Impact of spatial metabolomics on immune-microenvironment in oral cancer prognosis: a clinical report.

MALDI imaging for metabolites and immunohistochemistry for 38 immune markers was used to characterize the spatial biology of 2 primary oral tumours, one from a patient with an early recurrence (Tumour R), and the other from a patient with no recurrence 2 years after treatment completion (Tumour NR). Tumour R had an increased purine nucleotide metabolism in different regions of tumour and adenosine-mediated suppression of immune cells compared to Tumour NR. The differentially expressed markers in the different spatial locations in tumour R were CD33, CD163, TGF-ß, COX2, PD-L1, CD8 and CD20. These results suggest that altered tumour metabolomics concomitant with a modified immune microenvironment could be a potential marker of recurrence.

Overexpression of CD73 is associated with recurrence and poor prognosis of gingivobuccal oral cancer as revealed by transcriptome and deep immune profiling of paired tumor and margin tissues.

BACKGROUND: For various cancers, differences in response to treatment and subsequent survival period have been reported to be associated with variation in immune contextures. AIM: We sought to identify whether such association exists in respect of gingivobuccal oral cancer. MATERIALS AND METHODS: We performed deep immune profiling of tumor and margin tissues collected from 46 treatment naïve, Human Papillomavirus (HPV) negative, patients. Each patient was followed for 24 months and prognosis (recurrence/death) noted. Key findings were validated by comparing with TCGA-HNSC cohort data. RESULTS: About 28% of patients showed poor post-treatment prognosis. These patients exhibited a high probability of recurrence even within 1 year and death within 2 years. There was restricted immune cell infiltration in tumor, but not in margin, among these patients. Reduced expression of eight immune-related genes (IRGs) (NT5E, THRA, RBP1, TLR4, ITGA6, BMPR1B, ITGAV, SSTR1) in tumor strongly predicted better quality of prognosis, both in our patient cohort and in TCGA-HNSC cohort. Tumors of patients with better prognosis were associated with (a) lower CD73+ cells with concomitant lower expression level of NT5E/CD73, (b) higher proportions of CD4+ and CD8+ T cells, B cells, NK cells, M1 macrophages, (c) higher %Granzyme+ cells, (d) higher TCR and BCR repertoire diversities. CD73 expression in tumor was associated with low CD8+ and CD4+ T cells, low immune repertoire diversity, and advanced cancer stage. DISCUSSION AND CONCLUSION: High infiltration of anti-tumor immune cells in both tumors and margins results in good prognosis, while in patients with minimal infiltration in tumors in spite of high infiltration in margins results in poor prognosis. Targeted CD73 immune-checkpoint inhibition may improve clinical outcome.

Association of nasopharyngeal viruses and pathogenic bacteria in children and their parents with and without HIV.

BACKGROUND: Bacteria and respiratory viruses co-occur in the nasopharynx, and their interactions may impact pathogenesis of invasive disease. Associations of viruses and bacteria in the nasopharynx may be affected by HIV. METHODS: We conducted a nested case-control study from a larger cohort study of banked nasopharyngeal swabs from families with and without HIV in West Bengal India, to look at the association of viruses and bacteria in the nasopharynx of parents and children when they are asymptomatic. Quantitative polymerase chain reaction for 4 bacteria and 21 respiratory viruses was run on 92 random nasopharyngeal swabs from children--49 from children living with HIV (CLH) and 43 from HIV uninfected children (HUC)-- and 77 swabs from their parents (44 parents of CLH and 33 parents of HUC). RESULTS: Bacteria was found in 67% of children, viruses in 45%, and both in 27% of child samples. Staphylococcus aureus (53%) was the most common bacteria, followed by Streptococcus pneumoniae (pneumococcus) (37%) in children and parents (53, 20%). Regardless of HIV status, viruses were detected in higher numbers (44%) in children than their parents (30%) (p = 0.049), particularly rhinovirus (p = 0.02). Human rhinovirus was the most frequently found virus in both CLH and HUC. Children with adenovirus were at six times increased risk of also having pneumococcus (Odds ratio OR 6, 95% CI 1.12-31.9) regardless of HIV status. In addition, the presence of rhinovirus in children was associated with increased pneumococcal density (Regression coeff 4.5, 1.14-7.9). In CLH the presence of rhinovirus increased the risk of pneumococcal colonization by nearly sixteen times (OR 15.6, 1.66-146.4), and, pneumococcus and S. aureus dual colonization by nearly nine times (OR 8.7). CONCLUSIONS: Children more frequently carried viruses regardless of HIV status. In CLH the presence of rhinovirus, the most frequently detected virus, significantly increased co-colonization with pneumococcus and S. aureus.

Improved Mesenchymal Stem Cell Proliferation, Differentiation, Epithelial Transition, and Restrained Senescence on Hierarchically Patterned Porous Honey Silk Fibroin Scaffolds.

We report a significant improvement of adipose-derived mesenchymal stem cells' (ADMSCs) biocompatibility and proliferation on hierarchically patterned porous honey-incorporated silk fibroin scaffolds fabricated using a combination of soft lithography and freeze-drying techniques. Parametric variations show enhanced surface roughness, swelling, and degradation rate with good pore interconnectivity, porosity, and mechanical strength for soft-lithographically fabricated biomimetic microdome arrays on the 2% honey silk fibroin scaffold (PHSF2) as compared to its other variants, which eventually made PHSF2 more comparable to the native environment required for stem cell adhesion and proliferation. PHSF2 also exhibits sustained honey release with remarkable antibacterial efficacy against methicillin-resistant Staphylococcus aureus (MRSA). Honey incorporation (biochemical cue) influences microdome structural features, that is, biophysical cues (height, width, and periodicity), which further allows ADMSCs pseudopods (filopodia) to grasp the microdomes for efficient cell-cell communication and cell-matrix interaction and regulates ADMSCs behavior by altering their cytoskeletal rearrangement and thereby increases the cellular spreading area and cell sheet formation. The synergistic effect of biochemical (honey) and biophysical (patterns) cues on ADMSCs studied by the nitro blue tetrazolium assay and DCFDA fluorescence spectroscopy reveals limited free radical generation within cells. Molecular expression studies show a decrease in p53 and p21 expressions validating ADMSCs senescence inhibition, which is further correlated with a decrease in cellular senescence-associated ß galactosidase activity. We also show that an increase in CDH1 and CK19 molecular expressions along with an increase in SOX9, RUNX2, and PPARγ molecular expressions supported by PHSF2 justify the substrate's efficacy of underpinning mesenchymal to epithelial transition and multilineage trans-differentiation. This work highlights the fabrication of a naturally healing nutraceutical (honey)-embedded patterned porous stand-alone tool with the potential to be used as smart stem cells delivering regenerative healing implant.

Hypoxic Transformation of Immune Cell Metabolism Within the Microenvironment of Oral Cancers.

Oral squamous cell carcinoma (OSCC) includes tumors of the lips, tongue, gingivobuccal complex, and floor of the mouth. Prognosis for OSCC is highly heterogeneous, with overall 5-year survival of ~50%, but median survival of just 8-10 months for patients with locoregional recurrence or metastatic disease. A key feature of OSCC is microenvironmental oxygen depletion due to rapid growth of constituent tumor cells, which triggers hypoxia-associated signaling events and metabolic adaptations that influence subsequent tumor progression. Better understanding of leukocyte responses to tissue hypoxia and onco-metabolite expression under low-oxygen conditions will therefore be essential to develop more effective methods of diagnosing and treating patients with OSCC. This review assesses recent literature on metabolic reprogramming, redox homeostasis, and associated signaling pathways that mediate crosstalk of OSCC with immune cells in the hypoxic tumor microenvironment. The likely functional consequences of this metabolic interface between oxygen-starved OSCC and infiltrating leukocytes are also discussed. The hypoxic microenvironment of OSCC modifies redox signaling and alters the metabolic profile of tumor-infiltrating immune cells. Improved understanding of heterotypic interactions between host leukocytes, tumor cells, and hypoxia-induced onco-metabolites will inform the development of novel theranostic strategies for OSCC.

Evaluating the role of hsa-miR-200c in reversing the epithelial to mesenchymal transition in prostate cancer.

Deregulated epithelial-to-mesenchymal transition constitutes one of the major aspects of cancer progression. In this study, to identify key molecular principles of EMT pathway in prostate carcinogenesis, an elaborate gene expression profiling was conducted by qRT-PCR and Western blot analyses. A preponderance of mesenchymal trait was observed in the pathological samples of prostate cancer. To simulate an appropriate in vitro model, PC3 cell line was subjected to hypoxic stress, which resulted in elevated expression of vimentin along with EMT-mediating transcription factors Zeb1 and Slug. To conciliate this mesenchymal behavior of PC3 cells, hsa-miR-200c was deliberately overexpressed which led to a marked reduction of cell motility and expression of vimentin, N-cadherin, Zeb1 and Slug with concurrent increase in level of ß-catenin. hsa-miR-200c was demonstrated to appease hypoxia-aggravated changes in cellular morphology by coordinated repression of vimentin, Zeb1 and Slug. Mode of action for hsa-miR-200c was mediated through transcriptional repression of Zeb1 and Slug interacting with E-box sequences in the vimentin promoter as documented by promoter assay. This ability of hsa-miR-200c to reclaim epithelial traits leads to the anticipation that molecular reprogramming of Zeb1-Slug/vimentin axis may relieve aggressiveness of prostate cancer.

Identification of α-enolase as a prognostic and diagnostic precancer biomarker in oral submucous fibrosis.

AIMS: Diagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous cell carcinoma (OSCC) patients. Our goal is to identify proteomic signatures from biopsies that can be used as precancer diagnostic marker for patient suffering from OSF. METHODS: The high throughput techniques adopting de novo peptide sequencing (1D SDS-PAGE coupled nanoLC MALDI tandem mass spectrometry (MS/MS)-based peptide mass fingerprint), immunohistochemistry (IHC), Western blot (WB) and real-time PCR (RT-PCR) analysis are considered for such biomarker identification and multilevel validations. RESULTS: Alpha-enolase is identified as an overexpressed protein in biopsies of oral submucous fibrosis with dysplasia (OSFWD) compared with oral submucous fibrosis without dysplasia (OSFWT) and normal oral mucosa (NOM). Total proteome analysis of an overexpressed protein band around 47 kDa of OSFWD identifies 334 peptides corresponding to 61 human proteins. Among them α-enolase is identified as a prime protein with highest number of peptides (44 out of 334 peptides) and sequence coverage (66.4%). Furthermore, RT-PCR, WB and IHC analysis also show mRNA and tissue level upregulation of α-enolase in OSFWD validating α-enolase as precancer marker. CONCLUSIONS: This study for the first time identifies and validates α-enolase as a novel biomarker for early diagnosis of malignant potentiality of OSF. Hence, the identified protein marker, α-enolase can help in early therapeutic intervention of OSF patients leading to the reduction of patient's pain, treatment cost and enhancement of patient's quality of life.

Honey Extracted Polyphenolics Reduce Experimental Hypoxia in Human Keratinocytes Culture.

Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy cells/molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic cytotoxicity in this study. Different honey samples were physicochemically characterized to identify preferred (jamun) honey [pH 3.55 ± 0.04, conductivity (µs/cm) = 6.66 ± 0.14, water content % (w/w) = 14.70 ± 0.35, total solid content % (w/w) = 85.30 ± 0.35, phenol content (mg GAE/100 g) = 403.55 ± 0.35, flavonoid content (mg QE/100 g) = 276.76 ± 4.10, radical scavenging activity (% 500 µL) = 147.75 ± 3.13, catalase activity (absorbance at 620 nm) = 0.226 ± 0.01]. HEP was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and antioxidant assays. Cardinal molecular expressions such as cadherin-catenin-cytoskeleton complex (namely, E-cadherin, ß-catenin, and F-actin), hypoxia marker (Hif 1 α), proliferation marker (Ki67), and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and qRT-PCR. The 0.063 mg/mL HEP demonstrated better vitality and functionality of HaCaT cells as per viability assay (*, P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated restoration of cellular survival and homeostasis under 0.063 mg/mL HEP after hypoxic assault. Furthermore, major spectral changes for nucleic acid and membrane phospholipid reorganizations by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/mL HEP on hypoxic cells considering proliferation and cellular integrity. It was concluded that a specific dose of jamun HEP reduces hypoxic cytotoxicity.

Connecting cyto-nano-architectural attributes and epithelial molecular expression in oral submucous fibrosis progression to cancer.

OBJECTIVE: Problems in pre-cancer diagnosis complicate cancer theragnosis as well as life expectancy. There is uncertainty regarding malignant transformation of oral submucous fibrosis (OSF), an oral pre-cancer with dysplastic (OSFWD) and non-dysplastic (OSFWT) subtypes. Understanding the structural, molecular and physical aspects of epithelial homeostasis may be useful. MATERIALS AND METHODS: Histopathological grading of biopsy sections was performed using H&E staining. Alterations in epithelial surface architecture in different groups was evaluated using scanning electron microscopy (SEM). The expression of crucial epithelial genes (p63, CK-5/6, CK-10, E-cadherin and ß-catenin) was studied by immunohistochemistry, Western blot and RT-PCR analysis. RESULTS: SEM observations revealed that the surface epithelial ridge pattern became thick and dense, and pit pattern gradually decreased in OSFWD and oral squamous cell carcinoma (OSCC). p63, ΔNp63 and CK-5/6 were up-regulated in OSFWD and OSCC but down-regulated in OSFWT. CK-10 was down-regulated in OSFWD compared to OSFWT. Cytoplasmic expression of E-cadherin and ß-catenin was elevated in dysplastic and cancerous conditions. Moreover, statistical correlation between SEM features (ridges and pits) and molecular attributes demonstrated a significant positive relationship between the ridge-to-pit ratio and p63 population density (r=0.85) and the ridge-to-pit ratio and CK-5/6 intensity (r=0.63). CONCLUSIONS: Molecular changes related to epithelial progressive maturation and cellular proliferation are correlated with concomitant alteration of epithelial surface architecture which helps to predict the malignant potentiality of OSF.

Modulating prime molecular expressions and in vitro wound healing rate in keratinocyte (HaCaT) population under characteristic honey dilutions.

ETHNOPHARMACOLOGY RELEVANCE: In traditional medicines honey is known for healing efficacy and vividly used as "Anupan" in Ayurvedic medicines appreciating roles in dilutions. Validating efficacy of physico-chemically characterized honey in dilutions, studies on in vitro wound healing and attainment of cellular confluence epithelial cells including expressions of cardinal genes is crucial. To evaluate effects of characterized honey in varied dilutions on cellular viability, in vitro wound healing and modulation of prime epithelial gene expressions. MATERIALS AND METHODS: Six Indian honey-samples from different sources were physico-chemically characterized and optimal one was explored in dilutions (v/v%) through in vitro studies on human epithelial (HaCaT) cells for viability, wound healing and expressions of genes p63, E-cadherin, ß-catenin, GnT-III and GnT-V. RESULTS: Studied honey samples (i.e. A-F) depicted range of pH (2-4), water (12.48-23.95), electrical conductivity (2.57-14.34), carbohydrate (68.73-98.65), protein (.316-5.36) and antioxidant potential. Though sample A and F showed physico-chemical proximity, but overall bio-impact of the earlier was better, thus studied in 8-.1% (v/v) dilution range. Four dilutions (.01, .04, .1, .25 v/v%) augmented cellular viability but in vitro wound healing was fastest (p<.05) under .1%. Such efficacy was further documented for p63 up-regulation by immunocytochemistry and mRNA studies. The E-cadherin and ß-catenin mRNA-expressions were also up-regulated and their proteins were predominantly cytoplasmic. E-cadherin up-regulation was corroborative with down-regulation and up-regulation of GnT-III and GnT-V respectively. CONCLUSION: Present study illustrated efficacy of particular honey dilution (.1%) with characteristic free radical scavenging activity in facilitating cell proliferation and attainment of confluence towards faster wound healing and modulation of cardinal epithelial genes (viz. p63, E-cadherin, ß-catenin, Gnt-III and V).