A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression.

Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

The effect of side chain variations on quinazoline-pyrimidine G-quadruplex DNA ligands.

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

Emergence of hybrid states of stem-like cancer cells correlates with poor prognosis in oral cancer.

Cancer cell state transitions emerged as powerful mechanisms responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Here, by multiplexing phenotypic markers of stem-like cancer cells (SLCCs); CD44, CD24 and aldehyde dehydrogenase (ALDH), we characterized diversity among multiple oral tumor tissues and cell lines. Two distinct patterns of spontaneous transitions with stochastic bidirectional interconversions on 'ALDH-axis', and unidirectional non-interconvertible transitions on 'CD24-axis' were observed. Interestingly, plastic 'ALDH-axis' was harnessed by cells to adapt to a Cisplatin tolerant state. Furthermore, phenotype-specific RNA sequencing suggested the possible maintenance of intermediate hybrid cell states maintaining stemness within the differentiating subpopulations. Importantly, survival analysis with subpopulation-specific gene sets strongly suggested that cell-state transitions may drive non-genetic heterogeneity, resulting in poor prognosis. Therefore, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behavior in oral cancer; which may provide prerequisite knowledge for treatment strategies.

Two novel cell culture models of buccal mucosal oral cancer from patients with no risk-habits of tobacco smoking or chewing.

OBJECTIVE: Tobacco consumption is one of the major etiological factors for oral cancer, but it also develops in non-tobacco users, with unknown etiologies. Cellular models for tobacco associated oral cancer are available, however; reports of cellular models for studying non-tobacco associated oral cancer are limiting. We report here the establishment and characterization of two novel buccal mucosal cancer cell lines 'GBC02' and 'GBC035' derived from non-tobacco users. MATERIALS AND METHODS: Short tandem repeats (STR) profiling, Next-generation sequencing for whole-genome, exome and copy number alterations, immunofluorescence, flow-cytometry, proliferation, live-cell chemotaxis, 3D-spheroid formation, chemotherapy response, gene-expression microarray, gene-set enrichment analysis and xenograft development were performed. RESULTS: Sources of the established cultures were matched to their donors through STR profiling. Genome sequence analysis revealed somatic mutations in TP53, CASP8, CDKN2A for GBC02 with deletions and amplifications encompassing CDKN2A, FAT1 and CCND1, PIK3CA, SOX2, EGFR, MYC genes, respectively. GBC035 harbored mutations in FAT1, NOTCH1, HRAS, CDKN2A, HLA-B, HLA-A genes. While GBC035 cells showed higher E-Cadherin positive cell-cell junctions and collective cell migration in chemotaxis; GBC02 cells were vimentin-positive and demonstrated individual cell migration. Further, exhibiting their relevance to preclinical research, GBC02 3D-spheroids demonstrated enrichment of development-related gene-signatures in microarray transcriptome analysis and were resistant to Cisplatin, but showed sensitivity to cancer stem cells-targeting drug, Salinomycin. Additionally, tumorigenic ability of GBC02 was demonstrated. CONCLUSIONS: Altogether, we present here comprehensively characterized unique cell lines established from non-tobacco associated tumors, which may serve as models for preclinical investigations of oral cancers caused independent of tobacco usage.

Cancer associated fibroblasts: phenotypic and functional heterogeneity.

Cancer associated fibroblasts (CAFs) are the most abundant stromal cell-type in solid tumor-microenvironment (TME) and have emerged as key player in tumor progression. CAFs establish communication with cancer cells through paracrine mechanisms or via direct cell adhesion as well as influence the cancer cell behaviour indirectly by remodelling the extracellular matrix. Although numerous studies have strongly suggested the tumor promoting role of CAFs, few recent reports have revealed the heterogeneity in CAFs. Here, we have summarized the recent findings on the mechanisms related to the heterogeneous behaviour of CAFs serving as positive or negative regulator of tumor progression. Further, reports related to the targeted therapy against CAF-mediated mechanisms are also summarized briefly.

A subtype of cancer-associated fibroblasts with lower expression of alpha-smooth muscle actin suppresses stemness through BMP4 in oral carcinoma.

Cancer-associated fibroblasts (CAFs) demonstrate the characteristics of myofibroblast differentiation by often expressing the ultrastructure of alpha-smooth muscle actin (αSMA). However, heterogeneity among cancer-associated fibroblasts (CAFs), with respect to αSMA expression, has been demonstrated in several clinical studies of oral cancer. Like normal stem cells, stem-like cancer cells (SLCCs) are also regulated extrinsically by its microenvironment; therefore, we postulated that the heterogeneous oral-CAFs would differently regulate oral-SLCCs. Using transcriptomics, we clearly demonstrated that the gene expression differences between oral tumor-derived CAFs were indeed the molecular basis of heterogeneity. This also grouped these CAFs in two distinct clusters, which were named as C1 and C2. Interestingly, the oral-CAFs belonging to C1 or C2 clusters showed low or high αSMA-score, respectively. Our data with tumor tissues and in vitro co-culture experiments interestingly demonstrated a negative correlation between αSMA-score and cell proliferation, whereas, the frequency of oral-SLCCs was significantly positively correlated with αSMA-score. The oral-CAF-subtype with lower score for αSMA (C1-type CAFs) was more supportive for cell proliferation but suppressive for the self-renewal growth of oral-SLCCs. Further, we found the determining role of BMP4 in C1-type CAFs-mediated suppression of self-renewal of oral-SLCCs. Overall, we have discovered an unexplored interaction between CAFs with lower-αSMA expression and SLCCs in oral tumors and provided the first evidence about the involvement of CAF-expressed BMP4 in regulation of self-renewal of oral-SLCCs.

Grave Complication of Pharyngitis: Lemierre syndrome.

Fusobacterium necrophorum is the causative agent in the pharyngitis. After invasion of the oropharyngeal mucosa by the organism leads to the development of the grave complications like superficial thrombophlebitis of internal jugular vein, septic emboli in the lung and coagulopathy. A 23-year-old male presented with chief complaints of sore throat, headache and high grade fever with past history of upper respiratory tract infection one month ago. On examination, he had high grade fever, tachycardia, hypotension and tender cervical lymph nodes. Oropharyngeal mucosa appeared ooedematous and congested. Leucocytosis with raised ESR was present. Blood culture positive for Fusobacterium necrophorum. Dilated right IJV with the hypoechoic thrombosis seen on Ultrasonography & Doppler. Patient was diagnosed as a case of Lemierre syndrome secondary to oropharyngeal infection was made. He was treated with intravenous imipenem and metronidazole, and then shifted to oral amoxicillin. After completion of full course of the therapy of 6 weeks, patient was improved clinically and all routine blood investigations were normalized. Blood culture was negative for Fusobacterium necrophorum. No evidence of thrombosis within the right internal jugular vein on Doppler.