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Cancer patients undergo detrimental toxicities and ineffective treatments especially in the relapsed setting, due to failed treatment attempts. The development of a tool that predicts the clinical response of individual patients to therapy is greatly desired. We have developed a novel patient-derived 3D tissue engineered bone marrow (3DTEBM) technology that closely recapitulate the pathophysiological conditions in the bone marrow and allows ex vivo proliferation of tumor cells of hematologic malignancies. In this study, we used the 3DTEBM to predict the clinical response of individual multiple myeloma (MM) patients to different therapeutic regimens. We found that while no correlation was observed between in vitro efficacy in classic 2D culture systems of drugs used for MM with their clinical efficacious concentration, the efficacious concentration in the 3DTEBM were directly correlated. Furthermore, the 3DTEBM model retrospectively predicted the clinical response to different treatment regimens in 89% of the MM patient cohort. These results demonstrated that the 3DTEBM is a feasible platform which can predict MM clinical responses with high accuracy and within a clinically actionable time frame. Utilization of this technology to predict drug efficacy and the likelihood of treatment failure could significantly improve patient care and treatment in many ways, particularly in the relapsed and refractory setting. Future studies are needed to validate the 3DTEBM model as a tool for predicting clinical efficacy.

Drug resistance and dose-limiting toxicities are significant barriers for treatment of multiple myeloma (MM). Bone marrow microenvironment (BMME) plays a major role in drug resistance in MM. Drug delivery with targeted nanoparticles have been shown to improve specificity and efficacy and reduce toxicity. We aim to improve treatments for MM by (1) using nanoparticle delivery to enhance efficacy and reduce toxicity; (2) targeting the tumor-associated endothelium for specific delivery of the cargo to the tumor area, and (3) synchronizing the delivery of chemotherapy (bortezomib; BTZ) and BMME-disrupting agents (ROCK inhibitor) to overcome BMME-induced drug resistance. We find that targeting the BMME with P-selectin glycoprotein ligand-1 (PSGL-1)-targeted BTZ and ROCK inhibitor-loaded liposomes is more effective than free drugs, non-targeted liposomes, and single-agent controls and reduces severe BTZ-associated side effects. These results support the use of PSGL-1-targeted multi-drug and even non-targeted liposomal BTZ formulations for the enhancement of patient outcome in MM., The tumour microenvironment (TME) has a major role in chemoresistance in multiple myeloma. The authors show that a nanoparticle targeted to TME and loaded with bortezomib (BTZ) and Y27632 is more effective than free drugs, non-targeted and single-agent controls and reduces BTZ-related side effects.

Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

CD138 (also termed SDC1) has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug-resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down-regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138-negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

Radiation-induced lymphopenia (RIL) is associated with treatment of different tumors (lung, colon, pancreas, breast, sarcomas, and glioblastoma). It is a significant clinical problem affecting the survival of cancer patients. The biologic mechanisms leading to RIL are not clearly understood. In this study, we established a mouse model of RIL representing therapeutic clinical regimen for lung cancer. Flow cytometry was used to analyze circulating levels of T and B cells and bone marrow (BM) stem cells. We found that fractionated radiation to the thorax significantly reduced circulating T and B cells as well as BM stem cells. Ex-vivo irradiation of blood and autologous reinjection to mice also significantly induced lymphopenia. Furthermore, we found that mobilization of stem cells from the BM and autologous stem cell transplant rescued RIL in mice. Overall, our results suggest that RIL has not only direct effect on circulating lymphocytes, but also has indirect effect on circulating lymphocytes as well as stem cells in the nonirradiated BM. These results open a new window for investigating the direct and indirect biologic mechanisms leading to RIL, and provide a preclinical basis to test the effect of stem cell transplantation for treatment of RIL in cancer patients. Cancer Res; 75(17); 3442–5. ©2015 AACR.

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interactionin vivoled to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

Tumor hypoxia is a well-described phenomenon during the progression of solid tumors affecting cell signaling pathways and cell metabolism; however, its role in hematologic malignancies has not been given the same attention in the literature. Therefore, this review focuses on the comparative differences between solid and hematologic malignancies with emphasis on the role of hypoxia during tumorigenesis and progression. In addition, contribution of the bone marrow and angiogenic environment are also discussed. Insight is provided into the role of hypoxia in metastatic spread, stemness, and drug resistance in hematologic conditions. Finally, emerging therapeutic strategies such as small-molecule prodrugs and hypoxia-inducible factor (HIF) targeting approaches are outlined to combat hypoxic cells and/or adaptive mechanisms in the treatment of hematologic malignancies. Mol Cancer Res; 12(10); 1347–54. ©2014 AACR.

Blood vessel formation plays an essential role in many physiologic and pathologic processes, including normal tissue growth and healing, as well as tumor progression. Endothelial progenitor cells (EPC) are a subtype of stem cells with high proliferative potential that are capable of differentiating into mature endothelial cells, thus contributing to neovascularization in tumors. In response to tumor-secreted cytokines, EPCs mobilize from the bone marrow to the peripheral blood, home to the tumor site, and differentiate to mature endothelial cells and secrete proangiogenic factors to facilitate vascularization of tumors. In this review, we summarize the expression of surface markers, cytokines, receptors, adhesion molecules, proteases, and cell signaling mechanisms involved in the different steps (mobilization, homing, and differentiation) of EPC trafficking from the bone marrow to the tumor site. Understanding the biologic mechanisms of EPC cell trafficking opens a window for new therapeutic targets in cancer. Clin Cancer Res; 19(13); 3360–8. ©2013 AACR.

Background: Multiple Myeloma (MM) is the second most common hematological malignancy, and continues to be a fatal disease even with the development of novel therapies. Despite promising preclinical data in standard tissue culture models, most drugs fail in clinical trials and show lower efficacy in patients. This highlights the discrepancy between the current in vitro models, the pathophysiology of the disease in the patients, and the urgent need for better in vitro models for drug development and improved prediction of efficacy in patients. We have previously developed a patient-derived 3D-Tissue Engineered Bone Marrow (3DTEBM) culture model, which showed superior properties for proliferation of primary MM cells ex vivo, and better recapitulated drug resistance. The long-term goal of this study is to use the 3DTEBM model as a tool to perform drug screens on BM aspirates of MM patients and prospectively predict the efficacy of different therapies in individual patients, and help treatment providers develop personalized treatment plans for each individual patient. In the current study, we used the 3DTEBM model to, retrospectively, predict clinical responses of MM patients to therapy, as a proof of concept. Methods: We used whole-BM, viably frozen tissue banked samples from 20 MM patients with clear clinical response patterns of complete remission, and either very good partial response (sensitive) or progressive disease (non-sensitive). The BM aspirates were used to develop a 3DTEBM that represents each individual patient. The patient-derived 3DTEBM cultures were treated ex vivo with the same therapeutic regimen that the patient received in the clinic for 3 days. The treatment ex vivo was based on combinations at different concentrations which mimic the steady state concentrations (Css) of each drug. The efficacy of the treatment ex vivo was evaluated by digestion of the 3DTEBM matrix, extraction of the cells, and analysis for prevalence of MM cells in the treatment groups compared to the non-treated controls. Patients were defined "sensitive" if the effect reached 50% killing in the range of 10xCss. The ex vivo sensitivity data was then correlated with the clinical response outcomes. Results: We found that the 3DTEBM was predictive in approximately 80% of the cases (in about 85% of the combination therapy cases, and in about 70% of the single therapy cases). Broken down by individual drug, it was predictive in 80% of the cases treated with Bortezomib, 78% Lenalidomide, 84% Dexamethasone, 100% Daratumumab, 50% Carfilzomib, 50% Pomalidomide, and 100% Doxorubicin. Conclusions: The 3DTEBM is a more pathophysiologically relevant model which predicts clinical efficacy of drugs in multiple myeloma patients, retrospectively. This data provides the bases for future studies which will examine the ability of the 3DTEBM model to predict treatment efficacy, prospectively, for development of personalized treatment plans in individual multiple myeloma patients. Disclosures Jeske: Cellatrix LLC: Employment. Azab:Cellatrix LLC: Employment. De La Puente:Cellatrix LLC: Other: Co-founder. Vij:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Azab:Ach Oncology: Research Funding; Cellatrix LLC: Equity Ownership, Other: Founder and owner; Glycomimetics: Research Funding; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner.

Purpose Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. Experimental design: We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. Results 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. Conclusions 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients.

Waldenström macroglobulinemia, a rare and indolent type of non–Hodgkin lymphoma, is characterized by widespread lymphoplasmacytic B cells in the bone marrow. Previous studies have shown that hypoxic conditions play a key role in the dissemination of other hematologic malignancies. In this study, the effect of hypoxia was tested on the progression and spread of Waldenström macroglobulinemia. Interestingly, tumor progression correlated with hypoxia levels in Waldenström macroglobulinemia cells and other cells in the bone marrow and correlated with the number of circulating tumor cells in vivo. Mechanistic studies demonstrated that hypoxia decreased cell progression and cell cycle, did not induce apoptosis, and reduced the adhesion between Waldenström macroglobulinemia cells and bone marrow stroma, through downregulation of E-cadherin expression, thus explaining increased egress of Waldenström macroglobulinemia cells to the circulation. Moreover, hypoxia increased the extravasation and homing of Waldenström macroglobulinemia cells to new bone marrow niches in vivo, by increased CXCR4/SDF-1–mediated chemotaxis and maintaining the VLA4-mediated adhesion. Re-oxygenation of hypoxic Waldenström macroglobulinemia cells enhanced the rate of proliferation and cell cycle progression and restored intercellular adhesion between Waldenström macroglobulinemia cells and bone marrow stroma. This study suggests that targeting hypoxic response is a novel strategy to prevent dissemination of Waldenström macroglobulinemia. Implications: This study provides a better understanding of the biology of dissemination of Waldenström macroglobulinemia and opens new windows for investigation of new therapeutic targets in Waldenström macroglobulinemia based on tumor hypoxia mechanisms. Mol Cancer Res; 13(2); 263–72. ©2014 AACR.

Introduction: The presence of a minimal residual disease (MRD) that remains after treatment is one of the main reasons for recurrence and relapse in multiple myeloma (MM) patients. Diagnosis of MRD is becoming vital to assess the effectiveness of the treatment as well as to predict survival among patients with complete remission. CD138 (syndecan-1) is the gold standard marker for detecting MM cells using multiparametric flow cytometry analysis or immunohistochemistry (IHC) staining of BM biopsies. However, the presence of highly clonogenic, drug resistant and stem-cell like CD138-negative MM cells have been previously demonstrated. Moreover, hypoxia which is known to drive MM progression, drug resistance and metastasis was shown to significantly decrease CD138 expression in MM cells. In this study, we utilized a novel set of biomarkers to detect MRD in the bone marrow as well as circulating tumor cells (CTCs) in the blood, in order to predict progression free survival (PFS) in MM patients defined as complete remission according to the detection of CD138+ cells in their bone marrow. Methods: To detect myeloma cells, we utilized a novel set of biomarkers, independent of CD138 expression and hypoxic state of MM cells (CD38+/CD3-/CD19-/CD14-/CD16-/CD123-) by two-color flow cytometry. We detected MM cells in the bone marrow of 25 patients with complete remission and very good partial response, whose bone marrow was defined as a CD138-negative (less than 0.5%). In order to retrospectively correlate the involvement of MM cells (%) with PFS, we used 24 months as a cut-off and compared the results acquired with the new method to traditional CD138-based flow or histology. In addition, based on the percentage of detected MM cells with the 2% cut-off, we analyzed the time to progression (months). Moreover, we detected MM cells in the peripheral blood from matching MM patients and performed analogous analysis. Furthermore, we also detected CTCs in 7 newly diagnosed patients and compared to 7 patients with progressive disease. Results: A study conducted on 25 patients with their bone marrow defined as a CD138-negative (less than 0.5%) showed that the novel strategy to detect MM cells was more precise than CD138-based flow or histology in predicting PFS. Patients who relapsed in less than 24 months had an average (±SEM) of 3.53±0.82%, while patients who relapsed later than 24 months had an average of 1.15±0.27% MM cells in the bone marrow when detected by the new method (p=0.004). In addition, the median PFS with Conclusions: The novel flow cytometry-based set of biomarkers provides an alternative strategy to detect MRD in the bone marrow and CTCs in the peripheral blood of MM patients, and both allowed prediction of PFS in MM patients. Disclosures De La Puente: Cellatrix LLC: Other: Co-founder. Vij:Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Jazz: Consultancy; Shire: Consultancy; Karyopharma: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy. Azab:Targeted Therapeutics LLC: Other: Founder and owner; Cleave Bioscience: Research Funding; Verastem: Research Funding; Cell Works: Research Funding; Karyopharm: Research Funding; Vasculox: Research Funding; Glycomimetics: Research Funding; Cellatrix LLC: Other: Founder and owner; Selexys: Research Funding.

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy characterized by the continuous spread of MM cells in and out of the bone marrow (BM). Despite the introduction of novel therapies, cancer patients relapse due to the development of drug resistant cells, which are, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome drug resistance in MM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve the outcome of patients with solid tumors when combined with radiotherapy; however, it has not been tested in MM. We used TPZ for the first time in MM to target the drug resistant cancer cells and sensitize them to therapy. Methods: To test the effect of TPZ on tumor survival in vitro, MM cell lines (MM1.s, H929, OPM1, RPMI8226) were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 hours with different concentrations of TPZ in order to obtain an IC50, and cell survival was assessed using MTT assay. Also, a combination of bortezomib and carfilzomib with or without TPZ was tested on cell survival. For in vivo study, 5 x 106 MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. First, we tested the hypoxic status of mice treated with and without a high-dose bortezomib (1.5mg/kg). Pimonidazole (PIM) was injected intraperitoneally (IP) into mice and 4 hours later BM was harvested, stained with anti-PIM-APC antibody and followed by measuring PIM signal in MM1s-GFP+ cells using flow cytometry. Second, we developed drug resistant cells by treating mice with a high-dose bortezomib (1.5mg/kg), and then treated with (1) bortezomib only (0.5mg/kg; n=3), or (2) bortezomib and TPZ (40mg/kg; n=3), all administered IP sequentially twice a week. The number of residual MM1s-GFP+ cells was calculated by flow cytometry. Results: We found that TPZ was active in a dose-dependent manner only in hypoxic conditions in MM cell lines. We showed that residual MM cells in the BM after high-dose bortezomib are hypoxic, as demonstrated by PIM staining. The combination of TPZ with bortezomib and carfilzomib resensitized cancer cells to death in hypoxia, overcoming hypoxia-induced drug resistance in vitro. Moreover, TPZ-treatment in combination with bortezomib further decreased residual MM cells in vivo. Conclusions: We reported that MRD was hypoxic and that TPZ, which was cytotoxic for MM cells only in hypoxic conditions, overcame hypoxia-induced drug resistance in vitro and killed bortezomib-resistant residual MM cells in vivo. This is the first study to show the efficacy of TPZ in MM. This data provides a preclinical basis for future clinical trials testing efficacy of TPZ in MM. Disclosures Azab: Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Verastem: Research Funding.

Introduction: Diagnosis, responses to treatment, minimal residual disease (MRD) and circulating tumor cells (CTC) in multiple myeloma (MM) are all detected by the gold-standard marker CD138 flow cytometry-based method. However, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 downregulation were shown previously. In this study, we found that CD138 is significantly downregulated in MRD and CTC populations in MM, thus we developed a novel two-color flow cytometry-based set of biomarkers (independent of CD138) to detect MRD and CTC in MM. Methods: We created a MRD mouse model by treating MM-bearing mice with a high dose of bortezomib. We then tested the effect of hypoxia and drug treatment on the expression of different markers, including CD138. Therefore, to detect MM cells we utilized CD38-APC antibody, followed by the exclusion of non-MM CD38-expressing cells such as T cells, monocytes, NK cells, B cells and dendritic cells using (CD3, CD14, CD16, CD19 and CD123)-V450 antibodies, respectively. To verify the ability of the new method to selectively detect MM cells, mononuclear cells from peripheral blood (PB) from healthy patients and MM cell lines (hypoxic and normoxic) were stained by the antibody cocktail and analyzed by flow cytometry. Next, we compared the sensitivity of the traditional CD138 method to the new method in detecting (a) normoxic or hypoxic MM cells spiked into bone marrow (BM) cells in vitro, (b) normoxic or hypoxic MM cells spiked into PB in vitro, (c) MM1s-GFP+ cells in mice with different tumor burden in the BM in vivo, (d) circulating MM1s-GFP+ in the PB in vivo, (e) MRD cells in the BM samples from 16 patients with complete remission (CR) or very good partial response (VGPR), and (F) CTC in 12 progressive MM patients. We further aimed to predict time to progression (TTP) in 16 patients with complete remission based on the detection of MRD in these patients using the new method and compared with flow cytometry-based CD138 or histology. Results: In vivo, we found that bortezomib-treated MRD cells were hypoxic, compared to a progressive vehicle-treated cells. CD138 expression in these cells was significantly decreased, but CD38 expression was unchanged. In vitro expression of CD138 was decreased due to hypoxia and bortezomib treatment, whereas CD38 expression was unchanged. Furthermore, we developed a new method using an antibody cocktail, where the MM cell population is defined as CD38+/CD3-/CD14-/CD16-/CD19-/CD123- (APC+ and V450-). In vitro, the new method detected 100% of hypoxic and normoxic MM cells, and less than 0.5% of mononuclear cells from the PB or BM of healthy donors. In contrast, CD138+ cells failed to detect 50% of hypoxic MM cells and 10-25% of normoxic cells. In vivo, the amount of cells detected by the new method directly correlated with the number of MM1s-GFP+ cells detected in the BM with a range between 0-60%, with a correlation coefficient (slope) of 0.99 and R2 of 0.999. The CD138 detected only a fraction of the MM1s-GFP+ population ( In patients, the new method detected 0.5-8% MRD cells in the BM of 16 patients with CR or VGPR (which were defined as CD138-negative), and 0.1-1.8% CTC in the PB of 12 progressive MM patients. In contrast, CD138 marker detected less than 0.5% of MRD cells in the BM of the CR and VGPR patients, and less than 0.1% of CTCs in the PB of the progressive patients. Furthermore, we found that, while CD138 and histology failed to predict recurrence in CR patients, the new method successfully detected CD138-negative MM population whose prevalence in the BM inversely correlated with TTP in MM patients defined as CR based on CD138 and histology. Conclusions: We confirmed that CD138 expression is variable on MM cells, and that it is downregulated in MRD and CTC populations, and that it was not effective in detecting these particular populations in MM. Furthermore, we developed a novel two-color flow cytometry-based biomarker-set to detect MM cells independent of CD138. The new methods detected close to a 100% of all MM cells in vitro and in vivo, including MRD and CTC. Moreover, the new method detected a CD138-negative MRD and CTC in MM patients, and the prevalence of this population inversely correlated with TTP in MM patients. Disclosures Vij: Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) associated with lytic bone disease. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance. Low oxygenation (hypoxia) in the BM microenvironment, which was shown to decrease beyond physiologic BM conditions during the progression of MM, together with cellular and acellular components, plays a critical role in drug resistance. Increased levels of the nuclear exporter of tumor suppressors, exportin 1 (XPO1), was demonstrated in MM, and XPO1 inhibition showed a broad anti-tumor activity. In this study, we tested inhibition of XPO1 using the novel oral, covalent, slowly reversible inhibitor of nuclear export selinexor currently in Phase I/II clinical trials on sensitizing the hypoxia-inducible drug resistant MM cells both in vitro and in vivo. Methods: MM cell lines (MM1s, H929, OPM2 and U266) were treated with increasing concentrations of selinexor in normoxic (21% O2) and hypoxic (1% O2) conditions with or w/o bortezomib or carfilzomib. Assays included MTT proliferation Annexin V/PI staining for apoptosis, PI staining and immuniblots for cell cycle analysis. We tested the effects of selinexor as a single agent on tumor initiation in vivo, where MM1s-Luc-GFP cells were injected intravenously (IV) into SCID mice and mice were treated instantaneously with selinexor (15mg/kg) or vehicle (PVP + F-68) three times a week. We also tested the effect of selinexor (15mg/kg) as a single agent on tumor progression and survival in mice with established MM tumor. Furthermore, we studied the effects of selinexor on sensitization to bortezomib-resistant MM cells in vivo. MM1s-Luc-GFP cells were injected IV into SCID mice and tumors were allowed to grow for three weeks. Animals were then treated with a high-dose of bortezomib (1.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were then randomly divided into 3 groups treated with (1) bortezomib (0.5mg/kg), (2) KPT-330 (10mg/kg), and (3) a combination of selinexor and bortezomib. Selinexor was administered by oral gavage three times a week (days 1, 3 and 5) and bortezomib was injected intraperitoneally twice a week (days 1 and 4). Tumor progression was imaged using bioluminescence imaging (BLI). Results: Selinexor, as a single agent, decreased proliferation, induced G1 cell cycle arrest and increased apoptosis of MM cells in both hypoxia and normoxia in a dose-dependent manner. Moreover, we found that the combination of selinexor with bortezomib or carfilzomib reversed the hypoxia-induced resistance in MM cells to proteasome inhibition and induced a synergistic effect on the inhibition of cell proliferation, G1 arrest and apoptosis, which was confirmed by response of respective cell signaling pathways as PI3K, MAPK and JNK. In vivo studies revealed that selinexor alone delayed tumor initiation and decreased by half existing tumor size as a single agent (p=0.0397), and significantly extended mice survival (53 days on vehicle versus 62 days on selinexor, p=000375). In addition, MM-bearing mice which developed resistance to bortezomib were resensitized by treatment of selinexor combined with bortezomib, decreasing tumor burden and showed considerably extended survival (half of the mice were alive at day 52) compared to mice treated with either selinexor (half of the mice were alive at day 49) or bortezomib alone (half of the mice were alive at day 40) (bortezomib versus selinexor p=0.0014, bortezomib versus combination treatment p=0.0004 as analyzed by Student t-test). Conclusions: The orally available inhibitor of nuclear export, selinexor, decreased survival of MM cells in vitro and delayed tumor initiation and tumor progression in vivo as a single agent. In addition, selinexor overcame hypoxia-induced resistance to proteasome inhibitors and resensitized MM cells to therapy both in vitro and in vivo, extending mice survival. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by inhibition of XPO1 with selinexor as well as combination studies of selinexor and proteasome inhibitors. A study of selinexor and carfilzomib is currently ongoing and early results that were published last year showed marked activity for this combination treatment in heavily pretreated MM patients. Disclosures Landesman: Karyopharm: Employment. Azab:Karyopharm: Research Funding; Selexys: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Cell Works: Research Funding.

Introduction: Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are lymphoplasmacytic malignancies which are characterized by continuous spread through cell trafficking in and out of the BM. We have recently shown that the spread process in these diseases is mediated by hypoxia. Moreover, despite the introduction of novel therapies, cancer patients relapse due to the development of a drug-resistant stem cell-like subpopulation, which we recently found to be, at least in part, promoted by hypoxia. Therefore, in this study we aimed to overcome spread and drug resistance in MM and WM by inhibition of the hypoxic responses in these cells. Tirapazamine (TPZ) is a hypoxia-activated pro-drug causing cell apoptosis, which has been shown to improve cancer patient outcome when combined with radiotherapy. We use TPZ for the first time in hematologic malignancies to target the drug-resistant cancer cells and sensitize them to therapy. Procedures: Cell survival was tested on MM (MM1.s, H929, OPM1, RPMI8226) and WM (BCWM.1, MWCL.1) cell lines exposed to normoxia (21% O2) or hypoxia (1% O2) for 24hrs in the presence of bortezomib and carfilzomib, different concentrations of TPZ (in order to obtain an IC50), or combination of those drugs using MTT assay. For adhesion assay, cancer cells were pre-labeled with calcein-AM, treated with TPZ, applied to unlabeled endothelial cells, non-adherent cells were washed after 1hr, and adherent cells were analyzed by a fluorescent reader. Chemotactic properties of cells were tested using a transwell chamber and the migrated cells were counted by flow cytometry. For in vivo study, 5 × 106 MM1s-Luc-GFP cells were injected i.v. into 6 SCID mice and tumor progression was monitored for 3 weeks by bioluminescent imaging. All mice were treated with (1) high-dose bortezomib (1.5mg/kg) only (n = 3), or (2) bortezomib and TPZ (40mg/kg; n = 3), all administered i.p. sequentially twice a week. Bone marrow and peripheral blood were collected and analyzed for the presence of GFP+ cells by flow cytometry. Summary of data: We found that hypoxia induced drug resistance to bortezomib and carfilzomib of MM and WM cells, and that TPZ was active in a dose-dependent manner only in hypoxic conditions. Combination of TPZ with bortezomib and carfilzomib, resensitized cancer cells to death in hypoxia. Addition of TPZ increased adhesion of cancer cells to endothelial cells, and decreased chemotactic properties of cancer cells in hypoxia in vitro. Moreover, post-treatment residual cancer cells were further decreased using TPZ, preventing recurrence of MM in vivo. Conclusion: We report that TPZ prevents hypoxia-induced metastasis and overcomes hypoxia-induced drug resistance in MM and WM. This is the first study to show the efficacy of TPZ in hematologic malignancies in general and in MM and WM in particular. This data provide a preclinical basis for future clinical trials testing efficacy of TPZ in MM and WM. Citation Format: Barbara Muz, Pilar de la Puente, Feda Azab, Micah Luderer, Abdel Kareem Azab. Tirapazamine as a strategy to prevent cell dissemination and overcome drug resistance. [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 5468. doi:10.1158/1538-7445.AM2015-5468

Introduction: Despite the introduction of novel therapies, majority of multiple myeloma (MM) patients relapses. The major reason for the relapse in MM is the presence of a minimal residual disease (MRD) population that remains after treatment. MM cells are defined as CD138+; however, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 decrease in expression were previously shown. We propose a novel set of biomarkers (independent of CD138) to detect MRD in MM in order to predict patient relapse. Procedures: We tested the effect of hypoxia on CD markers expression in MM cell lines. We developed new method to detect MM cells by flow cytometry based on CD38(APC+) gating and negatively selecting other leukocytes including T cells, B cells, monocytes, NK cells, and dendritic cells using CD3, CD19, CD14, CD16, and CD123, respectively (FITC-conjugated), the MM cells were defined as APC+/FITC-. We compared traditional method (CD138-based) with the new set of biomarkers (1) to detect normoxic and hypoxic MM cells, (2) to detect MM cells in the peripheral blood (PB) and the bone marrow (BM) from MM patients, confirming cell clonality by internal staining with Kappa/Lambda light chain antibodies, and (3) to predict relapse in MM patients whose BM was defined a CD138-negative. Summary of data: We found that hypoxia decreased the expression of CD138 by ∼50%, while CD38 was not altered by hypoxia in MM cells. The use of CD138+ as a marker for MM detected only 60-75% of hypoxic MM cells, whereas APC+/FITC- strategy detected close to a 100% of the hypoxic MM cells in vitro. In patient samples, all patients had a higher number of BM-derived and circulating MM cells as detected by APC+/FITC- strategy compared to CD138+. In addition, the clonality of the APC+/FITC- population in BM negative fractions was the same as the original disease (CD138+ cells). Moreover, studies conducted on 16 patients with BM defined as a CD138-negative, showed that the novel APC+/FITC- strategy was more successful than CD138-based method or histology in predicting recurrence and time to relapse. The median time-to-progression with Conclusion: CD138 cannot be used as a universal marker to detect MM cells, especially when they are hypoxic. A novel set of biomarkers CD38+/CD3-/CD19-/CD14-/CD16-/CD123- (APC+/FITC-) was designed to detect MM cells independent of CD138 expression and hypoxic status. This strategy was able to detect a clonal CD138-negative MM cell population in the BM and PB from MM patients. Moreover, this strategy predicted tumor recurrence and relapse in MM patients more accurately compared to traditional methods such as CD138-based or histology. Citation Format: Barbara Muz, Feda Azab, Pilar de la Puente, Justin King, Micah Luderer, Ravi Vij, Abdel Kareem Azab. Predicting relapse using CD138-independent strategy to detect residual myeloma plasma 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 5305. doi:10.1158/1538-7445.AM2015-5305

INTRODUCTION: Current methods to assess multiple myeloma (MM) cancer treatments are often inaccurate, in part, due to these methods cannot take into consideration the variability of every patient, and fail to recreate the bone marrow (BM) microenvironment. In this study we developed a patient-derived three-dimensional tissue-engineered BM (3DTEBM) niche as novel model that will allow better evaluation of interactions of MM cells and their microenvironment and how these interactions may affect MM drug resistance in the specific patient. METHODS & RESULTS: 3DTEBM cultures were made by crosslinking of patient-derived BM supernatants; the 3D-scaffold included MM cell lines or primary CD138+ MM cells and other accessory cells (stromal cells and endothelial cells, or CD138- cells). 3DTEBM cultures were studied for cell proliferation by flow cytometry, and compared with 2D cultures and other commercial 3D systems including PLGA microspheres, Algimatrix, and Matrigel, and imaged for 2 weeks using a confocal microscope. 3DTEBM cultures promoted the proliferation of MM cells more than 2D cultures and commercial 3D systems. 3DTEBM cultures supported the growth of fresh and frozen primary MM cells and allowed the use of these cells ex vivo at least 2 weeks. In addition, we found a natural development of polarization in the cellularity of the scaffold, with the bottom rich of stromal cells, top rich of endothelial cells, and MM cells through all the parts of the scaffold, reflecting the pathophysiological development of MM in the BM niche. The effect of 3DTEBM cultures on cytokine expression was determined by cytokine antibody arrays and 3D-tissue depth effects in drug gradients, oxygenation levels and proliferation (HIF-1α, PIM, Ki67, CXCR4, and CD138 expression) were determined using flow cytometry or IHC. 3DTEBM cultures showed that 23 out of 80 cytokines tested were highly expressed with at least 3-fold increase in the 3D cultures vs 2D cultures. Furthermore, 3DTEBM cultures recreated the BM niche drug gradients and oxygenation levels due to depth of tissue; MM cells showed lower drug uptake and higher hypoxia levels (PIM and HIF-1α) with depth of tissue, and hypoxia-associated decreased CD138 and increased CXCR4 expression. We also found that MM cells were quiescent (lower Ki67 expression) in the hypoxic niche. Finally, drug resistance of MM cell lines or primary MM cells in 3DTEBM cultures was analyzed by flow cytometry. 3DTEBM cultures induced drug resistance in MM cells more than 2D cultures and commercial 3D systems, and preliminary results indicate that drug resistance profile in the 3DTEBM matched the clinical manifestation of the disease in the specific patient, while 2D cultures did not. CONCLUSION: The patient-derived 3DTEBM is a more biologically relevant model for evaluation of interactions of MM cells and their microenvironment in the BM niche, especially in the context of drug resistance. Citation Format: Pilar de la Puente, Rebecca Gilson, Barbara Muz, Feda Azab, Justin King, Samuel Achilefu, Ravi Vij, Abdel Kareem Azab. 3D tissue-engineered bone marrow niche as novel method to study pathophysiology and drug resistance in multiple myeloma. [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 5356. doi:10.1158/1538-7445.AM2015-5356

Introduction: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM). Despite the introduction of novel therapies more than 70% of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role on proliferation of MM and that its inhibition with small-molecule inhibitors sensitized MM cells to therapy. However, these small molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. In this study, we tested inhibition of P-selectin and PSGL-1 using functionally blocking monoclonal antibodies to sensitize MM cells to therapy. Methods: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals. Endothelial cells, stromal cells derived from MM patients, and MM cell lines (MM1s, H929, OPM1 and RPMI8226) were used for in vitro expression, proliferation, apoptosis and immuno-blotting assays. The expression of P-selectin and PSGL-1 were tested by the interaction of SelG1 (5-20µg/mL) and SelK2 (5-20µg/mL) antibodies with endothelial cells, stromal cells and MM cells for 1hr, followed by addition of a secondary-FITC antibody and flow cytometry analysis. For adhesion assay, cells were treated with increasing concentrations of SelG1 and SelK2 for 1hr; pre-labeled MM cells were then applied to unlabeled endothelial or stromal cells for 1hr, non-adherent cells were washed, and adherent cells were analyzed by a fluorescent reader. For proliferation, MM1s-GFP+ cells cultured alone, with stroma, or with endothelial cells; and were treated with, or without, bortezomib and carfilzomib, in the presence, or absence, of SelG1 and SelK2 antibodies, and proliferation was determined by flow cytometry. Protein expression associated with survival, apoptosis and cell cycle signaling was analyzed by western blotting. For in vivo, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumor progression followed for 4 weeks. Anti-mouse-P-selectin antibody was used to inhibit P-selectin in the mouse endothelial cells and stroma. Likewise, SelK2 and anti-mouse PSGL-1 were used to inhibit PSGL-1 on human MM cells in the mouse microenvironment, respectively. Mice were divided into 6 groups (1) vehicle treated control, (2) anti-mouse-P-selectin (5mg/kg) alone, (3) SelK2 (5mg/kg) and anti-mouse-PSGL-1 (5mg/kg) alone, (4) bortezomib (0.5mg/kg) alone, (5) a combination of anti-mouse-P-selectin and bortezomib, and (6) a combination of SelK2 (5mg/kg), anti-mouse-PSGL-1 (5mg/kg) and bortezomib. Anti-mouse-P-selectin, anti-mouse–PSGL-1, SelK2 and bortezomib were administered intraperitoneally twice a week. Results: The half-life of SelG1 in a Phase I clinical study was previously shown to be 363 hours while the half-life of SelK2 in a primate study was approximately 100 hours. P-selectin expression was detected on endothelial and stromal cells using the SelG1 antibody, while no expression was found on MM cells. PSGL-1 was highly expressed on MM cells as well as on endothelial cells and stromal cells as detected by SelK2 monoclonal antibody. Inhibition of P-selectin and PSGL-1 with SelG1 or SelK2, respectively, decreased MM cell adhesion to endothelial and stromal cells, and decreased the proliferation of MM cells induced by stromal and endothelial cells. Similarly, inhibition of the interaction between P-selectin and PSGL-1 sensitized MM cells to bortezomib and carfilzomib in vitro. In vivo results demonstrated that inhibition of the P-selectin or PSGL-1 as single agents delay tumor growth compared to non-treated mice and that it enhances the effect of bortezomib. Conclusions: These results demonstrate that inhibition of P-selectin and PSGL-1 by SelG1 and SelK2 antibodies, respectively, disrupts the interaction between MM cells and BM microenvironment and decreases adhesion and proliferation of MM cells. Moreover, inhibition of P-selectin and PSGL-1 increased the sensitivity of MM cells to bortezomib and carfilzomib in vitro, and to bortezomib in vivo. These data provides a basis for future clinical trials for sensitization of refractory MM patients to therapy by inhibition of P-selectin and PSGL-1 using SelG1 and SelK2 monoclonal antibodies. Disclosures Rollins: Selexys: Employment. Alvarez:Selexys: Employment. Kawar:Selexys: Employment. Azab:Selexys: Research Funding.

INTRODUCTION: In vitro culturing of primary myeloma cells has been a major challenge because the lack of an in vitro technology capable of recreating the complicated bone marrow (BM) microenvironment which multiple myeloma (MM) cells depend on for its survival. While primary myeloma cells cannot grow ex vivo in SCID mice, they are able to growth in SCIDhu mice. However, animal models are expensive, time-consuming, and often have limited reproducibility. In addition, classic laboratory models cannot take into consideration the variability of disease in every patient, and the MM patient population is highly variable, both genetically and epigenetically, and the biological characteristics of patients are widely different, which demonstrates sensitivity of individual patients to different therapies. Typical two-dimensional (2D) models rely on a limited number of MM cell lines which cannot reflect the enormous heterogeneity and variations present in individual patients. The goal of this study is to create a patient derived three-dimensional tissue-engineered bone marrow (3DTEBM) culture system based on cross-linked MM-derived BM supernatant, including endogenous soluble growth factors and cytokines, and by incorporating mononuclear cells, including MM (CD138+ population) and accessory cells (CD138- population), from the same patient. METHODS: The 3DTEBMwas formed through calcium cross-linking of BM supernatants combined with the CD138+ and CD138- population from the same MM patient. We tested the growth of fresh primary CD138+ MM cells with CD138- cells in 2D vs patient-derived 3DTEBM cultures based on the original BM ratio (CD138-/CD138+) of the patient at 3 days by flow cytometry. We further selected a patient with not very aggressive tumor and evaluated the effect of CD138- increasing densities on CD138+ MM growth in 3DTEBM cultures at day 3 by flow cytometry. In addition, the effect of patient-derived 3DTEBM and 2D cultures with and without CD138- cells on CD markers expression of the CD138+ population was tested at day 3 by flow cytometry. Finally, the growth of frozen primary CD138+ MM cells with and without CD138- cells in 2D vs patient-derived 3DTEBM cultures was analyzed for 14 days by flow cytometry. RESULTS: We found that patient-derived 3DTEBM cultures support primary MM cell growth. CD138+ MM cells from patients with less aggressive tumors (high BM ratio CD138-/CD138+) showed better growth in 3DTEBM than in 2D systems at day 3. However, more aggressive tumors (low BM ratio CD138-/CD138+), in which MM cells are less dependent of accessory cells, the 3DTEBM cultures showed similar CD138+ MM growth as in 2D cultures. After that, we detected in the patient-derived 3DTEBM cultures a direct correlation between positive and negative fractions, with increased CD138- densities the CD138+ MM growth increased. Next, we analyzed the effect of 3D cultures in CD markers expression and we showed that CD138+ MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), reduction of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D cultures. Finally, patient-derived 3DTEBM supported the growth of frozen primary CD138+ MM cells better than 2D cultures, and the addition of CD138- cells enhanced even more CD138+ MM growth after 14 days in culture. CONCLUSIONS: Patient-derived 3DTEBM cultures support primary fresh and frozen CD138+ MM growth better than classic systems, and induced de-differentiation of MM cells while increased a stem-cell like phenotype. These results highlight the importance of the BM microenvironment (BM supernatant, including endogenous soluble growth factors and cytokines, and the CD138- population from the same patient) to facilitate primary MM cell growth in vitro. Therefore, patient-derived 3DTEBM cultures allowed long-term culture of primary fresh and frozen myeloma cells ex vivo, and these findings indicate that patient-derived 3DTEBM can be utilized for studying multiple myeloma biology and for testing patient-targeted therapy. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. The discrepancy between in vitro efficacy and clinical outcomes can be attributed to several limitations of the classic tissue culture drug screening models including: (1) most of the in vitro models use MM cell line cultures and neglect the vital role of the bone marrow (BM) microenvironment in MM progression, which promotes drug resistance. (2) The BM niche is a three-dimensional (3D) structure with a gradient of both oxygen and drug concentration as a function of distance from blood vessels. The classic two-dimensional (2D) in vitro tissue culture system cannot mimic oxygen and drug gradients in culture wells, making all cells highly oxygenated. Therefore, 2D cultures cannot accurately predict drug sensitivity in different parts of the BM niche due to lack of accurate effects throughout various tissue depths. The goal of this study is to develop an in vitro model that will allow for better evaluation of interactions of MM cells and their microenvironment in the BM niche in a 3D system and how these interactions may affect MM progression and drug resistance. METHODS: The 3D tissue-engineered bone marrow (3DTEBM)was formed through calcium cross-linking of BM supernatants from MM patients. MM cell lines (MM1s, H929 and RPMI) and BM microenvironment (BMM) components, including MM-derived BM stromal cells, HUVECs, and ECM component (fibronectin), were incorporated in 3DTEBM or 2D cultures. We tested by flow cytometry the growth of MM cell lines with and without BMM in 2D vs 3DTEBM cultures at 3 and 7 days. The effect of 3DTEBM and 2D cultures with and without BMM on cytokine expression was determined at day 3 by human cytokine antibody arrays. In addition, the effect of 3DTEBM and 2D cultures with and without BMM on CD markers expression was tested at day 3 by flow cytometry. Finally, drug uptake and hypoxia levels by MM cells in 3DTEBM of different depth of tissue and 2D cultures was determined using flow cytometry and immunohistochemistry, respectively. Then, drug resistance of MM cell lines with and without BMM in 2D vs 3DTEBM cultures after 24h drug treatment were analyzed by flow cytometry. RESULTS: We found that MM cells doubled within 3 days in 2D and 3DTEBM cultures with and without BMM, compared to day 0. In contrast, while in the 3DTEBM induced about 3-5-fold increase in 7 and 14 days, the 2D cultures showed lower growth. In addition, 3DTEBM expressed more cytokines than 2D media in absence of cells, and incorporation of MM cells and BMM induced higher cytokine expression in 3DTEBM than in 2D cultures of SDF-1, IL1-α, TNF-α, TNF-β, MIP-1-δ, PARC, angiopoietin, eotaxin 3 and osteoprotegin. MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), mildly reduced or no changes of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D without BMM. Finally, we found that MM cells in 3DTEBM exhibited 2-fold less drug uptake than MM cells grown in 2D cultures, and drug uptake of MM cells grown in the 3DTEBM was inversely correlated with the depth of the tissue; with increasing tissue depth the drug uptake by MM cells decreased. Accordingly, MM cells in the 3DTEBM exhibited higher hypoxia levels (MFI of PIM) compared to MM cells grown in 2D cultures, and anti-HIF-1α staining revealed that the cells at the lower area were more hypoxic that the cells at the upper area of the 3DTEBM. Therefore, we found that 3DTEBM cultures induced drug resistance in MM cells; for the same drug concentration about 50 and 35% of the MM cells were killed in 2D, and about 15 and 5% of the cells were killed in 3DTEBM without and with BMM, respectively. CONCLUSIONS: Our results suggest that 3DTEBM cultures promoted tumor growth, enhanced cytokine expression, and induced de-differentiation of MM cells, and that a stem-cell-like phenotype might be developing. The 3DTEBM recreated hypoxia and drug gradients by reproducing tissue-specific structural features, so the 3DTEBM cultures induced drug resistance in MM cells more accurately than 2D. Therefore, the 3DTEBM is a promising model for the study of multiple myeloma biology, for in vitro examination of anti-myeloma drugs, tumor microenvironment and interactions between myeloma cells and the BMM. Disclosures No relevant conflicts of interest to declare.

Introduction Waldenström Macroglobulinemia (WM) is a rare, low-grade B-cell lymphoma characterized by lymphoplasmacytic cells spread widely in the bone marrow (BM) and overproduction of monoclonal immunoglobulins M (IgM). Previous studies showed that tumor hypoxia develops in the BM of other hematologic malignancies and promotes dissemination. In this study, we tested the effect of hypoxia on cell proliferation, cell cycle and apoptosis; on egress and homing of WM cells from and into the BM; and on recovery and tumor colonization in the new BM niche. Methods We characterized the effect of tumor progression on generation of hypoxic conditions in the BM in vivo, by injecting BCWM1-mCherry cells to SCID mice, letting them grow for two weeks, analyzing the hypoxic state of the WM cells in the BM using pimonidazole, and testing the number of circulating cells. Moreover, we tested the effect of hypoxia on the homing of WM cells to the BM by injecting normoxic and hypoxic cells to mice and monitoring the number of the circulating WM cells in the blood at different time points by flow cytometry. Cancer cell colonization was assessed 1 and 3 days post IV injection of normoxic and hypoxic cells to mice; mononuclear cells were isolated from the BM, fixed, permeabilized and stained with antibodies for p-Rb and cyclin-E. The percentile of WM cells in the BM and the expression of cell cycle proteins were analyzed by flow cytometry. BCWM1 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) in vitro for 24hrs, and n some cases reoxygenated for 24hrs. The expression of E-cadherin, VLA-4 and CXCR4 was analyzed by western blot or flow cytometry. We tested the effect of hypoxia on adhesion of WM cells to BM stroma and fibronectin. We further tested the effect of hypoxia on chemotactic properties of WM cells towards SDF-1 using a transwell migration chamber. In addition, we tested the effect of hypoxia on WM cell survival (by MTT assay), apoptosis and cell cycle (by using AnnexinV-PI and PI, respectively), and signaling pathways associated with survival, apoptosis and cell cycle (by western blotting). Results Tumor progression was shown to increase hypoxic conditions in the BM in vivo. We found a direct correlation between the percent of WM cells in the BM to the level of hypoxia. The level of hypoxia was in a direct correlation with the number of circulating WM cells in vivo. Then we mimicked the hypoxic conditions in vitro and found that cell progression (MTT) and cell cycle (PI staining) were decreased, but apoptosis of WM cells was not affected (AnnexinV-PI staining). These results were confirmed by decreased activation of the PI3K signaling pathway (p-PI3K, p-AKT, p-GSK) and decreased expression of cell cycle proteins (p-Rb, CDK2, CDK4, cyclin-D1 and p-cyclin-E); however, no change was observed in apoptosis-related proteins (PARP, cleaved caspase-3, -8 and -9). Moreover, hypoxia decreased the expression of E-cadherin which contributed to reduction of adhesion of WM cells to the BM stromal cells. At the same time, hypoxic WM cells exhibited increased CXCR4 surface expression and augmented migratory abilities in the presence of SDF-1. Neither the expression of integrins (VLA-4) nor the adhesion of WM cells to fibronectin was affected by hypoxia. This data indicates the conservation of the homing machinery of the WM to the BM despite the hypoxic conditions accompanied by increased chemotactic ability. When hypoxic and normoxic cells were injected to naïve mice, hypoxic cells showed enhanced homing to the BM and tumor colonization. Similarly, hypoxic cells which were reoxygenated in vitro showed more proliferation, cell cycle and activation of proliferative signaling pathways compared to normoxic cells. Conclusions We report that WM tumor growth in the BM increases hypoxia, and that hypoxia induces cell cycle arrest, and less proliferation of cells with no apoptosis. At the same time, hypoxia induces egress of WM cells from the BM through reduction of E-cadherin expression and decreased adhesion. When in the circulation, previously hypoxic cells home more efficiently to the BM through increased expression of CXCR4 and chemotaxis, and through maintaining expression of integrins and adhesion to fibronectin. When in the new oxygenated BM niche, hypoxic WM cells recover and colonize the new niche better than normoxic cells, and reoxygenated hypoxic cells have faster cell cycle and proliferation rate. Disclosures: No relevant conflicts of interest to declare.

Introduction Multiple myeloma (MM) is the second most prevalent hematological malignancy, and it remains incurable with a median survival of 3 to 5 years. Despite the introduction of several novel drugs, only 60% of the patients respond to therapy, and more than 75% of patients relapse after 5 years. The interaction of MM cells with extracellular matrix (ECM) and stromal cells in the bone marrow (BM) milieu was shown to play a crucial role in MM progression and drug resistance. Therefore, an appropriate model that demonstrates the complex interactions between MM cells and their environment and predicts the response to treatment of MM patients is warranted. The goal of this study is to produce an in vitro model of BM microenvironment in MM, which will mimic the BM in the patient and have closest prediction of cell-behavior in vivo. Methods Scaffolds for the 3D tissue-engineered BM (3DBM) were prepared by crosslinking BM supernatants from MM patients with calcium chloride in the presence of the antifibrinolytic tranexamic acid. The concentration of the different components for scaffold formation and physico-chemical properties of the scaffold were optimized. Then, we incorporated cellular components (MM cell lines and stromal cell lines) and ECM components (fibronectin) to 3DBM scaffold. We tested the effect of different MM cell densities, fibronectin concentration, and presence of different densities of stromal cells in the scaffold on the proliferation of MM cells, which was analyzed by flow cytometry. After optimization of the cellular and ECM components, we tested the sensitivity of MM cells to chemotherapeutic agents used in MM, including melphalan, bortezomib and carfilzomib, in 3DBM scaffolds with and without the presence of stromal cells. We assessed the sensitivity of MM cells to the drugs in the 3DBM scaffolds compared with the sensitivity in two-dimensional (2D) classic in vitro tissue culture system, with or without the presence of stromal cells. Results We optimized the conditions for the production of a 3DBM from BM supernatants from MM patients. We found that calcium concentration was a critical factor for the formation of the 3DBM scaffold; and 1mg/ml calcium chloride was the optimal concentration for fastest and most stable scaffold formation. Moreover, we found that the presence of an antifibrinolytic agent was essential for the stabilization of the scaffold, and 4mg/ml of tranexamic acid was sufficient to stabilize the scaffold. We optimized the conditions for seeding of the cellular components and found that MM cells proliferated in the 3DBM scaffolds better than in regular 2D tissue culture plates, in which the expansion rate over 3 days was about 5-folds in the 3DBM, compared to about 2.4-folds in 2D classic in vitro tissue culture system. In addition, we optimized the co-culture condition of MM cells with stromal cells and found that the ratio of MM/stroma of 3/1 generated the highest proliferation of MM cells in the 3DBM scaffolds. Similarly, the addition of ECM components (fibronectin) increased the proliferation of MM in the 3DBM by about 30%. In addition, we tested the effect of the 3DBM scaffold on drug resistance of MM cells. In a 2D classic in vitro tissue culture system approximately about 40-50% of the MM cells were killed using the different chemotherapies, the culture of MM cells in 3DBM decreased the sensitivity of MM cells to treatment, and addition of stromal cells to the co-culture decreased the sensitivity of MM cells to treatment in both 3D and 2D. We found that the co-culture of MM cells with stromal cells in the 3DBM treatment and using the same concentrations of the different chemotherapies killed only 5-15% of the MM cells. Conclusions We produced and characterized a 3D scaffold made from supernatant of BM aspirates from MM patients. This 3DBM is all made from patient material (autologous), no synthetic scaffolds were used, and it could represent the closest in vitro model to the BM in MM. The 3DBM induced drug resistance in MM cells, especially when it contained stromal cells. The autologous 3DBM seems to be closer to the real BM microenvironment, therefore, a better drug screening method than 2D classic in vitro tissue culture systems. Disclosures: No relevant conflicts of interest to declare.