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Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2LMW) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2LMW-/- mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS+/CD206+ TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.
\n \n\n \n \nLittle is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.
\n \n\n \n \nLiver metastases from gastrointestinal and non-gastrointestinal malignancies remain a major cause of cancer-related mortality and a major clinical challenge. The liver has unique properties that facilitate metastatic expansion, including a complex immune system that evolved to dampen immunity to neoantigens entering the liver from the gut, through the portal circulation. In this review, we describe the unique microenvironment encountered by cancer cells in the liver, focusing on elements of the innate and adaptive immune response that can act as a double-edge sword, contributing to the elimination of cancer cells on the one hand and promoting their survival and growth, on the other. We discuss this microenvironment in a clinical context, particularly for colorectal carcinoma, and highlight how a better understanding of the role of the microenvironment has spurred an intense effort to develop novel and innovative strategies for targeting liver metastatic disease, some of which are currently being tested in the clinic.
\n \n\n \n \nPURPOSE: Predicting perioperative morbidity and mortality can be achieved by several risk predicting algorithms. In the UK, the National Emergency Laparotomy Audit, mandated for all patients undergoing emergency laparotomy, uses pPOSSUM as its risk prediction tool. However, there is no literature reporting the inter-operator variability in calculating the score. Inter-rater variability was assessed based on 10 real general surgical cases that went on to have an emergency laparotomy. METHODS: Forty clinicians, 10 each of registrars and consultants in anaesthetics and general surgery, were asked to calculate the pPOSSUM based on the clinical information typically available at the time of making the decision to proceed to emergency laparotomy for the same 10 National Emergency Laparotomy Audit cases. All participants were surveyed to assess their understanding and use of the pPOSSUM score. RESULTS: More than 80% of respondents stated that they use pPOSSUM in daily clinical practice. There was variability in the calculated scores between the groups analysed. Two subgroups were evident: one in which the calculated mean pPOSSUM was similar between participants but did not reflect the true value, and the other which was accurate, but demonstrated high inter-rater variability. CONCLUSIONS: This is the first study to investigate inter-operator variability in pPOSSUM scores. Previous reports on the validity of the tool fail to account for subjective variation. At a time where pPOSSUM has become a routine part of clinical practice, this variability needs to be accounted for and taken into consideration in the decision-making process.
\n \n\n \n \nGemcitabine constitutes one of the backbones for chemotherapy treatment in pancreatic ductal adenocarcinoma (PDAC), but patients often respond poorly to this agent. Molecular markers downstream of gemcitabine treatment in preclinical models may provide an insight into resistance mechanisms. Using cytokine arrays, we identified potential secretory biomarkers of gemcitabine resistance (response) in the transgenic KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) mouse model of PDAC. We verified the oncogenic role of the cytokine tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in primary pancreatic tumors and metastases using both in vitro techniques and animal models. We identified potential pathways affected downstream of TIMP1 using the Illumina Human H12 array. Our findings were validated in both primary and metastatic models of pancreatic cancer. Gemcitabine increased inflammatory cytokines including TIMP1 in the KPC mouse model. TIMP1 was upregulated in patients with pancreatic intraepithelial neoplasias grade 3 and PDAC lesions relative to matched normal pancreatic tissue. In addition, TIMP1 played a role in tumor clonogenic survival and vascular density, while TIMP1 inhibition resensitized tumors to gemcitabine and radiotherapy. We observed a linear relationship between TIMP-1 expression, liver metastatic burden, and infiltration by CD11b+Gr1+ myeloid cells and CD4+CD25+FOXP3+ Tregs, whereas the presence of tumor cells was required for immune cell infiltration. Overall, our results identify TIMP1 upregulation as a resistance mechanism to gemcitabine and provide a rationale for combining chemo/radiotherapy with TIMP1 inhibitors in PDAC. Cancer Res; 77(21); 5952-62. \u00a92017 AACR.
\n \n\n \n \nEmerging evidence suggests a role for radiation in eliciting anti-tumour immunity. We aimed to investigate the role of macrophages in modulating the immune response to radiation. Irradiation to murine tumours generated from colorectal (MC38) and pancreatic (KPC) cell lines induced colony-stimulating factor 1 (CSF-1). Coincident with the elevation in CSF-1, macrophages increased in tumours, peaking 5\u00a0days following irradiation. These tumour-associated macrophages (TAMs) were skewed towards an immunosuppressive phenotype. Macrophage depletion via anti-CSF (aCSF) reduced macrophage numbers, yet only achieved tumour growth delay when combined with radiation. The tumour growth delay from aCSF after radiation was abrogated by depletion of CD8 T cells. There was enhanced recognition of tumour cell antigens by T cells isolated from irradiated tumours, consistent with increased antigen priming. The addition of anti-PD-L1 (aPD-L1) resulted in improved tumour suppression and even regression in some tumours. In summary, we show that adaptive immunity induced by radiation is limited by the recruitment of highly immunosuppressive macrophages. Macrophage depletion partly reduced immunosuppression, but additional treatment with anti-PD-L1 was required to achieve tumour regression.
\n \n\n \n \nHepatic metastatic growth is dependent upon stromal factors including the matrisomal proteins that make up the extracellular matrix (ECM). Laminins are ECM glycoproteins with several functions relevant to tumour progression including angiogenesis. We investigated whether metastatic colon cancer cells produce the laminins required for vascular basement membrane assembly as a mechanism for the promotion of angiogenesis and liver metastasis growth. qPCR was performed using human-specific primers to laminin chains on RNA from orthotopic human colorectal liver metastases. Laminin \u03b15 (LAMA5) expression was inhibited in colon cancer cells using shRNA. Notch pathway gene expression was determined in endothelia from hepatic metastases. Orthotopic hepatic metastases expressed human laminin chains \u03b15, \u03b21 and \u03b31 (laminin 511), all of which are required for vascular basement membrane assembly. The expression of Laminin 511 was associated with reduced survival in several independent colorectal cancer cohorts and angiogenesis signatures or vessel density significantly correlated with LAMA5 expression. Colorectal cancer cells in culture made little LAMA5, but its levels were increased by culture in a medium conditioned by tumour-derived CD11b+ myeloid cells through TNF\u03b1/NF\u03baB pathway signalling. Down-regulation of LAMA5 in cancer cells impaired liver metastatic growth and resulted in reduced intra-tumoural vessel branching and increased the expression of Notch pathway genes in metastasis-derived endothelia. This data demonstrates a mechanism whereby tumour inflammation induces LAMA5 expression in colorectal cancer cells. LAMA5 is required for the successful growth of hepatic metastases where it promotes branching angiogenesis and modulates Notch signalling.
\n \n\n \n \nBACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3\u2009\u00d7\u20095\u2009Gy\u2009\u00b1\u2009anti-PD-L1. RESULTS: 3\u2009\u00d7\u20095\u2009Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3\u2009\u00d7\u20095\u2009Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3\u2009\u00d7\u20095\u2009Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3\u2009\u00d7\u20095\u2009Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
\n \n\n \n \nBACKGROUND: Risk assessment is relevant to predict postoperative outcomes in patients with gastro-oesophageal cancer. This cohort study aimed to assess body composition changes during neoadjuvant chemotherapy and investigate their association with postoperative complications. METHODS: Consecutive patients with gastro-oesophageal cancer undergoing neoadjuvant chemotherapy and surgery with curative intent between 2016 and 2019 were identified from a specific database and included in the study. CT images before and after neoadjuvant chemotherapy were used to assess the skeletal muscle index, sarcopenia, and subcutaneous and visceral fat index. RESULTS: In a cohort of 199 patients, the mean skeletal muscle index decreased during neoadjuvant therapy (from 51\u00b7187 to 49\u00b719\u2009cm2 /m2 ; P\u2009
\n \n\n \n \nAIM: Performance in the operating room is affected by a combination of individual, patient and environmental factors amongst others. Stress has a potential negative impact on performance with the quality of surgical practice and patient safety being affected as a result. In order to appreciate the level of stress encountered during surgical procedures both objective and subjective methods can be used. This study reports the use of a combined objective (physiological) and subjective (psychological) method for evaluating stress experienced by the operating surgeon. METHOD: Six consultant colorectal surgeons were evaluated performing eighteen anterior resections. Heart rate was recorded using a wireless chest strap at eight pre-determined operative steps. Heart Rate Variability indices were calculated offline using computerized software. Surgeon reported stress was collected using the State Trait Anxiety Inventory, a validated clinical stress scale. RESULTS: A significant increase in stress was demonstrated in all surgeons whilst operating as indicated by sympathetic tone (control: 4.02 \u00b1 2.28 vs operative: 11.42 \u00b1 4.63; P < 0.0001). Peaks in stress according to operative step were comparable across procedures and surgeons. There was a significant positive correlation with subjective reporting of stress across procedures (r = 0.766; P = 0.0005). CONCLUSION: This study demonstrates a significant increase in sympathetic tone in consultant surgeons measured using heart rate variability during elective colorectal resections. A significant correlation can be demonstrated between HRV measurements and perceived stress using the State Trait Anxiety Inventory. A combined approach to assessing operative stress is required to evaluate any effect on performance and outcomes.
\n \n\n \n \nPancreatic ductal adenocarcinoma (PDAC) is considered a non-immunogenic tumor, and immune checkpoint inhibitor monotherapy lacks efficacy in this disease. Radiotherapy (RT) can stimulate the immune system. Here, we show that treatment of KPC and Pan02 murine PDAC cells with RT and gemcitabine upregulated PD-L1 expression in a JAK/Stat1-dependent manner. In vitro, PD-L1 inhibition did not alter radio- and chemosensitivity. In vivo, addition of anti-PD-L1 to high (12, 5\u00a0\u00d7\u00a03, 20\u00a0Gy) but not low (6, 5\u00a0\u00d7\u00a02\u00a0Gy) RT doses significantly improved tumor response in KPC and Pan02 allografts. Radiosensitization after PD-L1 blockade was associated with reduced CD11b+Gr1+ myeloid cell infiltration and enhanced CD45+CD8+ T-cell infiltration with concomitant upregulation of T-cell activation markers including CD69, CD44, and FasL, and increased CD8:Treg ratio. Depletion of CD8+ T cells abrogated radiosensitization by anti-PD-L1. Blockade of PD-L1 further augmented the effect of high RT doses (12\u00a0Gy) in preventing development of liver metastases. Exploring multiple mathematical models reveals a mechanism able to explain the observed synergy between RT and anti-PD-L1 therapy. Our findings provide a rationale for testing the use of immune checkpoint inhibitors with RT in PDAC.
\n \n\n \n \nAIM: Radiologically assessed muscle mass has been suggested as a surrogate marker of functional status and frailty and may predict patients at risk of postoperative complications. We hypothesize that sarcopenia negatively impacts on postoperative recovery and is predictive of complications. METHOD: One hundred patients undergoing elective resection for colorectal carcinoma were included in this study. Lean muscle mass was estimated by measuring the cross-sectional area of the psoas muscle at the level of the third lumbar vertebra identified on a preoperative CT scan, normalizing for patient height. Perioperative morbidity was scored according to the Clavien-Dindo classification. All statistical data analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 20.0. RESULTS: Fifteen per cent of patients were identified as sarcopenic. There were no deaths in the study group. Sarcopenia was associated with a significantly increased risk of developing major complications (Grade 3 or greater, OR = 5.41, 95% CI: 1.45-20.15, P = 0.01). Sarcopenia did not predict length of stay, critical care dependency or time to mobilization. CONCLUSION: Sarcopenia, as a marker of frailty, is an important risk factor in surgical patients but difficult to estimate using bedside testing. CT scans, performed for preoperative staging, provide an opportunity to quantify lean muscle mass without additional cost or exposure to radiation and eliminate the inconvenience of further investigations.
\n \n\n \n \nCitrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.
\n \n\n \n \nWhile radical prostatectomy remains the mainstay of prostate cancer (PCa) treatment, 20 to 40% of patients develop postsurgical biochemical recurrence (BCR). A particularly challenging clinical cohort includes patients with intermediate-risk disease whose risk stratification would benefit from advanced approaches that complement standard-of-care diagnostic tools. Here, we show that imaging tumor lactate using hyperpolarized 13C MRI and spatial metabolomics identifies BCR-positive patients in two prospective intermediate-risk surgical cohorts. Supported by spatially resolved tissue analysis of established glycolytic biomarkers, this study provides the rationale for multicenter trials of tumor metabolic imaging as an auxiliary tool to support PCa treatment decision-making.
\n \n\n \n \nBACKGROUND: Understanding the role of circulating proteins in prostate cancer risk can reveal key biological pathways and identify novel targets for cancer prevention. METHODS: We investigated the association of 2,002 genetically predicted circulating protein levels with risk of prostate cancer overall, and of aggressive and early onset disease, using cis-pQTL Mendelian randomization (MR) and colocalization. Findings for proteins with support from both MR, after correction for multiple-testing, and colocalization were replicated using two independent cancer GWAS, one of European and one of African ancestry. Proteins with evidence of prostate-specific tissue expression were additionally investigated using spatial transcriptomic data in prostate tumor tissue to assess their role in tumor aggressiveness. Finally, we mapped risk proteins to drug and ongoing clinical trials targets. RESULTS: We identified 20 proteins genetically linked to prostate cancer risk (14 for overall [8 specific], 7 for aggressive [3 specific], and 8 for early onset disease [2 specific]), of which a majority were novel and replicated. Among these were proteins associated with aggressive disease, such as PPA2 [Odds Ratio (OR) per 1 SD increment = 2.13, 95% CI: 1.54-2.93], PYY [OR = 1.87, 95% CI: 1.43-2.44] and PRSS3 [OR = 0.80, 95% CI: 0.73-0.89], and those associated with early onset disease, including EHPB1 [OR = 2.89, 95% CI: 1.99-4.21], POGLUT3 [OR = 0.76, 95% CI: 0.67-0.86] and TPM3 [OR = 0.47, 95% CI: 0.34-0.64]. We confirm an inverse association of MSMB with prostate cancer overall [OR = 0.81, 95% CI: 0.80-0.82], and also find an inverse association with both aggressive [OR = 0.84, 95% CI: 0.82-0.86] and early onset disease [OR = 0.71, 95% CI: 0.68-0.74]. Using spatial transcriptomics data, we identified MSMB as the genome-wide top-most predictive gene to distinguish benign regions from high grade cancer regions that had five-fold lower MSMB expression. Additionally, ten proteins that were associated with prostate cancer risk mapped to existing therapeutic interventions. CONCLUSION: Our findings emphasize the importance of proteomics for improving our understanding of prostate cancer etiology and of opportunities for novel therapeutic interventions. Additionally, we demonstrate the added benefit of in-depth functional analyses to triangulate the role of risk proteins in the clinical aggressiveness of prostate tumors. Using these integrated methods, we identify a subset of risk proteins associated with aggressive and early onset disease as priorities for investigation for the future prevention and treatment of prostate cancer.
\n \n\n \n \nBACKGROUND AND AIMS: Excess energy intake can lead to metabolic dysfunction-associated steatotic liver disease (MASLD), but the relationship between dietary carbohydrate intake and liver fat content remains unclear. This study aimed to examine the associations between types and sources of dietary carbohydrates and liver fat content. METHODS: UK Biobank participants with no pre-existing diabetes, liver disease or cardiovascular disease reported dietary intake of types and sources of carbohydrates (total carbohydrates, free sugars, non-free sugars, starch from whole grains, starch from refined grains, and fibre) on at least two 24-h dietary assessments. In cross-sectional analyses, (n\u00a0=\u00a022,973), odds ratios (OR) of high liver fat content (defined as a score of \u2265\u00a036 in the hepatic steatosis index) by quintiles of carbohydrate intakes were estimated using multivariable logistic regression models. In prospective analyses, a second sample (n\u00a0=\u00a09268) had liver proton density fat fraction (PDFF) measured by magnetic resonance imaging (2014-2020). Multivariable linear regression models estimated geometric means of PDFF (%) by quintiles of carbohydrate intakes. Models were adjusted for demographic and lifestyle confounders, including total energy intake. RESULTS: In the cross-sectional analyses, 6894 cases of high liver fat content were identified. Inverse associations between intakes of fibre (OR of highest vs. lowest quintile 0.46 [95% CI: 0.41-0.52]), non-free sugars (0.63 [0.57-0.70]) and starch from whole grains (0.52 [0.47-0.57]) with liver fat were observed. There were positive associations between starch from refined grains and liver fat (1.33 [1.21-1.46]), but no association with free sugars (p=0.61). In prospective analyses, inverse associations with PDFF (%) were observed for intakes of fibre (-\u00a00.48 geometric mean difference between highest and lowest quintile of intake [-\u00a00.60 to -\u00a00.35]), non-free sugars (-\u00a00.37 [-\u00a00.49 to -\u00a00.25]) and starch from whole grains (-\u00a00.31 [-\u00a00.42 to -\u00a00.19]). Free sugars, but not starch from refined grains, were positively associated with PDFF (0.17 [0.05 to 0.28]). CONCLUSION: This study suggests that different carbohydrate types and sources have varying associations with liver fat, which may be important for MASLD prevention. Non-free sugars, fibre, and starch from whole grains could be protective, while associations with free sugars and starch from refined grains are less clear.
\n \n\n \n \nBACKGROUND: Incidentally elevated cardiac troponin I (cTnI) levels are common in acutely unwell older patients. However, little is known about how this impacts on the prognosis of these patients. OBJECTIVE: We aimed to investigate whether incidentally elevated cTnI levels (group 1) are associated with poorer outcome when compared to age- and sex-matched patients without an elevated cTnI level (group 2), and to patients diagnosed with acute coronary syndrome (group 3). PATIENTS AND METHODS: This prospective, matched cohort study placed patients \u226575 years old who were admitted to a University teaching hospital into groups 1-3, based on the cTnI levels and underlying diagnosis. Outcomes were compared between the groups using mixed-effects regression models and adjusted for renal function and C-reactive protein. All-cause mortality at discharge, at 1 month and 3 months, alongside the length of hospital stay (LOS), were recorded. RESULTS: In total, 315 patients were included, with 105 patients in each of the 3 groups. The mean age was 84.8 \u00b1 5.5 years, with 41.9% males. All patients were followed up for 3 months. The percent all-cause mortality at discharge and the LOS for groups 1, 2 and 3 were 12.4, 3.8 and 8.6% and 11.2, 8.5 and 7.7 days, respectively. Group 1 had significantly increased mortality at 3 months [odds ratio (OR) 2.80, 95% confidence interval (CI) 1.12-6.96; p = 0.040] and LOS (OR 1.39, 95% CI 1.08-1.79; p = 0.008) compared to group 2 and did not differ significantly when compared to 3-month mortality (OR 2.39, 95% CI 0.91-6.29; p = 0.079) or LOS (OR 1.26, 95% CI 0.96-1.66; p = 0.097) in group 3. CONCLUSION: There is a significant association between an incidental rise in cTnI level with mortality and LOS in older patients. Further research is required to evaluate whether a more systematic management of these patients would improve the prognosis.
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