The integrated molecular and histological analysis defines subtypes of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is highly heterogeneous. Our understanding of full molecular and immune landscape of ESCC remains limited, hindering the development of personalised therapeutic strategies. To address this, we perform genomic-transcriptomic characterizations and AI-aided histopathological image analysis of 120 Chinese ESCC patients. Here we show that ESCC can be categorized into differentiated, metabolic, immunogenic and stemness subtypes based on bulk and single-cell RNA-seq, each exhibiting specific molecular and histopathological features based on an amalgamated deep-learning model. The stemness subgroup with signature genes, such as WFDC2, SFRP1, LGR6 and VWA2, has the poorest prognosis and is associated with downregulated immune activities, a high frequency of EP300 mutation/activation, functional mutation enrichment in Wnt signalling and the highest level of intratumoural heterogeneity. The immune profiling by transcriptomics and immunohistochemistry reveals ESCC cells overexpress natural killer cell markers XCL1 and CD160 as immune evasion. Strikingly, XCL1 expression also affects the sensitivity of ESCC cells to common chemotherapy drugs. This study opens avenues for ESCC treatment and provides a valuable public resource to better understand ESCC.

The capability of heterogeneous γδ T cells in cancer treatment.

γδ T cells, a specialized subset of T lymphocytes, have garnered significant attention within the realm of cancer immunotherapy. Operating at the nexus between adaptive and innate immunological paradigms, these cells showcase a profound tumor discernment repertoire, hinting at novel immunotherapeutic strategies. Significantly, these cells possess the capability to directly identify and eliminate tumor cells without reliance on HLA-antigen presentation. Furthermore, γδ T cells have the faculty to present tumor antigens to αß T cells, amplifying their anti-tumoral efficacy.Within the diverse and heterogeneous subpopulations of γδ T cells, distinct immune functionalities emerge, manifesting either anti-tumor or pro-tumor roles within the tumor microenvironment. Grasping and strategically harnessing these heterogeneous γδ T cell cohorts is pivotal to their integration in tumor-specific immunotherapeutic modalities. The aim of this review is to describe the heterogeneity of the γδ T cell lineage and the functional plasticity it generates in the treatment of malignant tumors. This review endeavors to elucidate the intricate heterogeneity inherent to the γδ T cell lineage, the consequential functional dynamics in combating malignancies, the latest advancements from clinical trials, and the evolving landscape of γδ T cell-based oncological interventions, while addressing the challenges impeding the field.

Hypoxia-regulated secretion of IL-12 enhances antitumor activity and safety of CD19 CAR-T cells in the treatment of DLBCL.

CD19-targeted chimeric antigen receptor-modified T (CD19 CAR-T) cell therapy has been demonstrated as one of the most promising therapeutic strategies for treating B cell malignancies. However, it has shown limited treatment efficacy for diffuse large B cell lymphoma (DLBCL). This is, in part, due to the tumor heterogeneity and the hostile tumor microenvironment. Human interleukin-12 (IL-12), as a potent antitumor cytokine, has delivered encouraging outcomes in preclinical studies of DLBCL. However, potentially lethal toxicity associated with systemic administration precludes its clinical application. Here, an armed CD19 CAR expressing hypoxia-regulated IL-12 was developed (CAR19/hIL12ODD). In this vector, IL-12 secretion was restricted to hypoxic microenvironments within the tumor site by fusion of IL-12 with the oxygen degradation domain (ODD) of HIF1α. In vitro, CAR19/hIL12ODD-T cells could only secrete bioactive IL-12 under hypoxic conditions, accompanied by enhanced proliferation, robust IFN-γ secretion, increased abundance of CD4+, and central memory T cell phenotype. In vivo, adoptive transfer of CAR19/hIL12ODD-T cells significantly enhanced regression of large, established DLBCL xenografts in a novel immunodeficient Syrian hamster model. Notably, this targeted and controlled IL-12 treatment was without toxicity in this model. Taken together, our results suggest that armed CD19 CARs with hypoxia-controlled IL-12 (CAR19/hIL12ODD) might be a promising and safer approach for treating DLBCL.

A novel microenvironment regulated system CAR-T (MRS.CAR-T) for immunotherapeutic treatment of esophageal squamous carcinoma.

Chimeric antigen receptor T cell immunotherapy has achieved promising therapeutic effects in the treatment of hematological malignancies. However, there are still many obstacles, including on-target off-tumor antigen expression, that prevent successful application to solid tumors. We designed a tumor microenvironment (TME) regulated system chimeric antigen receptor T (MRS.CAR-T) which can only be auto-activated in the solid TME. B7-H3 was selected as the target antigen for esophageal carcinoma. An element comprising a human serum albumin (HSA) binding peptide and a matrix metalloproteases (MMPs) cleavage site was inserted between the 5' terminal signal peptide and single chain fragment variable (scFv) of the CAR skeleton. Upon administration, HSA bound the binding peptide in MRS.B7-H3.CAR-T effectively and promoted proliferation and differentiation into memory cells. MRS.B7-H3.CAR-T was not cytotoxic in normal tissues expressing B7-H3 as the antigen recognition site in the scFv was cloaked by HSA. The anti-tumor function of MRS.B7-H3.CAR-T was recovered once the cleavage site was cleaved by MMPs in the TME. The anti-tumor efficacy associated with MRS.B7-H3.CAR-T cells was improved compared to classic B7-H3.CAR-T cells in vitro and less IFN-γ was released, suggesting a treatment that may induce less extent of cytokine release syndrome-mediated toxicity. In vivo, MRS.B7-H3.CAR-T cells had strong anti-tumor activity and were safe. MRS.CAR-T represents a novel strategy to improve the efficacy and safety of CAR-T therapy in solid tumors.

Syrian hamster as an ideal animal model for evaluation of cancer immunotherapy.

Cancer immunotherapy (CIT) has emerged as an exciting new pillar of cancer treatment. Although benefits have been achieved in individual patients, the overall response rate is still not satisfactory. To address this, an ideal preclinical animal model for evaluating CIT is urgently needed. Syrian hamsters present similar features to humans with regard to their anatomy, physiology, and pathology. Notably, the histological features and pathological progression of tumors and the complexity of the tumor microenvironment are equivalent to the human scenario. This article reviews the current tumor models in Syrian hamster and the latest progress in their application to development of tumor treatments including immune checkpoint inhibitors, cytokines, adoptive cell therapy, cancer vaccines, and oncolytic viruses. This progress strongly advocates Syrian hamster as an ideal animal model for development and assessment of CIT for human cancer treatments. Additionally, the challenges of the Syrian hamster as an animal model for CIT are also discussed.

Oncolytic vaccinia virus and cancer immunotherapy.

Oncolytic virotherapy (OVT) is a promising form of cancer treatment that uses genetically engineered viruses to replicate within cancer cells and trigger anti-tumor immune response. In addition to killing cancer cells, oncolytic viruses can also remodel the tumor microenvironment and stimulate a long-term anti-tumor immune response. Despite achieving positive results in cellular and organismal studies, there are currently only a few approved oncolytic viruses for clinical use. Vaccinia virus (VACV) has emerged as a potential candidate due to its ability to infect a wide range of cancer cells. This review discusses the mechanisms, benefits, and clinical trials of oncolytic VACVs. The safety and efficacy of different viral backbones are explored, as well as the effects of oncolytic VACVs on the tumor microenvironment. The potential combination of oncolytic VACVs with immunotherapy or traditional therapies is also highlighted. The review concludes by addressing prospects and challenges in the field of oncolytic VACVs, with the aim of promoting further research and application in cancer therapy.

An effective therapeutic regime for treatment of glioma using oncolytic vaccinia virus expressing IL-21 in combination with immune checkpoint inhibition.

Glioblastoma (GBM) is the most common primary malignant tumor in the brain, accounting for 51.4% of all primary brain tumors. GBM has a highly immunosuppressive tumor microenvironment (TME) and, as such, responses to immunotherapeutic strategies are poor. Vaccinia virus (VV) is an oncolytic virus that has shown tremendous therapeutic effect in various tumor types. In addition to its directly lytic effect on tumor cells, it has an ability to enhance immune cell infiltration into the TME allowing for improved immune control over the tumor. Here, we used a new generation of VV expressing the therapeutic payload interleukin-21 to treat murine GL261 glioma models. After both intratumoral and intravenous delivery, virus treatment induced remodeling of the TME to promote a robust anti-tumor immune response that resulted in control over tumor growth and long-term survival in both subcutaneous and orthotopic mouse models. Treatment efficacy was significantly improved in combination with systemic α-PD1 therapy, which is ineffective as a standalone treatment but synergizes with oncolytic VV to enhance therapeutic outcomes. Importantly, this study also revealed the upregulation of stem cell memory T cell populations after the virus treatment that exert strong and durable anti-tumor activity.

Optimized Anchor-Modified Peptides Targeting Mutated RAS Are Promising Candidates for Immunotherapy.

RAS mutations occur in approximately 20% of all cancers and given their clonality, key role as driver mutation, association with poor prognosis and undruggability, they represent attractive targets for immunotherapy. We have identified immunogenic peptides derived from codon 12 mutant RAS (G12A, G12C, G12D, G12R, G12S and G12V), which bind to HLA-A*02:01 and HLA-A*03:01 and elicit strong peptide-specific CD8+ T cell responses, indicating that there is an effective CD8+ T-cell repertoire against these mutant RAS-derived peptides that can be mobilized. Alterations in anchor residues of these peptides enhanced their binding affinity to HLA-A*02:01 molecules and allowed generation of CD8+ T cells that responded to target cells pulsed with the anchor-modified and also with the original peptide. Cytotoxic T cells generated against these peptides specifically lysed tumor cells expressing mutant RAS. Vaccination of transgenic humanized HLA-A2/DR1 mice with a long peptide encompassing an anchor-modified 9-mer G12V epitope generated CD8+ T cells reactive to the original 9-mer and to a HLA-A*02:01-positive human cancer cell line harboring the G12V mutation. Our data provide strong evidence that mutant RAS can be targeted by immunotherapy.

MHC class II molecules on pancreatic cancer cells indicate a potential for neo-antigen-based immunotherapy.

MHC class II expression is a hallmark of professional antigen-presenting cells and key to the induction of CD4+ T helper cells. We found that these molecules are ectopically expressed on tumor cells in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC) and on several PDAC cell lines. In contrast to the previous reports that tumoral expression of MHC-II in melanoma enabled tumor cells to evade immunosurveillance, the expression of MHC-II on PDAC cells neither protected cancer cells from Fas-mediated cell death nor caused T-cell suppression by engagement with its ligand LAG-3 on activated T-cells. In fact and surprisingly, the MHC-II/LAG-3 pathway contributed to CD4+ and CD8+ T-cell cytotoxicity toward MHC-II-positive PDAC cells. By combining bioinformatic tools and cell-based assays, we identified a number of immunogenic neo-antigens that can be presented by the patients' HLA class II alleles. Furthermore, CD4+ T-cells stimulated with neo-antigens were capable of recognizing and killing a human PDAC cell line expressing the mutated genes. To expand this approach to a larger number of PDAC patients, we show that co-treatment with IFN-γ and/or MEK/HDAC inhibitors induced tumoral MHC-II expression on MHC-II-negative tumors that are IFN-γ-resistant. Taken together, our data point to the possibility of harnessing MHC-II expression on PDAC cells for neo-antigen-based immunotherapy.

Redirecting anti-Vaccinia virus T cell immunity for cancer treatment by AAV-mediated delivery of the VV B8R gene.

Immunotherapies, such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor-T (CAR-T) cells, are only efficient in a small proportion of tumor patients. One of the major reasons for this is the lack of immune cell infiltration and activation in the tumor microenvironment (TME). Recent research reported that abundant bystander CD8+ T cells targeting viral antigens exist in tumor infiltrates and that virus-specific memory T cells could be recalled to kill tumor cells. Therefore, virus-specific memory T cells may be effective candidates for tumor immunotherapy. In this study, we established subcutaneous tumor mice models that were pre-immunized with Vaccinia virus (VV) and confirmed that tumor cells with ectopic expression of the viral B8R protein could be recognized and killed by memory T cells. To create a therapeutic delivery system, we designed a recombinant adeno-associated virus (rAAV) with a modified tumor-specific promoter and used it to deliver VV B8R to tumor cells. We observed that rAAV gene therapy can retard tumor growth in VV pre-immunized mice. In summary, our study demonstrates that rAAV containing a tumor-specific promoter to restrict VV B8R gene expression to tumor cells is a potential therapeutic agent for cancer treatment in VV pre-immunized or VV-treated mice bearing tumors.

CEACAM7 Is an Effective Target for CAR T-cell Therapy of Pancreatic Ductal Adenocarcinoma.

PURPOSE: To investigate whether CEACAM7 represents a novel therapeutic target for treating pancreatic ductal adenocarcinoma (PDAC) and to generate CEACAM7-targeting CAR T cells to test this hypothesis. EXPERIMENTAL DESIGN: We identified CEACAM7 (CGM2), a member of the CEA family of proteins with expression restricted to the colon and pancreas, as a potential CAR T-cell target for PDAC. We probed a panel of PDAC tumor sections as well as patient-derived PDAC cell cultures for CEACAM7 expression. We generated CAR-targeting CEACAM7, and assessed antitumor efficacy of CEACAM7 CAR T cells using in vitro and in vivo models. RESULTS: We show here that CEACAM7 is expressed in a large subset of PDAC tumors, with low to undetectable expression in all normal tissues tested. CEACAM7 is also expressed in primary PDAC cultures isolated from patient-derived tumors, with high expression within the cancer stem cell-enriched subset. CAR T cells targeting CEACAM7 are capable of targeting antigen-expressing tumor cells, and mediate remission in patient-derived xenograft tumors. CONCLUSIONS: We identify CEACAM7 as a potential therapeutic target in PDAC and describe the development of CEACAM7-targeted CAR T cells with efficacy against PDAC.

A Tumor-Targeted Replicating Oncolytic Adenovirus Ad-TD-nsIL12 as a Promising Therapeutic Agent for Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers in China and existing therapies have been unable to significantly improve prognosis. Oncolytic adenoviruses (OAds) are novel promising anti-tumor drugs and have been evaluated in several cancers including ESCC. However, the antitumour efficacy of the first generation OAds (H101) as single agent is limited. Therefore, more effective OAds are needed. Our previous studies demonstrated that the novel oncolytic adenovirus Ad-TD-nsIL12 (human adenovirus type 5 with E1ACR2, E1B19K, E3gp19K-triple deletions)harboring human non-secretory IL-12 had significant anti-tumor effect, with no toxicity, in a Syrian hamster pancreatic cancer model. In this study, we evaluated the anti-tumor effect of Ad-TD-nsIL12 in human ESCC. The cytotoxicity of Ad-TD-nsIL12, H101 and cisplatin were investigated in two newly established patient-derived tumor cells (PDCs) and a panel of ESCC cell lines in vitro. A novel adenovirus-permissive, immune-deficient Syrian hamster model of PDCs subcutaneous xenograft was established for in vivo analysis of efficacy. The results showed that Ad-TD-nsIL12 was more cytotixic to and replicated more effectively in human ESCC cell lines than H101. Compared with cisplatin and H101, Ad-TD-nsIL12 could significantly inhibit tumor growth and tumor angiogenesis as well as enhance survival rate of animals with no side effects. These findings suggest that Ad-TD-nsIL12 has superior anti-tumor potency against human ESCC with a good safety profile.

Characterization of four subtypes in morphologically normal tissue excised proximal and distal to breast cancer.

Widespread mammographic screening programs and improved self-monitoring allow for breast cancer to be detected earlier than ever before. Breast-conserving surgery is a successful treatment for select women. However, up to 40% of women develop local recurrence after surgery despite apparently tumor-free margins. This suggests that morphologically normal breast may harbor early alterations that contribute to increased risk of cancer recurrence. We conducted a comprehensive transcriptomic and proteomic analysis to characterize 57 fresh-frozen tissues from breast cancers and matched histologically normal tissues resected proximal to (<2 cm) and distant from (5-10 cm) the primary tumor, using tissues from cosmetic reduction mammoplasties as baseline. Four distinct transcriptomic subtypes are identified within matched normal tissues: metabolic; immune; matrisome/epithelial-mesenchymal transition, and non-coding enriched. Key components of the subtypes are supported by proteomic and tissue composition analyses. We find that the metabolic subtype is associated with poor prognosis (p < 0.001, HR6.1). Examination of genes representing the metabolic signature identifies several genes able to prognosticate outcome from histologically normal tissues. A subset of these have been reported for their predictive ability in cancer but, to the best of our knowledge, these have not been reported altered in matched normal tissues. This study takes an important first step toward characterizing matched normal tissues resected at pre-defined margins from the primary tumor. Unlocking the predictive potential of unexcised tissue could prove key to driving the realization of personalized medicine for breast cancer patients, allowing for more biologically-driven analyses of tissue margins than morphology alone.

SNPnexus: a web server for functional annotation of human genome sequence variation (2020 update).

SNPnexus is a web-based annotation tool for the analysis and interpretation of both known and novel sequencing variations. Since its last release, SNPnexus has received continual updates to expand the range and depth of annotations provided. SNPnexus has undergone a complete overhaul of the underlying infrastructure to accommodate faster computational times. The scope for data annotation has been substantially expanded to enhance biological interpretations of queried variants. This includes the addition of pathway analysis for the identification of enriched biological pathways and molecular processes. We have further expanded the range of user directed annotation fields available for the study of cancer sequencing data. These new additions facilitate investigations into cancer driver variants and targetable molecular alterations within input datasets. New user directed filtering options have been coupled with the addition of interactive graphical and visualization tools. These improvements streamline the analysis of variants derived from large sequencing datasets for the identification of biologically and clinically significant subsets in the data. SNPnexus is the most comprehensible web-based application currently available and these new set of updates ensures that it remains a state-of-the-art tool for researchers. SNPnexus is freely available at https://www.snp-nexus.org.

Transient Inhibition of PI3Kδ Enhances the Therapeutic Effect of Intravenous Delivery of Oncolytic Vaccinia Virus.

Tumor-targeting oncolytic viruses such as vaccinia virus (VV) are attractive cancer therapeutic agents that act through multiple mechanisms to provoke both tumor lysis and anti-tumor immune responses. However, delivery of these agents remains restricted to intra-tumoral administration, which prevents effective targeting of inaccessible and disseminated tumor cells. In the present study we have identified transient pharmacological inhibition of the leukocyte-enriched phosphoinositide 3-kinase δ (PI3Kδ) as a novel mechanism to potentiate intravenous delivery of oncolytic VV to tumors. Pre-treatment of immunocompetent mice with the PI3Kδ-selective inhibitor IC87114 or the clinically approved idelalisib (CAL-101), prior to intravenous delivery of a tumor-tropic VV, dramatically improved viral delivery to tumors. This occurred via an inhibition of viral attachment to, but not internalization by, systemic macrophages through perturbation of signaling pathways involving RhoA/ROCK, AKT, and Rac. Pre-treatment using PI3Kδ-selective inhibitors prior to intravenous delivery of VV resulted in enhanced anti-tumor efficacy and significantly prolonged survival compared to delivery without PI3Kδ inhibition. These results indicate that effective intravenous delivery of oncolytic VV may be clinically achievable and could be useful in improving anti-tumor efficacy of oncolytic virotherapy.

Author Correction: TGF-ß induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

'Multi-omic' data analysis using O-miner.

Innovations in -omics technologies have driven advances in biomedical research. However, integrating and analysing the large volumes of data generated from different high-throughput -omics technologies remain a significant challenge to basic and clinical scientists without bioinformatics skills or access to bioinformatics support. To address this demand, we have significantly updated our previous O-miner analytical suite, to incorporate several new features and data types to provide an efficient and easy-to-use Web tool for the automated analysis of data from '-omics' technologies. Created from a biologist's perspective, this tool allows for the automated analysis of large and complex transcriptomic, genomic and methylomic data sets, together with biological/clinical information, to identify significantly altered pathways and prioritize novel biomarkers/targets for biological validation. Our resource can be used to analyse both in-house data and the huge amount of publicly available information from array and sequencing platforms. Multiple data sets can be easily combined, allowing for meta-analyses. Here, we describe the analytical pipelines currently available in O-miner and present examples of use to demonstrate its utility and relevance in maximizing research output. O-miner Web server is free to use and is available at http://www.o-miner.org.

A HIF-LIMD1 negative feedback mechanism mitigates the pro-tumorigenic effects of hypoxia.

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-ß subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.

SNPnexus: assessing the functional relevance of genetic variation to facilitate the promise of precision medicine.

Broader functional annotation of genetic variation is a valuable means for prioritising phenotypically-important variants in further disease studies and large-scale genotyping projects. We developed SNPnexus to meet this need by assessing the potential significance of known and novel SNPs on the major transcriptome, proteome, regulatory and structural variation models. Since its previous release in 2012, we have made significant improvements to the annotation categories and updated the query and data viewing systems. The most notable changes include broader functional annotation of noncoding variants and expanding annotations to the most recent human genome assembly GRCh38/hg38. SNPnexus has now integrated rich resources from ENCODE and Roadmap Epigenomics Consortium to map and annotate the noncoding variants onto different classes of regulatory regions and noncoding RNAs as well as providing their predicted functional impact from eight popular non-coding variant scoring algorithms and computational methods. A novel functionality offered now is the support for neo-epitope predictions from leading tools to facilitate its use in immunotherapeutic applications. These updates to SNPnexus are in preparation for its future expansion towards a fully comprehensive computational workflow for disease-associated variant prioritization from sequencing data, placing its users at the forefront of translational research. SNPnexus is freely available at http://www.snp-nexus.org.