Downregulation of Manic fringe impedes angiogenesis and cell migration of renal carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a highly angiogenic cancer. Manic fringe (MFng) is elevated in ccRCC compared to the normal kidney. However, its role in ccRCC tumour angiogenesis remains elusive. This study seeks to determine the expression pattern of MFng in ccRCC blood vessels and its role in angiogenesis. The association between MFng and the blood vessels was established through online compendia, immunohistochemistry and qPCR analyses. The anti-angiogenic potential of lentiviral-mediated MFng knockdown in endothelial cells (EC shMFng) was assessed for viability, proliferation, apoptosis, migration, adhesion, cell cycle, vessel sprouting, and molecular expression of adhesion and apoptosis markers. Finally, EC shMFng were co-cultured with 786-0 renal cancer cells to determine their impact on cancer cell migration. The online dataset analyses and immunostaining on ccRCC tissues revealed high expression of MFng in ECs. MFng and CD31/PECAM-1 genes were up-regulated in ccRCC tissue samples compared to normal kidney tissues. EC shMFng demonstrated decreased cell viability due to G1 cell cycle arrest and reduced Ki-67 protein expression. In addition, shMFng down-regulated endothelial adhesion molecules and hindered EC migration, network formation and sprouting, compared to their respective empty vector (EV) controls. Co-culture assay of EC shMFng with 786-0 renal cancer cells inhibited cancer cell migration. These findings underscore the potential role of MFng in ECs in influencing renal cancer cell migration, thus opening an avenue for anti-angiogenic strategy targeting MFng to treat ccRCC.

ICOS-Positive Regulatory T Cells in Hepatocellular Carcinoma: The Perspective from Digital Pathology Analysis.

INTRODUCTION: Inducible co-stimulator (ICOS), an important co-stimulatory receptor on effector T cells (Teffs), may also contribute to tumor growth due to its high expression on regulatory T cells (Tregs). This study explored the clinical significance of ICOS-expressing Tregs in hepatocellular carcinoma (HCC). METHODS: Tumor tissues from HCC patients who received curative hepatectomy were obtained at a referral center. Dual immunohistochemistry was performed to evaluate the expression of ICOS and Foxp3. The cell densities and proximities between stained cells in regions of interest were measured by digital pathology and the associations with clinical outcome were analyzed. RESULTS: A total of 142 patients (male:female = 112: 30, median age of 61.0 years) were enrolled. Among them, 87 (61.3%) had chronic hepatitis B virus infection and 33 (23.2%) had chronic hepatitis C infection. Low α-fetoprotein level (<20 ng/mL) and early-stage were significantly associated with improved overall survival (OS). The density of ICOS+Foxp3+ cells and the ratio of ICOS+Foxp3+/total Foxp3+ cells were significantly higher (p < 0.001) in the tumor center than in the peritumor area. Patients with a high density of ICOS+Foxp3+ cells or a high ratio of ICOS+Foxp3+/total Foxp3+ cells in the tumor center trended to have a shorter OS. A shorter distance between ICOS+Foxp3+ cells and ICOS+Foxp3- cells (likely Teffs) in the tumor center was significantly associated with a shorter OS (p = 0.030), suggesting active immunosuppression of ICOS+ Tregs on ICOS+ Teffs. CONCLUSION: An increased abundance of ICOS+ Tregs in the tumor center in comparison to the peritumor area indicates a strong immunosuppressive tumor microenvironment of HCC. A high proportion of ICOS+Foxp3+ cells and a shorter distance between ICOS+ Tregs and other ICOS+ cells were associated with a poor OS, suggesting that depleting ICOS+ Tregs might provide clinical benefit for patients with HCC.

In utero ultrafine particulate matter exposure causes offspring pulmonary immunosuppression.

Early life exposure to fine particulate matter (PM) in air is associated with infant respiratory disease and childhood asthma, but limited epidemiological data exist concerning the impacts of ultrafine particles (UFPs) on the etiology of childhood respiratory disease. Specifically, the role of UFPs in amplifying Th2- and/or Th17-driven inflammation (asthma promotion) or suppressing effector T cells (increased susceptibility to respiratory infection) remains unclear. Using a mouse model of in utero UFP exposure, we determined early immunological responses to house dust mite (HDM) allergen in offspring challenged from 0 to 4 wk of age. Two mice strains were exposed throughout gestation: C57BL/6 (sensitive to oxidative stress) and BALB/C (sensitive to allergen exposure). Offspring exposed to UFPs in utero exhibited reduced inflammatory response to HDM. Compared with filtered air (FA)-exposed/HDM-challenged mice, UFP-exposed offspring had lower white blood cell counts in bronchoalveolar lavage fluid and less pronounced peribronchiolar inflammation in both strains, albeit more apparent in C57BL/6 mice. In the C57BL/6 strain, offspring exposed in utero to FA and challenged with HDM exhibited a robust response in inflammatory cytokines IL-13 and Il-17. In contrast, this response was lost in offspring exposed in utero to UFPs. Circulating IL-10 was significantly up-regulated in C57BL/6 offspring exposed to UFPs, suggesting increased regulatory T cell expression and suppressed Th2/Th17 response. Our results reveal that in utero UFP exposure at a level close to the WHO recommended PM guideline suppresses an early immune response to HDM allergen, likely predisposing neonates to respiratory infection and altering long-term pulmonary health.

Potential sea-level rise from Antarctic ice-sheet instability constrained by observations.

Large parts of the Antarctic ice sheet lying on bedrock below sea level may be vulnerable to marine-ice-sheet instability (MISI), a self-sustaining retreat of the grounding line triggered by oceanic or atmospheric changes. There is growing evidence that MISI may be underway throughout the Amundsen Sea embayment (ASE), which contains ice equivalent to more than a metre of global sea-level rise. If triggered in other regions, the centennial to millennial contribution could be several metres. Physically plausible projections are challenging: numerical models with sufficient spatial resolution to simulate grounding-line processes have been too computationally expensive to generate large ensembles for uncertainty assessment, and lower-resolution model projections rely on parameterizations that are only loosely constrained by present day changes. Here we project that the Antarctic ice sheet will contribute up to 30 cm sea-level equivalent by 2100 and 72 cm by 2200 (95% quantiles) where the ASE dominates. Our process-based, statistical approach gives skewed and complex probability distributions (single mode, 10 cm, at 2100; two modes, 49 cm and 6 cm, at 2200). The dependence of sliding on basal friction is a key unknown: nonlinear relationships favour higher contributions. Results are conditional on assessments of MISI risk on the basis of projected triggers under the climate scenario A1B (ref. 9), although sensitivity to these is limited by theoretical and topographical constraints on the rate and extent of ice loss. We find that contributions are restricted by a combination of these constraints, calibration with success in simulating observed ASE losses, and low assessed risk in some basins. Our assessment suggests that upper-bound estimates from low-resolution models and physical arguments (up to a metre by 2100 and around one and a half by 2200) are implausible under current understanding of physical mechanisms and potential triggers.

Recent Antarctic Peninsula warming relative to Holocene climate and ice-shelf history.

Rapid warming over the past 50 years on the Antarctic Peninsula is associated with the collapse of a number of ice shelves and accelerating glacier mass loss. In contrast, warming has been comparatively modest over West Antarctica and significant changes have not been observed over most of East Antarctica, suggesting that the ice-core palaeoclimate records available from these areas may not be representative of the climate history of the Antarctic Peninsula. Here we show that the Antarctic Peninsula experienced an early-Holocene warm period followed by stable temperatures, from about 9,200 to 2,500 years ago, that were similar to modern-day levels. Our temperature estimates are based on an ice-core record of deuterium variations from James Ross Island, off the northeastern tip of the Antarctic Peninsula. We find that the late-Holocene development of ice shelves near James Ross Island was coincident with pronounced cooling from 2,500 to 600 years ago. This cooling was part of a millennial-scale climate excursion with opposing anomalies on the eastern and western sides of the Antarctic Peninsula. Although warming of the northeastern Antarctic Peninsula began around 600 years ago, the high rate of warming over the past century is unusual (but not unprecedented) in the context of natural climate variability over the past two millennia. The connection shown here between past temperature and ice-shelf stability suggests that warming for several centuries rendered ice shelves on the northeastern Antarctic Peninsula vulnerable to collapse. Continued warming to temperatures that now exceed the stable conditions of most of the Holocene epoch is likely to cause ice-shelf instability to encroach farther southward along the Antarctic Peninsula.

Regulation of the anti-tumour immune response by cancer-associated fibroblasts.

The microenvironment of established tumours is often immunosuppressed, and this allows tumours to grow and disseminate without being eliminated by the patient's immune system. The recent FDA approval of immunotherapies such as ipilimumab and sipuleucel-T that directly activate the adaptive and innate immune responses has triggered interest in developing other novel anti-cancer approaches that modulate the immune system. Understanding how the different constituents of the tumour microenvironment influence the immune system is thus crucial and is expected to generate a plethora of factors that can be targeted to boost immunity and trigger long lasting anti-tumour efficacy. Cancer associated fibroblasts (CAFs) are a crucial component of the tumour microenvironment. Through secretion of multiple growth factors, cytokines and proteases, CAFs are known to be key effectors for tumour progression and can promote cancer cell growth, invasiveness and angiogenesis. However, recent publications have also linked CAF biology to innate and adaptive immune cell recruitment and regulation. Here, we review recent findings on how CAFs can influence the immune status of tumours through direct and indirect interaction with immune cells and other key components of the tumour microenvironment.

A Phase 1 First-in-Human Study of FS118, a Tetravalent Bispecific Antibody Targeting LAG-3 and PD-L1 in Patients with Advanced Cancer and PD-L1 Resistance.

PURPOSE: This phase 1 study (NCT03440437) evaluated the safety, tolerability, pharmacokinetics (PK), and activity of FS118, a bispecific antibody-targeting LAG-3 and PD-L1, in patients with advanced cancer resistant to anti-PD-(L)1 therapy. PATIENTS AND METHODS: Patients with solid tumors, refractory to anti-PD-(L)1-based therapy, received intravenous FS118 weekly with an accelerated dose titration design (800 µg to 0.3 mg/kg) followed by 3+3 ascending dose expansion (1 to 20 mg/kg). Primary objectives were safety, tolerability, and PK. Additional endpoints included antitumor activity, immunogenicity, and pharmacodynamics. RESULTS: Forty-three patients with a median of three prior regimens in the locally advanced/metastatic setting, including at least one anti-PD-(L)1 regimen, received FS118 monotherapy. FS118 was well tolerated, with no serious adverse events relating to FS118 reported. No dose-limiting toxicities (DLT) were observed, and an MTD was not reached. The recommended phase 2 dose of FS118 was established as 10 mg/kg weekly. The terminal half-life was 3.9 days. Immunogenicity was transient. Pharmacodynamic activity was prolonged throughout dosing as demonstrated by sustained elevation of soluble LAG-3 and increased peripheral effector cells. The overall disease control rate (DCR) was 46.5%; this disease control was observed as stable disease, except for one late partial response. Disease control of 54.8% was observed in patients receiving 1 mg/kg or greater who had acquired resistance to PD-(L)1-targeted therapy. CONCLUSIONS: FS118 was well tolerated with no DLTs observed up to and including 20 mg/kg QW. Further studies are warranted to determine clinical benefit in patients who have become refractory to anti-PD-(L)1 therapy. See related commentary by Karapetyan and Luke, p. 835.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.

Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes as thrombin inhibitors.

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.

TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype.

Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial "tip cell" phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFkappaB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFkappaB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression.

The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells (Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOShigh T cells but was also associated with increased secretion of proinflammatory cytokines from ICOSlow effector T cells (Teff). In syngeneic mouse tumor models, KY1044 depleted ICOShigh Treg and increased the intratumoral TEff:Treg ratio, resulting in increased secretion of IFNγ and TNFα by TEff cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.

Anti-Dll4 therapy: can we block tumour growth by increasing angiogenesis?

Since the early 1970s, the dogma postulating that blocking tumour angiogenesis can inhibit tumour growth has been accepted widely and has resulted in the generation of a variety of successful anti-angiogenic therapies. More recently, new signalling pathways, such as the Dll4-Notch signalling pathway, have been shown to regulate angiogenesis during development. In pathological conditions, such as cancer, Dll4 is up-regulated strongly in the tumour vasculature. Based on this expression pattern, different molecules have been generated to block Dll4 signalling. Unexpectedly, these blocking agents inhibited tumour growth in vivo by triggering excessive but nonfunctional angiogenesis. Altogether, these molecules constitute a new category of pro-angiogenic yet anticancer agents and offer an exciting alternative to previously described vascular targeting molecules.

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 2: N-acetamidoimidazoles.

Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.

Development and validation of a method for fipronil residue determination in ovine plasma using 96-well plate solid-phase extraction and gas chromatography-tandem mass spectrometry.

Fipronil, a phenylpyrazole insecticide introduced for pest control on a broad range of crops, undergoes a reinforcement of the regulation within the European Union (2007/52/EC directive) due to its potential effects on environment and human health. In order to assess the plasmatic concentrations of fipronil residues (sulfone, sulfide, fipronil, desulfinyl and amide) in ovine, a methodology based on gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was developed and validated according to the European standard (2002/657/EC). The proposed method allows a large number of samples to be treated concurrently (n=80) using a reduced sample amounts (0.2 mL), and consents to reach a level of quantification of 0.1 pg microL(-1). The sample preparation consisted of a single solid-phase extraction (SPE) purification on a 96-well plate filled with a styrene-divinyl-benzene phase. Linearity was demonstrated all along the investigated range of concentrations, i.e. from 0.25 to 2000 pg microL(-1), with coefficient of determination (R(2)) from 0.977 to 0.994, depending on target analytes. Calculated decision limit (CCalpha) and detection capability (CCbeta) for fipronil, sulfone and sulphide were in the range 0.05-0.16 and 0.28-0.73 pg microL(-1) respectively.

Hypoxia-regulated differentiation: let's step it up a Notch.

Although hypoxia is widely associated with adult pathologies such as cancer, it is also a physiological process that regulates cell differentiation during organogenesis. In an attempt to characterize the molecular mechanisms that are involved in hypoxia-regulated cell fate, a recent publication by Gustafsson and colleagues elegantly demonstrated that hypoxia blocks cell differentiation through the regulation of Notch signalling. This study showed that hypoxia-inducible factor (HIF)-1alpha interacts and acts in synergy with the Notch intracellular domain (NIC) and subsequently activates transcription of Notch targets. The identification of this crosstalk between pathways that are often deregulated in cancer is groundbreaking and opens up new areas for cancer research.

Analytical challenges in the confirmative identification of dipyrone as an adulterant in illicit drug samples.

Dipyrone is an analgesic and antipyretic drug that is sometimes encountered as an adulterant in illicit drug samples, particularly illicit fentanyl containing samples. It undergoes thermal decomposition to aminopyrine and 4-methylaminoantipyrine during analysis via gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). During analysis via high pressure liquid chromatography (HPLC) and high pressure liquid chromatography-mass spectrometry (HPLC-MS), it undergoes hydrolytic decomposition solely to 4-methylaminoantipyrine. Given that mass spectrometry is a widely used confirmatory analytical technique, these instabilities present challenges for the forensic chemist seeking to confirm the presence of dipyrone. Studies were conducted to determine rigorous confirmative protocols for the identification of dipyrone in multicomponent illicit drug samples.

Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth.

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.

Optoacoustic Detection of Early Therapy-Induced Tumor Cell Death Using a Targeted Imaging Agent.

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU).Experimental Design: C2Am was labeled with a NIR fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU.Results: Three-dimensional (3D) MSOT images of probe distribution showed development of tumor contrast within 3 hours of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 hours. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated (R2 = 0.97, P value < 0.01) with a marker of apoptotic cell death measured in histologic sections obtained post mortem.Conclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic. Clin Cancer Res; 23(22); 6893-903. ©2017 AACR.

Rational Selection of Syngeneic Preclinical Tumor Models for Immunotherapeutic Drug Discovery.

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.