Bimodal protein expression, characterized by the distribution of protein expression with two modes, is linked to phenotypic variation across various biological systems. Whereas previous studies focused on RNA expression data, we developed a bimodality model tailored for proteomics to enhance the identification of cancer-associated biomarkers and targets, facilitating precision oncology. We analyzed proteomics data from various cancer types and identified 2401 tumor-associated bimodal proteins. These proteins were evaluated for pathway enrichment, revealing significant associations with critical cancer pathways, such as metabolism of non-essential amino acids, interaction between the extracellular matrix and its receptors on the cell surface, and central carbon metabolism in cancer. Utilizing an AI-enhanced knowledge graph, we further delineated common patterns among pan-cancer tumor-associated bimodal proteins. A case study on the bimodal expression of TROP2 in colon adenocarcinoma highlighted upregulation of MYC and WNT/β-catenin signaling pathways and down-regulation of inflammatory and interferon-related pathways in the TROP2-high group. The biological difference between TROP2-high and TROP2-low groups underscored its significance in determining cancer heterogeneity and differences in cancer vulnerability, which can inform treatment decisions. Our findings show the value of proteomics in uncovering novel biomarkers and advancing precision medicine, setting a precedent for further multi-omics integration and clinical validation. ### Competing Interest Statement The authors have declared no competing interest.

Background: Vocal biomarker-based machine learning approaches have shown promising results in the detection of various health conditions, including respiratory diseases, such as asthma. Objective: This study aimed to determine whether a respiratory-responsive vocal biomarker (RRVB) model platform initially trained on an asthma and healthy volunteer (HV) data set can differentiate patients with active COVID-19 infection from asymptomatic HVs by assessing its sensitivity, specificity, and odds ratio (OR). Methods: A logistic regression model using a weighted sum of voice acoustic features was previously trained and validated on a data set of approximately 1700 patients with a confirmed asthma diagnosis and a similar number of healthy controls. The same model has shown generalizability to patients with chronic obstructive pulmonary disease, interstitial lung disease, and cough. In this study, 497 participants (female: n=268, 53.9%; <65 years old: n=467, 94%; Marathi speakers: n=253, 50.9%; English speakers: n=223, 44.9%; Spanish speakers: n=25, 5%) were enrolled across 4 clinical sites in the United States and India and provided voice samples and symptom reports on their personal smartphones. The participants included patients who are symptomatic COVID-19 positive and negative as well as asymptomatic HVs. The RRVB model performance was assessed by comparing it with the clinical diagnosis of COVID-19 confirmed by reverse transcriptase-polymerase chain reaction. Results: The ability of the RRVB model to differentiate patients with respiratory conditions from healthy controls was previously demonstrated on validation data in asthma, chronic obstructive pulmonary disease, interstitial lung disease, and cough, with ORs of 4.3, 9.1, 3.1, and 3.9, respectively. The same RRVB model in this study in COVID-19 performed with a sensitivity of 73.2%, specificity of 62.9%, and OR of 4.64 (P<.001). Patients who experienced respiratory symptoms were detected more frequently than those who did not experience respiratory symptoms and completely asymptomatic patients (sensitivity: 78.4% vs 67.4% vs 68%, respectively). Conclusions: The RRVB model has shown good generalizability across respiratory conditions, geographies, and languages. Results using data set of patients with COVID-19 demonstrate its meaningful potential to serve as a prescreening tool for identifying individuals at risk for COVID-19 infection in combination with temperature and symptom reports. Although not a COVID-19 test, these results suggest that the RRVB model can encourage targeted testing. Moreover, the generalizability of this model for detecting respiratory symptoms across different linguistic and geographic contexts suggests a potential path for the development and validation of voice-based tools for broader disease surveillance and monitoring applications in the future.

Supplementary Table from Cancer-Associated Fibroblasts Suppress CD8+ T-cell Infiltration and Confer Resistance to Immune-Checkpoint Blockade

Supplementary Figure Legends and Tables 1-2 from Soluble IGF2 Receptor Rescues ApcMin/+ Intestinal Adenoma Progression Induced by Igf2 Loss of Imprinting

Supplementary Figure S1: Summary of experiments Supplementary Figure S2: Gating strategy Supplementary Figure S3:Profiling by array CGH, whole-exome and targeted sequencing Supplementary Figure S4: Comparison of mutational profiles of murine syngeneic tumor cell lines and TCGA patient tumors Supplementary Figure S5: Differentially-expressed gene-sets in lymph node and spleen Supplementary Table S1: Cell line details Supplementary Table S2: Copy Number Variation Supplementary Table S3: List of 64 genes investigated by targeted sequencing Supplementary Table S4: Fluorescent antibodies used Supplementary Methods: Linear mixed-effect model

Supplementary dataset containing raw data from targeted sequencing, whole exome sequencing, array CGH and transcriptomic analysis

Supplementary Figure 3 from Soluble IGF2 Receptor Rescues <i>Apc</i>^<i>Min/</i>+ Intestinal Adenoma Progression Induced by <i>Igf2</i> Loss of Imprinting

Supplementary Figure from Cancer-Associated Fibroblasts Suppress CD8+ T-cell Infiltration and Confer Resistance to Immune-Checkpoint Blockade

Supplementary Figure 3 from Soluble IGF2 Receptor Rescues <i>Apc</i>^<i>Min/</i>+ Intestinal Adenoma Progression Induced by <i>Igf2</i> Loss of Imprinting

Abstract Immune-checkpoint blockade (ICB) promotes antitumor immune responses and can result in durable patient benefit. However, response rates in breast cancer patients remain modest, stimulating efforts to discover novel treatment options. Cancer-associated fibroblasts (CAF) represent a major component of the breast tumor microenvironment and have known immunosuppressive functions in addition to their well-established roles in directly promoting tumor growth and metastasis. Here we utilized paired syngeneic mouse mammary carcinoma models to show that CAF abundance is associated with insensitivity to combination αCTLA4 and αPD-L1 ICB. CAF-rich tumors exhibited an immunologically cold tumor microenvironment, with transcriptomic, flow cytometric, and quantitative histopathologic analyses demonstrating a relationship between CAF density and a CD8+ T-cell–excluded tumor phenotype. The CAF receptor Endo180 (Mrc2) is predominantly expressed on myofibroblastic CAFs, and its genetic deletion depleted a subset of αSMA-expressing CAFs and impaired tumor progression in vivo. The addition of wild-type, but not Endo180-deficient, CAFs in coimplantation studies restricted CD8+ T-cell intratumoral infiltration, and tumors in Endo180 knockout mice exhibited increased CD8+ T-cell infiltration and enhanced sensitivity to ICB compared with tumors in wild-type mice. Clinically, in a trial of melanoma patients, high MRC2 mRNA levels in tumors were associated with a poor response to αPD-1 therapy, highlighting the potential benefits of therapeutically targeting a specific CAF subpopulation in breast and other CAF-rich cancers to improve clinical responses to immunotherapy. Significance: Paired syngeneic models help unravel the interplay between CAF and tumor immune evasion, highlighting the benefits of targeting fibroblast subpopulations to improve clinical responses to immunotherapy.

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti–PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo. Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

Oncolytic virus (OV) therapy is an emerging approach with the potential to redefine treatment options across a range of cancer indications and in patients who remain resistant to existing standards of care, including immuno-oncology (IO) drugs. MEDI5395, a recombinant Newcastle disease virus (NDV), engineered to express granulocyte–macrophage colony-stimulating factor (GM-CSF), exhibits potent oncolytic activity. It was hypothesized that activation of immune cells by MEDI5395, coupled with its oncolytic activity, would enhance the priming of antitumor immunity. Using MEDI5395 and recombinant NDVs encoding fluorescent reporter genes, we demonstrated preferential virus uptake and non-productive infection in myeloid cells, including monocytes, macrophages, and dendritic cells (DCs). Infection resulted in immune-cell activation, with upregulation of cell surface activation markers (e.g., CD80, PD-L1, HLA-DR) and secretion of proinflammatory cytokines (IFN-α2a, IL-6, IL-8, TNF-α). Interestingly, in vitro M2-polarized macrophages were more permissive to virus infection than were M1-polarized macrophages. In a co-culture system, infected myeloid cells were effective virus vectors and mediated the transfer of infectious NDV particles to tumor cells, resulting in cell death. Furthermore, NDV-infected DCs stimulated greater proliferation of allogeneic T cells than uninfected DCs. Antigens released after NDV-induced tumor cell lysis were cross-presented by DCs and drove activation of tumor antigen-specific autologous T cells. MEDI5395 therefore exhibited potent immunostimulatory activity and an ability to enhance antigen-specific T-cell priming. This, coupled with its tumor-selective oncolytic capacity, underscores the promise of MEDI5395 as a multimodal therapeutic, with potential to both enhance current responding patient populations and elicit de novo responses in resistant patients.

Abstract Oncolytic viruses offer the potential to be transformative in the treatment of cancers through their ability to selectively infect, replicate in and kill tumors whilst able to potently activate the immune system. Their activity can be further augmented through the addition of transgenes leading to tumor selective gene expression. Our proprietary Newcastle Disease virus (NDV) backbone has previously been demonstrated to have broad oncolytic activity whilst inducing potent anti tumor immune effects in preclinical models. We set out to test whether the incorporation of an IL-12 transgene into the NDV backbone could result in transgene dependent enhancement in anti tumor efficacy. In vitro, the addition of IL-12 into the NDV genome did not alter NDV infectivity in tumor or non-cancer cells compared to the backbone virus and infection of cancer cells resulted in the production of bioactive IL-12. In an immune-deficient xenograft model, a single systemic administration of NDVhuIL-12 resulted in tumor selective replication and local IL-12 production within the tumor microenvironment (TME). In syngeneic tumor models, local tumoral administration of NDVmuIL-12 resulted in the generation of an elevated and more durable type II interferon response compared to NDV-GMCSF; though similar pharmacokinetic profiles were observed. This difference in cytokine response correlated with superior single agent efficacy of NDVmuIL-12 compared to NDV alone in the B16F10 model. Furthermore, NDVmuIL-12 demonstrated anti tumor efficacy in several NDV refractory models, suggesting tumor expression of IL-12 was necessary to drive anti tumor activity in some circumstances. Across all models tested significant changes in the tumor immune microenvironment, previously associated with enhanced anti tumor immunity were observed in NDVmuIL-12 tumours compared to backbone virus alone. There is an abundance of pre-clinical data demonstrating that IL-12 can exert potent anti tumor activity, but its clinical application has been hindered through its toxicity when administered systemically. These data provide evidence that recombinant NDV could be utilised to deliver IL-12 to the TME when administered systemically and that the addition of IL-12 potentiates the anti tumor activity of NDV. Citation Format: James Harper, Andrew Leinster, Nicola Rath, Shannon Burke, Kathy Mulgrew, Hong Jin, Robert W. Wilkinson. A recombinant Newcastle disease virus expressing IL-12 has potent pre-clinical immunomodulatory and anti tumor properties [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 6535.

Abstract Oncolytic viruses (OV) are defined by their intrinsic or engineered tropism for tumor cells. Understanding the drivers of susceptibility to infection as well as the downstream intracellular signaling and immune activation profile is critical in identifying the patients that will respond to OV therapy. Newcastle Disease Virus (NDV) is a negative stranded RNA avian paramyxovirus, here modified to encode human GM-CSF or IL-12. Patient derived xenografts (PDX) can more accurately reflect the genetic, epigenetic, and architectural heterogeneity of human tumors and were therefore selected to model the in vivo activity of NDV:GM-CSF. Eighty-nine independent models derived from patients with Bladder, Colorectal, Head and Neck, Kidney, Liver, Lung, Ovarian, Pancreatic, and Breast tumors were studied. Mice were dosed intravenously with NDV:GM-CSF, monitored for pharmacodynamic and pharmacokinetic changes 48 hours after the first dose, and followed for tumor growth inhibition. Objective response was observed in 68% of treated mice, with responses observed in all indications. Using total stranded RNAseq, we were able to assess changes in human tumor, murine immune infiltrate, and viral RNA. Results showed that NDV:GM-CSF treatment leads to increases in transcripts of inflammatory chemokines and cytokines such as RANTES and CXCL9, with induction directly correlated to level of viral replication. Additionally, we saw increases in NKp46 and IL-12R, markers of NK cells. To assess if IL-12 would augment immune activation, we treated surgically resected human tumors with NDV:IL-12. NDV:IL-12 was capable of infecting tumor cells, generating IL-12 protein, and driving a strong IFNγ response. Here we show that NDV can infect a wide variety of human tumors across multiple indications and drive a potent immune activation which can be modulated by transgenes. This data provides insight into what types of tumors could be treated with NDV:GM-CSF or NDV:IL-12. Citation Format: Nicholas M. Durham, Kelly McGlinchey, Shannon Burke, Todd Creasy, Nicola Rath, Noel Monks, Ravinder Tammali, Kevin Schifferli, Nick Holoweckyj, Susie Hayes, Emma Jones, Elizabeth J. Kelly, Danielle Carroll, James Harper, Katie Streicher. Newcastle Disease Virus in human derived tumors: Understanding immune recruitment and activation via virally delivered IL-12 [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 5544.

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

In the wake of the success of modern immunotherapy, oncolytic viruses (OVs) are currently seen as a potential therapeutic option for patients with cancer who do not respond or fail to achieve durable responses following treatment with immune checkpoint inhibitors. OVs offer a multifaceted therapeutic platform because they preferentially replicate in tumour cells, can be engineered to express transgenes that augment their cytotoxic and immunostimulatory activities, and modulate the tumour microenvironment to optimize immune-mediated tumour eradication, both at locoregional and systemic sites of disease. Lysis of tumour cells releases tumour-specific antigens that trigger both the innate and adaptive immune systems. OVs also represent attractive combination partners with other systemically delivered agents by virtue of their highly favourable safety profiles. Rational combinations of OVs with different immune modifiers and/or antitumour agents, based on mechanisms of tumour resistance to immune-mediated attack, may benefit the large, currently underserved, population of patients who respond poorly to immune checkpoint inhibition.

MEDI5395 is a genetically modified attenuated Newcastle disease virus (NDV). A reverse genetic system has overcome environmental and regulatory concerns by uncoupling oncolytic potency from avian pathogenicity. MEDI5395 has the intrinsic ability to infect and kill tumor cells and has been inserted with a GM-CSF transgene to potentiate a stronger adaptive immune response. Described here is an extensive in vitro and in vivo pharmacology package that reveals the broad oncolytic activity and immune-modulatory properties of MEDI5395 in a variety of pre-clinical models.In vitro, MEDI5395 lytic activity in human tumor cells is associated with elevated levels of tumor selective viral replication and expression of the GM-CSF transgene. MEDI5395 infection of immune cells indicated preferential uptake of virus and subsequent self-limiting replication in myeloid cells. Infected myeloid cells expressed cell surface activation markers (e.g. PD-L1) after 24 hours and were effective carriers of virus and mediated the transfer of infectious NDV particles to tumor cells resulting in death through oncolysis. Further mechanistic studies, indicated NDV-killed tumor cells released antigens that were capable of cross-presentation by dendritic cells driving activation of tumor antigen-specific autologous T cells.In murine models, IV delivery of NDV leads to long lasting tumor selective replication and transgene expression. MEDI5395 administration results in significant anti-tumor activity, observed in patient-derived xenograft models, and in murine syngeneic cancer models. Together, the results suggest that MEDI5395 may act to positively transform the tumor microenvironment. This, coupled to its tumor-selective oncolytic capacity, further underscore NDV as a promising multimodal cancer therapeutic platform and why FTIH studies are planned to start in 2019.Citation Format: James A. Harper, Shannon Burke, Andrew Leinster, Nicola Rath, Xing Cheng, Hong Jin, Robert W. Wilkinson, Danielle Carroll. MEDI5395: A recombinant oncolytic virus with oncolytic and immune modulatory properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1456.

Abstract Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell lysis occurs as a natural consequence of the viral life cycle and released virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of releasing multiple tumour antigens that may further induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of preclinical cancer models and in PhI clinical studies as an oncolytic agent, oncolysate or whole cell vaccine. Using reverse genetics, we have generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. Furthermore we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). We have evaluated the biological characteristics of recNDVGM-CSF (MEDI5395) in vivo and in vitro. MEDI5395 selectively replicates in and kills a wide variety of human and mouse tumour cell lines. Additionally infection of cancer cells with MEDI5395 results in the increased production and secretion of pro-inflammatory cytokines and chemokines which are able to recruit mediators of both the innate and adaptive immune responses. MEDI5395 is a potent activator of the type I interferon response. In vivo, using a range of syngeneic and xenograft models we have demonstrated that NDV treatment has robust anti-tumour activity. In a HT1080 fibrosarcoma xenograft model a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. In syngeneic mouse tumour models, which support minimal viral replication MEDI5395 treatment causes significant changes in the local immune suppressive microenvironment and results in long-lasting anti-tumour immune responses. These responses are further enhanced in models that permit greater replication and also when combined with immune checkpoint blockade. The inherent properties of NDV (self-propagation, tumour-selective replication, and immunostimulatory properties) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: Danielle Carroll, James Harper, Travers Jon, Shannon Burke, Ruth Franks, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin. MEDI5395: An armed oncolytic Newcastle disease virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2017-4556

Abstract Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has significant oncolytic activity against mammalian cancers. It is multi-modal in its anti-tumour activity and offers a tumour selective, self-propagating therapeutic with oncolytic activity and immunostimulatory properties. Within the tumour itself, NDV infection is able to modulate the immune suppressive microenvironment and induce positive anti-tumour inflammatory responses. Additionally, we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). To better understand the immune modulatory mechanisms by which NDV infection is able to alter the tumour microenvironment we investigated the responses of normal human PBMCs and isolated immune cell populations following NDV exposure. Using flow cytometry, cytokine and gene expression analysis we demonstrated activation of the innate immune cells and a robust type I IFN and pro-inflammatory response. 24 hours post infection, innate immune cells (macrophages, natural killer cells and dendritic cells) had upregulated cell surface activation markers and secreted high levels of cytokines in a dose-dependent fashion. Furthermore, by using NDV constructs encoding fluorescent proteins we showed, in a specific subset of myeloid cells, the preferential uptake of virus and subsequent self-limiting viral replication. In a co-culture system, these infected myeloid cells were able to function as ‘virus cellular carriers’ and were able to mediate the efficient transfer of infectious NDV to tumour cells resulting in their oncolytic death. To further investigate the ability of NDV to infect, spread and kill tumour cells in vivo, we utilised tissue slice cultures of fresh patient samples. These studies demonstrated virus replication and transgene expression in tumour slices, as well as the uptake of virus in a small fraction of specific cells in slices of normal liver. Moreover, evidence that NDV was able to alter the tumour microenvironment could be demonstrated in such cultures by sampling the culture supernatants over time. By investigating the immune modulatory properties of NDV in vitro and in vivo we will gain greater insight and understanding of the anti-cancer properties of NDV. This work will also help guide the selection of transgenes for next generation approaches to augment the inherent immunostimulatory properties of NDV, and help inform clinical dosing options such as intravenous infusion or cell-based delivery. Citation Format: Shannon Burke, Ruth Franks, James Harper, Jon Travers, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin, Danielle Carroll. Exploring the immune stimulatory properties of oncolytic Newcastle Disease Virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4562. doi:10.1158/1538-7445.AM2017-4562

Abstract 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.