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Magdalena Massalska,1 Marzena Ciechomska,1 Ewa Kuca-Warnawin,1 Tomasz Burakowski,1 Anna Kornatka,1 Anna Radzikowska,1 Dariusz Pawlak,2 Barbara Muz,3 Adrianna Loniewska-Lwowska,4 Andrzej Palucha,5 Pawel Maldyk,6,7 Wlodzimierz Maslinski1 1Department of Pathophysiology and Immunology, National Institute of Geriatrics, Rheumatology, and Rehabilitation (NIGRiR), Warsaw, 02-637, Poland; 2Department of Pharmacodynamics, Medical University of Bialystok, Bialystok, 15-222, Poland; 3Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO, 63108, USA; 4Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, 02-106, Poland; 5Genomed S.A. Ponczowa 12, Warsaw, 02-971, Poland; 6Department of Rheumoorthopaedic Surgery, National Institute of Geriatrics, Rheumatology, and Rehabilitation (NIGRiR), Warsaw, 02-637, Poland; 7Clinical Department of Orthopaedic and Traumatology of Locomotor System, Enfant-Jesus Clinical Hospital, Warsaw, 02-005, PolandCorrespondence: Magdalena Massalska, Department of Pathophysiology and Immunology, National Institute of Geriatrics, Rheumatology, and Rehabilitation, Spartanska 1, Warsaw, 02-637, Poland, Tel/Fax +48 22 670 94 94, Email magdalena.massalska@spartanska.plBackground: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery.Methods: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls.Results: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls.Conclusion: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.Keywords: rheumatoid arthritis, bone marrow, CTLA-4-Fc, indoleamine 2,3- dioxygenase, CD4+Foxp3+, monocytes

Barbara Muz,1 Rachel Nicole Ghazarian,1,2 Monica Ou,1,3 Micah John Luderer,1 Hubert Daniel Kusdono,1,2 Abdel Kareem Azab1 1Department of Radiation Oncology, Cancer Biology Division, Washington University in St Louis School of Medicine, 2Department of Pharmaceutical and Administrative Sciences, St Louis College of Pharmacy, 3Department of Biology, St Louis University, St Louis, MO, USA Abstract: Despite the significant therapeutic advances achieved with proteasome inhibitors (PIs) such as bortezomib and carfilzomib in prolonging the survival of patients with multiple myeloma, the development of drug resistance, peripheral neuropathy, and pharmacokinetic limitations continue to pose major challenges when using these compounds. Ixazomib is a second-generation PI with improved activity over other PIs. Unlike bortezomib and carfilzomib, which are administered by injection, ixazomib is the first oral PI approved by US Food and Drug Administration. This review discusses the biochemical properties, mechanisms of action, preclinical efficacy, and clinical trial results leading to the US Food and Drug Administration approval of ixazomib. Keywords: proteasome inhibitor, oral administration, biological mechanism, clinical trials

Multiple myeloma (MM) is the cancer of plasma cells within the bone marrow and remains incurable. Tumor-associated macrophages (TAMs) within the tumor microenvironment often display a pro-tumor phenotype and correlate with tumor proliferation, survival, and therapy resistance. IL-10 is a key immunosuppressive cytokine that leads to recruitment and development of TAMs. In this study, we investigated the role of IL-10 in MM TAM development as well as the therapeutic application of IL-10/IL-10R/STAT3 signaling inhibition. We demonstrated that IL-10 is overexpressed in MM BM and mediates M2-like polarization of TAMs in patient BM, 3D co-cultures in vitro, and mouse models. In turn, TAMs promote MM proliferation and drug resistance, both in vitro and in vivo. Moreover, inhibition of IL-10/IL-10R/STAT3 axis using a blocking IL-10R monoclonal antibody and STAT3 protein degrader/PROTAC prevented M2 polarization of TAMs and the consequent TAM-induced proliferation of MM, and re-sensitized MM to therapy, in vitro and in vivo. Therefore, our findings suggest that inhibition of IL-10/IL-10R/STAT3 axis is a novel therapeutic strategy with monotherapy efficacy and can be further combined with current anti-MM therapy, such as immunomodulatory drugs, to overcome drug resistance. Future investigation is warranted to evaluate the potential of such therapy in MM patients., (© 2024. The Author(s).)

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4 + Foxp3 + Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery., Methods: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4 + CD69 + and CD4 + Foxp3 + percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls., Results: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4 + T cells activation without influencing CD8 + cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls., Conclusion: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA., Competing Interests: The authors declare no conflict of interest., (© 2022 Massalska et al.)

Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs., (© 2022. The Author(s).)

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM., (© 2022. The Author(s).)

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow (BM) and represents the second most common hematological malignancy in the world. The MM tumor microenvironment (TME) within the BM niche consists of a wide range of elements which play important roles in supporting MM disease progression, survival, proliferation, angiogenesis, as well as drug resistance. Together, the TME fosters an immunosuppressive environment in which immune recognition and response are repressed. Macrophages are a central player in the immune system with diverse functions, and it has been long established that macrophages play a critical role in both inducing direct and indirect immune responses in cancer. Tumor-associated macrophages (TAMs) are a major population of cells in the tumor site. Rather than contributing to the immune response against tumor cells, TAMs in many cancers are found to exhibit protumor properties including supporting chemoresistance, tumor proliferation and survival, angiogenesis, immunosuppression, and metastasis. Targeting TAM represents a novel strategy for cancer immunotherapy, which has potential to indirectly stimulate cytotoxic T cell activation and recruitment, and synergize with checkpoint inhibitors and chemotherapies. In this review, we will provide an updated and comprehensive overview into the current knowledge on the roles of TAMs in MM, as well as the therapeutic targets that are being explored as macrophage-targeted immunotherapy, which may hold key to future therapeutics against MM., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The synthesis of d-glucoheptose derivative containing a boronic moiety is described herein. Starting from benzyl 6,7-dideoxy-2,3,4-tri-O-benzyl-β-d-gluco-ept-6-enopyranoside, the introduction of the boronic acid was performed through a metathesis reaction by using MIDA vinyl boronic acid and the 2nd generation Grubbs catalyst. Hydrogenation led to the final product in only two reaction steps. This new sugar-containing boronic acid in the skeleton could mimic carbohydrate behavior and follow the glucose uptake in living cells. The in vitro toxicity tests performed in fibroblasts and glioma tumor cell lines showed minimal toxicity. Boron uptake measured using ICP-MS was minimal in fibroblasts, while in glioma cells showed a value of 6 ng of total boron accumulation per mg of cells, implying that compound 1a is able to accumulate selectively in the tumor tissues compared to normal., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Immunotherapy is an attractive approach for treating cancer. T-cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T-cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T-cell exhaustion., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) are hematological malignancies that remain incurable despite novel treatments. In order to improve current treatments and clinical efficacy, there remains a need for more complex in vitro models that mimic the intricate human leukemic microenvironment. This study aimed to use 3D tissue engineered plasma cultures (3DTEPC) derived from CML, AML and CLL patients to promote proliferation of leukemic cells for use as a drug screening tool for treatment. 3DTEPC supported the growth of primary CML, AML and CLL cells and also induced significantly more drug resistance in CML, AML and CLL cell lines compared to 2D. The 3DTEPC created a more physiologically relevant environment for leukemia cell proliferation, provided a reliable model for growing leukemia patient samples, and serves as a relevant tool for drug screening and personalized medicine.

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., (© 2021. The Author(s).)

CD47 is a surface glycoprotein expressed by host cells to impede phagocytosis upon binding to macrophage SIRPα, thereby represents an immune checkpoint known as the "don't-eat-me" signal. However, accumulating evidence shows that solid and hematologic tumor cells overexpress CD47 to escape immune surveillance. Thus, targeting the CD47-SIRPa axis by limiting the activity of this checkpoint has emerged as a key area of research. In this review, we will provide an update on the landscape of CD47-targeting antibodies for hematological malignancies, including monoclonal and bi-specific antibodies, with a special emphasis on agents in clinical trials and novel approaches to overcome toxicity., Competing Interests: All authors state no conflict of interest., (© 2021 The Authors. Published by Elsevier Ltd.)

Acute myeloid leukemia (AML) is the most common type of leukemia and has a 5-year survival rate of 25%. The standard-of-care for AML has not changed in the past few decades. Promising immunotherapy options are being developed for the treatment of AML; yet, these regimens require highly laborious and sophisticated techniques. We create nanoTCEs using liposomes conjugated to monoclonal antibodies to enable specific binding. We also recreate the bone marrow niche using our 3D culture system and use immunocompromised mice to enable use of human AML and T cells with nanoTCEs. We show that CD33 is ubiquitously present on AML cells. The CD33 nanoTCEs bind preferentially to AML cells compared to Isotype. We show that nanoTCEs effectively activate T cells and induce AML killing in vitro and in vivo . Our findings suggest that our nanoTCE technology is a novel and promising immuno-therapy for the treatment of AML and provides a basis for supplemental investigations for the validation of using nanoTCEs in larger animals and patients., Competing Interests: CONFLICTS OF INTEREST AKA and KA have filed a patent with regards to the T cell engagers described in this study. AKA is the founder and owner of Cellatrix LLC and Targeted Therapeutics LLC. Some of the experiments were performed using 3DTEBM products supplied by Cellatrix LLC; however, both companies had no role in the study. Other authors state no conflicts of interest., (Copyright: © 2021 Alhallak et al.)

T-cell-based immunotherapy, such as CAR-T cells and bispecific T-cell engagers (BiTEs), has shown promising clinical outcomes in many cancers; however, these therapies have significant limitations, such as poor pharmacokinetics and the ability to target only one antigen on the cancer cells. In multiclonal diseases, these therapies confer the development of antigen-less clones, causing tumor escape and relapse. In this study, we developed nanoparticle-based bispecific T-cell engagers (nanoBiTEs), which are liposomes decorated with anti-CD3 monoclonal antibodies (mAbs) targeting T cells, and mAbs targeting the cancer antigen. We also developed a nanoparticle that targets multiple cancer antigens by conjugating multiple mAbs against multiple cancer antigens for T-cell engagement (nanoMuTEs). NanoBiTEs and nanoMuTEs have a long half-life of about 60 h, which enables once-a-week administration instead of continuous infusion, while maintaining efficacy in vitro and in vivo. NanoMuTEs targeting multiple cancer antigens showed greater efficacy in myeloma cells in vitro and in vivo, compared to nanoBiTEs targeting only one cancer antigen. Unlike nanoBiTEs, treatment with nanoMuTEs did not cause downregulation (or loss) of a single antigen, and prevented the development of antigen-less tumor escape. Our nanoparticle-based immuno-engaging technology provides a solution for the major limitations of current immunotherapy technologies., (© 2021. The Author(s).)

Purpose: Cervical cancer represents the fourth most frequent malignancy in the world among women, and mortality has remained stable for the past 4 decades. Intravenous cisplatin with concurrent radiation therapy is the standard-of-care for patients with local and regional cervical cancer. However, cisplatin induces serious dose-limiting systemic toxicities and recurrence frequently occurs. In this study, we aimed to develop an intracervical drug delivery system that allows cisplatin release directly into the tumor and minimize systemic side effects., Methods and Materials: Twenty patient biopsies and 5 cell lines treated with cisplatin were analyzed for platinum content using inductively coupled plasma mass spectrometry. Polymeric implants loaded with cisplatin were developed and evaluated for degradation and drug release. The effect of local or systemic cisplatin delivery on drug biodistribution as well as tumor burden were evaluated in vivo, in combination with radiation therapy., Results: Platinum levels in patient biopsies were 6-fold lower than the levels needed for efficacy and radiosensitization in vitro. Cisplatin local delivery implant remarkably improved drug specificity to the tumor and significantly decreased accumulation in the blood, kidney, and other distant normal organs, compared with traditional systemic delivery. The localized treatment further resulted in complete inhibition of tumor growth., Conclusions: The current standard-of-care systemic administration of cisplatin provides a subtherapeutic dose. We developed a polymeric drug delivery system that delivered high doses of cisplatin directly into the cervical tumor, while lowering drug accumulation and consequent side effects in normal tissues. Moving forward, these data will be used as the basis of a future first-in-human clinical trial to test the efficacy of localized cisplatin as adjuvant or neoadjuvant chemotherapy in local and regional cervical cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

E-selectin is a vascular adhesion molecule expressed mainly on endothelium, and its primary role is to facilitate leukocyte cell trafficking by recognizing ligand surface proteins. E-selectin gained a new role since it was demonstrated to be involved in cancer cell trafficking, stem-like properties and therapy resistance. Therefore, being expressed in the tumor microenvironment, E-selectin can potentially be used to eradicate cancer. Uproleselan (also known as GMI-1271), a specific E-selectin antagonist, has been tested on leukemia, myeloma, pancreatic, colon and breast cancer cells, most of which involve the bone marrow as a primary or as a metastatic tumor site. This novel therapy disrupts the tumor microenvironment by affecting the two main steps of metastasis-extravasation and adhesion-thus blocking E-selectin reduces tumor dissemination. Additionally, uproleselan mobilized cancer cells from the protective vascular niche into the circulation, making them more susceptible to chemotherapy. Several preclinical and clinical studies summarized herein demonstrate that uproleselan has favorable safety and pharmacokinetics and is a tumor microenvironment-disrupting agent that improves the efficacy of chemotherapy, reduces side effects such as neutropenia, intestinal mucositis and infections, and extends overall survival. This review highlights the critical contribution of E-selectin and its specific antagonist, uproleselan, in the regulation of cancer growth, dissemination, and drug resistance in the context of the bone marrow microenvironment.

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.

Objectives: Despite substantial research linking adverse childhood experiences (ACEs) and health, it is unclear how associations differ by veteran status and military service era (draft, volunteer era). The current study evaluated differences in ACEs and health by veteran status and era, increasing understanding important for service provision as the volunteer era veteran population increases. Methods: Behavioral Risk Factor Surveillance System 2012 data were used in univariate and weighted multivariable logistic regression models to assess associations among veteran status, service era, ACEs, and health. Results: Volunteer era veterans experienced the most ACEs (M = 2.42); draft era veterans experienced the fewest (M = 1.04). Individuals reporting 3 or more ACEs were 3.67 times (95% CI = 3.22-4.19) more likely to endorse depression, 1.32 times (95% CI = 1.17-1.48) more likely to report poorer general health, and 1.77 times (95% CI = 1.58-1.97) more likely to endorse poorer physical health, compared to those reporting none. Volunteer era veterans were 2.43 times more likely to report poorer physical health (95% CI = 1.49-3.97) than draft era veterans, adjusting for ACEs. Conclusions: ACEs were associated with poorer health independent of veteran status and service era. Volunteer era veterans experienced more ACEs; need for trauma-informed services supporting whole health may increase.

Background: Multiple sclerosis adult day programs (MSADPs) offer life-enhancing services for individuals and informal caregivers affected by multiple sclerosis (MS), including medical care, rehabilitation therapies, nutrition therapy, cognitive training, tailored education, exercise programs, and social interaction. The purpose of this study was to examine the effects of MSADPs on health-related quality of life (HRQOL) and health care utilization of persons with MS and HRQOL and well-being of informal caregivers., Methods: Using a quasi-experimental design, outcomes between baseline and 1-year follow-up in persons with MS and informal caregivers who used MSADP services and a comparison group of similar persons with MS and caregivers who did not use MSADP services were compared. For persons with MS, outcomes included standardized measures of physical and mental HRQOL and health care utilization. For caregivers, outcomes included physical and mental HRQOL and well-being. Changes in outcomes between baseline and follow-up were examined using propensity score-weighted difference-in-differences regression analysis., Results: For persons with MS, MSADP use had a significant positive effect on 12-Item Short Form Health Survey physical component scores, although the difference was not clinically meaningful. Use of MSADPs did not have effects on any other outcomes for persons with MS or caregivers., Conclusions: Use of MSADPs did not show a clinically meaningful effect on HRQOL for persons with MS or informal caregivers. The MSADPs do not seem to offer sustained benefits to persons with MS or caregivers, but the possibility of initial short-term benefits cannot be ruled out., Competing Interests: The authors declare no conflicts of interest., (© 2020 Consortium of Multiple Sclerosis Centers.)

Multiple myeloma (MM) remains to be incurable despite recent therapeutic advances. CD47, an immune checkpoint known as the "don't eat me" signal, is highly expressed on the surface of various cancers, allowing cancer cells to send inhibitory signals to macrophages and impede phagocytosis and immune response. In this study, we hypothesized that blocking the "don't eat me" signaling using an anti-CD47 monoclonal antibody will induce killing of MM cells. We report that CD47 expression was directly correlated with stage of the disease, from normal to MGUS to MM. Moreover, MM cells had remarkably higher CD47 expression than other cell populations in the bone marrow. These findings indicate that CD47 is specifically expressed on MM and can be used as a potential therapeutic target. Further, blocking of CD47 using an anti-CD47 antibody induced immediate activation of macrophages, which resulted in induction of phagocytosis and killing of MM cells in the 3D-tissue engineered bone marrow model, as early as 4 hours. These results suggest that macrophage checkpoint immunotherapy by blocking the CD47 "don't eat me" signal is a novel and promising strategy for the treatment of MM, providing a basis for additional studies to validate these effects in vivo and in patients., Competing Interests: Dr. Azab received research support from Vasculox Inc. to conduct this study. He also received funding from GlycoMimetic Inc.; however, this has no contribution to this study. In addition, Dr. Azab is the founder and owner of Cellatrix LLC that has an exclusive license for the 3DTEBM technology described in part of the experiments in this paper. Moreover, Dr. Azab is the founder and owner of Targeted Therapeutics LLC; however, this has no contribution to this study. Other authors state no conflicts of interest.

Objective :  Waldenström Macroglobulinemia (WM) is a rare B-cell malignancy characterized by secretion of immunoglobulin M and cancer infiltration in the bone marrow. Chemokine receptor such as CXCR4 and hypoxic condition in the bone marrow play crucial roles in cancer cell trafficking, homing, adhesion, proliferation, survival, and drug resistance. Herein, we aimed to use CXCR4 as a potential biomarker to detect hypoxic-metastatic WM cells in the bone marrow and in the circulation by using CXCR4-detecting radiopharmaceutical. Methods : We radiolabeled a CXCR4-inhibitor (AMD3100) with 64 Cu and tested its binding to WM cells with different levels of CXCR4 expression using gamma counter in vitro . The accumulation of this radiopharmaceutical tracer was tested in vivo in subcutaneous and intratibial models using PET/CT scan. In addition, PBMCs spiked with different amounts of WM cells ex vivo were detected using gamma counting. Results : In vitro , 64 Cu-AMD3100 binding to WM cell lines demonstrated a direct correlation with the level of CXCR4 expression, which was increased in cells cultured in hypoxia with elevated levels of CXCR4, and decreased in cells with CXCR4 and HIF-1α knockout. Moreover, 64 Cu-AMD3100 detected localized and circulating CXCR4 high WM cells with high metastatic potential. Conclusions : In conclusion, we developed a molecularly targeted system, 64 Cu-AMD3100, which binds to CXCR4 and specifically detects WM cells with hypoxic phenotype and metastatic potential in the subcutaneous and intratibial models. These preliminary findings using CXCR4-detecting PET radiopharmaceutical tracer indicate a potential technology to predict high-risk patients for the progression to WM due to metastatic potential.

Purpose: Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation requires sufficient tumor boron delivery while minimizing nonspecific accumulation., Methods: Thermal sensitive liposomes (TSLs) were designed to have a stable drug payload at physiological temperatures but engineered to have high permeability under mild hyperthermia., Results: We found that TSLs improved the tumor-specific delivery of boronophenylalanine (BPA) and boronated 2-nitroimidazole derivative B-381 in D54 glioma cells. Uniquely, the 2-nitroimidazole moiety extended the tumor retention of boron content compared to BPA., Conclusion: This is the first study to show the delivery of boronated compounds using TSLs for BNCT, and these results will provide the basis of future clinical trials using TSLs for BNCT.

Overall survival of patients with newly diagnosed glioblastoma (GBM) remains dismal at 16 months with state-of-the-art treatment that includes surgical resection, radiation, and chemotherapy. GBM tumors are highly heterogeneous, and mechanisms for overcoming tumor resistance have not yet fully been elucidated. An injectable chitosan hydrogel capable of releasing chemotherapy (temozolomide [TMZ]) while retaining radioactive isotopes agents (iodine, [ 131 I]) was used as a vehicle for localized radiation and chemotherapy, within the surgical cavity. Release from hydrogels loaded with TMZ or 131 I was characterized in vitro and in vivo and their efficacy on tumor progression and survival on GBM tumors was also measured. The in vitro release of 131 I was negligible over 42 days, whereas the TMZ was completely released over the first 48 h. 131 I was completely retained in the tumor bed with negligible distribution in other tissues and that when delivered locally, the chemotherapy accumulated in the tumor at 10-fold higher concentrations than when delivered systemically. We found that the tumors were significantly decreased, and survival was improved in both treatment groups compared to the control group. Novel injectable chemo-radio-hydrogel implants may potentially improve the local control and overall outcome of aggressive, poor prognosis brain tumors., (Copyright © 2018. Published by Elsevier Inc.)

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. We have showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through BTZ diffusion from the surface and endocytosed NPs, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells when BTZ was administered through anti-CD38 chitosan NPs. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. The anti-CD38 chitosan NPs showed low toxicity profile allowing enhancement of proteasome-inhibitory activity and specificity of BTZ by endocytosis-mediated uptake of CD38 representing a promising therapy in MM., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Using photoacoustic microscopy (PAM), we evaluated non-invasively oxygenation and vascularization in vivo due to multiple myeloma (MM) progression. Mice injected with MM.1S-GFP were monitored with a fluorescence microscope for tumor progression. In vivo PAM of the cerebral bone marrow quantified the total oxygen saturation (sO 2 ). At 28 days after the MM cell injection, the total sO 2 had decreased by half in the developing tumor regions, while in the non-tumor regions it had decreased by 20% compared with the value at one day post MM injection. The blood vessel density was reduced by 35% in the developing tumor regions, while in the non-tumor regions it was reduced by 8% compared with the value at one day post MM injection. Hence, PAM corroborated the development of hypoxia due to MM progression and demonstrated decreased vascularization surrounding the tumor areas.

Purpose: Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells., Methods: B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo., Results: B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively., Conclusions: B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on hypoxic tumor metabolism. Further studies are warranted to evaluate B-381 and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma.

Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by Tris (Dibenzylideneacetone) dipalladium (Tris DBA). Tris DBA reduces proliferation, induces G1 arrest and apoptosis in MM cells. Tris DBA showed additive effect with proteasome inhibitors reducing proliferation, cell cycle signaling, and increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to proteasome inhibitors by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma.

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., (© 2016 John Wiley & Sons Ltd.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Current treatment options as well as clinical efficacy are limited for chronic myelogenous leukemia (CML), Ph+ acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). In response to the pressing need for more efficacious treatment approaches and strategies to override drug resistance in advanced stage CML, Ph+ ALL, and AML, we investigated the effects of inhibition of ILK as a potentially novel and effective approach to treatment of these challenging malignancies. Using the small molecule ILK inhibitor, Cpd22, and ILK knockdown, we investigated the importance of ILK in the growth and viability of leukemia. Our results suggest that the ILK inhibition may be an effective treatment for CML, Ph+ ALL, and AML as a single therapy, with ILK expression levels positively correlating with the efficacy of ILK inhibition. The identification of ILK as a novel target for leukemia therapy warrants further investigation as a therapeutic approach that could be of potential clinical benefit in both acute and chronic myeloid leukemias., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: 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., (©2014 American Association for Cancer Research.)

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 interaction in vivo led 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., (©2014 American Association for Cancer Research.)

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

The phosphatidylinositide 3-kinase (PI3K) pathway is activated and correlated with drug resistance in multiple myeloma (MM). In the present study we investigated the role of PI3KCA (PI3K-α) in the progression and drug resistance in MM. We showed that the gene expression of PI3KCA isoform was higher in MM compared to normal subjects. BYL719, a novel and specific PI3KCA inhibitor inhibited the survival of primary MM cells and cell lines but not normal peripheral blood mononuclear cells. BYL719 induced the apoptosis of MM cells and inhibited their cell cycle by causing G1 arrest. BYL719 inhibited PI3K signalling, decreased proliferation and cells cycle signalling, and induced apoptosis signalling in MM cells. Finally, BYL719 synergized with bortezomib and carfilzomib, and overcame drug resistance induced by bone marrow stroma. These results were confirmed using in silico simulation of MM cell lines, BYL719 and bortezomib, and showed similar trends in survival, proliferation, apoptosis, cell signalling and synergy with drugs. In conclusion, PI3KCA plays a major role in proliferation and drug resistance of MM cells, the effects of which were inhibited with BYL719. These results provide a preclinical basis for a future clinical trial of BYL719 in MM as a single agent or in combination with other drugs., (© 2014 John Wiley & Sons Ltd.)

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. Novel agents are currently in development for the treatment of relapsed/refractory MM, including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, cell signaling targeted therapies, and strategies targeting the tumor microenvironment. We have previously reviewed in detail the contemporary immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies therapies for MM. Therefore, in this review, we focused on the role of molecular targeted therapies in the treatment of relapsed/refractory multiple myeloma, including cell signaling targeted therapies (HDAC, PI3K/AKT/mTOR, p38 MAPK, Hsp90, Wnt, Notch, Hedgehog, and cell cycle) and strategies targeting the tumor microenvironment (hypoxia, angiogenesis, integrins, CD44, CXCR4, and selectins). Although these novel agents have improved the therapeutic outcomes for MM patients, further development of new therapeutic agents is warranted.

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., (©2013 AACR.)

Objective: Rheumatoid arthritis (RA) is characterized by hypoxia and the expression of hypoxia-inducible transcription factors (HIFs), which coordinate cellular responses to hypoxia. The objective of this study was to analyze the expression and regulation of prolyl hydroxylase domain (PHD) enzymes and factor-inhibiting HIF-1α (FIH-1), which regulate cellular HIF levels, and to study the roles of these enzymes in RA fibroblast-like synoviocytes (RA FLS)., Methods: The expression of PHD and FIH and downstream target genes was assessed by quantitative polymerase chain reaction and Western blotting. A small interfering RNA (siRNA) approach and an in vitro endothelial cell angiogenesis assay were used to analyze the roles of HIF hydroxylases., Results: In human RA FLS, knockdown of PHD-2, but not knockdown of PHD-1 or FIH-1, dramatically augmented HIF-1α expression, modestly increased HIF-2α protein expression under normoxic conditions, and up-regulated HIF-dependent gene expression. In contrast, silencing of PHD-3 up-regulated HIF-2α but reduced HIF-1α, thereby decreasing the expression of HIF-regulated genes. A similar effect of PHD-2 knockdown was observed in osteoarthritis FLS (OA FLS) but not in nondiseased primary human dermal fibroblasts. These findings correlated with the induction of in vitro angiogenesis by supernatants from RA FLS and OA FLS transfected with siPHD-2 but not by supernatants from nondiseased fibroblasts or from siPHD-3-transfected cells., Conclusion: Our data suggest that PHD-2 is the major hydroxylase regulating HIF levels and the expression of angiogenic genes in arthritic cells. PHD-2 appears to regulate responses relevant to arthritis via HIF-α, highlighting the major importance of this enzyme in hypoxia- and angiogenesis-dependent inflammatory diseases such as RA., (Copyright © 2012 by the American College of Rheumatology.)

Introduction: Hypoxia and T-helper cell 1 (Th1) cytokine-driven inflammation are key features of rheumatoid arthritis (RA) and contribute to disease pathogenesis by promoting angiogenesis. The objective of our study was to characterise the angiogenic gene signature of RA fibroblast-like synoviocytes (FLS) in response to hypoxia, as well as Th1 and T-helper cell 2 (Th2) cytokines, and in particular to dissect out effects of combined hypoxia and cytokines on hypoxia inducible transcription factors (HIFs) and angiogenesis., Methods: Human angiogenesis PCR arrays were used to screen cDNA from RA FLS exposed to hypoxia (1% oxygen) or dimethyloxalylglycine, which stabilises HIFs. The involvement of HIF isoforms in generating the angiogenic signature of RA FLS stimulated with hypoxia and/or cytokines was investigated using a DNA-binding assay and RNA interference. The angiogenic potential of conditioned media from hypoxia-treated and/or cytokine-treated RA FLS was measured using an in vitro endothelial-based assay., Results: Expression of 12 angiogenic genes was significantly altered in RA FLS exposed to hypoxia, and seven of these were changed by dimethyloxalylglycine, including ephrin A3 (EFNA3), vascular endothelial growth factor (VEGF), adipokines angiopoietin-like (ANGPTL)-4 and leptin. These four proangiogenic genes were dependent on HIF-1 in hypoxia to various degrees: EFNA3 >ANGPTL-4 >VEGF >leptin. The Th1 cytokines TNFα and IL-1β induced HIF-1 but not HIF-2 transcription as well as activity, and this effect was additive with hypoxia. In contrast, Th2 cytokines had no effect on HIFs. IL-1β synergised with hypoxia to upregulate EFNA3 and VEGF in a HIF-1-dependent fashion but, despite strongly inducing HIF-1, TNFα suppressed adipokine expression and had minimal effect on EFNA3. Supernatants from RA FLS subjected to hypoxia and TNFα induced fewer endothelial tubules than those from FLS subjected to TNFα or hypoxia alone, despite high VEGF protein levels. The Th2 cytokine IL-4 strongly induced ANGPTL-4 and angiogenesis by normoxic FLS and synergised with hypoxia to induce further proangiogenic activity., Conclusion: The present work demonstrates that Th1 cytokines in combination with hypoxia are not sufficient to induce angiogenic activity by RA FLS despite HIF-1 activation and VEGF production. In contrast, Th2 cytokines induce angiogenic activity in normoxia and hypoxia, despite their inability to activate HIFs, highlighting the complex relationships between hypoxia, angiogenesis and inflammation in RA.