Nanobugs as Drugs: Bacterial Derived Nanomagnets Enhance Tumor Targeting and Oncolytic Activity of HSV-1 Virus.

The survival strategies of infectious organisms have inspired many therapeutics over the years. Indeed the advent of oncolytic viruses (OVs) exploits the uncontrolled replication of cancer cells for production of their progeny resulting in a cancer-targeting treatment that leaves healthy cells unharmed. Their success against inaccessible tumors however, is highly variable due to inadequate tumor targeting following systemic administration. Coassembling herpes simplex virus (HSV1716) with biocompatible magnetic nanoparticles derived from magnetotactic bacteria enables tumor targeting from circulation with magnetic guidance, protects the virus against neutralizing antibodies and thereby enhances viral replication within tumors. This approach additionally enhances the intratumoral recruitment of activated immune cells, promotes antitumor immunity and immune cell death, thereby inducing tumor shrinkage and increasing survival in a syngeneic mouse model of breast cancer by 50%. Exploiting the properties of such a nanocarrier, rather than tropism of the virus, for active tumor targeting offers an exciting, novel approach for enhancing the bioavailability and treatment efficacy of tumor immunotherapies for disseminated neoplasms.

Targeting circulating monocytes with CCL2-loaded liposomes armed with an oncolytic adenovirus.

Oncolytic viruses (OVs) selectively replicate in and destroy cancer cells resulting in anti-tumor immunity. However, clinical use remains a challenge because of virus clearance upon intravenous delivery. OV packaging using a nanomedicine approach could overcome this. Here we encapsulate an oncolytic adenovirus (Ad[I/PPT-E1A]) into CCL2-coated liposomes in order to exploit recruitment of CCR2-expressing circulating monocytes into tumors. We demonstrate successful encapsulation of Ad[I/PPT-E1A] into CCL2-coated liposomes that were preferentially taken up by CCR2-expressing monocytes. No complex-related toxicities were observed following incubation with prostate tumor cells and the encapsulation did not affect virus oncolytic activity in vitro. Furthermore, intravenous administration of our nanomedicine resulted in a significant reduction in tumor size and pulmonary metastasis in prostate cancer-bearing mice whereby a 1000-fold less virus was needed compared to Ad[I/PPT-E1A] alone. Taken together our data provide an opportunity to target OVs via circulation to inaccessible tumors using liposome-assisted drug delivery.

The Autoimmune Susceptibility Gene C5orf30 Regulates Macrophage-Mediated Resolution of Inflammation.

Genetic variants in C5orf30 have been associated with development of the autoimmune conditions primary biliary cirrhosis and rheumatoid arthritis. In rheumatoid arthritis, C5orf30 expression is cell-specific, with highest expression found in macrophages and synovial fibroblasts. C5orf30 is highly expressed in inflamed joints and is a negative regulator of tissue damage in a mouse model of inflammatory arthritis. Transcriptomic analysis from ultrasound-guided synovial biopsy of inflamed joints in a well characterized clinical cohort of newly diagnosed, disease-modifying antirheumatic drugs-naive rheumatoid arthritis patients was used to determine the clinical association of C5orf30 expression with disease activity. A combined molecular and computational biology approach was used to elucidate C5orf30 function in macrophages both in vitro and in vivo. Synovial expression of C5orf30 is inversely correlated with both clinical measures of rheumatoid arthritis disease activity and with synovial TNF mRNA expression. C5orf30 plays a role in regulating macrophage phenotype and is differentially turned over in inflammatory and anti-inflammatory macrophages. Inhibition of C5orf30 reduces wound healing/repair-associated functions of macrophages, reduces signaling required for resolution of inflammation, and decreases secretion of anti-inflammatory mediators. In an animal model of wound healing (zebrafish), C5orf30 inhibition increases the recruitment of macrophages to the wound site. Finally, we demonstrate that C5orf30 skews macrophage immunometabolism, demonstrating a mechanism for C5orf30-mediated immune regulation.

Cell-Based Tracers as Trojan Horses for Image-Guided Surgery.

Surgeons rely almost completely on their own vision and palpation to recognize affected tissues during surgery. Consequently, they are often unable to distinguish between different cells and tissue types. This makes accurate and complete resection cumbersome. Targeted image-guided surgery (IGS) provides a solution by enabling real-time tissue recognition. Most current targeting agents (tracers) consist of antibodies or peptides equipped with a radiolabel for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), magnetic resonance imaging (MRI) labels, or a near-infrared fluorescent (NIRF) dye. These tracers are preoperatively administered to patients, home in on targeted cells or tissues, and are visualized in the operating room via dedicated imaging systems. Instead of using these 'passive' tracers, there are other, more 'active' approaches of probe delivery conceivable by using living cells (macrophages/monocytes, neutrophils, T cells, mesenchymal stromal cells), cell(-derived) fragments (platelets, extracellular vesicles (exosomes)), and microorganisms (bacteria, viruses) or, alternatively, 'humanized' nanoparticles. Compared with current tracers, these active contrast agents might be more efficient for the specific targeting of tumors or other pathological tissues (e.g., atherosclerotic plaques). This review provides an overview of the arsenal of possibilities applicable for the concept of cell-based tracers for IGS.

Specific Targeting of PEGylated Liposomal Doxorubicin (Doxil®) to Tumour Cells Using a Novel TIMP3 Peptide.

Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers.

C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis.

The variant rs26232, in the first intron of the chromosome 5 open reading frame 30 (C5orf30) locus, has recently been associated with both risk of developing rheumatoid arthritis (RA) and severity of tissue damage. The biological activities of human C5orf30 are unknown, and neither the gene nor protein show significant homology to any other characterized human sequences. The C5orf30 gene is present only in vertebrate genomes with a high degree of conservation, implying a central function in these organisms. Here, we report that C5orf30 is highly expressed in the synovium of RA patients compared with control synovial tissue, and that it is predominately expressed by synovial fibroblast (RASF) and macrophages in the lining and sublining layer of the tissue. These cells play a central role in the initiation and perpetuation of RA and are implicated in cartilage destruction. RASFs lacking C5orf30 exhibit increased cell migration and invasion in vitro, and gene profiling following C5orf30 inhibition confirmed up-regulation of genes involved in cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, because inhibition of C5orf30 in the collagen-induced arthritis model markedly accentuated joint inflammation and tissue damage. Our study reveal C5orf30 to be a previously unidentified negative regulator of tissue damage in RA, and this protein may act by modulating the autoaggressive phenotype that is characteristic of RASFs.

Histone deacetylase 1 regulates tissue destruction in rheumatoid arthritis.

Emerging evidence implicates epigenetic mechanisms in the pathogenesis of rheumatoid arthritis (RA). In this study, we have investigated the role of histone deacetylase (HDAC) enzymes in RA synovial fibroblasts (RASFs), a key cellular mediator of cartilage and bone destruction and determined effects of HDAC1 inhibition on both RASF phenotype in vitro, and joint inflammation and damage in the collagen-induced arthritis (CIA) model. Expression of HDACs 1-11 messenger ribonucleic acid (mRNA) was compared between RASFs and osteoarthritic synovial fibroblast (OASFs) using quantitative polymerase chain reaction. HDAC1 expression in RASFs was inhibited using small interfering RNA (siRNA) technology to assess effects on invasiveness, migration, proliferation and apoptosis. Effects of HDAC1 knockdown (KD) on the transcriptome were assessed using gene microarrays. The effects of siRNA-mediated HDAC(KD) on clinical scores, tissue inflammation and damage were assessed on CIA up to 47 days following immunization. Expression of HDAC1 was significantly higher in RASFs than OASFs. HDAC1(KD) resulted in reduced proliferation, invasion and migration in vitro and transcriptome profiling revealed effects on expression of genes regulating proliferation migration and inflammation. Furthermore, inhibition of HDAC1 in CIA resulted in reduced joint swelling, cartilage and bone damage and lower tumor necrosis factor in joint tissue. These results implicate HDAC1 as an important mediator of tissue damage in RA and support the potential therapeutic utility of inhibitors of this enzyme.

A peptide derived from TIMP-3 inhibits multiple angiogenic growth factor receptors and tumour growth and inflammatory arthritis in mice.

The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the surface of vascular endothelial cells stimulates many steps in the angiogenic pathway. Inhibition of this interaction is proving of value in moderating the neovascularization accompanying age-related macular degeneration and in the treatment of cancer. Tissue inhibitor of metalloproteinases-3 (TIMP-3) has been shown to be a natural VEGFR-2 specific antagonist-an activity that is independent of its ability to inhibit metalloproteinases. In this investigation we localize this activity to the C-terminal domain of the TIMP-3 molecule and characterize a short peptide, corresponding to part of this domain, that not only inhibits all three VEGF-family receptors, but also fibroblast growth factor and platelet-derived growth factor receptors. This multiple-receptor inhibition may explain why the peptide was also seen to be a powerful inhibitor of tumour growth and also a partial inhibitor of arthritic joint inflammation in vivo.

In-vitro assays for immuno-oncology drug efficacy assessment and screening for personalized cancer therapy: scopes and challenges.

INTRODUCTION: Immunotherapies have revolutionized cancer treatment, but often fail to produce desirable therapeutic outcomes in all patients. Due to the inter-patient heterogeneity and complexity of the tumor microenvironment, personalized treatment approaches are gaining demand. Researchers have long been using a range of in-vitro assays including 2D models, organoid co-cultures, and cancer-on-a-chip platforms for cancer drug screening. A comparative analysis of these assays with their suitability, high-throughput capacity, and clinical translatability is required for optimal translational use. AREAS COVERED: The review summarized in-vitro platforms with their comparative advantages and limitations including construction strategies, and translational potential for immuno-oncology drug efficacy assessment. We also discussed end-point analysis strategies so that researchers can contextualize their usefulness and optimally design experiments for personalized immunotherapy efficacy prediction. EXPERT OPINION: Researchers developed several in-vitro platforms that can provide information on personalized immunotherapy efficacy from different angles. Image-based assays are undoubtedly more suitable to gather a wide range of information including cellular morphology and phenotypical behaviors but need significant improvement to overcome issues including background noise, sample preparation difficulty, and long duration of experiment. More studies and clinical trials are needed to resolve these issues and validate the assays before they can be used in real-life scenarios.

The role of histone deacetylases in rheumatoid arthritis fibroblast-like synoviocytes.

RA (rheumatoid arthritis) is an inflammatory disease of synovial joints affecting approximately 1% of the population. One of the main cell types involved in damage to RA joint tissue is the FLSs (fibroblast-like synoviocytes). These have a semi-transformed, auto-aggressive phenotype typified by loss of contact inhibition, reduced apoptosis and the production of matrix-degrading enzymes. The mechanisms involved in the development of this phenotype are unclear; however, increasing evidence implicates alterations in the epigenetic regulation of gene expression. Reduced acetylation of amino acids in the tails of histone proteins is an epigenetic mark associated with transcriptional repression and is controlled by the HDAC (histone deacetylase) enzyme family. To date, evidence has implicated HDACs in the auto-aggressive phenotype of FLSs, and administration of HDAC inhibitors to both animal models of RA and individuals with juvenile arthritis has shown efficacy in attenuating inflammation and tissue damage. This highlights a role for HDACs in disease pathogenesis and, more importantly, that HDACs are potential novel therapeutic targets.

Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.

The dual immunoregulatory roles of stress proteins.

Stress proteins (SPs) from the heat shock and glucose-regulated protein families are abundant intracellular molecules that have powerful extracellular roles as immune modulators. Mammalian immune cells encounter both identical (self) SPs and non-identical SPs derived from invading pathogens. Although such extracellular SPs can function as powerful immunological adjuvants, SPs, including Hsp60 and Hsp70, can also attenuate inflammatory disease via apparent effects on immunoregulatory T cell populations. It therefore seems that the immunostimulatory and immunosuppressive potential of extracellular SPs depends on the context in which they are encountered by the cellular immune-response network. Conclusions regarding the immunobiology of these powerful immunomodulatory molecules must therefore take into account their dichotomous properties and their physiological role and importance must be interpreted in the context of the complex in vivo microenvironments in which these proteins exist.

Exercise to transform tumours from cold to hot and improve immunotherapy responsiveness.

Exercise provides significant health benefits to patients diagnosed with cancer including improved survival outcomes, quality of life and reduced cancer recurrence. Across multiple murine cancer models, aerobic exercise and resistance training has exhibited anti-tumour properties illustrated by inhibited tumour growth, reduced metastatic potential and modulation of the tumour microenvironment to allow the recognition and destruction of cancer cells. Clinical studies have demonstrated the rapid mobilisation and circulatory release of mature lymphoid populations, myokines and cytokines that occurs with exercise along with tumour vasculature normalisation. Tumour microenvironments enriched with immune cells with anti-cancer potential, such as CD8+ T cells, are termed 'hot', whilst those favouring an immunosuppressive environment and lacking in effector immune cells are classed as 'cold'. Pre-clinical evidence suggests exercise training has the potential to reprogramme cold tumours to become hot, although this requires validation in clinical studies. This hot environment could potentiate immunotherapy responsiveness, improving survival outcomes of patients undergoing cancer immunotherapy and allow those with typically cold tumours to benefit from immunotherapy. This review discusses the complex interactions between exercise and cancer, including exercise-induced alterations within the tumour microenvironment and systemic immunity. The potential role exercise may play in improving cancer immunotherapy responsiveness is explored. This review also highlights the need for translational studies exploring the role of exercise in patients with cancer with the potential to widen the spectrum of tumours that derive significant benefit from immunotherapy.

Microfluidic-Assisted ZIF-Silk-Polydopamine Nanoparticles as Promising Drug Carriers for Breast Cancer Therapy.

Metal-organic frameworks (MOFs) are heralded as potential nanoplatforms for biomedical applications. Zeolitic imidazolate framework-8 (ZIF-8), as one of the most well known MOFs, has been widely applied as a drug delivery carrier for cancer therapy. However, the application of ZIF-8 nanoparticles as a therapeutic agent has been hindered by the challenge of how to control the release behaviour of anti-cancer zinc ions to cancer cells. In this paper, we designed microfluidic-assisted core-shell ZIF-8 nanoparticles modified with silk fibroin (SF) and polydopamine (PDA) for sustained release of zinc ions and curcumin (CUR) and tested these in vitro in various human breast cancer cells. We report that microfluidic rapid mixing is an efficient method to precisely control the proportion of ZIF-8, SF, PDA, and CUR in the nanoparticles by simply adjusting total flow rates (from 1 to 50 mL/min) and flow rate ratios. Owing to sufficient and rapid mixing during microfluidic-assisted nanoprecipitation, our designer CUR@ZIF-SF-PDA nanoparticles had a desired particle size of 170 nm with a narrow size distribution (PDI: 0.08), which is much smaller than nanoparticles produced using traditional magnetic stirrer mixing method (over 1000 nm). Moreover, a properly coated SF layer successfully enhanced the capability of ZIF-8 as a reservoir of zinc ions. Meanwhile, the self-etching reaction between ZIF-8 and PDA naturally induced a pH-responsive release of zinc ions and CUR to a therapeutic level in the MDA-MB-231, SK-BR-3, and MCF-7 breast cancer cell lines, resulting in a high cellular uptake efficiency, cytotoxicity, and cell cycle arrest. More importantly, the high biocompatibility of designed CUR@ZIF-SF-PDA nanoparticles remained low in cytotoxicity on AD-293 non-cancer cells. We demonstrate the potential of prepared CUR@ZIF-SF-PDA nanoparticles as promising carriers for the controlled release of CUR and zinc ions in breast cancer therapy.

HSV1716 Prevents Myeloma Cell Regrowth When Combined with Bortezomib In Vitro and Significantly Reduces Systemic Tumor Growth in Mouse Models.

Multiple myeloma remains largely incurable due to refractory disease; therefore, novel treatment strategies that are safe and well-tolerated are required. Here, we studied the modified herpes simplex virus HSV1716 (SEPREHVIR®), which only replicates in transformed cells. Myeloma cell lines and primary patient cells were infected with HSV1716 and assessed for cell death using propidium iodide (PI) and Annexin-V staining and markers of apoptosis and autophagy by qPCR. Myeloma cell death was associated with dual PI and Annexin-V positivity and increased expression of apoptotic genes, including CASP1, CASP8, CASP9, BAX, BID, and FASL. The combination of HSV1716 and bortezomib treatments prevented myeloma cell regrowth for up to 25 days compared to only transient cell growth suppression with bortezomib treatment. The viral efficacy was tested in a xenograft (JJN-3 cells in NSG mice) and syngeneic (murine 5TGM1 cells in C57BL/KaLwRijHsd mice) systemic models of myeloma. After 6 or 7 days, the post-tumor implantation mice were treated intravenously with the vehicle or HSV1716 (1 × 107 plaque forming units/1 or 2 times per week). Both murine models treated with HSV1716 had significantly lower tumor burden rates compared to the controls. In conclusion, HSV1716 has potent anti-myeloma effects and may represent a novel therapy for multiple myeloma.

Development of a Personalised Device for Systemic Magnetic Drug Targeting to Brain Tumours.

Delivering therapies to deeply seated brain tumours (BT) is a major clinical challenge. Magnetic drug targeting (MDT) could overcome this by rapidly transporting magnetised drugs directly into BT. We have developed a magnetic device for application in murine BT models using an array of neodymium magnets with a combined strength of 0.7T. In a closed fluidic system, the magnetic device trapped magnetic nanoparticles (MNP) up to distances of 0.8cm. In mice, the magnetic device guided intravenously administered MNP (<50nm) from the circulation into the brain where they localised within mouse BT. Furthermore, MDT of magnetised Temozolomide (TMZmag+) significantly reduced tumour growth and extended mouse survival to 48 days compared to the other treatment groups. Using the same principles, we built a proof of principle scalable magnetic device for human use with a strength of 1.1T. This magnetic device demonstrated trapping of MNP undergoing flow at distances up to 5cm. MDT using our magnetic device provides an opportunity for targeted delivery of magnetised drugs to human BT.

Inconsistencies in Modeling the Efficacy of the Oncolytic Virus HSV1716 Reveal Potential Predictive Biomarkers for Tolerability.

Treatment with HSV1716 via intralesional administration has proven successful for melanoma patients with the hope that oncolytic virotherapy would become another weapon in the systemic anticancer therapy (SACT) arsenal. In addition to challenges surrounding the systemic delivery of oncolytic viruses (OVs), problems associated with its in vivo modeling have resulted in low predictive power, contributing to the observed disappointing clinical efficacy. As OV's efficacy is elicited through interaction with the immune system, syngeneic orthotopic mouse models offer the opportunity to study these with high reproducibility and at a lower cost; however, inbred animals display specific immune characteristics which may confound results. The systemic delivery of HSV1716 was, therefore, assessed in multiple murine models of breast cancer. Tolerability to the virus was strain-dependent with C57/Bl6, the most tolerant and Balb/c experiencing lethal side effects, when delivered intravenously. Maximum tolerated doses were not enough to demonstrate efficacy against tumor growth rates or survival of Balb/c and FVB mouse models; therefore; the most susceptible strain (Balb/c mice) was treated with immunomodulators prior to virus administration in an attempt to reduce side effects. These studies demonstrate the number of variables to consider when modeling the efficacy of OVs and the complexities involved in their interpretation for translational purposes. By reporting these observations, we have potentially revealed a role for T-cell helper polarization in viral tolerability. Importantly, these findings were translated to human studies, whereby a Th1 cytokine profile was expressed in pleural effusions of patients that responded to HSV1716 treatment for malignant pleural mesothelioma with minimal side effects, warranting further investigation as a biomarker for predictive response.

Norovirus: An Overview of Virology and Preventative Measures.

Norovirus (NoV) is an enteric non-enveloped virus which is the leading cause of gastroenteritis across all age groups. It is responsible for around 200,000 deaths annually and outbreaks are common in small communities such as educational and care facilities. 40% of all NoV outbreaks occur in long-term and acute-care facilities, forming the majority of outbreaks. Nosocomial settings set ideal environments for ease of transmission, especially due to the presence of immunocompromised groups. It is estimated to cost global economies around £48 billion a year, making it a global issue. NoV is transmitted via the faecal-oral route and infection with it results in asymptomatic cases or gastrointestinal disease. It has high mutational rates and this allows for new variants to emerge and be more resistant. The classification system available divides NoV into 10 genogroups and 49 genotypes based on whole amino acid sequencing of VP1 capsid protein and partial sequencing of RdRp, respectively. The most predominant genotypes which cause gastroenteritis in humans include GI.1 and GII.4, where GII.4 is responsible for more extreme clinical implications such as hospitalisation. In addition, GII.4 has been responsible for 6 pandemic strains, the last of which is the GII.4 Sydney (2012) variant. In recent years, the successful cultivation of HuNoV was reported in stem cell-derived human intestinal enteroids (HIEs), which promises to assist in giving a deeper understanding of its underlying mechanisms of infection and the development of more personalized control measures. There are no specific control measures against NoV, therefore common practices are used against it such as hand washing. No vaccine is available, but the HIL-214 candidate passed clinical phase 2b and shows promise.

Understanding Immune Responses to Viruses-Do Underlying Th1/Th2 Cell Biases Predict Outcome?

Emerging and re-emerging viral diseases have increased in number and geographical extent during the last decades. Examples include the current COVID-19 pandemic and the recent epidemics of the Chikungunya, Ebola, and Zika viruses. Immune responses to viruses have been well-characterised within the innate and adaptive immunity pathways with the outcome following viral infection predominantly attributed to properties of the virus and circumstances of the infection. Perhaps the belief that the immune system is often considered as a reactive component of host defence, springing into action when a threat is detected, has contributed to a poorer understanding of the inherent differences in an individual's immune system in the absence of any pathology. In this review, we focus on how these host factors (age, ethnicity, underlying pathologies) may skew the T helper cell response, thereby influencing the outcome following viral infection but also whether we can use these inherent biases to predict patients at risk of a deviant response and apply strategies to avoid or overcome them.

TRIB1 regulates tumor growth via controlling tumor-associated macrophage phenotypes and is associated with breast cancer survival and treatment response.

Molecular mechanisms that regulate tumor-associated macrophage (TAM) phenotype and function are incompletely understood. The pseudokinase TRIB1 has been reported as a regulator of macrophage phenotypes, both in mouse and human systems. Methods: Bioinformatic analysis was used to investigate the link between TRIB1 expression in breast cancer and therapeutic response to chemotherapy. In vivo models of breast cancer included immune-competent mice to characterize the consequences of altered (reduced or elevated) myeloid Trib1 expression on tumor growth and composition of stromal immune cell populations. Results: TRIB1 was highly expressed by TAMs in breast cancer and high TRIB1 expression correlated with response to chemotherapy and patient survival. Both overexpression and knockout of myeloid Trib1 promote mouse breast tumor growth, albeit through different molecular mechanisms. Myeloid Trib1 deficiency led to an early acceleration of tumor growth, paired with a selective reduction in perivascular macrophage numbers in vivo and enhanced oncogenic cytokine expression in vitro. In contrast, elevated levels of Trib1 in myeloid cells led to an increased late-stage mammary tumor volume, coupled with a reduction of NOS2 expressing macrophages and an overall reduction of macrophages in hypoxic tumor regions. In addition, we show that myeloid Trib1 is a previously unknown, negative regulator of the anti-tumor cytokine IL-15, and that increased myeloid Trib1 expression leads to reduced IL-15 levels in mammary tumors, with a consequent reduction in the number of T-cells that are key to anti-tumor immune responses. Conclusions: Together, these results define a key role for TRIB1 in chemotherapy responses for human breast cancer and provide a mechanistic understanding for the importance of the control of myeloid TRIB1 expression in the development of this disease.