Flow cytometry study of the role of superoxide anion and hydrogen peroxide in cellular photodestruction with 5-aminolevulinic acid-induced protoporphyrin IX.

Pancreatic cancer is the fourth most common cause of cancer death in the western world. The disease is very resistant to radiotherapy and chemotherapy. One reason for that is the resistance of pancreatic cancer cells to apoptosis. Among the current investigational approaches, targeting human epidermal growth factor receptor (HER-1/EGFR) and interstitial photodynamic therapy (PDT) show promises. When used alone or together, these new approaches might provide an alternative modality to treat pancreatic cancer. This study examined and compared cytotoxic effects of antibody C225 (an anti-HER-1/EGFR monoclonal antibody) and Photofrin-mediated PDT on two human pancreatic cancer cell lines (BxPc-3, HPAF-II). Preliminary in vitro data indicated that these treatments could block various proliferation pathways of pancreatic cancer cells through different mechanisms. For instance, PDT could induce early apoptosis. C225 could induce G1 arrest. These findings might help to design new strategies such as the combination of PDT and immunotherapy for the treatment of pancreatic cancer.

The epithelial-mesenchymal transition allows cancer cells to remodel the extracellular matrix and invade tissues. However, invasive squamous cell carcinoma (SCC) cells do not lose their epithelial markers, which led Gaggioli et al . to investigate how these cells invaded tissues. The authors devised a three-dimensional culture system consisting of a matrix block, containing mainly collagen and laminin, placed in culture medium with its upper surface exposed. SCC cells placed on this surface did not invade the matrix when cultured alone but did so when cultured with fibroblasts from tumors, as determined by immunohistochemical staining. Invasion by SCC cells was blocked when a thin sheet of matrix was placed between the fibroblasts and the SCC cells. Differential fluorescent labeling of SCC cells and fibroblasts showed that a chain of invading SCC cells in the matrix was always preceded by a fibroblast. Electron and reflectance microscopy revealed that "tracks" in the matrix generated by fibroblasts contained deposits of fibronectin and evidence of matrix remodeling. Inhibitor studies showed that, whereas the invasion of the matrix by fibroblasts was dependent on the activities of matrix metalloproteinases, matrix remodeling was dependent on the small guanosine triphosphatase (GTPase) Rho and Rho-associated kinase (ROCK). Antibodies against the α3 and α5 integrins also inhibited matrix remodeling by fibroblasts. Inhibiting Rho or ROCK activity in SCC cells had no effect on their ability to follow fibroblasts; however, inhibition of the GTPase Cdc42 prevented invasion. Immunohistochemical analysis of sections from head and neck SCCs showed invading groups of SCC cells in close association with fibroblasts and fibronectin deposits. In commentary, Radisky discusses how the invasion process revealed in this study can help us in our understanding of metastasis. C. Gaggioli, S. Hooper, C. Hidalgo-Carcedo, R. Grosse, J. F. Marshall, K. Harrington, E. Sahai, Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat. Cell Biol. 9 , 1392-1400 (2007). [PubMed] D. C. Radisky, Leading the charge. Nat. Cell Biol . 9 , 1341-1342 (2007). [PubMed]

Proteasome Inhibitor PS-341 Induces Apoptosis in Cisplatin-resistant Squamous Cell Carcinoma Cells by Induction of Noxa

A Novel Recognition Site on Laminin for the α3β1 Integrin

De Novo PITX1 Expression Controls Bi-Stable Transcriptional Circuits to Govern Self-Renewal and Differentiation in Squamous Cell Carcinoma.

BackgroundCELF2 belongs to the CELF RNA‐binding protein family and exhibits antitumor activity in various tumor models. Analysis of the pan‐cancer TCGA database reveals that CELF2 expression strongly correlates with favorable prognosis among cancer patients. The function of CELF2 in nonmelanoma skin cancer has not been studied.MethodsWe used shRNA‐mediated knockdown (KD) of CELF2 expression in human squamous cell carcinoma (SCC) cells to investigate how CELF2 impacted SCC cell proliferation, survival, and xenograft tumor growth. We determined CELF2 expression in human SCC tissues and adjacent normal skin using immunofluorescence staining. Additionally, we investigated the changes in CELF2 and its target gene expression during UV‐induced and chemical‐induced skin tumorigenesis by western blotting.ResultsCELF2 KD significantly increased SCC cell proliferation, colony growth, and SCC xenograft tumor growth in immunodeficient mice. CELF2 KD in SCC cells led to activation of KRT80 and GDF15, which can potentially promote cell proliferation and tumor growth. While control SCC cells were sensitive to anticancer drugs such as doxorubicin, SCC cells with CELF2 KD became resistant to drug‐induced tumor growth retardation. Finally, we found CELF2 expression diminished during both UV‐ and chemical‐induced skin tumorigenesis in mice, consistent with reduced CELF2 expression in human SCC tumors compared to adjacent normal skin.ConclusionThis study shows for the first time that CELF2 loss occurs during skin tumorigenesis and increases drug resistance in SCC cells, highlighting the possibility of targeting CELF2‐regulated pathways in skin cancer prevention and therapies.

To determine if treatment with recombinant human interferon gamma (rHuIFN-gamma) increases the adhesion to, and lysis of, head and neck squamous cell carcinoma (SCC) cells by lymphokine-activated killer (LAK) and peripheral blood mononuclear (PBM) effector cells in vitro and to evaluate the role of cell surface adhesion molecules in these processes. Two human SCC cell lines, JHU-020-SCC and JHU-022-SCC, were used. Lymphokine-activated killer cells were generated by interleukin-2 stimulation of PBM cells obtained from the hemapheresis blood donor packs of healthy individuals. Adhesion assays were performed to assess the level of binding of both effector populations to SCC cells, which were treated with either fresh media or rHuIFN-gamma (100 U/mL). Binding was measured by flow cytometric detection of effector cells labeled with fluorescein-conjugated anti-CD45 monoclonal antibody. Monoclonal antibodies to the cell adhesion molecules HLA-DR, lymphocyte function-associated antigen 1, and intercellular adhesion molecule 1 were used in blocking experiments to determine their contribution to the process of effector-SCC cell adhesion. Cytotoxicity experiments were performed using a colorimetric assay to determine the cytotoxic response generated by LAK and PBM cells against SCC cells, with and without prior rHuIFN-gamma treatment of the tumor cells. Effector cell binding level and percent cytotoxicity of SCC cells. Recombinant human interferon gamma treatment of JHU-020-SCC cells resulted in increased adhesion to both LAK cells and PBM cells (P < .001). The presence of anti-lymphocyte function-associated antigen 1 antibody resulted in elimination of the enhanced adhesion seen with rHuIFN-gamma pretreatment of SCC cells (P =.03), but antibody to intercellular adhesion molecule 1 and HLA-DR did not reduce the level of effector binding. The greatest cytotoxic response against both JHU-020-SCC and JHU-022-SCC was seen with LAK cells (P < or = .001). Pretreatment of tumor targets by rHuIFN-gamma (100 U/mL) resulted in no enhancement of cytotoxic response by either LAK or PBM cells; at the effector-target ratio of 30:1, there was a significant decrease in LAK cell-mediated cytotoxic response against rHuIFN-gamma-treated SCC cells (P < or = .02). Recombinant human interferon gamma treatment of head and neck SCC cells does increase binding of both LAK cells and PBM cells to tumor cells, in part via the lymphocyte function-associated antigen 1 ligand mechanism. The cytotoxic effect mediated by LAK cells against head and neck SCC cells is reduced after rHuIFN-gamma treatment, suggesting that the activity of this cytokine may be more important in regulating antigen-specific cytotoxic response mediated by cytotoxic T-lymphocytes.

Glioblastoma (GB) is one of the most aggressive brain tumors, characterized by high infiltrative capacity that enables tumor cells to invade healthy brain tissue and evade complete surgical resection. This invasiveness contributes to resistance against conventional therapies and a high recurrence rate. Strategies capable of eliminating residual tumor cells are urgently needed. Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA), an FDA- and EMA-approved compound, induces selective accumulation of the photosensitizer protoporphyrin IX (PpIX) in metabolically active tumor cells, enabling targeted cytotoxicity through light activation. A major limitation to its clinical application is the unclear variation in the cytotoxic effect of PDT according to individual tumoral differences. In this study, we propose and validate an in vivo model of patient-derived GB initiating cells (GICs) and brain organoids to test the effects of PDT. First, patient-derived GICs were molecularly characterized by flow cytometry and copy number variation profiling using OncoScan CNV Assays, then co-cultured with human brain organoids to generate a hybrid model recapitulating key aspects of the tumor microenvironment. 5-ALA photodynamic therapy (PDT) efficacy was assessed in vitro by GFP-based viability measurements, LDH release assays, and TUNEL staining. Then, a murine model was generated to study PDT in vivo, based on a heterotopic (renal subcapsular engraftment) xenograft of the GICs-human brain organoid co-culture. PDT was tested in the model; in each subject, one kidney tumoral engraftment was treated and the contralateral served as a control. Immunofluorescence analysis was used to study the cell composition of the brain organoid-tumoral engraftment after PDT, and the effects on non-GIC cells. The antitumoral effect was determined by the degree of cell death analysis with the TUNEL technique. The GICs-brain organoid co-culture resulted in tumoral growth and infiltration both in vitro and in vivo. The pattern of growth and infiltration varied according to the tumoral genetic profile. 5-ALA PDT resulted in a reduction in the number of GICs and an increase in apoptotic cells in all four lines tested in vitro. A correlation was found between the induced phototoxicity in vivo with the molecular typification of GICs cell lines in vitro. There were no changes in the number or distribution of neuronal cells after the application of PDT, while a reduction in active astrocytes was observed. 5-ALA PDT could be effective in eradicating GICs with a heterogeneous molecular profile. The hybrid human-murine model presented here could be useful in investigating adjuvant therapies in GB, under the concept of personalized medicine.

Squamous cell carcinoma (SCC) cells of the head and neck specifically express collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the expression of which correlates with their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 and collagenase-1 (MMP-1) expression and invasion of SCC cells via p38 mitogen-activated protein kinase. Here, we have examined the role of Smad signaling in regulating MMP-13 expression and in invasion of head and neck SCC cells. Treatment with TGF-beta resulted in activation of Smad2 and Smad3 in SCC cells, but had no effect on their proliferation or viability. Basal activation of Smad3 and p38 was noted in SCC cells without exogenous TGF-beta stimulation, and adenoviral delivery of Smad7 and dominant-negative Smad3 inhibited p38 activation in these cells. Adenoviral overexpression of Smad3 augmented the upregulatory effect of TGF-beta on MMP-13 expression by SCC cells. Disruption of Smad signaling by adenoviral expression of kinase-defective TGF-beta type I receptor (activin-receptor-like kinase-5), Smad7, and dominant-negative Smad3 potently suppressed the basal and TGF-beta-induced expression of MMP-13 and MMP-1 in SCC cells, and inhibited their basal and TGF-beta-induced invasion through Matrigel and type I collagen. Adenoviral overexpression of Smad7 in cutaneous and oral SCC cells significantly inhibited their implantation in skin of SCID mice and growth of xenografts in vivo, as compared to LacZ adenovirus-transduced control cells. Together, these results show that Smad signaling plays an important role in promoting the invasive phenotype of human head and neck SCC cells by upregulating their collagenase expression.

Characteristics of immunosuppressive regulatory T cells in cutaneous squamous cell carcinomas and role in metastasis

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Enhancement of the photodynamic effects on human oral squamous cell carcinoma cell lines by treatment with calcipotriol

126 cAMP response element-binding protein 1 (CREB) is a β-catenin-regulated transcription factor in squamous cell carcinoma (SCC) cells

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2′,3′-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner.

We attempted to find a specific antigen of oral squamous cell carcinoma (SCC) cells that could be safely applied to gene therapy in the conservative clinical treatment of oral cancer. We performed subtraction using normal human keratinocyte cells, followed by selection using four oral SCC cell lines. We isolated three clones from poorly differentiated SCC cells and four from well-differentiated SCC cells. These seven clones adsorbed to the oral SCC cells at rates 10-100 times those of normal human keratinocyte cells. The three clones from the poorly differentiated SCC cells showed the same peptide sequence (LAPRTHP). Of the four clones from the well-differentiated SCC cells, three showed the same peptide sequence (FGTLPGT) and the fourth showed a different one (VTPNSTP). Each peptide sequence may recognize the material that exists specifically on the oral SCC cell cortex. We can expect applications not only for tumor-targeting treatment using a gene therapy virus vector but also for diagnosis using, as a tumor marker, the peculiar SCC surface material that these peptides recognize.