The spatial distribution of intermediate fibroblasts and myeloid-derived cells dictate lymph node metastasis dynamics in oral cancer.

BACKGROUND: Oral cancer poses a significant health challenge due to limited treatment protocols and therapeutic targets. We aimed to investigate the invasive margins of gingivo-buccal oral squamous cell carcinoma (GB-OSCC) tumors in terms of the localization of genes and cell types within the margins at various distances that could lead to nodal metastasis. METHODS: We collected tumor tissues from 23 resected GB-OSCC samples for gene expression profiling using digital spatial transcriptomics. We monitored differential gene expression at varying distances between the tumor and its microenvironvent (TME), and performed a deconvulation study and immunohistochemistry to identify the cells and genes regulating the TME. RESULTS: We found that the tumor-stromal interface (a distance up to 200 µm between tumor and immune cells) is the most active region for disease progression in GB-OSCC. The most differentially expressed apex genes, such as FN1 and COL5A1, were located at the stromal ends of the margins, and together with enrichment of the extracellular matrix (ECM) and an immune-suppressed microenvironment, were associated with lymph node metastasis. Intermediate fibroblasts, myocytes, and neutrophils were enriched at the tumor ends, while cancer-associated fibroblasts (CAFs) were enriched at the stromal ends. The intermediate fibroblasts transformed into CAFs and relocated to the adjacent stromal ends where they participated in FN1-mediated ECM modulation. CONCLUSION: We have generated a functional organization of the tumor-stromal interface in GB-OSCC and identified spatially located genes that contribute to nodal metastasis and disease progression. Our dataset might now be mined to discover suitable molecular targets in oral cancer.

Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma.

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

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.

Biosynthesized CdS Nanoparticle Induces ROS-dependent Apoptosis in Human Lung Cancer Cells.

BACKGROUND: The World Health Organization (WHO) estimated that the number of cancer-related deaths was 9.6 million in 2018 and 2.09 million deaths occurred by lung cancer. The American Institute for Cancer Research (AICR) also observed gender preferences in lung cancer, common in men than women. Since the past decade, nanoparticles have now been widely documented for their anti-cancer properties, which signifies that the development of nanotechnology would be a future diagnosis and treatment strategy for lung cancer. OBJECTIVE: The current study aimed to investigate the role of biosynthesized CdS nanoparticles (CdS NPs) in lung cancer cells (A549). Therefore, whether the CdS NP induces lung cancer cell death and the underlying mechanism is yet to be elucidated. METHODS: Literature was searched from various archives of biomedical and life science journals. Then, CdS NPs were biosynthesized and characterized by traditional and cutting-edge protocols. The CdS NP-mediated cell death was elucidated following standard protocols. RESULTS: CdS NPs induced cytotoxicity towards A549 cells in a dose-dependent manner. However, such a death mechanism does not go through necrosis. Intracellular reactive oxygen species (ROS) accumulation and mitochondrial membrane depolarization demonstrated that cell death is associated with intracellular ROS production. Furthermore, increased sub-G1 population, Bax expression, and decreased Bcl-2 expression revealed that the death was caused by apoptosis. CONCLUSION: CdS NPs promote apoptosis-mediated lung cancer cell death through ROS production.

The Hard-To-Close Window of T-Type Calcium Channels.

T-Type calcium channels (TTCCs) are key regulators of membrane excitability, which is the reason why TTCC pharmacology is subject to intensive research in the neurological and cardiovascular fields. TTCCs also play a role in cancer physiology, and pharmacological blockers such as tetralols and dihydroquinazolines (DHQs) reduce the viability of cancer cells in vitro and slow tumor growth in murine xenografts. However, the available compounds are better suited to blocking TTCCs in excitable membranes rather than TTCCs contributing window currents at steady potentials. Consistently, tetralols and dihydroquinazolines exhibit cytostatic/cytotoxic activities at higher concentrations than those required for TTCC blockade, which may involve off-target effects. Gene silencing experiments highlight the targetability of TTCCs, but further pharmacological research is required for TTCC blockade to become a chemotherapeutic option.

Bafilomycin-A1 and ML9 Exert Different Lysosomal Actions to Induce Cell Death.

OBJECTIVE: Bafilomycin-A1 and ML9 are lysosomotropic agents, irrespective of cell types. However, the mechanisms of lysosome targeting either bafilomycin-A1 or ML9 are unclear. METHODS: The present research has been carried out by different molecular and biochemical analyses like western blot, confocal imaging and FACS studies, as well as molecular docking. RESULTS: Our data shows that pre-incubation of neonatal cardiomyocytes with ML9 for 4h induced cell death, whereas a longer period of time (24h) with bafilomycin-A1 was required to induce an equivalent effect. Neither changes in ROS nor ATP production is associated with such death mechanisms. Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9. CONCLUSION: Bafilomycin-A1 and ML9 are lysosomotropic agents, involved in cell death events. But such death events are not associated with ATP and ROS production. Furthermore, both the drugs target lysosomes through different mechanisms. For the latter, cell death is likely due to lysosomal membrane permeabilization and release of lysosomal proteases into the cytosol.

T-type Ca2+ Channels: T for Targetable.

In the past decade, T-type Ca2+ channels (TTCC) have been unveiled as key regulators of cancer cell biology and thus have been proposed as chemotherapeutic targets. Indeed, in vitro and in vivo studies indicate that TTCC pharmacologic blockers have a negative impact on the viability of cancer cells and reduce tumor size, respectively. Consequently mibefradil, a TTCC blocker approved in 1997 as an antihypertensive agent but withdrawn in 1998 because of drug-drug interactions, was granted 10 years later the orphan drug status by the FDA to investigate its efficacy against brain, ovary, and pancreatic cancer. However, the existence of different channel isoforms with distinct physiologic roles, together with the lack of selective pharmacologic agents, has hindered a conclusive chemotherapeutic evaluation. Here, we review the available evidence on TTCC expression, value as prognostic markers, and effectiveness of their pharmacologic blockade on cancer cells in vitro and in preclinical models. We additionally summarize the status of clinical trials using mibefradil against glioblastoma multiforme. Finally, we discuss the future perspectives and the importance of further development of multidisciplinary research efforts on the consideration of TTCCs as biomarkers or targetable molecules in cancer. Cancer Res; 78(3); 603-9. ©2018 AACR.

Effect of m-calpain in PKCalpha-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain.

There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate. PKC activity using an assay kit and assay of Na+/K+ ATPase activity spectrophotometrically. We purified m-calpain and PKCalpha by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCalpha by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCalpha (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCalpha in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCalpha increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2]i-mCalpain-PKCalpha signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.