Agonist Antibodies for Cancer Immunotherapy: History, Hopes, and Challenges.

Immunotherapy is among the most promising new treatment modalities to arise over the last two decades; antibody drugs are delivering immunotherapy to millions of patients with many different types of cancer. Initial success with antibody therapeutics came in the form of direct targeting or cytotoxic antibodies, such as rituximab and trastuzumab, which bind directly to tumor cells to elicit their destruction. These were followed by immunomodulatory antibodies that elicit antitumor responses by either stimulating immune cells or relieving tumor-mediated suppression. By far the most successful approach in the clinic to date has been relieving immune suppression, with immune checkpoint blockade now a standard approach in the treatment of many cancer types. Despite equivalent and sometimes even more impressive effects in preclinical models, agonist antibodies designed to stimulate the immune system have lagged behind in their clinical translation. In this review, we document the main receptors that have been targeted by agonist antibodies, consider the various approaches that have been evaluated to date, detail what we have learned, and consider how their anticancer potential can be unlocked.

The role of three-dimensional in vitro models in modelling the inflammatory microenvironment associated with obesity in breast cancer.

Obesity is an established risk factor for breast cancer in postmenopausal women. However, the underlying biological mechanisms of how obesity contributes to breast cancer remains unclear. The inflammatory adipose microenvironment is central to breast cancer progression and has been shown to favour breast cancer cell growth and to reduce efficacy of anti-cancer treatments. Thus, it is imperative to further our understanding of the inflammatory microenvironment seen in breast cancer patients with obesity. Three-dimensional (3D) in vitro models offer a key tool in increasing our understanding of such complex interactions within the adipose microenvironment. This review discusses some of the approaches utilised to recapitulate the breast tumour microenvironment, including various co-culture and 3D in vitro models. We consider how these model systems contribute to the understanding of breast cancer research, with particular focus on the inflammatory tumour microenvironment. This review aims to provide insight and prospective future directions on the utility of such model systems for breast cancer research.

Serum immunoglobulin and the threshold of Fc receptor-mediated immune activation.

Antibodies can mediate immune recruitment or clearance of immune complexes through the interaction of their Fc domain with cellular Fc receptors. Clustering of antibodies is a key step in generating sufficient avidity for efficacious receptor recognition. However, Fc receptors may be saturated with prevailing, endogenous serum immunoglobulin and this raises the threshold by which cellular receptors can be productively engaged. Here, we review the factors controlling serum IgG levels in both healthy and disease states, and discuss how the presence of endogenous IgG is encoded into the functional activation thresholds for low- and high-affinity Fc receptors. We discuss the circumstances where antibody engineering can help overcome these physiological limitations of therapeutic antibodies. Finally, we discuss how the pharmacological control of Fc receptor saturation by endogenous IgG is emerging as a feasible mechanism for the enhancement of antibody therapeutics.

Obesity Is Associated with Immunometabolic Changes in Adipose Tissue That May Drive Treatment Resistance in Breast Cancer: Immune-Metabolic Reprogramming and Novel Therapeutic Strategies.

White adipose tissue (WAT) represents an endocrinologically and immunologically active tissue whose primary role is energy storage and homeostasis. Breast WAT is involved in the secretion of hormones and proinflammatory molecules that are associated with breast cancer development and progression. The role of adiposity and systemic inflammation in immune responses and resistance to anti-cancer treatment in breast cancer (BC) patients is still not clear. Metformin has demonstrated antitumorigenic properties both in pre-clinical and clinical studies. Nevertheless, its immunomodulating properties in BC are largely unknown. This review aims to evaluate the emerging evidence on the crosstalk between adiposity and the immune-tumour microenvironment in BC, its progression and treatment resistance, and the immunometabolic role of metformin in BC. Adiposity, and by extension subclinical inflammation, are associated with metabolic dysfunction and changes in the immune-tumour microenvironment in BC. In oestrogen receptor positive (ER+) breast tumours, it is proposed that these changes are mediated via a paracrine interaction between macrophages and preadipocytes, leading to elevated aromatase expression and secretion of pro-inflammatory cytokines and adipokines in the breast tissue in patients who are obese or overweight. In HER2+ breast tumours, WAT inflammation has been shown to be associated with resistance to trastuzumab mediated via MAPK or PI3K pathways. Furthermore, adipose tissue in patients with obesity is associated with upregulation of immune checkpoints on T-cells that is partially mediated via immunomodulatory effects of leptin and has been paradoxically associated with improved responses to immunotherapy in several cancers. Metformin may play a role in the metabolic reprogramming of tumour-infiltrating immune cells that are dysregulated by systemic inflammation. In conclusion, evidence suggests that body composition and metabolic status are associated with patient outcomes. To optimise patient stratification and personalisation of treatment, prospective studies are required to evaluate the role of body composition and metabolic parameters in metabolic immune reprogramming with and without immunotherapy in patients with BC.

Tri-Lineage Differentiation Potential of Osteosarcoma Cell Lines and Human Bone Marrow Stromal Cells from Different Anatomical Locations.

The bone cancer osteosarcoma, found mainly in adolescents, routinely forms around the growth plate/metaphysis of long bones. Bone marrow composition changes with age, shifting from a more hematopoietic to an adipocyte-rich tissue. This conversion occurs in the metaphysis during adolescence, implicating a link between bone marrow conversion and osteosarcoma initiation. To assess this, the tri-lineage differentiation potential of human bone marrow stromal cells (HBMSCs) isolated from the femoral diaphysis/metaphysis (FD) and epiphysis (FE) was characterized and compared to two osteosarcoma cell lines, Saos-2 and MG63. Compared to FE-cells, FD-cells showed an increase in tri-lineage differentiation. Additionally, differences were found between the Saos-2 cells exhibiting higher levels of osteogenic differentiation, lower adipogenic differentiation, and a more developed chondrogenic phenotype than MG63, with the Saos-2 being more comparable to FD-derived HBMSCs. The differences found between the FD and FE derived cells are consistent with the FD region containing more hematopoietic tissue compared to the FE. This may be related to the similarities between FD-derived cells and Saos-2 cells during osteogenic and chondrogenic differentiation. These studies reveal distinct differences in the tri-lineage differentiations of 'hematopoietic' and 'adipocyte rich' bone marrow, which correlate with specific characteristics of the two osteosarcoma cell lines.

ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance.

Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Cyclophosphamide depletes tumor infiltrating T regulatory cells and combined with anti-PD-1 therapy improves survival in murine neuroblastoma.

The outcome for children with high-risk neuroblastoma is poor despite intensive multi-modal treatment protocols. Toxicity from current treatments is significant, and novel approaches are needed to improve outcome. Cyclophosphamide (CPM) is a key component of current chemotherapy regimens and is known to have immunomodulatory effects. However, this has not been investigated in the context of tumor infiltrating lymphocytes in neuroblastoma. Using murine models of neuroblastoma, the immunomodulatory effects of low-dose CPM were investigated using detailed immunophenotyping. We demonstrated that CPM resulted in a specific depletion of intratumoral T regulatory cells by apoptosis, and when combined with anti-PD-1 antibody therapy, this resulted in improved therapeutic efficacy. CPM combined with anti-PD-1 therapy was demonstrated to be an effective combinational therapy, with metronomic CPM found to be more effective than single dosing in more resistant tumor models. Overall, this pre-clinical data strongly support clinical evaluation of such combination strategies in neuroblastoma.

FcγRIIB controls antibody-mediated target cell depletion by ITIM-independent mechanisms.

Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.

Prognostic significance of crown-like structures to trastuzumab response in patients with primary invasive HER2 + breast carcinoma.

Obesity can initiate, promote, and maintain systemic inflammation via metabolic reprogramming of macrophages that encircle adipocytes, termed crown-like structures (CLS). In breast cancer the presence of CLS has been correlated to high body mass index (BMI), larger mammary adipocyte size and postmenopausal status. However, the prognostic significance of CLS in HER2 + breast cancer is still unknown. We investigated the prognostic significance of CLS in a cohort of 69 trastuzumab-naïve and 117 adjuvant trastuzumab-treated patients with primary HER2 + breast cancer. Immunohistochemistry of tumour blocks was performed for CLS and correlated to clinical outcomes. CLS were more commonly found at the adipose-tumour border (B-CLS) (64.8% of patients). The presence of multiple B-CLS was associated with reduced time to metastatic disease (TMD) in trastuzumab treated patients with BMI ≥ 25 kg/m2 but not those with BMI < 25 kg/m2. Phenotypic analysis showed the presence of CD32B + B-CLS was strongly correlated to BMI ≥ 25 kg/m2 and reduced TMD in trastuzumab treated patients. Multivariable analysis suggested that CD32B + B-CLS positive tumours are associated with shorter TMD in trastuzumab-treated patients (HR 4.2 [95%CI, (1.01-17.4). This study indicates adipose-tumour border crown-like structures that are CD32B + potentially represent a biomarker for improved personalisation of treatment in HER2-overexpressed breast cancer patients.

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy.

BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering.

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

Fc-null anti-PD-1 monoclonal antibodies deliver optimal checkpoint blockade in diverse immune environments.

BACKGROUND: Despite extensive clinical use, the mechanisms that lead to therapeutic resistance to anti-programmed cell-death (PD)-1 monoclonal antibodies (mAbs) remain elusive. Here, we sought to determine how interactions between the Fc region of anti-PD-1 mAbs and Fcγ receptors (FcγRs) affect therapeutic activity and how these are impacted by the immune environment. METHODS: Mouse and human anti-PD-1 mAbs with different Fc binding profiles were generated and characterized in vitro. The ability of these mAbs to elicit T-cell responses in vivo was first assessed in a vaccination setting using the model antigen ovalbumin. The antitumor activity of anti-PD-1 mAbs was investigated in the context of immune 'hot' MC38 versus 'cold' neuroblastoma tumor models, and flow cytometry performed to assess immune infiltration. RESULTS: Engagement of activating FcγRs by anti-PD-1 mAbs led to depletion of activated CD8 T cells in vitro and in vivo, abrogating therapeutic activity. Importantly, the extent of this Fc-mediated modulation was determined by the surrounding immune environment. Low FcγR-engaging mouse anti-PD-1 isotypes, which are frequently used as surrogates for human mAbs, were unable to expand ovalbumin-reactive CD8 T cells, in contrast to Fc-null mAbs. These results were recapitulated in mice expressing human FcγRs, in which clinically relevant hIgG4 anti-PD-1 led to reduced endogenous expansion of CD8 T cells compared with its engineered Fc-null counterpart. In the context of an immunologically 'hot' tumor however, both low-engaging and Fc-null mAbs induced long-term antitumor immunity in MC38-bearing mice. Finally, a similar anti-PD-1 isotype hierarchy was demonstrated in the less responsive 'cold' 9464D neuroblastoma model, where the most effective mAbs were able to delay tumor growth but could not induce long-term protection. CONCLUSIONS: Our data collectively support a critical role for Fc:FcγR interactions in inhibiting immune responses to both mouse and human anti-PD-1 mAbs, and highlight the context-dependent effect that anti-PD-1 mAb isotypes can have on T-cell responses. We propose that engineering of Fc-null anti-PD-1 mAbs would prevent FcγR-mediated resistance in vivo and allow maximal T-cell stimulation independent of the immunological environment.

Remodeling the Tumor Myeloid Landscape to Enhance Antitumor Antibody Immunotherapies.

Among the diverse tumor resident immune cell types, tumor-associated macrophages (TAMs) are often the most abundant, possess an anti-inflammatory phenotype, orchestrate tumor immune evasion and are frequently associated with poor prognosis. However, TAMs can also be harnessed to destroy antibody-opsonized tumor cells through the process of antibody-dependent cellular phagocytosis (ADCP). Clinically important tumor-targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing macrophages to eliminate tumor cells via ADCP. For IgG mAb, this is mediated by antibody-binding activating Fc gamma receptors (FcγR), with resultant phagocytic activity impacted by the level of co-engagement with the single inhibitory FcγRIIb. Approaches to enhance ADCP in the tumor microenvironment include the repolarization of TAMs to proinflammatory phenotypes or the direct augmentation of ADCP by targeting so-called 'phagocytosis checkpoints'. Here we review the most promising new strategies targeting the cell surface molecules present on TAMs, which include the inhibition of 'don't eat me signals' or targeting immunostimulatory pathways with agonistic mAb and small molecules to augment tumor-targeting mAb immunotherapies and overcome therapeutic resistance.

On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation.

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of 'on-target hexamerisation', whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC.

Prognostic significance of FCGR2B expression for the response of DLBCL patients to rituximab or obinutuzumab treatment.

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP.

Activation of GABA(A) receptors inhibits T cell proliferation.

BACKGROUND: The major sites for fast synaptic inhibition in the central nervous system (CNS) are ion channels activated by γ-aminobutyric acid (GABA). These receptors are referred as GABA(A) receptors (GABA(A)R). Recent evidence indicates a role of GABA(A)R in modulating the immune response. This work aimed to discern the role of GABA and GABA(A)Rs in human and mouse T cell activity. METHODS: Mouse splenocytes or human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 antibodies and the proliferation of both CD8+ and CD4+ T cells assessed through flow cytometry. Subsequently, the effects on T cell proliferation of either GABA(A)R modulation by diazepam that is also capable of activating mitochondrial based translocator protein (TSPO), alprazolam and allopregnanolone or inhibition by bicucculine methiodide (BMI) and (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) were assessed. RESULTS: Positive modulation of GABA(A)Rs either by benzodiazepines or the neurosteroid allopregnanolone inhibits both mouse and human T cell proliferation. GABAergic inhibition of T cell proliferation by benzodiazepines could be rescued by GABA(A)R blocking. Our data suggest that benzodiazepines influence T cell proliferation through both TSPO and GABA(A)Rs activation. CONCLUSIONS: We conclude that activation of GABA(A)Rs provides immunosuppression by inhibiting T cell proliferation.

Fc-Engineering for Modulated Effector Functions-Improving Antibodies for Cancer Treatment.

The majority of monoclonal antibody (mAb) therapeutics possess the ability to engage innate immune effectors through interactions mediated by their fragment crystallizable (Fc) domain. By delivering Fc-Fc gamma receptor (FcγR) and Fc-C1q interactions, mAb are able to link exquisite specificity to powerful cellular and complement-mediated effector functions. Fc interactions can also facilitate enhanced target clustering to evoke potent receptor signaling. These observations have driven decades-long research to delineate the properties within the Fc that elicit these various activities, identifying key amino acid residues and elucidating the important role of glycosylation. They have also fostered a growing interest in Fc-engineering whereby this knowledge is exploited to modulate Fc effector function to suit specific mechanisms of action and therapeutic purposes. In this review, we document the insight that has been generated through the study of the Fc domain; revealing the underpinning structure-function relationships and how the Fc has been engineered to produce an increasing number of antibodies that are appearing in the clinic with augmented abilities to treat cancer.

Immune characterization of pre-clinical murine models of neuroblastoma.

Immunotherapy offers a potentially less toxic, more tumor-specific treatment for neuroblastoma than conventional cytotoxic therapies. Accurate and reproducible immune competent preclinical models are key to understanding mechanisms of action, interactions with other therapies and mechanisms of resistance to immunotherapy. Here we characterized the tumor and splenic microenvironment of two syngeneic subcutaneous (NXS2 and 9464D), and a spontaneous transgenic (TH-MYCN) murine model of neuroblastoma, comparing histological features and immune infiltrates to previously published data on human neuroblastoma. Histological sections of frozen tissues were stained by immunohistochemistry and immunofluorescence for immune cell markers and tumor architecture. Tissues were dissociated by enzymatic digestion, stained with panels of antibodies to detect and quantify cancer cells, along with lymphocytic and myeloid infiltration by flow cytometry. Finally, we tested TH-MYCN mice as a feasible model for immunotherapy, using prior treatment with cyclophosphamide to create a therapeutic window of minimal residual disease to favor host immune development. Immune infiltration differed significantly between all the models. TH-MYCN tumors were found to resemble immune infiltration in human tumors more closely than the subcutaneous models, alongside similar GD2 and MHC class I expression. Finally, TH-MYCN transgenic mice were administered cyclophosphamide alone or in combination with an anti-GD2 or anti-4-1BB monoclonal antibody, which resulted in increase in survival in both combination therapies. The TH-MYCN transgenic mouse is a promising in vivo model for testing immunotherapy compounds and combination therapy in a preclinical setting.

The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma.

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

Developing a 3D B Cell Lymphoma Culture System to Model Antibody Therapy.

Diffuse large cell B cell lymphoma (DLBCL) accounts for approximately 30%-40% of all non-Hodgkin lymphoma (NHL) cases. Current first line DLBCL treatment results in long-term remission in more than 60% of cases. However, those patients with primary refractory disease or early relapse exhibit poor prognosis, highlighting a requirement for alternative therapies. Our aim was to develop a novel model of DLBCL that facilitates in vitro testing of current and novel therapies by replicating key components of the tumor microenvironment (TME) in a three-dimensional (3D) culture system that would enable primary DLBCL cell survival and study ex vivo. The TME is a complex ecosystem, comprising malignant and non-malignant cells, including cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAM) whose reciprocal crosstalk drives tumor initiation and growth while fostering an immunosuppressive milieu enabling its persistence. The requirement to recapitulate, at least to some degree, this complex, interactive network is exemplified by the rapid cell death of primary DLBCL cells removed from their TME and cultured alone in vitro. Building on previously described methodologies to generate lymphoid-like fibroblasts from adipocyte derived stem cells (ADSC), we confirmed lymphocytes, specifically B cells, interacted with this ADSC-derived stroma, in the presence or absence of monocyte-derived macrophages (MDM), in both two-dimensional (2D) cultures and a 3D collagen-based spheroid system. Furthermore, we demonstrated that DLBCL cells cultured in this system interact with its constituent components, resulting in their improved viability as compared to ex-vivo 2D monocultures. We then assessed the utility of this system as a platform to study therapeutics in the context of antibody-directed phagocytosis, using rituximab as a model immunotherapeutic antibody. Overall, we describe a novel 3D spheroid co-culture system comprising key components of the DLBCL TME with the potential to serve as a testbed for novel therapeutics, targeting key cellular constituents of the TME, such as CAF and/or TAM.