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Supplementary Figure 1. Hypoxia regulates WM cell formation of clumps in an E-cadherin-dependent manner. The effect of E-cadherin blocking antibody (5 μg/mL) on the size of colonies created by BCWM.1 cells in normoxia or hypoxia normalized to normoxic untreated cells (A); and on the colony formation by BCWM.1 cells as shown by representative pictures (scale bar represents 1 micrometer) (B).

Supplementary Figure 2. Re-oxygenation induces WM cell clump-formation. The effect of hypoxia (1% O2, 48hrs) and re-oxygenation (hypoxia for 24hrs followed by normoxia for 24hrs) on BCWM.1 size of colonies normalized to hypoxic cells (A), and on the creation of clumps monitored and captured using light microscopy (scale bar represents 1 micrometer) (B).

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

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.

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.

Multiple myeloma (MM) is the cancer of plasma cells within the bone marrow (BM) 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. Thus, TAMs have become an emerging target of interest. 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 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 pathway using a blocking IL-10R antibody 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 axis is a novel immunotherapy strategy with monotherapy efficacy and can be further combined with current anti-MM therapy to overcome drug resistance. Future investigation is warranted to evaluate the potential of such therapy in MM patients.

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.

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

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.

ICB therapy has transformed cancer treatment; durable responses in difficult-to-treat cancers have been observed. Despite this most patients (pts) don’t respond to ICB (primary resistance), and many pts who initially respond eventually relapse (acquired resistance). Primary resistance is associated with tumor cell extrinsic factors, e.g., the immunosuppressive nature of the tumor microenvironment (TME), while acquired resistance is associated with tumor intrinsic factors, e.g., downregulation of MHC1, preventing T cell recognition. Treatments to overcome ICB resistance are necessary. WU-NK-101 is a PBMC-derived, cytokine-reprogrammed, expanded, and cryopreserved off-the-shelf memory NK cell product. WU-NK-101 cells capture the memory-like NK cell biology of cytokine-induced memory-like (CIML) NK cells, exhibiting enhanced cytotoxicity, metabolic fitness/flexibility, and resistance to TME immunosuppression (Muth et al. EHA 2022; Rutella et al. ESMO 2022). Bone marrow biopsies, collected from R/R AML pts who received CIML-NK cells (NCT01898793), were interrogated using immune gene expression (GE) profiling and spatially resolved proteomics (IO360® panel, n = 740 genes, and GeoMx® DSP; NanoString Technologies). Higher T-cell infiltration was noted post CIML-NK. CIBERSORT deconvoluted GE data inferred higher abundance of macrophages, γδT cells and activated dendritic cells on day 28 post-treatment. GE signatures showed downregulation of NFIL3 and FAM30A (log2 fold-change Citation Format: Tom A. Leedom, Barbara Muz, Jaykumar Vadakekolathu, John J. Muth, Xiao-Hua Li, Gregory Watson, Kristaan Magee, Ryan P. Sullivan, Melissa Berrien-Elliott, Todd Fenigher, Sergio Rutella, Matthew L. Cooper, Ayman Kabakibi, Jan K. Davidson-Moncada. xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6418.

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.

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, CD4Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4We 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 CD4This 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.

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.

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.

Objective: Waldenstrom 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 CXCR...

Boron Neutron Capture Therapy (BNCT) is a binary therapy that promises to be suitable in treating many non-curable cancers. To that, the discovery of new boron compounds able to accumulate selectively in the tumour tissue is still required. Hypoxia, a deficiency of oxygen in tumor tissue, is a great challenge in the conventional treatment of cancer, because hypoxic areas are resistant to conventional anticancer treatments. 2-Nitroimidazole derivatives are known to be hypoxia markers due to their enrichment by bioreduction in hypoxic cells. In the present work, 2-nitroimidazole was chosen as the starting point for the synthesis of a new boron-containing compound based on a 1,3,5-triazine skeleton. Two o-carborane moieties were inserted to achieve a high ratio of boron on the molecular weight, exploiting a short PEG spacer to enhance the polarity of the compound and outdistance the active part from the core. The compound showed no toxicity on normal human primary fibroblasts, while it showed noteworthy toxicity in multiple myeloma cells together with a consistent intracellular boron accumulation.

Simple Summary This review focuses on eradicating cancer by targeting a surface protein expressed on the endothelium—E-selectin—with a novel drug, uproleselan (GMI-1271). Blocking E-selectin in the tumor microenvironment acts on multiple levels; uproleselan was shown (i) to inhibit cancer cell tethering, rolling and extravasating, i.e., cancer dissemination, (ii) to reduce adhesion and lose stem cell-like properties, (iii) to mobilize cancer cells to circulation where they are more susceptible to chemotherapy, which altogether contributes (iv) to overcome drug resistance. Uproleselan has been tested effective in leukemia, myeloma, pancreatic, colon and breast cancer cells, all of which can be found in the bone marrow as a primary or as a metastatic tumor site. In addition, uproleselan has a good safety profile in patients. It improves the efficacy of chemotherapy, reduces side effects such as neutropenia, intestinal mucositis and infections, and extends overall survival. Abstract 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.

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.

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.

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.

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) remains to be incurable despite recent therapeutic advances. CD47, an immune checkpoint known as the &ldquo, don&rsquo, t eat me&rdquo, 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 &ldquo, 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 &ldquo, 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.

Cancer immunotherapy has advanced significantly in recent years due to its promising clinical outcomes in a variety of cancer malignancies and holds great promise in becoming the “cure” for cancer. Cancer immunotherapy is the treatment that stimulates a person’s own immune system to recognize, target, and eliminate cancer cells. As the field progresses with emerging and novel strategies, the ability to manipulate the immune system while mitigating toxicities becomes the hurdle for clinical translation. To control for both efficacy and safety, biomaterials have been incorporated into immunotherapies to achieve tissue- and/or cell-specific immunomodulation, overcome immunosuppression, and address tumor microenvironment heterogeneity. This chapter reviews different immune-based treatment modalities and how biomaterials (such as polymer scaffolds, nanoparticles, and engineered cells) are used to improve upon these major strategies. This chapter focuses on discussing examples of biomaterials in noncellular, artificial cellular, cellular, and gene-based immunotherapies, as well as their advantages and opportunities to create future options for cancer patients.

Introduction: Multiple myeloma (MM) is the cancer of plasma cells within the bone marrow (BM) and represents the second most common hematologic malignancy. Although therapeutic options have broadened over the years, the disease is challenged by frequent relapses. Relapsed/refractory MM (RRMM) often becomes non-responsive to previous lines of treatment and has significantly poorer survival outcome. Physicians are faced with a difficult task to choose a right treatment regimen. Thus, a precision medicine tool that predicts the clinical response of individual patients to therapy is greatly desired. We have previously developed a novel 3D tissue-engineered BM (3DTEBM) culture model, which is patient derived, closely recapitulates the pathophysiological conditions in the BM, and allows ex vivo proliferation of primary cells of various hematologic malignancies. In this study, we conducted a retrospective study that tests the ability of the ex vivo 3DTEBM platform to predict the clinical response in individual MM patients, to help decision-making process for RRMM. We hypothesized that the 3DTEBM will be able to predict clinical responses of RRMM patients. Methods: We first performed a literature search to examine the clinical efficacious concentrations (Css) for 10 MM drugs. We then experimentally determined the in vitro efficacious concentrations (IC50) of these drugs in MM cell lines, in both 2D and 3DTEBM cultures. The IC50 values were then correlated with their respective clinical Css values to evaluate how well each culture system reproduce drug efficacy. For the retrospective trial, we used viably frozen whole BM samples from 19 RRMM patients with known clinical responsiveness to the regimen they received. 3DTEBM cultures were developed for each patient with BM biopsies obtained prior to the start of clinical regimen. Cultures were treated ex vivo with the same treatment regimen each patient received clinically, at increasing concentrations (0X, 3X and 10X of Css of individual drugs). After 4 days, cultures were digested and cells were retrieved for flow cytometry analysis. Primary cells were stained Leukocyte-/CD38+ and counted against counting beads. Survival was determined as % of untreated control, and ex vivo responsiveness was analyzed by ANOVA. Finally, the clinical team correlated the ex vivo response with the clinical response for each patient. Results: To demonstrate this discrepancy between drug efficacy in laboratory settings and clinical outcomes, we compared the in vitro IC50 to the clinical Css of 10 drugs used for the treatment of MM. We found that there was no correlation between the IC50 in classic 2D culture systems and the clinical Css (R 2=0.019) (Figure 1A). In contrast, the IC50 in the 3DTEBM directly correlated with the clinical Css (R 2=0.993) (Figure 1B). We then conducted a retrospective clinical trial to determine if the 3DTEBM platform is able to predict each patient's clinical response by recreating the same treatment regimen ex vivo (Figure 1C). The 3DTEBM was able to predict the response in 89% of the MM patient cohort across multiple treatment regimens, with no false positives (Figure 1D). Conclusions: Our retrospective clinical trial demonstrated that the 3DTEBM technology is a feasible platform for predicting therapeutic responses in MM with a high predictive accuracy within a clinically actionable time frame. Such platforms can provide precise clinical insight about the efficacy of different treatment plans and assist physicians to propose the best choice of therapy for their individual patients. Future prospective studies are needed to validate these significant findings by testing prospective prediction ability of 3DTEBM to improve therapy response in hematologic malignancies. Figure 1 Figure 1. Disclosures De La Puente: Cellatrix LLC: Other: Co-founder. Azab: Cellatrix LLC: Current Employment. Vij: BMS: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; BMS: Honoraria; GSK: Honoraria; Oncopeptides: Honoraria; Karyopharm: Honoraria; CareDx: Honoraria; Legend: Honoraria; Biegene: Honoraria; Adaptive: Honoraria; Harpoon: Honoraria. Azab: Cellatrix, LLC: Current Employment, Current holder of individual stocks in a privately-held company.

Background CD138 has been the gold-standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug resistant minimal-residual disease (MRD) and circulating tumor cells (CTCs) were shown to have lower expression of this marker. We previously published that residual MM cells following treatment in vivo were hypoxic and the combination of hypoxia and chemotherapy such as bortezomib downregulated CD138 expression, thereby making this marker unsuitable for MM detection. Needless to say, accurate number of MM cells is critical in diagnosis, autologous transplantation, MRD and drug efficiency assessment. Moreover, CTCs are considered an unfavorable prognostic factor and indicate an aggressive form of the disease, and therefore detecting CTCs can be used as a powerful prognostic tool for MM. Methods We used an alternative biomarker-set using flow cytometry defining MM cells as any cell that expresses CD38 but excluding T cell (CD3), B cell (CD19), NK cell (CD16), monocyte/macrophage (CD14), neutrophil/eosinophil (CD16), and basophil/dendritic cell (CD123). We demonstrated previously that this approach it widely available due to accessibility of flow cytometry, inexpensive, and works independently of hypoxic-, CD138 expression- and treatment status. We analyzed primary patient samples (n=50) with complete response or very good partial response for MRD and CTCs and correlated the numbers of detected MM with patients’ time-to-progression (TTP) obtained from a clinical data base. Results We found that the alternative biomarker-set identifies MM cells more precisely and at higher numbers than CD138 marker by flow or histology. Moreover, we found a correlation between the number of MM cells detected and TTP in these patients: the amount of MM cells detected by the new method ranged between 0.5-7.3%, and patients who progressed sooner than two years had 2.5-fold higher percentage of MM cells compared to patients who relapsed later than 2 years. Similarly, patients who relapsed sooner than 3 years had 4-fold higher number of MM cells than patients who relapsed later than 3 years. We further found that, among all patients who had more than 2% of MM cells detected by the new method had a mean TPP of about 20 months, while patients who had less than 2% had a mean TPP of about 38 months. Testing the prevalence of CTCs in MM patients with progressive disease using CD138 or the new method, demonstrated that CD138 detected minimal amounts of MM cells in all patients (less than 0.1%), while the new method detected a range between 0.1 - 1.8% of MM cells in the peripheral blood. Conclusion These results suggest that the alternative biomarker-set detected MM cells which correlated with relapse in MM patients. Therefore, the amount of residual cells in the bone marrow and circulating myeloma cells can be used as a prognostic marker in MM patients. Further examination to characterize this population and its role in MM relapse is warranted.

Background Multiple myeloma (MM) is a cancer of plasma cells within the bone marrow (BM) and remains to be incurable. Tumor-associated macrophages (TAMs) are a major immunosuppressive component in the tumor microenvironment and display the pro-tumor M2 phenotype, supporting tumor proliferation, survival, and drug resistance. Targeting TAMs to block their pro-tumor functions represent a promising class of cancer immunotherapy. IL10 is a key immunosuppressive cytokine that leads to recruitment and development of TAMs. In this study, we investigate the role of IL10 in MM TAM development, and hypothesize that inhibition of IL10/IL10R signaling in TAMs will reprogram them for MM killing and overcome drug resistance. Methods Macrophage (MPP) polarization to M1 (CD80+) or M2 (CD163+) is determined by antibody staining and flow cytometry. In vitro and ex vivo studies were carried out in our lab’s proprietary 3D tissue-engineered BM model (3DTEBM). First, we compared TAM M2/M1 ratio as well as IL10 cytokine levels in the BM of healthy and MM subjects. To study the role of IL10 in TAM development, we co-cultured MΦ with increasing MM cells or rhIL10 in the 3DTEBM for 3 days and analyzed the resulting M2/M1 ratio. To determine the effect of IL10R inhibition, MM and MΦ co-cultures, or unsorted patient BM were treated with or without 5ug/ml α-IL10R monoclonal antibody (mAb), and TAM phenotype was analyzed after 3 days. Additionally, humanized huCD34-NCG mice were inoculated with MM cells and treated with α-IL10R mAb 3 times/week for 2 weeks; mice femurs were flushed and BM cells were analyzed for TAM phenotype. Finally, we investigated α-IL10R mAb as combination treatment with chemotherapy in MM-GFP and MΦ co-cultures; MM survival was determined by count of GFP+ cells and apoptosis was determined by Annexin/PI staining with flow cytometry. Results Compared to healthy subjects, M2/M1 ratio in MM patient TAMs were 3-fold higher, and BM IL10 level was 3.8-fold higher. Additionally, increasing MM cell ratio in co-cultures induced TAM polarization to M2, and was contributed by IL10. Importantly, α-IL10R mAb was able to reverse MM induced M2 TAM phenotype in vitro, ex vivo, and in vivo. Moreover, the presence of TAMs resulted in significant MM proliferation and chemotherapy resistance toward lenalidomide and dexamethasone. However, combination treatment with α-IL10R mAb completely reversed the drug resistance and abrogated cancer cells in vitro, partially due to induction of apoptosis in MM cells. Conclusions In summary, we have shown that MM induced M2 polarization of TAMs in an IL-10 dependent fashion, and disruption of IL10/IL10R signaling reversed M2 phenotype in TAMs, and overcame TAM-supported drug resistance. Future studies are warranted to examine mechanism behind overcoming drug resistance as well as the effect of α-IL10R immunotherapy in vivo and in patients.

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.

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.

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. Thermal sensitive liposomes (TSLs) were designed to have a stable drug payload at physiological temperatures but engineered to have high permeability under mild hyperthermia. 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. 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.

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.

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy localized in the bone marrow (BM) characterized by the continuous metastasis. Despite the introduction of novel therapies, MM patients relapse due to the development of drug resistance that is, at least in part, promoted by hypoxia (insufficient oxygen). MM cells develop a hypoxic phenotype, leading to cellular adaptations that cause metastasis, angiogenesis, stemness and resistance to drugs, such as carfilzomib, promoted by a hypoxia-inducible factor-1α (HIF-1α) transcription factor. Herein, we explored the mechanisms underlying HIF pathway inhibition using for the first time in MM a HIF-1α selective small molecule inhibitor, PX-478, both in vitro and in vivo. Methods: In vitro, to test the effect of PX-478 (0 - 50 µM) in combination with carfilzomib on MM cell survival exposed to normoxia (21% O2) or hypoxia (1% O2) was assessed using MTT assay. Cell adhesion to endothelial cells (HUVECs), and cell migration to stromal cells of prelabeled MM cells treated with PX-478 was measured by fluorescent spectrophotometer and flow cytometry, respectively. Tube-like formation of HUVECs as well as survival was tested in the presence of PX-478. For in vivo study, MM.1S-Luc-GFP cells were injected intravenously (i.v.) into 40 SCID mice; 3 weeks post injection the mice were divided into 4 groups and treated with vehicle (PBS), carfilzomib, PX-478, and a combination of PX-478 and carfilzomib. Tumor progression and weight was monitored weekly by bioluminescent imaging, and survival was monitored daily. At day 28, 3 mice from each group were randomly taken: (i) to test the number of circulating tumor cells (MM-GFP+) in the peripheral blood counted by flow cytometry; (ii) to test the MM apoptosis in the femurs by TUNEL staining; and (iii) to test extramedullar metastasis of MM in the kidney, spleen and the liver using immunohistochemistry. Additionally, tumor vasculature was demonstrated in the skull using photoacoustic imaging as well as tumor involvement using fluorescent microscopy. Moreover, we tested the drug delivery by injecting fluorescent large molecule (Dextran-AF405 Mw=70,000) i.v. in MM-bearing mice treated with and without PX-478. Lastly, we tested the effect of PX-478 on prelabeled MM cell retention in the blood and homing to the BM one hour post-MM injection in naïve mice. Results: We found that PX-478 reversed the hypoxia-induced resistance of MM cells to carfilzomib, inhibited metastasis-related cell processes such as adhesion and migration, and reduced MM-mediated tube-like formation of HUVECs in vitro. In vivo, in MM-bearing mice PX-478 decreased the number of MM circulating cells, suppressed tumor metastasis, improved vascularization of the tumor thus delivery of chemotherapy, and as a result re-sensitized MM cells to carfilzomib by increasing tumor apoptosis thus completely abrogating tumor growth and significantly extending mice survival. Conclusions: This is the first study to show the efficacy of PX-478 in MM demonstrating that PX-478 is acting as a pleiotropic molecule in which it inhibited many different hypoxia-induced biological processes - migration, angiogenesis and drug resistance. By overcoming these cancer adaptations, PX-478 has a clear advantage over using agents that carry an effect against one of these processes. This data provides a preclinical basis for future clinical trials testing efficacy of PX-478 in MM. Disclosures No relevant conflicts of interest to declare.

We have previously demonstrated that expression of the integrin β3 (β3) subunit is consistently increased in breast cancer (BC) bone metastases across clinical samples and preclinical models. Bone metastases frequently exhibit resistance to chemotherapy. Based on evidence that integrin β3 can function as a promoter of tumor survival and metastasis, we asked whether increased β3 signaling in bone metastases promotes chemoresistance. In established murine bone metastases, the proportion of integrin β3+ tumor cells was increased after docetaxel administration. Genetic deletion of β3 in either MMTV-PyMT-derived BC cells (BO1-FL-GFP, modeling luminal B disease) or 4T1 BC cells (4T1-FL-GFP, modeling triple-negative disease) yielded bone metastases with increased sensitivity to docetaxel treatment in vivo. Retroviral rescue of β3 expression in β3-/- BO1-FL-GFP cells by a signaling-competent human integrin β3 construct (hβ3) restored relative docetaxel chemoresistance in bone metastases. By contrast, expression of a signaling-deficient mutant β3 (Δβ3) failed to restore chemoresistance, suggesting that signaling through β3 is critical to its capacity to promote docetaxel chemoresistance in bone-residing BC cells. Mechanistically, RNAseq analysis of docetaxel response in β3-/- and hβ3-rescued cells revealed β3-mediated enrichment in transcriptional pathways associated with oxidative phosphorylation and metabolism. Direct imaging of mitochondria by super-resolution microscopy and extracellular flux analysis of oxygen consumption rate further confirmed an alternative metabolic response to docetaxel in resistant, β3-expressing tumor cells. mTORC1 plays an important role in mitochondrial biogenesis and cellular metabolism, and can be activated downstream of integrin signaling in certain contexts. Based on transcriptomic data identifying enhanced mTORC1 activity as a possible mediator of β3-dependent metabolic changes, we asked whether mTOR inhibition could restore chemosensitivity in BC bone metastases. In mice bearing chemoresistant, β3-WT metastases, combination of the mTORC1 inhibitor rapamycin with docetaxel yielded synergistic, site-specific attenuation of bone tumor burden. Taken together, our data 1) establish integrin β3 as a mediator of chemoresistance in breast cancer bone metastases, 2) demonstrate a mechanistic link between integrin β3 expression and an alternative metabolic response to docetaxel in resistant cells, and 3) suggest mTORC1 inhibition as a candidate for rational combination with chemotherapy to interrupt treatment resistance in breast cancer bone metastases. Citation Format: Gregory C. Fox, Michael H. Ross, Xinming Su, Yalin Xu, Alison Esser, Elizabeth Cordell, Elizabeth Wilson, Barbara Muz, Ha Dang, Christopher A. Maher, Abdel Kareem Azab, Deborah Veis, Samuel Achilefu, Gregory M. Lanza, Katherine N. Weilbaecher. Integrin beta-3 signaling links chemoresistance and mitochondrial metabolism in breast cancer bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2613.

Introduction: Endothelial progenitor cells (EPCs) are a subtype of stem cells capable of differentiating into mature endothelial cells. Tumor-derived paracrine signals activate the bone marrow-residing EPCs to home to the tumor to promote angiogenesis. The aim of this study was to utilize the EPCs and their specific pathophysiologic homing to tumors as a biologically targeted drug-delivery system, as a novel “Trojan horse” cellular immunotherapy in cancer. Methods: To test the specificity of EPCs’ homing to cancer, chemotaxis of EPCs towards conditioned media derived from a series of cancer cell lines or towards nonconditioned media was performed in vitro using Boyden Chamber. In vivo biodistribution of EPCs was studied in diverse tumor models (localized and disseminated) of different cancers (myeloma, lymphoma, leukemia, breast, lung, pancreatic and glioma) after injecting EPCs intravenously, by analysis of the accumulation of EPCs in the tumors and other organs. To enhance the ability of EPCs to home to tumors, EPCs were “primed” in vitro by exposing them to conditioned media from hypoxic (1% O2) tumor cells, followed by testing the homing of “primed” EPCs to tumors, both in vitro and in vivo. Next, EPCs were loaded with LS-542 (NIR imaging dye), and with Titanocene (Tc) (a nontoxic compound used in phototherapy that can be activated by positrons to become toxic) and tested for the effect of the loading on viability and functionality of EPCs, using MTT and migration assays, respectively. Also, we tested the effect of Tc-loaded EPCs on killing of cancer cells when activated by 18FDG/PET. Results: We found that EPCs specifically homed to tumors in vitro and in vivo, in tumor size-dependent and time-dependent manner, and that EPCs selectively accumulated in tumor tissues, with significantly negligible amounts in other organs. Moreover, EPCs homed to primary tumors and followed the metastatic pattern of the tumor. Hypoxia “priming” of EPCs significantly improved their homing to cancer in vitro and in vivo. Loading EPCs with LS-542 or with Tc did not affect their viability or functionality. Additionally, Tc-loaded EPCs, when activated by 18FDG, induced a dose-dependent killing of cancer cells. Conclusions: EPCs have a highly specific and efficient biologic machinery to target tumors in as a pan-cancer phenomenon, especially when “primed” with hypoxic tumor media. Loading EPCs with LS-542 or Tc did not change their viability and functionality; thus, both are promising molecules for imaging and therapy of cancer. These results provide a proof of concept that EPCs can be used as a novel cellular immunotherapy for specific and efficacious delivery of theranostic agents in cancer. Also, unlike engineered/targeted immunotherapies such as cellular (CAR-T cells), bispecific T cell engagers, or monoclonal antibodies that target specific molecules on a specific tumor type, EPCs can be used as a pan-cancer “Trojan horse” drug-delivery platform due to the universality of (angiogenesis) in all cancer types. Citation Format: Barbara Muz, Pilar de la Puente, Shruti Shah, Cinzia Federico, Feda Azab, Justin King, Mark Fiala, Rui Tang, Partha Karmakar, Ravi Vij, Samuel Achilefu, Abdel Kareem Azab. Endothelial progenitor cells as drug-delivery Trojan horses for treatment and imaging of cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A27.

Introduction: CD47 is an immune checkpoint highly expressed on the surface of various cancer cells, allowing cells to escape innate immune response by sending inhibitory signals to macrophages, which further hinders the antitumor response by the adaptive immune system. This CD47 “don’t-eat-me signal” has been identified as a novel therapeutic target in cancers. Targeted reprogramming of this interaction using anti-CD47 monoclonal antibodies (mAbs) represents a new class of checkpoint inhibitors that attack tumors via coordinating the innate and adaptive immune systems. Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow (BM). The objective of this study is to investigate the inhibition of CD47 using anti-CD47 mAbs as a novel checkpoint immunotherapy for the treatment of MM. Methods: We performed analysis of CD47 mRNA expression in healthy, MGUS (pre-condition of MM), and MM subjects. We measured CD47 protein expression on MM cell lines by flow cytometry, labeling MM cells with Vx1000R (anti-human CD47 mAb). We also investigated the effect of hypoxia (1% O2), stromal cells, and 3D tissue engineered bone marrow (3DTEBM) cultures on CD47 expression in MM cells. Next, we tested the cytotoxic activity of Vx1000R on MM cells alone by MTT assay. Additionally, we investigated the effect of Vx1000R on phagocytosis and killing of MM cells (GFP+) by primary macrophages (labeled with DiD dye) at 4 and 24 hours with or without Vx1000R, in 2D cultures and in 3DTEBM cultures. Phagocytosis was determined as GFP+ DiD+ cells and MM survival was analyzed by counting GFP+ by flow cytometry. Moreover, we performed real-time live confocal imaging on 3DTEBM cultures of GFP-MM cells and DiD-macrophages, with or without treatment of Vx1000R, to further visualize the effect of Vx1000R. Results: CD47 gene expression was increased with MM progression (healthy Conclusions: Myeloma cells express high levels of CD47, and this expression is not affected by hypoxia or TME in 2D, but significantly increases in 3DTEBM cultures. Blocking CD47 on MM cells with anti-CD47 mAbs enhanced MM phagocytosis and killing by macrophages, especially in 3DTEBM. These results suggest that anti-CD47 mAbs are applicable for MM treatment, and further studies are warranted to examine the effect of anti-CD47 mAbs as a novel checkpoint immunotherapy to target MM in vivo and in patients. This abstract is also being presented as Poster A88. Citation Format: Jennifer Sun, Barbara Muz, Kinan Alhallak, Christopher Egbulefu, Justin King, Danny Kohnen, Mark Fiala, Benjamin Cappocia, Pamela Manning, Samuel Achilefu, Ravi Vij, Abdel Kareem Azab. Targeting CD47 as a novel immunotherapy for multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR06.

Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Chimeric antigen receptor (CAR)-T cells and bispecific T-cell engagers targeting BCMA, CS1, and CD38 are promising approaches for MM. However, using these targets individually limits the efficacy of these immunotherapy options due to the variable expression levels of these markers, leading to inefficient eradication of MM. Studies have attempted to develop CAR-T cells and T-cell engagers with multispecific targeting; however, these are still technically very challenging and are highly expensive. In this study, we developed nanoparticles with monovalent or multivalent specificity against MM targets and engage T cells, by chemical conjugation of monoclonal antibodies against each molecule to the surface of the liposome. Bi-specific nanoparticles with anti CD38/CD3, BCMA/CD3 or CS1/CD3 were termed nanoBiTEs, while the multispecific particles with anti BCMA/CS1/CD38/CD3 were termed nanoMuTEs. Methods: Liposomes were prepared using the thin-film hydration method followed by extrusion. nanoBiTEs and nanoMuTEs were developed by chemical conjugation of monoclonal antibodies against BCMA, CS1, and/or CD38; together with anti CD3 onto the liposomes. We have tested the expression of BCMA, CS1, and CD38 antigens in MM cell lines and primary MM patient samples, and we analyzed the binding of the CD38/CD3, BCMA/CD3, and CS1/CD3 nanoBiTEs, as well as the BCMA/CS1/CD38/CD3 nanoMuTEs to these cells by flow cytometry and confocal microscopy. Binding specificity was assessed by blocking the target with corresponding free antibodies. CD25 expression, a T-cell activation marker, was assessed for CD8 and CD4 T cells when incubated with or without nanoBiTEs and nanoMuTEs. Moreover, we tested the efficacy of the nanoBiTEs and nanoMuTEs against MM cells in vitro using the 3D tissue engineered bone marrow (3DTEBM) myeloma model. Finally, we tested the efficacy of the nanoBiTEs and nanoMuTE against MM tumors in vivo in xenograft MM models. Results: The expression of BCMA, CS1 and CD38 was variable in MM cell lines and patient samples, and so was the binding of each nanoBiTEs, while the binding of the nanoMuTE was higher and more universal than each nanoBiTE. Similar patterns were observed in activation of CD8 T cells (no significant activation was observed in CD4 T cells) and in eradication of MM cell lines and primary patient samples in vitro. In vivo, the nanoBiTEs extended the survival and reduced the tumor burden in the MM bearing mice compared to untreated and T cell alone groups, and nanoMuTE experiments are still ongoing. Conclusions: The nanoBiTEs and nanoMuTEs were bound specifically to MM cells and initiated CD8 T-cell activation that resulted in MM eradication in vitro and in vivo, while the effect of the nanoMuTEs was more universal and efficacious than each nanoBiTE. These nanoparticles are an inexpensive method of engaging T cells for the treatment of MM and represent a new class of immunotherapy that can be expanded to other tumors. Citation Format: Kinan Alhallak, Jennifer Sun, Barbara Muz, Christopher Egbulefu, Pilar de la Puente, Cinzia Federico, Justin King, Danny Kohnen, Ravi Vij, Samuel Achilefu, Abdel Kareem Azab. Nanoparticle multispecific T-cell engagers for the treatment of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B01.

In cancer research, regions of increasingly lowered oxygenation in tissue (hypoxia), which are due to tumor development, are considered to play an important role in activating various signaling pathways that facilitate further development of the cancer. However, devising a minimally invasive method to monitor tissue oxygenation has remained a challenge. Photoacoustic microscopy has been posed as a solution in a variety of preclinical research studies. Here, using optical-resolution photoacoustic microscopy (OR-PAM), for the first time, we non-invasively measured oxygenation and vascularization in vivo caused by multiple myeloma (MM) progression. Mice injected with MM cells tagged with green fluorescent protein were monitored with a fluorescence microscope for tumor progression over the course of 28 days. OR-PAM evaluated the oxygen saturation (sO2) and the blood vessel density in the cerebral bone marrow, where MM cells home. At 28 days after the injection of MM cells, the total sO2 had dropped by 50% in the developing tumor regions, while in the non-tumor developing regions it had dropped by 20% compared with the value at one day after MM injection. The blood vessel density had dropped by 35% in the tumor developing regions, while in the non-tumor developing regions it had dropped by 8% compared with the value at one day after MM injection. In summary, non-invasive measurement by OR-PAM correlated the development of hypoxia with to MM progression. It revealed decreased vascularization surrounding the tumor areas, which we feel can be ascribed to the rapid tumor progression.

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.

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.

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.

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.

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.