Defining the post-chemotherapeutic immune landscape in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the 5th most common cause of cancer death in the western world, with a 5-year survival of <7% [1, 2]. Surgical resection remains the best treatment option, although the 5-year survival remains <25% [3] .Most patients are ineligible for resection as they present with metastatic disease. These patients undergo systemic chemotherapy, which offers only a modest improvement in survival [4]. Compared to similar solid tumours, PDAC is a relatively poorly characterised disease, with few treatment improvements. This is due, in part, to its complex, heterogenous landscape, defined by a dense fibrotic stroma, low immunogenicity and low mutational burden. These factors make it highly chemo resistant and offers few options for targeted treatments. Of the few treatment improvements, the switch from Gemcitabine based to FOLFIRINOX based chemotherapy, offers a paradigm shift by doubling survival to almost 12 months in high performance patients [3]. Similarly, the introduction of neoadjuvant therapy in locally advanced and borderline resectable disease has resulted in improved prognosis [4, 5]. The tumour microenvironment is relatively well established in pancreatic cancer, with studies predominantly focused on naïve patients. Both an anti-tumorigenic and pro-tumorigenic role has been reported in pancreatic cancer. This is highly dependent on the types of immune and stromal cells present [6]. Traditionally, T helper and cytotoxic T cells are associated with immunosurveillance, increased tumour cell death and improved prognosis [7, 8]. Whereas, macrophages, fibroblasts and Tregs tend to inhibit the immune response and are primarily associated with poor prognosis [9, 10]. Furthermore, B cells fall into both the pro and anti-tumour categories due to contradictory reports [11-13]. Until recently, the number of immune cells investigated at one time was limited due to technology. This has resulted in the majority of studies reporting density-based metrics. The introduction of spatial biology and deep phenotyping assays has resulted in studies focused on co-expression, and inter-phenotypic distance relationships being established. Carstens et al reported one of the first upfront resected PDAC studies focused on single cell deep spatial phenotyping [7]. They found cytotoxic T cells within 20μm of cancer cells exhibited increased anti-tumour effects and correlated positively with increased survival. Immunohistochemistry (IHC) based studies demonstrate an immunogenic switch in neoadjuvant therapy patients. A depletion of pro-tumorigenic immune cells, recruitment of anti-tumour immune cells and alteration in the functional states in subsets of immune cells has been reported [6, 14, 15]. Again, these studies predominantly rely on single-plex technologies, with no consideration to spatial relationships within the tumour microenvironment. Furthermore, little is known regarding the biological pathways responsible for this immunogenic switch. Characterization of pancreatic ductal adenocarcinoma in treatment naïve and neoadjuvant patients represents a niche research field with limited associated literature. The main aim of this thesis was to address this issue. The primary aim was to establish the protein immune cell landscape in treatment naïve and neoadjuvant human pancreatic cancer in terms of content, cellular density and spatial orientation of different phenotypes. The first step was to confirm the IHC prognostic benefit of the most common prognostic associated immune cells. Elevated CD3 (p=0.015) and CD8 (p=0.043) cells positively correlated with improved disease specific survival (DSS) in naïve PDAC tissue microarrays (TMAs). Subsequently, deep spatial phenotyping was initially separately established in treatment naïve and neoadjuvant setting, then compared. The immune cells explored included T cells, macrophages, fibroblasts and epithelial cells. Improved DSS in naïve patients correlated with increased CD3 T cell (p=0.004) and reduced CD68 (p=0.008) macrophage density. Additionally, increased proximity from CD68 macrophages to tumour cells (p=0.005), and decreased proximity from CD68 macrophages to CD3 T cells (p<0.001) also presented in longer survivors. Contradictory to the hypothesis, improved DSS in neoadjuvant patients correlated with reduced CD3 T cells (p=0.004) and CD68 macrophages (p=0.001). Furthermore, increased proximity from CD68 macrophages to PanCk (p=0.001), increased proximity from CD3CD8 cytotoxic T cells to CD3 T cells (p=0.018), and reduced proximity to FOXP3CD3 from CD3CD8 (p<0.001) correlated with survival. Additionally, this assay established distinct immune differences across chemotherapy versus chemoradiotherapy, and FOLFIRINOX treated versus Gemcitabine treated patients. The deep phenotyping assay lacked functional markers, prompting use of a larger regional protein assay, revealing a prognostically relevant, epithelial compartment specific immune checkpoint marker, B7-H3 (p=0.026). Subsequent Spatial Transcriptomic characterisation was established in order to gain insight into underlying immune related biological mechanisms, something severely lacking in PDAC. Naïve intra-segment heterogeneity demonstrated two unique epithelial signatures, with a non-significant prognostic trend. A variety of potentially targetable significant genes and pathways appeared when integrating mIF findings into Spatial Transcriptomics. These included angiotensin, type I INF, JAK/STAT and IL-2 pathways, which also suggest potential mechanisms responsible for the immune phenotypes observed. Furthermore, transcriptomic B7-H3 expression validated the regional protein result, and was replicated in the neoadjuvant cohort, demonstrating distinct signature profiles between the ranked expression. Interest is growing within the cancer field regarding B7-H3 expression as an immune checkpoint marker [16]. This molecule has, reportedly, limited expression in normal tissue, and high expression in pancreatic cancer, with elevated expression correlating with poor survival and metastasis [17-19]. The results demonstrate potential targetable treatment options for PDAC. Three main immune cell estimates were repeatedly associated with the better outcome group. These were T cells, B cells and dendritic cells. Taking into consideration variable protein translation from RNA, these results were investigated using a single cell ultra-high plex CosMx assay, with only CD4 and CD8 cell clusters validated. In-depth B7-H3 clustering demonstrated a range of immune cell and epithelial markers co-expressing with B7-H3 across naïve and neoadjuvant patients, with naïve exhausted T cell cluster 12 (p=0.003) and neoadjuvant T cell cluster 27 (p=0.022) negatively correlating with survival. In conclusion, comprehensive protein and transcriptomic characterisation of pancreatic cancer spanning both naive and neoadjuvant setting reveals novel patterns. This established inter-phenotypic spatial relations, demonstrated significant differences between naïve and neoadjuvant patients, and has begun to explore complex biological mechanisms within PDAC. These results, if validated, represent potential novel predictive biomarkers, and novel targetable therapies, developments critically needed in pancreatic cancer

Cytotoxicity of ZnO Nanoparticles Can Be Tailored by Modifying Their Surface Structure: A Green Chemistry Approach for Safer Nanomaterials

ZnO nanoparticles (NP) are extensively used in numerous nanotechnology applications; however, they also happen to be one of the most toxic nanomaterials. This raises significant environmental and health concerns and calls for the need to develop new synthetic approaches to produce safer ZnO NP, while preserving their attractive optical, electronic, and structural properties. In this work, we demonstrate that the cytotoxicity of ZnO NP can be tailored by modifying their surface-bound chemical groups, while maintaining the core ZnO structure and related properties. Two equally sized (9.26 ± 0.11 nm) ZnO NP samples were synthesized from the same zinc acetate precursor using a forced hydrolysis process, and their surface chemical structures were modified by using different reaction solvents. X-ray diffraction and optical studies showed that the lattice parameters, optical properties, and band gap (3.44 eV) of the two ZnO NP samples were similar. However, FTIR spectroscopy showed significant differences in the surface structures and surface-bound chemical groups. This led to major differences in the zeta potential, hydrodynamic size, photocatalytic rate constant, and more importantly, their cytotoxic effects on Hut-78 cancer cells. The ZnO NP sample with the higher zeta potential and catalytic activity displayed a 1.5-fold stronger cytotoxic effect on cancer cells. These results suggest that by modifying the synthesis parameters/conditions and the surface chemical structures of the nanocrystals, their surface charge density, catalytic activity, and cytotoxicity can be tailored. This provides a green chemistry approach to produce safer ZnO NP

Genomic and molecular analyses identify molecular subtypes of pancreatic cancer recurrence

No abstract available

JAK/STAT3 represents a therapeutic target for colorectal cancer patients with stromal-rich tumors

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression

Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data

PURPOSE Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. EXPERIMENTAL DESIGN Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. RESULTS Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. CONCLUSIONS Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples

Photoinduced transformation of waste-derived soluble bio-based substances

[EN] Waste-derived, soluble bin-based substances (SBO), are effective low-cost photosensitizers that could find application in pollutant photodegradation. For this reason, it is important to understand if and to what extent irradiation could modify their properties. The exposure of SBO to simulated sunlight induced important spectral and structural modifications. Both the whole material and its acid-soluble fraction were characterized, highlighting several properties in common with humic and fulvic substances, including absorption spectra, specific absorbance and fluorescence behavior. The latter was described with a three-component model using PARAFAC analysis. Irradiation induced SBO photobleaching, but the absorbance of the acid-soluble fraction increased with irradiation. This finding suggests a progressive photochemical solubilization of SBO, which is confirmed by the increase of the carboxylic groups. In addition to absorbance, the fluorescence of whole SBO was also decreased by irradiation, thereby suggesting that both chromophores and fluorophores were photodegraded. The increasingly hydrophilic character given to SBO by irradiation also accounted for the photoinduced decrease of the surfactant properties of the material. (C) 2015 Elsevier B.V. All rights reserved.This work was funded by the 7thFP IRSES-2010-269128-EnvironBos Marie Curie Action and by Ministero delle Politiche Agricole e Forestali (Agrienergia project). The authors are grateful to the following private and/or public Italian institutions: (a) Acea Pinerolese Spa in Pinerolo (TO) for supplying the SBO sourcing materials; (b) Studio Chiono ed Associati in Rivarolo Canavese (TO) for making available pilot equipment and services for the production of the SBO.Avetta, P.; Berto, S.; Bianco Prevot, A.; Minella, M.; Montoneri, E.; Persico, D.; Vione, D.... (2015). Photoinduced transformation of waste-derived soluble bio-based substances. Chemical Engineering Journal. 274:247-255. https://doi.org/10.1016/j.cej.2015.03.126S24725527

A disease-associated gene desert directs macrophage inflammation through ETS2

Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22—which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis3–6—we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities

Application of UV absorbance and fluorescence indicators to assess the formation of biodegradable dissolved organic carbon and bromate during ozonation

This study examined the significance of changes of UV absorbance and fluorescence of dissolved organic matter (DOM) as surrogate indicators for assessing the formation of bromate and biodegradable dissolved organic carbon (BDOC) during the ozonation of surface water and wastewater effluent. Spectroscopic monitoring was carried out using benchtop UV/Vis and fluorescence spectrophotometers and a newly developed miniature LED UV/fluorescence sensor capable of rapidly measuring UVA280 and protein-like and humic-like fluorescence. With the increase of O3/DOC mass ratio, the plots of BDOC formation were characterized of initial lag, transition slope and final plateau. With the decrease of UV absorbance and fluorescence, BDOC concentrations initially increased slowly and then rose more noticeably. Inflection points in plots of BDOC versus changes of spectroscopic indicators were close to 35 e45% loss of UVA254 or UVA280 and 75e85% loss of humic-like fluorescence. According to the data from size exclusion chromatography (SEC) with organic carbon detection and 2D synchronous correlation analyses, DOM fractions assigned to operationally defined large biopolymers (apparent molecular weight, AMW>20 kDa) and medium AMW humic substances (AMW 5.5e20 kDa) were transformed into medium-size building blocks (AMW 3e5.5 kDa) and other smaller AMW species (AMW<3 kDa) associated with BDOC at increasing O3/DOC ratios. Appreciable bromate formation was observed only after the values of UVA254, UVA280 and humic-like fluorescence in O3-treated samples were decreased by 45 e55%, 50e60% and 86e92% relative to their respective initial levels. No significant differences in plots of bromate concentrations versus decreases of humic-like fluorescence were observed for surface water and wastewater effluent samples. This was in contrast with the plots of bromate concentration versus UVA254 and UVA280 which exhibited sensitivity to varying initial bromide concentrations in the investigated water matrixes. These results suggest that measurements of humic-like fluorescence can provide a useful supplement to UVA indices for characterization of ozonation processes

A disease-associated gene desert directs macrophage inflammation through ETS2

Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22—which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis3,4,5,6—we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities

Visible Light Responsive Photocatalyst Induces Progressive and Apical-Terminus Preferential Damages on Escherichia coli Surfaces

BACKGROUND: Recent research shows that visible-light responsive photocatalysts have potential usage in antimicrobial applications. However, the dynamic changes in the damage to photocatalyzed bacteria remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Facilitated by atomic force microscopy, this study analyzes the visible-light driven photocatalyst-mediated damage of Escherichia coli. Results show that antibacterial properties are associated with the appearance of hole-like structures on the bacteria surfaces. Unexpectedly, these hole-like structures were preferentially induced at the apical terminus of rod shaped E. coli cells. Differentiating the damages into various levels and analyzing the percentage of damage to the cells showed that photocatalysis was likely to elicit sequential damages in E. coli cells. The process began with changing the surface properties on bacterial cells, as indicated in surface roughness measurements using atomic force microscopy, and holes then formed at the apical terminus of the cells. The holes were then subsequently enlarged until the cells were totally transformed into a flattened shape. Parallel experiments indicated that photocatalysis-induced bacterial protein leakage is associated with the progression of hole-like damages, further suggesting pore formation. Control experiments using ultraviolet light responsive titanium-dioxide substrates also obtained similar observations, suggesting that this is a general phenomenon of E. coli in response to photocatalysis. CONCLUSION/SIGNIFICANCE: The photocatalysis-mediated localization-preferential damage to E. coli cells reveals the weak points of the bacteria. This might facilitate the investigation of antibacterial mechanism of the photocatalysis