Cytotoxic response of tumor-infiltrating lymphocytes of head and neck cancer slice cultures under mitochondrial dysfunction.

Background: Head and neck squamous cell carcinomas (HNSCC) are highly heterogeneous tumors. In the harsh tumor microenvironment (TME), metabolic reprogramming and mitochondrial dysfunction may lead to immunosuppressive phenotypes. Aerobic glycolysis is needed for the activation of cytotoxic T-cells and the absence of glucose may hamper the full effector functions of cytotoxic T-cells. To test the effect of mitochondrial dysfunction on cytotoxic T cell function, slice cultures (SC) of HNSCC cancer were cultivated under different metabolic conditions. Methods: Tumor samples from 21 patients with HNSCC were collected, from which, SC were established and cultivated under six different conditions. These conditions included high glucose, T cell stimulation, and temporarily induced mitochondrial dysfunction (MitoDys) using FCCP and oligomycin A with or without additional T cell stimulation, high glucose and finally, a control medium. Over three days of cultivation, sequential T cell stimulation and MitoDys treatments were performed. Supernatant was collected, and SC were fixed and embedded. Granzyme B was measured in the supernatant and in the SC via immunohistochemistry (IHC). Staining of PD1, CD8/Ki67, and cleaved-caspase-3 (CC3) were performed in SC. Results: Hematoxylin eosin stains showed that overall SC quality remained stable over 3 days of cultivation. T cell stimulation, both alone and combined with MitoDys, led to significantly increased granzyme levels in SC and in supernatant. Apoptosis following T cell stimulation was observed in tumor and stroma. Mitochondrial dysfunction alone increased apoptosis in tumor cell aggregates. High glucose concentration alone had no impact on T cell activity and apoptosis. Apoptosis rates were significantly lower under conditions with high glucose and MitoDys (p=0.03). Conclusion: Stimulation of tumor-infiltrating lymphocytes in SC was feasible, which led to increased apoptosis in tumor cells. Induced mitochondrial dysfunction did not play a significant role in the activation and function of TILs in SC of HNSCC. Moreover, high glucose concentration did not promote cytotoxic T cell activity in HNSCC SC.

Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization.

BACKGROUND: Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment. METHODS: We examined CD45-/CD31-/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR. RESULTS: CD45-/CD31-/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs. CONCLUSION: Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

CHANGES IN SYSTEMIC LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AFTER INTRAVITREAL INJECTION OF AFLIBERCEPT OR BROLUCIZUMAB FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To analyze and compare the effects of intravitreal brolucizumab versus aflibercept on systemic vascular endothelial growth factor (VEGF)-A levels in patients with neovascular age-related macular degeneration. METHODS: In this prospective interventional case series study, brolucizumab (6.0 mg/50 µL) or aflibercept (2.0 mg/50 µL) was injected intravitreally in 30 patients each. Blood samples were drawn at baseline and 7 days and 28 days after the first injection. Systemic VEGF-A levels were measured using enzyme-linked immunosorbent assay. Thirty healthy individuals served as controls. RESULTS: The median baseline systemic VEGF-A levels in the brolucizumab, aflibercept, and control groups were 10.8 (8.0-13.2), 12.0 (8.0-18.5), and 10.0 (8.0-15.1) pg/mL, respectively (P = 0.315). In the brolucizumab group, VEGF-A levels significantly decreased to 8.0 (8.0-11.5) pg/mL on Day 7 (P = 0.0254) and to 8.0 (8.0-8.0) pg/mL on Day 28 (P < 0.001). In the aflibercept group, VEGF-A levels significantly decreased to 8.0 (8.0-8.0) pg/mL on Day 7 (P < 0.001) but returned to the baseline level, 12.5 (8.5-14.6) pg/mL, on Day 28 (P = 0.120). Vascular endothelial growth factor-A levels were significantly different between the treatment groups after 28 days (P < 0.001). CONCLUSION: Intravitreal brolucizumab resulted in a sustained reduction of systemic VEGF-A levels until 28 days posttreatment, which raises concerns regarding its safety and long-term effects.

Effects of Intravitreal Aflibercept on the Systemic Insulin-like Growth Factor-I and Vascular Endothelial Growth Factor-A in Patients with Diabetic Retinopathy and Age-Related Macular Degeneration.

PURPOSE: To analyze the effect of intravitreal aflibercept injections on systemic levels of insulin-like growth factor-1 and vascular endothelial growth factor-A in patients with diabetic retinopathy and age-related macular degeneration. METHODS: Vascular endothelial growth factor-A and insulin-like growth factor-1 levels were determined before and one week and four weeks after intravitreal injection of aflibercept (2.0 mg/50 µl) for 19 patients with age-related macular degeneration (mean age, 76 ± 11 years) and 18 patients with diabetic retinopathy (mean age, 64 ± 14 years). Twenty-two healthy individuals were enrolled as controls. RESULTS: A significant decline in systemic vascular endothelial growth factor-A level, from 43 (30-57) pg/ml at baseline to 8 (8-8) pg/ml (p < 0.001) at week one and 17 (8-25) pg/ml (p=0.0054) at week four, was observed in the age-related macular degeneration group. In the diabetic retinopathy group, vascular endothelial growth factor-A levels declined from 53 (35-117) pg/ml to 2 (1-5) pg/ml (p < 0.0001) one week after injection and 16 (13-22) pg/ml four weeks after injection (p=0.0327). At baseline, systemic insulin-like growth factor-1 concentration was higher in the diabetic retinopathy group (57 [37-99] pg/ml) than in the age-related macular degeneration group (35 [24-51] pg/ml) (p=0.0056). A subgroup analysis showed that patients in the proliferative diabetic retinopathy subgroup had significantly higher systemic insulin-like growth factor-1 concentrations (71 [44.7-243] pg/ml) than those in the nonproliferative diabetic retinopathy subgroup (43 [29-66] pg/ml) (p=0.0048). CONCLUSIONS: The difference between the baseline systemic insulin-like growth factor-1 levels of the age-related macular degeneration and diabetic retinopathy groups and the higher insulin-like growth factor-1 levels in the proliferative diabetic retinopathy subgroup one week after aflibercept therapy suggest that insulin-like growth factor-1 may play a role in the pathomechanism of diabetic retinopathy.

Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing.

BACKGROUND: Adipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. However, much less is known about ASC and adipocytes outside classical fat tissue depots and their necessity in tissue remodeling after injury. Therefore, we investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing. METHODS: Identification of human wound tissue adipocytes was determined by immunohistochemical staining of granulation tissue sections from patients undergoing surgical debridement. Stromal cell fractions from granulation tissue and subcutaneous fat tissue were generated by collagenase type II-based protocols. Pro- and anti-inflammatory wound bed conditions were mimicked by THP1- and CD14+ monocyte-derived macrophage models in vitro. Effects of macrophage secretome on ASC differentiation and metabolism were determined by immunoblotting, flow cytometry, and microscopy assessing early and late adipocyte differentiation states. Functional rescuing experiments were conducted by lentiviral transduction of wildtype PPARG, IL1RA, and N-chlorotaurine (NCT) treatment. RESULTS: Single and clustered adipocyte populations were detected in 11 out of 13 granulation tissue specimens and single-cell suspensions from granulation tissue showed adipogenic differentiation potential. Pro-inflammatory signaling by IFNG/LPS-stimulated macrophages (M (IFNG/LPS)) inhibited the maturation of lipid droplets in differentiated ASC. In contrast, anti-inflammatory IL4/IL13-activated macrophages (M (IL4/IL13)) revealed minor effects on adipocyte development. The M (IFNG/LPS)-induced phenotype was associated with a switch from endogenous fatty acid synthesis to glycolysis-dominated cell metabolism and increased pro-inflammatory cytokine production. Impaired adipogenesis was associated with increased, but seemingly non-functional, CEBPB levels, which failed to induce downstream PPARG and CEBPA. Neither transgenic PPARG overexpression, nor inhibition of IL1B was sufficient to rescue the anti-adipogenic effects induced by IFNG/LPS-activated macrophages. Instead, macrophage co-treatment during stimulation with NCT, a mild oxidant produced by activated granulocytes present in human wounds in vivo, significantly attenuated the anti-adipogenic effects. CONCLUSIONS: In conclusion, the appearance of adipocytes in wound tissue indicates a prevailing anti-inflammatory environment that could be promoted by NCT treatment and may be associated with improved healing outcomes.

Systemic counterregulatory response of angiopoietin-2 after aflibercept therapy for nAMD: a potential escape mechanism.

PURPOSE: To analyse the effect of intravitreal aflibercept injections on systemic angiopoietin-2 (Ang2) and vascular endothelial growth factor (VEGF)-A levels in patients with neovascular age-related macular degeneration (nAMD). METHODS: In a prospective, randomized study, aflibercept (2.0 mg/50 µl) or ranibizumab (0.5 mg/50 µl) was administered intravitreally to 38 treatment-naive patients. Blood samples were taken before, 7 days after, and 28 days after the first intravitreal therapy. Cytokine levels were measured by enzyme-linked immunosorbent assay. Twenty-two age- and sex-matched individuals served as controls. RESULTS: At baseline, there were no significant differences of systemic Ang2 and VEGF-A levels among the treatment and control groups. After intravitreal aflibercept administration, median (interquartile range: IQR) systemic Ang2 was significantly upregulated from 1819 pg/ml (1262-3099) to 2123 pg/ml (1441-3769; p = 0.011) 7 days after the drug injection and remained non-significantly elevated at 1944 pg/ml (1431-2546 pg/ml; p = 0.653) 28 days after the drug injection. Median (IQR) systemic VEGF-A levels were significantly reduced from 43 pg/ml (30-57) to 8 pg/ml (8-8; p < 0.0001) 7 days and 16 pg/ml (8-26; p = 0.001) 28 days after the injection in the aflibercept group. There were no significant effects on systemic VEGF-A and Ang2 levels in the ranibizumab group at any time point following the first injection. CONCLUSION: In this study, we report significant systemic upregulation of Ang2 after intravitreal aflibercept administration. This counterregulatory response may represent a potential escape mechanism from antiangiogenic therapy.

Proton beam radiation for iris melanoma: case series and review of literature.

OBJECTIVE: Purpose of this study is to analyse the visual outcomes, the complication and eye retention rate as well as tumour control data of patients treated with proton beam radiation therapy (PBRT) for iris melanoma. METHODS: Retrospective case series and review based on patients' records. All tumours were categorised according to the American Joint Committee of Cancer staging criteria for primary iris melanoma und underwent either sectorial or whole anterior segment PBRT. RESULTS: Thirteen cases were identified of which five received PBRT of the whole anterior segment and eight received sectorial PBRT. Local tumour control after a mean follow-up of 25 months was 92%. Complications after PBRT included cataract (46%), secondary glaucoma (31%), superficial keratitis (15%) and madarosis (8%). Complications were more common in patients necessitating irradiation of the entire anterior segment than in patients which received sectorial irradiation. Eye retention was achieved in all cases. No statistically significant difference in the mean best corrected visual acuity (BCVA) and intraocular pressure (IOP) was found before and after treatment. Comparison of mean BCVA and IOP between different treatment groups (complete anterior segment vs sectorial irradiation) at the last follow-up visit were also not significantly different. No patient developed metastatic disease during follow-up. CONCLUSION: PBRT is a safe and vision preserving therapeutic modality for iris melanoma. Complete irradiation of the anterior segment is associated with higher complication rates.

En-face analysis of the human limbal lymphatic vasculature.

PURPOSE: To describe the location and morphometric characteristics of the human limbal lymphatic vasculature and its relation to the marginal corneal vascular arcades (MCA). METHODS: Ex vivo confocal microscopic (CM) imaging and immunofluorescence double staining for CD-31 and D2-40 of histological en-face sections using 12 preserved human cadaveric corneoscleral discs were performed, followed by a semi-automated morphometric analysis of the two-dimensional vascular network architecture. RESULTS: Ex vivo CM confirmed the presence of 2 distinct vascular networks. The haematic limbal vascular complex (HLVC) extended further into the cornea, forming typical MCAs. The lymphatic limbal vascular complex (LLVC) was peripheral from the termination of Bowman's layer and was also found to be peripheral to and deeper than the HLVC. LLVC and HLVC were significantly different with respect to vessel diameter, segment length and wall thickness. CONCLUSION: The lymphatic vasculature of the human corneoscleral limbal region displays specific morphometric features that allow its differentiation from haematic vessels using CM.

N-chlorotaurine Inactivates Acanthamoeba and Candida albicans in the Porcine Ex Vivo Corneal Infection Model.

PURPOSE: N-chlorotaurine (NCT) is an anti-infective belonging to the class of chloramines and an investigative drug for the topical treatment of keratoconjunctivitis. The aim of the present study was to demonstrate its efficacy against Acanthamoeba and Candida in corneas infected ex vivo. METHODS: Corneal buttons from porcine eyes were contaminated with Acanthamoeba castellanii trophozoites or Candida albicans Centraalbureau voor Schimmelcultures 5982 and incubated for 7 and 3 days, respectively. Subsequently, they were treated with 1% NCT for 5 to 120 minutes. After further incubation for 2 days in the absence of NCT in tests with A. castellanii, the buttons were homogenized, and the amoebae grown for a further 5 days before they were counted in a light microscope. For C. albicans, quantitative cultures were performed from corneal homogenates. RESULTS: Incubation of 120 minutes in NCT completely inhibited the regrowth of A. castellanii and reduced the number of C. albicans colony-forming unit counts by 4 log10. In addition, at 60 minutes, significant reductions of both pathogens could be observed. Histology showed penetration of pathogens into the stroma of the corneal buttons. CONCLUSIONS: NCT inactivates A. castellanii and C. albicans in corneal tissue.

Reduced Levels of ATP Synthase Subunit ATP5F1A Correlate with Earlier-Onset Prostate Cancer.

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) to aerobic glycolysis occurs in many types of tumors. However, the significance of energy metabolism for the development of prostate carcinoma is poorly understood. We investigated the expression of OXPHOS complexes in 94 human prostate carcinomas and paired benign tissue using immunohistochemistry. Overall mitochondrial mass was upregulated in carcinomas compared to benign prostate tissue in all Gleason grades. A significant direct correlation between the expression of OXPHOS complexes I, II, and V and the Gleason score was observed. However, 17% of prostate carcinomas and 18% of benign prostate tissues showed isolated or combined deficiency of OXPHOS complexes (one deficiency in 12% of the tumors, combined deficiencies in 5%). Complex I was absent in 9% of the samples, with only parts of the tumor affected. ATP5F1A, a complex V protein, was the most frequently affected subunit, in 10% of tumors and 11% of benign prostate tissues (but not both tissues in any single patient). A possible role of complex V in prostate cancer development is suggested by the significant positive correlation of ATP5F1A levels with earlier-onset prostate cancer (age at diagnosis and at prostatectomy) and free PSA percentage. The relatively high percentage (17%) of prostate carcinomas with regional foci of partial OXPHOS complex deficiencies could have important therapeutic implications.

The involvement of NK1 and Y2 receptor in the development of laser-induced CNVs in C57Bl/6N mice.

PURPOSE: to explore whether the NK1 and Y2 receptors are involved in the pathogenesis of laser-induced CNV (choroidal neovascularization) in C57Bl/6N mice. METHODS: CNV was induced by laser damage of Bruch's membrane and the CNV volume was determined by OCT and/or flatmount preparation. First, the development of the CNV volume over time was evaluated. Second, the CNV development in NK1- and Y2 KO mice was analyzed. Third, the effect on the development as well as the regression of CNV by intravitreal injections of the NK1 antagonist SR140333 and the Y2 antagonist BIIEO246 separately and each in combination with Eylea®, was investigated. Furthermore, flatmount CNV volume measurements were correlated to volumes obtained by the in vivo OCT technique. RESULTS: CNV volume peak was observed at day 4 after laser treatment. Compared to wild type mice, NK1 and Y2 KO mice showed significantly smaller CNV volumes. Eylea® and the Y2 antagonist significantly reduced the volume of the developing CNV. In contrast to Eylea® there was no effect of either antagonist on the regression of CNV, additionally no additive effect upon combined Eylea®/antagonist treatment was observed. There was a strong positive correlation between CNV volumes obtained by OCT and flatmount. CONCLUSION: NK1 and Y2 receptors mediate the development of laser-induced CNVs in mice. They seem to play an important role at the developmental stage of CNVs, whereas VEGF via VEGF receptor may be an important mediator throughout the CNV existence. In vivo OCT correlates with flatmount CNV volume, representing a useful tool for in vivo evaluations of CNV over time.

Effects of Intravitreal Aflibercept on Galectin-1 and Vascular Endothelial Growth Factor-A Plasma Levels in Patients with Diabetic Retinopathy.

PURPOSE: To analyze the interaction between aflibercept and galectin-1 and evaluate the plasma levels of galectin-1 and vascular endothelial growth factor (VEGF)-A after intravitreal injection of aflibercept in patients with diabetic retinopathy (DR). METHODS: Interaction of galectin-1 with aflibercept was determined via immunoprecipitation. Seventeen patients with type 2 diabetes and diabetic macular edema (DME) were each treated with a single intravitreal injection of aflibercept (2.0 mg, 50 µL) monthly for three consecutive months. Plasma galectin-1 and VEGF-A levels were measured just before an injection was administered, 1 week after the first injection, and 2 months after the last injection. Nineteen age- and sex-matched healthy participants served as controls. RESULTS: Irrespective of the tested galectin-1 concentration, 24% of added galectin-1 was precipitated by aflibercept. Baseline plasma concentrations of galectin-1 were 22.0 and 23.0 ng/mL in the control and aflibercept-treated groups, respectively. Systemic galectin-1 levels increased to 27.0 and 24.0 ng/mL at 7 days and 4 weeks, respectively, after treatment. At week 8, plasma galectin-1 levels significantly increased to 36.0 ng/mL. This level persisted for 20 weeks. Systemic VEGF-A levels significantly reduced to below the minimum detectable dose in 16 DME patients at 7 days after treatment. This level persisted for 4 weeks. Plasma VEGF-A levels were reduced at weeks 8 (p = 0.099) and 20 (p = 0.023). Decreased plasma VEGF-A levels were observed in all patients after treatment. CONCLUSION: We confirmed that physiological aflibercept levels precipitate galectin-1 in in vitro assays. Additionally, systemic upregulation of galectin-1 might be induced by intravitreal aflibercept, which may be relevant in the clinical outcomes of DR treatment.

Morphometric characterisation of pterygium associated with corneal stromal scarring using high-resolution anterior segment optical coherence tomography.

AIMS: To investigate the role of high-resolution anterior segment optical coherence tomography (HR-ASOCT) in the assessment of pterygia. METHODS: Single centre cross-sectional study. Patients with primary pterygium and/or pingueculae were included. Clinical assessment included HR-ASOCT, colour photography, keratometry followed by histology. Associations were tested between HR-ASOCT features of the pterygium and the degree of corneal scarring and elastotic degeneration, astigmatism and best-corrected visual acuity. RESULTS: 29 eyes of 26 patients with pterygium and 6 patients with pinguecula were included. Apical anterior stromal scarring was found in 23 cases (79.3%) reaching a mean depth of 68.8±21.7 µm (minimum: 33 µm, maximum: 126 µm). Increased stromal scarring and subepithelial elastotic degenerative tissue was significantly associated with HR-ASOCT features of flat bridging of the corneoscleral transition zone (p<0.01) reduced thickness of the pterygium head (p=0.01), and a greater degree of corneal astigmatism (p=0.04). CONCLUSIONS: HR-ASOCT is a useful tool for the assessment and monitoring of pterygia in clinical practice. Features associated with increased stromal scarring and astigmatism are reduced thickness of the head of the pterygium and flat bridging of the corneoscleral transition zone.

Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases. METHODS: We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base). RESULTS: We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p<0.001). Non-synonymous heteroplasmic variants were enriched among cancerous tissues. The proportions of somatic and inherited variants in a given gene region were strongly correlated (r = 0.85; p<0.001). Half of the patients shared mutations between benign and cancerous tissue samples. Low level heteroplasmies (<10%) were more frequent in benign samples compared to tumor samples, where heteroplasmies >10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases. CONCLUSIONS: We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.

MALDI-MS tissue imaging identification of biliverdin reductase B overexpression in prostate cancer.

New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue. BIOLOGICAL SIGNIFICANCE: In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target.

Enhanced inhibition of prostate tumor growth by dual targeting the androgen receptor and the regulatory subunit type iα of protein kinase a in vivo.

Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysis revealed that both ODNs were effective in reducing their target proteins when administered alone or in combination. In addition, treatment with the ODNs was associated with an induction of apoptosis. Our data suggest that dual targeting of the AR and PKARIα is more effective in inhibiting LNCaP and LNCaPabl tumor growth than single treatment and may give a treatment benefit, especially in castration-resistant prostate cancers.

Characterization of transcriptional changes in ERG rearrangement-positive prostate cancer identifies the regulation of metabolic sensors such as neuropeptide Y.

ERG gene rearrangements are found in about one half of all prostate cancers. Functional analyses do not fully explain the selective pressure causing ERG rearrangement during the development of prostate cancer. To identify transcriptional changes in prostate cancer, including tumors with ERG gene rearrangements, we performed a meta-analysis on published gene expression data followed by validations on mRNA and protein levels as well as first functional investigations. Eight expression studies (n = 561) on human prostate tissues were included in the meta-analysis. Transcriptional changes between prostate cancer and non-cancerous prostate, as well as ERG rearrangement-positive (ERG+) and ERG rearrangement-negative (ERG-) prostate cancer, were analyzed. Detailed results can be accessed through an online database. We validated our meta-analysis using data from our own independent microarray study (n = 57). 84% and 49% (fold-change>2 and >1.5, respectively) of all transcriptional changes between ERG+ and ERG- prostate cancer determined by meta-analysis were verified in the validation study. Selected targets were confirmed by immunohistochemistry: NPY and PLA2G7 (up-regulated in ERG+ cancers), and AZGP1 and TFF3 (down-regulated in ERG+ cancers). First functional investigations for one of the most prominent ERG rearrangement-associated genes - neuropeptide Y (NPY) - revealed increased glucose uptake in vitro indicating the potential role of NPY in regulating cellular metabolism. In summary, we found robust population-independent transcriptional changes in prostate cancer and first signs of ERG rearrangements inducing metabolic changes in cancer cells by activating major metabolic signaling molecules like NPY. Our study indicates that metabolic changes possibly contribute to the selective pressure favoring ERG rearrangements in prostate cancer.

High expression of prostate-specific membrane antigen in the tumor-associated neo-vasculature is associated with worse prognosis in squamous cell carcinoma of the oral cavity.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed in prostate cancer as well as in the neo-vasculature of nonprostatic solid tumors. Here, we determined the expression pattern of PSMA in the vasculature of oral squamous cell carcinoma. Using a previously validated antibody, PSMA staining distribution and cyclooxygenase 2 (COX2) expression status was evaluated in a cohort of patients with squamous cell carcinoma of the oral cavity (n=96) using immunohistochemistry and was correlated with clinicopathological features as well as outcome. Twenty-four (25%) cases showed no detectable PSMA staining, 48 (50%) demonstrated positive immunoreactivity for PSMA in less than 50% of microvessels and 24 (25%) cases showed strong endothelial PSMA expression in more than 50% of tumor-associated microvessels. High endothelial PSMA expression was associated with greatly reduced survival (18.2 vs 77.3 months; P=0.0001) and maintained prognostic significance after adjusting for grade and stage in multivariate analysis (hazard ratio=2.19, P=0.007). Furthermore, we observed a strong association between endothelial PSMA and cancer cell-specific COX2 expression. In conclusion, we provide the first evidence for the prognostic significance of endothelial PSMA expression in oral squamous cell carcinoma and, suggest a potential interaction between arachidonic acid metabolites and endothelial PSMA expression in the tumor neo-vasculature.

Accumulation of mutations over the entire mitochondrial genome of breast cancer cells obtained by tissue microdissection.

The occurrence of heteroplasmy and mixtures is technically challenging for the analysis of mitochondrial DNA. More than that, observed mutations need to be carefully interpreted in the light of the phylogeny as mitochondrial DNA is a uniparental marker reflecting human evolution. Earlier attempts to explain the role of mtDNA in cancerous tissues led to substantial confusion in medical genetics mainly due to the presentation of low sequence data quality and misinterpretation of mutations representing a particular haplogroup background rather than being cancer-specific. The focus of this study is to characterize the extent and level of mutations in breast cancer samples obtained by tissue microdissection by application of an evaluated full mtDNA genome sequencing protocol. We amplified and sequenced the complete mitochondrial genomes of microdissected breast cancer cells of 15 patients and compared the results to those obtained from paired non-cancerous breast tissue derived from the same patients. We observed differences in the heteroplasmic states of substitutions between cancerous and normal cells, one of which was affecting a position that has been previously reported in lung cancer and another one that has been identified in 16 epithelial ovarian tumors, possibly indicating functional relevance. In the coding region, we found full transitions in two cancerous mitochondrial genomes and 12 heteroplasmic substitutions as compared to the non-cancerous breast cells. We identified somatic mutations over the entire mtDNA of human breast cancer cells potentially impairing the mitochondrial OXPHOS system.

Somatic mutations throughout the entire mitochondrial genome are associated with elevated PSA levels in prostate cancer patients.

The genetic etiology of prostate cancer, the most common form of male cancer in western countries, is complex and the interplay of disease genes with environmental factors is far from being understood. Studies on somatic mitochondrial DNA (mtDNA) mutations have become an important aspect of cancer research because these mutations might have functional consequences and/or might serve as biosensors for tumor detection and progression. We sequenced the entire mitochondrial genome (16,569 bp) from 30 prospectively collected pairs of macrodissected cancerous and benign cells from prostate cancer patients and compared their genetic variability. Given recent concerns regarding the authenticity of newly discovered mtDNA mutations, we implemented a high-quality procedure for mtDNA whole-genome sequencing. In addition, the mitochondrial genes MT-CO2, MT-CO3, MT-ATP6, and MT-ND6 were sequenced in further 35 paired samples from prostate cancer patients. We identified a total of 41 somatic mutations in 22 out of 30 patients: the majority of these mutations have not previously been observed in the human phylogeny. The presence of somatic mutations in transfer RNAs (tRNAs) was found to be associated with elevated PSA levels (14.25 ± 5.44 versus 7.15 ± 4.32 ng/ml; p = 0.004). The level and degree of heteroplasmy increased with increasing tumor activity. In summary, somatic mutations in the mitochondrial genome are frequent events in prostate cancer. Mutations mapping to mitochondrial tRNAs, ribosomal RNAs, and protein coding genes might impair processes that occur within the mitochondrial compartment (e.g., transcription, RNA processing, and translation) and might finally affect oxidative phosphorylation.