Tumour invasiveness, the local and systemic environment and the basis of staging systems in colorectal cancer

background: The present study aimed to examine the relationship between tumour invasiveness (T stage), the local and systemic environment and cancer-specific survival (CSS) of patients with primary operable colorectal cancer. methods: The tumour microenvironment was examined using measures of the inflammatory infiltrate (Klintrup-Makinen (KM) grade and Immunoscore), tumour stroma percentage (TSP) and tumour budding. The systemic inflammatory environment was examined using modified Glasgow Prognostic Score (mGPS) and neutrophil:lymphocyte ratio (NLR). A 5-year CSS was examined. results: A total of 331 patients were included. Increasing T stage was associated with colonic primary, N stage, poor differentiation, margin involvement and venous invasion (P<0.05). T stage was significantly associated with KM grade (P=0.001), Immunoscore (P=0.016), TSP (P=0.006), tumour budding (P<0.001), and elevated mGPS and NLR (both P<0.05). In patients with T3 cancer, N stage stratified survival from 88 to 64%, whereas Immunoscore and budding stratified survival from 100 to 70% and from 91 to 56%, respectively. The Glasgow Microenvironment Score, a score based on KM grade and TSP, stratified survival from 93 to 58%. conclusions: Although associated with increasing T stage, local and systemic tumour environment characteristics, and in particular Immunoscore, budding, TSP and mGPS, are stage-independent determinants of survival and may be utilised in the staging of patients with primary operable colorectal cancer

Lactate signalling regulates fungal β-glucan masking and immune evasion

AJPB: This work was supported by the European Research Council (STRIFE, ERC- 2009-AdG-249793), The UK Medical Research Council (MR/M026663/1), the UK Biotechnology and Biological Research Council (BB/K017365/1), the Wellcome Trust (080088; 097377). ERB: This work was supported by the UK Biotechnology and Biological Research Council (BB/M014525/1). GMA: Supported by the CNPq-Brazil (Science without Borders fellowship 202976/2014-9). GDB: Wellcome Trust (102705). CAM: This work was supported by the UK Medical Research Council (G0400284). DMM: This work was supported by UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC/K000306/1). NARG/JW: Wellcome Trust (086827, 075470,101873) and Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology (097377). ALL: This work was supported by the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPostprin

Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer

Tumor–stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of β-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets β-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing β-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses β-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking β-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting β-catenin in MDSCs

Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy

RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function.

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle

Genome-wide RNA-Sequencing analysis reveals a distinct fibrosis gene signature in the conjunctiva after glaucoma surgery

Fibrosis-related events play a part in most blinding diseases worldwide. However, little is known about the mechanisms driving this complex multifactorial disease. Here we have carried out the first genome-wide RNA-Sequencing study in human conjunctival fibrosis. We isolated 10 primary fibrotic and 7 non-fibrotic conjunctival fibroblast cell lines from patients with and without previous glaucoma surgery, respectively. The patients were matched for ethnicity and age. We identified 246 genes that were differentially expressed by over two-fold and p < 0.05, of which 46 genes were upregulated and 200 genes were downregulated in the fibrotic cell lines compared to the non-fibrotic cell lines. We also carried out detailed gene ontology, KEGG, disease association, pathway commons, WikiPathways and protein network analyses, and identified distinct pathways linked to smooth muscle contraction, inflammatory cytokines, immune mediators, extracellular matrix proteins and oncogene expression. We further validated 11 genes that were highly upregulated or downregulated using real-time quantitative PCR and found a strong correlation between the RNA-Seq and qPCR results. Our study demonstrates that there is a distinct fibrosis gene signature in the conjunctiva after glaucoma surgery and provides new insights into the mechanistic pathways driving the complex fibrotic process in the eye and other tissues

Th Inducing POZ-Kruppel Factor (ThPOK) Is a Key Regulator of the Immune Response since the Early Steps of Colorectal Carcinogenesis

We purposed to evaluate the role of Th inducing POZ-Kruppel Factor (ThPOK), a transcriptional regulator of T cell fate, in tumour-induced immune system plasticity in colorectal carcinogenesis. The amounts of CD4+, CD8+ and CD56+ and ThPOK+ cells infiltrate in normal colorectal mucosa (NM), in dysplastic aberrant crypt foci (microadenomas, MA), the earliest detectable lesions in colorectal carcinogenesis, and in colorectal carcinomas (CRC), were measured, and the colocalization of ThPOK with the above-mentioned markers of immune cells was evaluated using confocal microscopy. Interestingly, ThPOK showed a prominent increase since MA. A strong colocalization of ThPOK with CD4 both in NM and in MA was observed, weaker in carcinomas. Surprisingly, there was a peak in the colocalization levels of ThPOK with CD8 in MA, which was evident, although to a lesser extent, in carcinomas, too. In conclusion, according to the data of the present study, ThPOK may be considered a central regulator of the earliest events in the immune system during colorectal cancer development, decreasing the immune response against cancer cells

Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune). © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Cellular metabolism constrains innate immune responses in early human ontogeny

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny