B cell-dependent subtypes and treatment-based immune correlates to survival in stage 3 and 4 lung adenocarcinomas.

Lung cancer is the leading cause of cancer-related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non-small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late-stage NSCLC. The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA-Seq) with deconvolution of RNA-Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B-cell rich patient group had increased expression of CXCL13 and greater abundance of PD1+ CD8 T cells. The presence of B cells and PD1+ CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS). We then assessed the predictive and prognostic utility of these cell types and TLS within publicly available stage 3 and 4 lung adenocarcinoma (LUAD) RNA-Seq datasets. As previously described by others, pre-treatment expression of intratumoral 12-chemokine TLS gene signature is associated with progression free survival (PFS) in patients who receive treatment with immune checkpoint inhibitors (ICI). Notably and unexpectedly pre-treatment percentages of intratumoral B cells are associated with PFS in patients who receive surgery, chemotherapy, or radiation. Further studies to confirm these findings would allow for more effective patient selection for both ICI and non-ICI treatments.

Conserved pan-cancer microenvironment subtypes predict response to immunotherapy.

The clinical use of molecular targeted therapy is rapidly evolving but has primarily focused on genomic alterations. Transcriptomic analysis offers an opportunity to dissect the complexity of tumors, including the tumor microenvironment (TME), a crucial mediator of cancer progression and therapeutic outcome. TME classification by transcriptomic analysis of >10,000 cancer patients identifies four distinct TME subtypes conserved across 20 different cancers. The TME subtypes correlate with patient response to immunotherapy in multiple cancers, with patients possessing immune-favorable TME subtypes benefiting the most from immunotherapy. Thus, the TME subtypes act as a generalized immunotherapy biomarker across many cancer types due to the inclusion of malignant and microenvironment components. A visual tool integrating transcriptomic and genomic data provides a global tumor portrait, describing the tumor framework, mutational load, immune composition, anti-tumor immunity, and immunosuppressive escape mechanisms. Integrative analyses plus visualization may aid in biomarker discovery and the personalization of therapeutic regimens.

Single-cell Spatial Proteomic Revelations on the Multiparametric MRI Heterogeneity of Clinically Significant Prostate Cancer.

PURPOSE: Multiparametric MRI (mpMRI) has become an indispensable radiographic tool in diagnosing prostate cancer. However, mpMRI fails to visualize approximately 15% of clinically significant prostate cancer (csPCa). The molecular, cellular, and spatial underpinnings of such radiographic heterogeneity in csPCa are unclear. EXPERIMENTAL DESIGN: We examined tumor tissues from clinically matched patients with mpMRI-invisible and mpMRI-visible csPCa who underwent radical prostatectomy. Multiplex immunofluorescence single-cell spatial imaging and gene expression profiling were performed. Artificial intelligence-based analytic algorithms were developed to examine the tumor ecosystem and integrate with corresponding transcriptomics. RESULTS: More complex and compact epithelial tumor architectures were found in mpMRI-visible than in mpMRI-invisible prostate cancer tumors. In contrast, similar stromal patterns were detected between mpMRI-invisible prostate cancer and normal prostate tissues. Furthermore, quantification of immune cell composition and tumor-immune interactions demonstrated a lack of immune cell infiltration in the malignant but not in the adjacent nonmalignant tissue compartments, irrespective of mpMRI visibility. No significant difference in immune profiles was detected between mpMRI-visible and mpMRI-invisible prostate cancer within our patient cohort, whereas expression profiling identified a 24-gene stromal signature enriched in mpMRI-invisible prostate cancer. Prostate cancer with strong stromal signature exhibited a favorable survival outcome within The Cancer Genome Atlas prostate cancer cohort. Notably, five recurrences in the 8 mpMRI-visible patients with csPCa and no recurrence in the 8 clinically matched patients with mpMRI-invisible csPCa occurred during the 5-year follow-up post-prostatectomy. CONCLUSIONS: Our study identified distinct molecular, cellular, and structural characteristics associated with mpMRI-visible csPCa, whereas mpMRI-invisible tumors were similar to normal prostate tissue, likely contributing to mpMRI invisibility.

Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases.

Mitochondria-targeted antioxidant SKQ1 protects cornea from oxidative damage induced by ultraviolet irradiation and mechanical injury.

BACKGROUND: Cornea protects the eye against natural and anthropogenic ultraviolet (UV) damage and mechanical injury. Corneal incisions produced by UV lasers in ophthalmic surgeries are often complicated by oxidative stress and inflammation, which delay wound healing and result in vision deterioration. This study trialed a novel approach to prevention and treatment of iatrogenic corneal injuries using SkQ1, a mitochondria-targeted antioxidant approved for therapy of polyethiological dry eye disease. METHODS: Rabbit models of UV-induced and mechanical corneal damage were employed. The animals were premedicated or treated with conjunctival instillations of 7.5 µM SkQ1. Corneal damage was assessed by fluorescein staining and histological analysis. Oxidative stress in cornea was monitored by measuring malondialdehyde (MDA) using thiobarbituric acid assay. Total antioxidant activity (AOA) was determined using hemoglobin/H2O2/luminol assay. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were measured using colorimetric assays. RESULTS: In both models corneas exhibited fluorescein-stained lesions, histologically manifesting as basal membrane denudation, apoptosis of keratocytes, and stromal edema, which were accompanied by oxidative stress as indicated by increase in lipid peroxidation and decline in AOA. The UV-induced lesions were more severe and long healing as corneal endothelium was involved and GPx and SOD were downregulated. The treatment inhibited loss of keratocytes and other cells, facilitated re-epithelialization and stromal remodeling, and reduced inflammatory infiltrations and edema thereby accelerating corneal healing approximately 2-fold. Meanwhile the premedication almost completely prevented development of UV-induced lesions. Both therapies reduced oxidative stress, but only premedication inhibited downregulation of the innate antioxidant activity of the cornea. CONCLUSIONS: SkQ1 efficiently prevents UV-induced corneal damage and enhances corneal wound healing after UV and mechanical impacts common to ocular surgery. Its therapeutic action can be attributed to suppression of mitochondrial oxidative stress, which in the first case embraces all corneal cells including epitheliocytes, while in the second case affects residual endothelial cells and stromal keratocytes actively working in wound healing. We suggest SkQ1 premedication to be used in ocular surgery for preventing iatrogenic complications in the cornea.

Mitochondria-Targeted Antioxidant SkQ1 Prevents Anesthesia-Induced Dry Eye Syndrome.

Dry eye syndrome (DES) is an age-related condition increasingly detected in younger people of risk groups, including patients who underwent ocular surgery or long-term general anesthesia. Being a multifactorial disease, it is characterized by oxidative stress in the cornea and commonly complicated by ocular surface inflammation. Polyetiologic DES is responsive to SkQ1, a mitochondria-targeted antioxidant suppressing age-related changes in the ocular tissues. Here, we demonstrate safety and efficacy of topical administration of SkQ1 at a dosage of 7.5 µM for the prevention of general anesthesia-induced DES in rabbits. The protective action of SkQ1 improves clinical state of the ocular surface by inhibiting apoptotic and prenecrotic changes in the corneal epithelium. The underlying mechanism involves the suppression of the oxidative stress supported by the stimulation of intrinsic antioxidant activity and the activity of antioxidant enzymes, foremost glutathione peroxidase and glutathione reductase, in the cornea. Furthermore, SkQ1 increases antioxidant activity and stability of the tear film and produces anti-inflammatory effect exhibited as downregulation of TNF-α and IL-6 and pronounced upregulation of IL-10 in tears. Our data suggest novel features of SkQ1 and point to its feasibility in patients with DES and individuals at risk for the disease including those subjected to general anesthesia.

The cancer-retina antigen recoverin as a potential biomarker for renal tumors.

The renal cell carcinoma is the ninth most common cancer with an increasing occurrence and mortality. Recoverin is the first retina-specific photoreceptor protein that was shown to undergo aberrant expression, due to its promoter demethylation, as a cancer-retina antigen in a number of malignant tumors. In this work, we demonstrated that recoverin is indeed expressed in 68.4 % of patients with different subtypes of renal cell carcinoma, and this expression has tendency to correlate with tumor size. Interestingly, 91.7 % of patients with the benign renal tumor, oncocytoma, express recoverin as well in their tumor. Epigenetic analysis of the recoverin gene promoter revealed a stable mosaic methylation pattern with the predominance of the methylated state, with the exception of -80 and 56 CpG dinucleotides (CpGs). While the recoverin expression does not correlate withoverall survival of the tumor patients, the methylation of the recoverin gene promoter at -80 position is associated with better overall survival of the patients. This work is the first report pointing towards the association of overall survival of renal cell carcinoma (RCC) patients with promoter methylation of a cancer-retina antigen. Taken together, these data allow to consider recoverin as a potential therapeutic target and/or marker for renal tumors.

Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions.

Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1-5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca(2+)-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol-disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from -160 to -155 mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.