Reproducibility of Laser Doppler Flowmetry in gingival microcirculation. A study on six different protocols.

OBJECTIVES: Laser Doppler Flowmetry (LDF) is a non-invasive technique for the assessment of tissue blood flow, but increased reproducibility would facilitate longitudinal studies. The aim of the study was to assess the interday reproducibility of Laser Doppler Flowmetry (LDF) at rest, at elevated local temperatures, and with the use of the vasodilator Methyl Nicotinate (MN) in six interconnected protocols for the measurement of the blood supply to the microvascular bed of the gingiva. METHODS: Ten healthy volunteers were included. Interweek LDF measurements with custom-made acrylic splints were performed. Six protocols were applied in separate regions of interest (ROI): 1; basal LDF, 2; LDF with thermoprobe 42 °C, 3; LDF with thermoprobe 45 °C, 4; LDF with thermoprobe 42 °C and MN, 5; LDF with thermoprobe 45 °C and MN and 6; LDF with MN. RESULTS: Intra-individual reproducibility was assessed by the within-subject coefficient of variation (wCV) and the intraclass correlation coefficient (ICC). Basal LDF measurements demonstrated high reproducibility with wCV 11.1 in 2 min and 10.3 in 5 min. ICC was 0.9 and 0.92. wCV after heat and MN was 4.9-10.3 and ICC 0.82-0.93. The topically applied MN yielded increased blood flow. CONCLUSION: This is the first study evaluating the reproducibility of basal LDF compared to single or multiple vasodilatory stimuli in gingiva. Multiple collector fibers probe and stabilizing acrylic splints are recommended. Vasodilatory stimulation showed a tendency toward higher reproducibility. Furthermore, MN yields vasodilation in gingiva.

Tumor-matched and unmatched cancer associated fibroblasts exhibit differential effect on proliferation and FMOD and MMP9 gene expression in head and neck squamous cell carcinoma cells when cocultured in spheroids.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response. METHODS: Three HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRß, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results. RESULTS: The proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs. CONCLUSION: Our results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients' tumor.

Tailorable Membrane-Penetrating Nanoplatform for Highly Efficient Organelle-Specific Localization.

Given the breadth of currently arising opportunities and concerns associated with nanoparticles for biomedical imaging, various types of nanoparticles have been widely exploited, especially for cellular/subcellular level probing. However, most currently reported nanoparticles either have inefficient delivery into cells or lack specificity for intracellular destinations. The absence of well-defined nanoplatforms remains a critical challenge hindering practical nano-based bio-imaging. Herein, the authors elaborate on a tailorable membrane-penetrating nanoplatform as a carrier with encapsulated actives and decorated surfaces to tackle the above-mentioned issues. The tunable contents in such a versatile nanoplatform offer huge flexibility to reach the expected properties and functions. Aggregation-induced emission luminogen (AIEgen) is applied to achieve sought-after photophysical properties, specific targeting moieties are installed to give high affinity towards different desired organelles, and critical grafting of cell-penetrating cyclic disulfides (CPCDs) to promote cellular uptake efficiency without sacrificing the specificity. Hereafter, to validate its practicability, the tailored nano products are successfully applied to track the dynamic correlation between mitochondria and lysosomes during autophagy. The authors believe that the strategy and described materials can facilitate the development of functional nanomaterials for various life science applications.

CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. METHODS: Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. RESULTS: The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. CONCLUSION: Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer.

BACKGROUND: Head and Neck Squamous Cell Carcinoma (HNSCC) tumors are often resistant to therapies. Therefore searching for predictive markers and new targets for treatment in clinically relevant in vitro tumor models is essential. Five HNSCC-derived cell lines were used to assess the effect of 3D culturing compared to 2D monolayers in terms of cell proliferation, response to anti-cancer therapy as well as expression of EMT and CSC genes. METHODS: The viability and proliferation capacity of HNSCC cells as well as induction of apoptosis in tumor spheroids cells after treatment was assessed by MTT assay, crystal violet- and TUNEL assay respectively. Expression of EMT and CSC markers was analyzed on mRNA (RT-qPCR) and protein (Western blot) level. RESULTS: We showed that HNSCC cells from different tumors formed spheroids that differed in size and density in regard to EMT-associated protein expression and culturing time. In all spheroids, an up regulation of CDH1, NANOG and SOX2 was observed in comparison to 2D but changes in the expression of EGFR and EMT markers varied among the cell lines. Moreover, most HNSCC cells grown in 3D showed decreased sensitivity to cisplatin and cetuximab (anti-EGFR) treatment. CONCLUSIONS: Taken together, our study points at notable differences between these two cellular systems in terms of EMT-associated gene expression profile and drug response. As the 3D cell cultures imitate the in vivo behaviour of neoplastic cells within the tumor, our study suggest that 3D culture model is superior to 2D monolayers in the search for new therapeutic targets.

Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

BACKGROUND: The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. MATERIALS AND METHODS: The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. RESULTS: A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. CONCLUSION: High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.

Hypoxia Mediates Differential Response to Anti-EGFR Therapy in HNSCC Cells.

Despite advances in the head and neck squamous cell carcinoma (HNSCC) treatment modalities, drug resistance and cancer recurrence are often reported. Hypoxia signaling through hypoxia-inducible factor 1 (HIF-1) promotes angiogenesis and metastasis by inducing epithelial-mesenchymal-transition (EMT). The aim of this study was to evaluate the impact of hypoxia on response to therapy as well as EMT and expression of stem cell markers in HNSCC cells. Five HNSCC cell lines (UT-SCC-2, UT-SCC-14, LK0412, LK0827, and LK0923) were selected for this study. The treatment sensitivity for radiation, cisplatin, cetuximab, and dasatinib was assessed by crystal violet assay. Gene expression of EMT and cancer stem cell (CSC) markers as well as protein level of EGFR signaling molecules were analyzed by qPCR and western blotting, respectively. Unlike UT-SCC-14 and LK0827, the LK0412 cell line became significantly more sensitive to cetuximab in hypoxic conditions. This cetuximab sensitivity was efficiently reversed after suppression of HIF-1α with siRNA. Additionally, hypoxia-induced EMT and expression of stem cell markers in HNSCC cells was partially revoked by treatment with cetuximab or knockdown of HIF-1α. In summary, our study shows that hypoxia might have a positive influence on the anti-EGFR therapy effectiveness in HNSCC. However, due to heterogeneity of HNSCC lesions, targeting HIF-1α may not be sufficient to mediate such a response. Further studies identifying a trait of hypoxia-specific response to cetuximab in HNSCC are advisable.

The relationship between EMT, CD44high /EGFRlow phenotype, and treatment response in head and neck cancer cell lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) tumors are often therapy resistant and may originate from cancer stem cells or tumor cells with an epithelial-to-mesenchymal transition (EMT) phenotype. The aim of this study was to characterize HNSCC cell lines with regard to EMT profile and to investigate the influence of EMT on the response to treatment. METHODS: mRNA expression of the EMT-associated genes CDH1 (E-cadherin), CDH2 (N-cadherin), FOXC2, TWIST1, VIM (vimentin), and FN1 (fibronectin) was determined using quantitative real-time PCR. Cell morphology and migration were investigated by phase-contrast microscopy and Boyden chamber assay, respectively. The cell surface expression of CD44 and epidermal growth factor receptor (EGFR) was examined by flow cytometry. The response to radiotherapy, cetuximab, and dasatinib was assessed by crystal violet staining. RESULTS: A total of 25 cell lines investigated differed greatly with regard to EMT phenotype. Cell lines with an EMT expression profile showed a mesenchymal morphology and a high migratory capacity. In addition, they exhibited a high cell surface expression of CD44 and a low expression of EGFR, a pattern previously associated with stemness. When the EMT inducer transforming growth factor-ß (TGF-ß) was added to non-EMT cells, changes in treatment responses were observed. Moreover, the expression of TWIST1 was found to correlate with radioresistance. CONCLUSIONS: The data presented in this report suggest that EMT is associated with a CD44high /EGFRlow phenotype and possibly negative impact on radiotherapy response in HNSCC cell lines.

Epidermal growth factor is a potential biomarker for poor cetuximab response in tongue cancer cells.

BACKGROUND: Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines. METHODS: The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry. RESULTS: EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive. CONCLUSIONS: Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development.

The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer- and anti-ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia-induced damage. However, initial clinical studies on apoptosis-modulating drugs led to unexpected results in different clinical conditions and this may have been due to co-effects on non-apoptotic interconnected cell death mechanisms and the 'yin-yang' role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2-family members and p53). We also briefly highlight stress-induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation-induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.

Strong expression of survivin is associated with positive response to radiotherapy and improved overall survival in head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that is associated with severe mortality despite advances in therapy. Today's standard treatment most commonly includes radiotherapy, often combined with chemotherapy or surgery. There are so far no established biomarkers to predict response to radiation, and thus the aim of this study was to investigate a series of markers that could potentially identify HNSCC patients who would benefit from radiotherapy. The selected markers, both proteins (epidermal growth factor receptor, survivin and p53), and single nucleotide polymorphisms (SNPs) in the genes of XRCC3, XRCC1, XPC, XPD, MDM2, p53 and FGFR4 were correlated to the response to radiotherapy and overall survival. Investigations were performed on pretreatment tumor biopsies from patients classified as responders or nonresponders to radiotherapy. Protein expression was examined using immunohistochemistry and the genotyping of specific SNPs was analyzed using PCR-RFLP or pyrosequencing. We found that survivin expression was significantly stronger in the responder group (p = 0.003) and that patients with a strong survivin expression had a significantly better overall survival (p < 0.001). Moreover, downregulation of survivin by siRNA in two HNSCC cell lines significantly decreased their sensitivity to radiation. Among the SNPs analyzed, patients with the XPD Lys751Gln SNP had a significantly shorter overall survival (p = 0.048), and patients with the FGFR4 Gly388Arg SNP had a significantly longer overall survival (p = 0.010). In conclusion, our results suggest that survivin plays an important role in the response to radiotherapy and may be a useful marker for predicting radiotherapy response in patients with HNSCC.

Differentiation-promoting culture of competent and noncompetent keratinocytes identifies biomarkers for head and neck cancer.

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines.

BACKGROUND: Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. METHODS: Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. RESULTS: Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). CONCLUSIONS: Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

Feasibility of Coacervate-Like Nanostructure for Instant Drug Nanoformulation.

Despite the enormous advancements in nanomedicine research, a limited number of nanoformulations are available on the market, and few have been translated to clinics. An easily scalable, sustainable, and cost-effective manufacturing strategy and long-term stability for storage are crucial for successful translation. Here, we report a system and method to instantly formulate NF achieved with a nanoscale polyelectrolyte coacervate-like system, consisting of anionic pseudopeptide poly(l-lysine isophthalamide) derivatives, polyethylenimine, and doxorubicin (Dox) via simple "mix-and-go" addition of precursor solutions in seconds. The coacervate-like nanosystem shows enhanced intracellular delivery of Dox to patient-derived multidrug-resistant (MDR) cells in 3D tumor spheroids. The results demonstrate the feasibility of an instant drug formulation using a coacervate-like nanosystem. We envisage that this technique can be widely utilized in the nanomedicine field to bypass the special requirement of large-scale production and elongated shelf life of nanomaterials.

Hypoxia induces radioresistance, epithelial­mesenchymal transition, cancer stem cell­like phenotype and changes in genes possessing multiple biological functions in head and neck squamous cell carcinoma.

Hypoxia has been linked with increased resistance to treatment in various solid tumors, including head and neck squamous cell carcinoma (HNSCC). The aim of the present study was to identify genes involved in hypoxia­mediated responses to radiotherapy in HNSCC. A total of three HNSCC cell lines with an epithelial phenotype were selected for this study and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. The sensitivity of the HNSCC cells to radiotherapy was assessed by a crystal violet assay. Western blotting (for protein expression), cDNA microarrays and reverse transcription­quantitative PCR (for gene expression) were also applied. Small interfering RNA silencing was used to knock down target genes. The results revealed that hypoxia negatively affected the response of HNSCC cells to radiotherapy. Of note, increased levels of N­cadherin, vimentin and fibronectin, as well as stem cell­associated transcription factors, were observed under hypoxia. The microarray analysis revealed a number of hypoxia­regulated genes that were involved in multiple biological functions. However, downregulation of hypoxia­regulated genes did not affect sensitivity to radiotherapy of the investigated cell lines. Taken together, the present findings indicated several important pathways and genes that were involved in hypoxia and radiotherapy resistance. It is hypothesized that panels of reported hypoxia­regulated genes may be useful for the prediction of radiotherapy responses in patients with HNSCC.

Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines.

BACKGROUND: Radiotherapy is the main therapy for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems, highlighting the need for predictive markers. In this study, we evaluated selected proteins, mutations, and single nucleotide polymorphisms (SNPs) involved in apoptosis, cell proliferation, and DNA repair alone or combined as predictive markers for radioresponse in 42 HNSCC cell lines. METHODS: The expression of epidermal growth factor receptor, survivin, Bax, Bcl-2, Bcl-X(L) , cyclooxygenase-2 (COX-2), and heat shock protein 70 was analyzed by ELISA. Furthermore, mutations and SNPs in the p53 gene as well as SNPs in the MDM2, XRCC1, and XRCC3 genes were analyzed for their relation to radioresponse. To enable the evaluation of the predictive value of several factors combined, each cell line was allocated points based on the number of negative points (NNP) system, and the NNP sum was correlated with radioresponse. RESULTS: Survivin was the only factor that alone was significantly correlated with the intrinsic radiosensitivity (IR; r = 0.36, P = 0.02). The combination of survivin, Bax, Bcl-2, Bcl-X(L) , COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse (r = 0.553, P < 0.001). CONCLUSION: These data indicate that the IR of 42 HNSCC cell lines can be predicted by a panel of factors on both the protein and gene levels. Moreover, among the investigated factors, survivin was the most promising biomarker of radioresponse.

Cancer-Associated Fibroblasts Modulate Transcriptional Signatures Involved in Proliferation, Differentiation and Metastasis in Head and Neck Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAFs) are known to increase tumor growth and to stimulate invasion and metastasis. Increasing evidence suggests that CAFs mediate response to various treatments. HNSCC cell lines were co-cultured with their patient-matched CAFs in 2D and 3D in vitro models, and the tumor cell gene expression profiles were investigated by cDNA microarray and qRT-PCR. The mRNA expression of eight candidate genes was examined in tumor biopsies from 32 HNSCC patients and in five biopsies from normal oral tissue. Differences in overall survival (OS) were tested with Kaplan-Meier long-rank analysis. Thirteen protein coding genes were found to be differentially expressed in tumor cells co-cultured with CAFs in 2D and 81 in 3D when compared to tumor cells cultured without CAFs. Six of these genes were upregulated both in 2D and 3D (POSTN, GREM1, BGN, COL1A2, COL6A3, and COL1A1). Moreover, two genes upregulated in 3D, MMP9 and FMOD, were significantly associated with the OS. In conclusion, we demonstrated in vitro that CAF-derived signals alter the tumor cell expression of multiple genes, several of which are associated with differentiation, epithelial-to-mesenchymal transition (EMT) phenotype, and metastasis. Moreover, six of the most highly upregulated genes were found to be overexpressed in tumor tissue compared to normal tissue.

Regulation of apoptosis-associated lysosomal membrane permeabilization.

Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on an early release of cathepsins, while under other circumstances LMP occurs late in the cell death process and contributes to amplification of the death signal. The mechanism underlying LMP is still incompletely understood; however, a growing body of evidence suggests that LMP may be governed by several distinct mechanisms that are likely engaged in a death stimulus- and cell-type-dependent fashion. In this review, factors contributing to permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described. Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-dependent cell death are also discussed.

Multiple genotypic aberrances associate to terminal differentiation-deficiency of an oral squamous cell carcinoma in serum-free culture.

Oral squamous cell carcinoma (OSCC) lines proliferative in the serum-free conditions devised for normal oral keratinocytes (NOK) are virtually absent, complicating studies of carcinogenesis. A tongue squamous cell carcinoma generated under conditions for normal cell culture an apparently immortal line (termed LK0412) that has undergone >or=200 population doublings from over a year in culture. LK0412 exhibited epithelial morphology, intermediate filaments, desmosomes, and cytokeratin. Soft agar growth and tumorigenicity in athymic nude mice indicated the malignant phenotype. Compared with NOK, LK0412 exhibited increased indices for proliferation and apoptosis, and a decreased terminal differentiation index. Fetal bovine serum inhibited growth and increased apoptosis but failed to induce terminal differentiation of LK0412; the latter outcome differed clearly from that in NOK. Gene ontology assessment of transcript profiles implicated multiple alterations in biological processes, molecular functions, and cellular components in LK0412. Genetic changes, some that were confirmed to the protein level, included previously proposed OSCC markers, i.e., BAX, CDC2, and TP53, as well as multiple cancer-associated genes not considered for OSCC, e.g., BST2, CRIP1, ISG15, KLRC1, NEDD9, NNMT, and TWIST1. Elevation of p53 protein agreed with a missense mutation detectable in both the LK0412 line and the original tumor specimen. Moderate differentiation characterized the original tumor as well as tumors generated from inoculation of LK0412 in mice. Overall, the results suggest that the LK0412 cell line represent a subgroup of OSCC with unique genomic and phenotypic profiles. LK0412 should be useful to exploration of OSCC development, particularly the deregulated growth and differentiation responsiveness to serum factors.

In vitro measurement of glucose uptake after radiation and cetuximab treatment in head and neck cancer cell lines using 18F-FDG, gamma spectrometry and PET/CT.

The standard treatment for head and neck squamous cell carcinoma (HNSCC) is radiotherapy, often in combination with chemotherapy or surgery. However, a novel monoclonal antibody, cetuximab (Erbitux®), has also been approved for patient therapy. The aim of present study was to develop an in vitro method for the measurement of 18F-fluoro-2deoxy-D-glucose (FDG) to determine if cellular 18F-FDG uptake is associated with response to radiotherapy or cetuximab treatment. In the current study, HNSCC cell lines were treated with radiation or with cetuximab. Next, the uptake of 18F-FDG was measured using a gamma spectrometer (GS). Thereafter, uptake after radiation was measured first with GS and then compared with positron emission tomography (PET)/computed tomography (CT) imaging. Furthermore, the mRNA expression of glucose transporter 1 (GLUT1) was measured following cetuximab treatment via reverse transcription-quantitative PCR. A study protocol was developed to measure the cellular uptake of 18F-FDG via gamma-ray spectrometry and comparable results were obtained with those of clinical PET/CT. The results revealed a decrease in 18F-FDG after radiation and cetuximab treatment. The uptake of 18F-FDG following cetuximab treatment was significantly lower in the cetuximab-sensitive cell line UT-SCC-14 compared with the cetuximab-resistant cell lines UT-SCC-2 and UT-SCC-45. Furthermore, after treatment with cetuximab for 24 and 48 h, a significant increase in GLUT1 expression was detected in the sensitive cell line compared with the two resistant cell lines. In conclusion, a novel yet reliable method for the measurement of intracellular 18F-FDG via GS has been developed, and our results indicate that 18F-FDG uptake is associated with radiation and cetuximab response in HNSCC.