DrugMap: A quantitative pan-cancer analysis of cysteine ligandability.

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy.

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Stroma-specific gene expression signature identifies prostate cancer subtype with high recurrence risk.

Current prognostic tools cannot clearly distinguish indolent and aggressive prostate cancer (PC). We hypothesized that analyzing individual contributions of epithelial and stromal components in localized PC (LPC) could improve risk stratification, as stromal subtypes may have been overlooked due to the emphasis on malignant epithelial cells. Hence, we derived molecular subtypes of PC using gene expression analysis of LPC samples from prostatectomy patients (cohort 1, n = 127) and validated these subtypes in two independent prostatectomy cohorts (cohort 2, n = 406, cohort 3, n = 126). Stroma and epithelium-specific signatures were established from laser-capture microdissection data and non-negative matrix factorization was used to identify subtypes based on these signatures. Subtypes were functionally characterized by gene set and cell type enrichment analyses, and survival analysis was conducted. Three epithelial (E1-E3) and three stromal (S1-S3) PC subtypes were identified. While subtyping based on epithelial signatures showed inconsistent associations to biochemical recurrence (BCR), subtyping by stromal signatures was significantly associated with BCR in all three cohorts, with subtype S3 indicating high BCR risk. Subtype S3 exhibited distinct features, including significantly decreased cell-polarity and myogenesis, significantly increased infiltration of M2-polarized macrophages and CD8 + T-cells compared to subtype S1. For patients clinically classified as CAPRA-S intermediate risk, S3 improved prediction of BCR. This study demonstrates the potential of stromal signatures in identification of clinically relevant PC subtypes, and further indicated that stromal characterization may enhance risk stratification in LPC and may be particularly promising in cases with high prognostic ambiguity based on clinical parameters.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Insulin-like growth factor 1 associated with altered immune responses in preterm infants and pigs.

BACKGROUND: Preterm infants show low blood levels of insulin-like growth factor 1 (IGF-1), known to be negatively correlated with Interleukin-6 (IL-6). We hypothesized that circulating IGF-1 is associated with systemic immune-markers following preterm birth and that exogenous IGF-1 supplementation modulates immune development in preterm pigs, used as model for preterm infants. METHODS: Plasma levels of IGF-1 and 29 inflammatory markers were measured in very preterm infants (n = 221). In preterm pigs, systemic immune development, assessed by in vitro challenge, was compared between IGF-1 treated (2.25 mg/kg/day) and control animals. RESULTS: Preterm infants with lowest gestational age and birth weight showed the lowest IGF-1 levels, which were correlated not only with IL-6, but a range of immune-markers. IGF-1 supplementation to preterm pigs reduced plasma IL-10 and Interferon-γ (IFN-γ), IL-2 responses to challenge and reduced expression of genes related to Th1 polarization. In vitro addition of IGF-1 (100 ng/mL) further reduced the IL-2 and IFN-γ responses but increased IL-10 response. CONCLUSIONS: In preterm infants, plasma IGF-1 correlated with several immune markers, while supplementing IGF-1 to preterm pigs tended to reduce Th1 immune responses. Future studies should document whether IGF-1 supplementation to preterm infants affects immune development and sensitivity to infection. IMPACT: Supplementation of insulin-like growth factor 1 (IGF-1) to preterm infants has been proposed to promote postnatal growth, but its impact on the developing immune system is largely unknown. In a cohort of very preterm infants, low gestational age and birth weight were the primary predictors of low plasma levels of IGF-1, which in turn were associated with plasma immune markers. Meanwhile, in immature preterm pigs, experimental supplementation of IGF-1 reduced Th1-related immune responses in early life. Supplementation of IGF-1 to preterm infants may affect the developing immune system, which needs consideration when evaluating overall impact on neonatal health.

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability.

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap , an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFκB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

Spatial whole transcriptome profiling of primary tumor from patients with metastatic prostate cancer.

Prostate cancer (PCa) is a highly heterogeneous disease in terms of its molecular makeup and clinical prognosis. The prostate tumor microenvironment (TME) is hypothesized to play an important role in driving disease aggressiveness, but precise mechanisms remain elusive. In our study, we used spatial transcriptomics to explore for the first time the spatial gene expression heterogeneity within primary prostate tumors from patients with metastatic disease. In total, we analyzed 5459 tissue spots from three PCa patients comprising castration-resistant (CRPC) and neuroendocrine (NEPC) disease stages. Within CRPC, we identified a T cell cluster whose activity might be impaired by nearby regulatory T cells, potentially mediating the aggressive disease phenotype. Moreover, we identified Hallmark signatures of epithelial-mesenchymal transition in a cancer-associated fibroblast (CAF) cluster, indicating the aggressive characteristic of the primary TME leading to metastatic dissemination. Within NEPC, we identified active immune-stroma cross-talk exemplified by significant ligand-receptor interactions between CAFs and M2 macrophages. Further, we identified a malignant cell population that was associated with the down-regulation of an immune-related gene signature. Lower expression of this signature was associated with higher levels of genomic instability in advanced PCa patients (SU2C cohort, n = 99) and poor recurrence free survival in early-stage PCa patients (TCGA cohort, n = 395), suggesting prognostic biomarker potential. Taken together, our study reveals the importance of whole transcriptome profiling at spatial resolution for biomarker discovery and for advancing our understanding of tumor biology.

Developmental Expression of the Cell Cycle Regulator p16INK4a in Retinal Glial Cells: A Novel Marker for Immature Ocular Astrocytes?

Retinal astrocytes are vital for neuronal homeostasis in the retina. Together with Müller glia, they provide retinal cells with neurotrophic factors, antioxidative support, and defense mechanisms such as the formation of the blood-retinal barrier. Substantial heterogeneity of astrocyte morphology and function represents a challenge for identification of distinct subtypes which may be potential targets for therapeutic purposes. Hence, identification of novel markers of astrocyte subpopulations is highly relevant to better understand the molecular mechanisms involved in retinal development, homeostasis, and pathology. In this study, we observed that the cell cycle regulator, p16INK4a, is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16INK4a expression in the optic nerve of wild-type mice from 3 days to 3 months of age and in the nerve fiber layer of the adult mouse retina. Colocalization of p16INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16INK4a expression and vimentin (immature astrocyte marker) remains high in the optic nerve from the early postnatal period to adulthood. The observations from this study provide a valuable tool for further investigations of ocular astrocytes in the developing retina as well as in degenerative retinopathies.

Epigenetic Silencing of LRP2 Is Associated with Dedifferentiation and Poor Survival in Multiple Solid Tumor Types.

More than 80% of human cancers originate in epithelial tissues. Loss of epithelial cell characteristics are hallmarks of tumor development. Receptor-mediated endocytosis is a key function of absorptive epithelial cells with importance for cellular and organismal homeostasis. LRP2 (megalin) is the largest known endocytic membrane receptor and is essential for endocytosis of various ligands in specialized epithelia, including the proximal tubules of the kidney, the thyroid gland, and breast glandular epithelium. However, the role and regulation of LRP2 in cancers that arise from these tissues has not been delineated. Here, we examined the expression of LRP2 across 33 cancer types in The Cancer Genome Atlas. As expected, the highest levels of LRP2 were found in cancer types that arise from LRP2-expressing absorptive epithelial cells. However, in a subset of tumors from these cancer types, we observed epigenetic silencing of LRP2. LRP2 expression showed a strong inverse correlation to methylation of a specific CpG site (cg02361027) in the first intron of the LRP2 gene. Interestingly, low expression of LRP2 was associated with poor patient outcome in clear cell renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, papillary thyroid carcinoma, and invasive breast carcinoma. Furthermore, loss of LRP2 expression was associated with dedifferentiated histological and molecular subtypes of these cancers. These observations now motivate further studies on the functional role of LRP2 in tumors of epithelial origin and the potential use of LRP2 as a cancer biomarker.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design.

BACKGROUND: Cancer immunotherapies such as bispecific T-cell engagers have seen limited adoption in prostate cancer (PC), possibly due to differing levels of cancer receptor expression and effector T-cell infiltration between patients and inherent defects in T-cell engager design. METHODS: CD8+ T-cell infiltration and PSMA expression were determined by RNA sequencing of primary PC tissue samples from 126 patients with localised PC and 17 patients with metastatic PC. Prognostic value was assessed through clinical parameters, including CAPRA-S risk score. A panel of albumin-fused anti-CD3 × anti-PSMA T-cell engagers with different neonatal Fc receptor (FcRn) affinity were characterised by flow cytometry, Bio-Layer Interferometry and functional cellular assays. RESULTS: A subset of patients with localised (30/126 = 24%) and metastatic (10/17 = 59%) PC showed both high PSMA expression and high CD8+ T-cell enrichment. The High/High phenotype in localised PC associated with a clinically high-risk cancer subtype, confirmed in an external patient cohort (n = 550, PRAD/TCGA). The T-cell engagers exhibited tunable FcRn-driven cellular recycling, CD3 and PSMA cellular engagement, T-cell activation and PSMA level-dependent cellular cytotoxicity. CONCLUSION: This work presents an albumin-fused bispecific T-cell engager with programmable FcRn engagement and identifies a high-risk PC patient subset as candidates for treatment with the T-cell engager class of immuno-oncology biologics.

Predicting Grade group 2 or higher cancer at prostate biopsy by 4Kscore in blood and uCaP microRNA model in urine.

Elevated prostate-specific antigen (PSA) levels often lead to unnecessary and possibly harmful transrectal ultrasound guided biopsy, e.g. when the biopsy is negative or contains only low-grade insignificant cancer, unlikely to become symptomatic in the man's normal lifespan. A model based on four-kallikrein markers in blood (commercialized as 4Kscore) predicts risk of Grade group 2 or higher prostate cancer at biopsy, reducing unnecessary biopsies. We assessed whether these results extend to a single institution prostate biopsy cohort of Danish men and are enhanced by three microRNAs from urine (referred to as uCaP). The 4Kscore measured in cryopreserved blood from 234 men referred for 10+ core biopsy to Aarhus University Hospital, 29 with PSA > 25 ng/ml. We explored uCaP in urine from 157 of these men. Combined with age and DRE findings, both 4Kscore and uCaP could accurately predict Grade group 2 or higher prostate cancer (all patients: AUC = 0.802 and 0.797; PSA ≤ 25: AUC = 0.763 and 0.759). There was no additive effect when combining the 4Kscore and uCaP. Limitations include a study cohort with higher risk than commonly reported for biopsy cohorts. Our findings further support the clinical use of the 4Kscore to predict Grade group 2 or higher cancers in men being considered for biopsy.

A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.

Microbiota of the prostate tumor environment investigated by whole-transcriptome profiling.

BACKGROUND: With over 350,000 estimated deaths worldwide in 2018, prostate cancer (PCa) continues to be a major health concern and a significant cause of cancer-associated mortality among men. While cancer in general is considered a disease of the human genome, there is a growing body of evidence suggesting that changes to the healthy microbiota could play a vital role in cancer development, progression, and/or treatment outcome. METHODS: Using a metatranscriptomic approach, we annotated the microbial reads obtained from total RNA sequencing of 106 prostate tissue samples from 94 PCa patients (discovery cohort). We investigated microbial dysbiosis associated with PCa by systematically comparing the microbiomes between benign and malignant tissue samples, between less vs. more-aggressive PCa, and between patients who had biochemical recurrence as opposed to those who did not. We further performed differential gene expression and cell type enrichment analysis to explore the host transcriptomic and cellular responses to selected microbial genera. A public dataset (GSE115414) of total RNA sequencing reads from 24 prostate tissue samples (8 benign and 16 malignant) served as the validation cohort. RESULTS: We observed decreased species diversity and significant under-representation of Staphylococcus saprophyticus and Vibrio parahaemolyticus, as well as significant over-abundance of Shewanella in malignant as compared to benign prostate tissue samples in both the discovery (p < 0.01) and validation (p < 0.05) cohorts. In addition, we identified Microbacterium species (p < 0.01) to be significantly over-abundant in pathologically advanced T3 tumors compared to T2 in the discovery cohort. Malignant samples having high vs. low Shewanella counts were associated with downregulated Toll-like receptor signaling pathways and decreased enrichment of dendritic cells. Malignant samples having low vs. high V. parahaemolyticus counts were enriched for olfactory transduction and drug metabolism pathways. Finally, malignant samples were enriched for M1 and M2 macrophages as compared to benign tissue samples. CONCLUSIONS: The results from this exploratory study support the existence of an important biological link between the prostate microbiota and PCa development/progression. Our results highlight Shewanella, V. parahaemolyticus, and Microbacterium sp. as interesting candidates for further investigation of their association with PCa.

Independent validation of a pre-specified four-kallikrein marker model for prediction of adverse pathology and biochemical recurrence.

BACKGROUND: Accurate markers for prostate cancer (PC) risk stratification could aid decision-making for initial management strategies. The 4Kscore has an undefined role in predicting outcomes after radical prostatectomy (RP). METHODS: We included 1476 patients with 4Kscore measured prior to RP at two institutions. The 4Kscore was assessed for prediction of adverse pathology at RP and biochemical recurrence (BCR) relative to a clinical model. We pre-specified that all analyses would be assessed in biopsy Grade Group 1 (GG1) or 2 (GG2) PC patients, separately. RESULTS: The 4Kscore increased discrimination for adverse pathology in all patients (delta area under the receiver operative curve (AUC) 0.009, 95% confidence interval (CI) 0.002, 0.016; clinical model AUC 0.767), driven by GG1 (delta AUC 0.040, 95% CI 0.006, 0.073) rather than GG2 patients (delta AUC 0.005, 95% CI -0.012, 0.021). Adding 4Kscore improved prediction of BCR in all patients (delta C-index 0.014, 95% CI 0.007, 0.021; preop-BCR nomogram C-index 0.738), again with larger changes in GG1 than in GG2. CONCLUSIONS: This study validates prior investigations on the use of 4Kscore in men with biopsy-confirmed PC. Men with GG1 PC and a high 4Kscore may benefit from additional testing to guide treatment selection. Further research is warranted regarding the value of the 4Kscore in men with biopsy GG2 PC.

Association of Cycling With All-Cause and Cardiovascular Disease Mortality Among Persons With Diabetes: The European Prospective Investigation Into Cancer and Nutrition (EPIC) Study.

Importance: Premature death from all causes and cardiovascular disease (CVD) causes is higher among persons with diabetes. Objective: To investigate the association between time spent cycling and all-cause and CVD mortality among persons with diabetes, as well as to evaluate the association between change in time spent cycling and risk of all-cause and CVD mortality. Design, Setting, and Participants: This prospective cohort study included 7459 adults with diabetes from the European Prospective Investigation into Cancer and Nutrition study. Questionnaires regarding medical history, sociodemographic, and lifestyle information were administered in 10 Western European countries from 1992 through 2000 (baseline examination) and at a second examination 5 years after baseline. A total of 5423 participants with diabetes completed both examinations. The final updated primary analysis was conducted on November 13, 2020. Exposures: The primary exposure was self-reported time spent cycling per week at the baseline examination. The secondary exposure was change in cycling status from baseline to the second examination. Main Outcomes and Measures: The primary and secondary outcomes were all-cause and CVD mortality, respectively, adjusted for other physical activity modalities, diabetes duration, and sociodemographic and lifestyle factors. Results: Of the 7459 adults with diabetes included in the analysis, the mean (SD) age was 55.9 (7.7) years, and 3924 (52.6%) were female. During 110 944 person-years of follow-up, 1673 deaths from all causes were registered. Compared with the reference group of people who reported no cycling at baseline (0 min/wk), the multivariable-adjusted hazard ratios for all-cause mortality were 0.78 (95% CI, 0.61-0.99), 0.76 (95% CI, 0.65-0.88), 0.68 (95% CI, 0.57-0.82), and 0.76 (95% CI, 0.63-0.91) for cycling 1 to 59, 60 to 149, 150 to 299, and 300 or more min/wk, respectively. In an analysis of change in time spent cycling with 57 802 person-years of follow-up, a total of 975 deaths from all causes were recorded. Compared with people who reported no cycling at both examinations, the multivariable-adjusted hazard ratios for all-cause mortality were 0.90 (95% CI, 0.71-1.14) in those who cycled and then stopped, 0.65 (95% CI, 0.46-0.92) in initial noncyclists who started cycling, and 0.65 (95% CI, 0.53-0.80) for people who reported cycling at both examinations. Similar results were observed for CVD mortality. Conclusion and Relevance: In this cohort study, cycling was associated with lower all-cause and CVD mortality risk among people with diabetes independent of practicing other types of physical activity. Participants who took up cycling between the baseline and second examination had a considerably lower risk of both all-cause and CVD mortality compared with consistent noncyclists.

Immune cell analyses of the tumor microenvironment in prostate cancer highlight infiltrating regulatory T cells and macrophages as adverse prognostic factors.

Improved risk stratification is needed for patients with localized prostate cancer. This study characterized and assessed the prognostic potential of distinct immune cell infiltration patterns in the prostate tumor microenvironment. Using tissue microarrays, multiplex immunohistochemistry/immunofluorescence, and automated digital pathology, we analyzed radical prostatectomy specimens from two large patient cohorts (training: n = 470; validation: n = 333) to determine infiltration levels of seven immune cell types in malignant versus benign prostate tissue: CD3+ CD8- FoxP3- T helper cells, CD3+ CD8+ FoxP3- cytotoxic T cells (CTLs), CD3+ CD8- FoxP3+ regulatory T cells (Tregs ), CD20+ B cells, CD68+ CD163- M1 macrophages, CD68+ CD163+ M2 macrophages, and tryptase+ mast cells. Results were further validated by cell type enrichment analyses of bulk tumor RNAseq data from a third independent patient cohort (n = 99). Prognostic potential was assessed by Kaplan-Meier and uni-/multi-variate Cox regression analyses. Clinical endpoint was biochemical recurrence. All seven immune cell types were enriched in prostate cancer versus benign stroma, while there was selective enrichment for B cells, Tregs , M1 and M2 macrophages, and depletion of mast cells and CTLs in prostate cancer epithelium. In all three cohorts, high levels of infiltrating Tregs , M1, and M2 macrophages in stroma and/or epithelium were associated with biochemical recurrence (p < 0.05; log-rank test). After adjustment for routine clinical variables, Tregs and M2 macrophages remained significant adverse predictors of biochemical recurrence (p < 0.05; multivariate Cox regression). Our comprehensive analyses of immune cell infiltration patterns in the prostate tumor microenvironment highlight infiltrating Tregs , M1, and M2 macrophages as adverse predictors of prostate cancer outcome. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Label-Free Sensing of Biorecognition on Liposomes.

Nanometer-sized liposomes decorated with macromolecules are increasingly used as drug delivery vehicles due to their long lifetimes and target cell specificity, but surface characterization methods often change their properties, which leads to incorrect results. Ligand binding is commonly applied for characterizing these surface modifications. Here, we use a nanofluidic-based label-free sensor for real-time sensing of ligands binding to liposomes. The liposomes are trapped in a nanochannel with a salt concentration gradient, and as the trapping position depends on the liposomes' zeta potential, it changes when charged ligands bind to the liposomes. Our sensing method does not require immobilization of the liposomes or labeling of the ligands with fluorophores, which may both affect the sensing. The zeta potential sensing is demonstrated by measuring hybridization of DNA targets with complementary DNA probes on liposome surfaces. DNA hybridization is monitored for both ensembles and individual liposomes, the latter allows for analysis of ensemble heterogeneity, and we demonstrate sensitivity to changes in surface charge down to 1.5%. DNA hybridization is used to demonstrate label-free sensing, but the method also has potential applications within exosome characterization, where biorecognition of, e.g., surface DNA, proteins, and antibodies is a promising candidate for early stage cancer diagnostics.

Size and surface charge characterization of nanoparticles with a salt gradient.

Exosomes are nanometer-sized lipid vesicles present in liquid biopsies and used as biomarkers for several diseases including cancer, Alzheimer's, and central nervous system diseases. Purification and subsequent size and surface characterization are essential to exosome-based diagnostics. Sample purification is, however, time consuming and potentially damaging, and no current method gives the size and zeta potential from a single measurement. Here, we concentrate exosomes from a dilute solution and measure their size and zeta potential in a one-step measurement with a salt gradient in a capillary channel. The salt gradient causes oppositely directed particle and fluid transport that trap particles. Within minutes, the particle concentration increases more than two orders of magnitude. A fit to the spatial distribution of a single or an ensemble of exosomes returns both their size and surface charge. Our method is applicable for other types of nanoparticles. The capillary is fabricated in a low-cost polymer device.

Differentially expressed marker genes and glycogen levels in pectoralis major of Ross308 broilers with wooden breast syndrome indicates stress, inflammation and hypoxic conditions.

The occurrence of wooden breast (WB) in broiler production is increasing, but onset of its development is only described in part. In this study, we determined the regulation of marker genes related to oxidative stress in Ross308 broilers categorized as no-, mild- or severe-WB, on days 21 and 30 of production. The biochemical parameters, lactate dehydrogenase and pro- and macro-glycogen, were also determined. On day 21, breast meat from birds affected severely by WB had increased mRNA abundances of heat-shock protein 70, heme-oxygenase 1, cyclooxygenase 2, tumor necrosis factor 1, and hypoxia inducible factors as well as higher pH and lower dry matter contents. On day 30, breast meat from both mild and severely affected birds had increased mRNA for heme oxygenase 1, lactate dehydrogenase, and hypoxia inducible factor. Moreover, pro- and micro-glycogen, as well as the total pool of glycogen, were decreased compared with the non-WB birds. In conclusion, this study indicates oxidative stress, inflammation and hypoxic conditions in WB.