Assays (ELISA). The CAM assay was used to study the effect of ZA on a co-culture system of BC- and stromal cells. SCP2 cells were inoculated on the CAM with and without Hs27a cells on day 6 of embryonic development. ZA (200 μM) or PBS was added on the CAM on day 10 and tumors were harvested at day 14. Immunohistochemistry (IHC) for pan-cytokeratin was performed to identify BC cells in the tumors. Results - ZA (4 days continuous incubation) did not induce a direct anti BC effect in vitro (IC50&amp;gt;100 μM), but strongly affected stromal cell survival (IC50 8 μM). ZA treatment decreased TGFβ excretion by Hs27a cells dose dependently (ZA 10, 50, 100 or 500 μM for 48 hours) from 538±63 pg/ml (control) to 446±57 (ns), 285±98 (p&amp;lt;.01), 198±47 (p&amp;lt;.01) and 186±60 pg/ml (p&amp;lt;.001). In the co-culture, ZA (10, 50, 100 or 500 μM for 48 hours) also decreased TGFβ excretion dose dependently from 625±60 pg/ml (control) to 613±33 (ns), 535±83 (ns), 389±48 (p&amp;lt;.01) and 244±55 (p&amp;lt;.001) pg/ml. No TGFβ secretion by sole MCF-7 cells was detectable (&amp;lt;0±60 pg/ml). ZA had no effect on VEGF-A excretion by BC or stromal cells. On the CAM, co-cultures were sensitive to ZA treatment (73±8.27 vs 32±13.15 mm3 (p&amp;lt;.001)), whereas BC cells without stromal cells were resistant to ZA (tumor size: 22±14.35 vs 33±15 mm3 (ns)). In the co-cultures, tumor cell percentage evaluated by IHC tended to be smaller in the ZA treated group compared to control (31±15 vs 12±5 (ns)). Conclusion - Effects of therapeutic interventions on tumors by microenvironment modulation can be studied using the CAM assay. Our data suggest an indirect anti-BC effect of ZA in vivo via stromal cells in the BC microenvironment. This is accompanied by decreased TGFβ excretion, which may therefore function as biomarker for ZA treatment effect. Supported by Dutch Cancer Society grant RUG 2010-4739 Citation Format: Hilde H. Nienhuis, Marlous Arjaans, Hetty Timmer-Bosscha, Elisabeth G.E. de Vries, Carolina P. Schröder. Targeting a breast cancer xenograft by modulation of the microenvironment with zoledronic acid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4950. doi:10.1158/1538-7445.AM2013-4950

Effect of intravenous coadministration of human stroma cell lines on engraftment of long-term repopulating clonal myelodysplastic syndrome cells in immunodeficient mice

Engraftment of clonal hematopoietic precursor cells from patients with myelodysplastic syndrome (MDS) in immunodeficient mice has been difficult to achieve by intravenous (i.v.) injection. We used i.v. coadministration of the human marrow stroma cell line HS27a with CD34+ MDS cells in Nod.cg-Prkdcscid Il2rgtm1wjll (NSG) mice to provide signals that would facilitate engraftment. Hematopoietic cells from 24 MDS patients were transplanted. Cells from all patients were engrafted, and engraftment was documented in 44 of 46 evaluable mice (95%). Immunohistochemistry revealed human HS27a stroma colocalizing with human hematopoietic cells in mouse spleens. Human CD34+ precursors harvested from marrow and spleen of primary murine recipients, when combined with HS27a cells, were also engrafted successfully in secondary NSG recipients, showing persistence of the original clonal characteristics. This observation supports the concept that clonal markers were present in long-term repopulating cells. We suggest that HS27a stroma cells ‘traveled' in direct contact with hematopoietic precursors and enabled their propagation. An essential signal for engraftment appears to be CD146, which is prominently expressed on HS27a cells. This xenotransplantation model will allow to further dissect signals that control engraftment of MDS cells and should be amenable to in vivo treatment studies.