Endostatin induces proliferation of oral carcinoma cells but its effect on invasion is modified by the tumor microenvironment.

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen-fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment.

Macrophages modulate migration and invasion of human tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

Anti-heparanase aptamers as potential diagnostic and therapeutic agents for oral cancer.

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.

Cathepsin K is present in invasive oral tongue squamous cell carcinoma in vivo and in vitro.

OBJECTIVES: Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens. MATERIALS AND METHODS: OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated. RESULTS: Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively). CONCLUSIONS: Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.

Arresten, a collagen-derived angiogenesis inhibitor, suppresses invasion of squamous cell carcinoma.

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement membrane collagen IV α1 chain. As the mere prevention of tumor angiogenesis leads to hypoxia that can result in selection of more aggressive cell types and reduces the efficacy of chemotherapy, we aimed here to elucidate how arresten influences the aggressive human carcinoma cells. Arresten efficiently inhibited migration and invasion of HSC-3 tongue carcinoma cells in culture and in an organotypic model. Subcutaneous Arr-HSC xenografts grew markedly more slowly in nude mice and showed reduced tumor cell proliferation, vessel density and local invasiveness. In the organotypic assay, HSC-3 cells overproducing arresten (Arr-HSC) showed induction of cell death. In monolayer culture the Arr-HSC cells grew in aggregated cobblestone-like clusters and, relative to the control cells, showed increased expression and localization of epithelial marker E-cadherin in cell-cell contacts. Application of electric cell-substrate impedance sensing (ECIS) further supported our observations on altered morphology and motility of the Arr-HSC cells. Administration of a function-blocking α1 integrin antibody abolished the impedance difference between the Arr-HSC and control cells suggesting that the effect of arresten on promotion of HSC-3 cell-cell contacts and cell spreading is at least partly mediated by α1ß1 integrin. Collectively, our data suggest novel roles for arresten in the regulation of oral squamous carcinoma cell proliferation, survival, motility and invasion through the modulation of cell differentiation state and integrin signaling.

Type I and III collagen degradation products in serum predict patient survival in head and neck squamous cell carcinoma.

Cancer invasion induces extracellular matrix remodeling and collagen degradation. The aim of this study was to assess whether serum levels of type I and III collagen degradation products were associated with patient survival in head and neck squamous cell carcinoma (HNSCC). A novel enzyme immunoassay was developed for measuring type III collagen N-terminal telopeptide (IIINTP) in human serum samples. In addition, type I collagen C-terminal telopeptide (ICTP), matrix metalloprotease-8 (MMP-8) and tissue inhibitor of metalloproteases-1 (TIMP-1) were assessed in 205 blood samples from HNSCC patients. High levels of serum ICTP and IIINTP and plasma TIMP-1 were associated with poor survival. The concentration of ICTP was associated with levels of IIINTP and TIMP-1. The plasma concentration of MMP-8 was associated with tumor stage, but not with survival or levels of ICTP, IIINTP or TIMP-1 suggesting that other collagenases/proteases are responsible for the cleavage of type I and type III collagens. The rate of type I and type III collagen degradation is associated with patient survival and can be used as a prognostic marker in HNSCC.

Molecular crosstalk between cancer cells and tumor microenvironment components suggests potential targets for new therapeutic approaches in mobile tongue cancer.

We characterized tumor microenvironment (TME) components of mobile tongue (MT) cancer patients in terms of overall inflammatory infiltrate, focusing on the protumorigenic/anti-inflammatory phenotypes and on cancer-associated fibroblasts (CAFs) in order to determine their interrelations and associations with clinical outcomes. In addition, by culturing tongue carcinoma cells (HSC-3) on a three-dimensional myoma organotypic model that mimics TME, we attempted to investigate the possible existence of a molecular crosstalk between cancer cells and TME components. Analysis of 64 cases of MT cancer patients revealed that the overall density of the inflammatory infiltrate was inversely correlated to the density of CAFs (P = 0.01), but that the cumulative density of the protumorigenic/anti-inflammatory phenotypes, including regulatory T cells (Tregs, Foxp3+), tumor-associated macrophages (TAM2, CD163+), and potentially Tregs-inducing immune cells (CD80+), was directly correlated with the density of CAFs (P = 0.01). The hazard ratio (HR) for recurrence in a TME rich in CD163+ Foxp3+ CD80+ was 2.9 (95% CI 1.03-8.6, P = 0.043 compared with low in CD163+ Foxp3+ CD80+). The HR for recurrence in a TME rich in CAFs was 4.1 (95% confidence interval [CI] 1.3-12.8, P = 0.012 compared with low in CAFs). In vitro studies showed cancer-derived exosomes, epithelial-mesenchymal transition process, fibroblast-to-CAF-like cell transdifferentiation, and reciprocal interrelations between different cytokines suggesting the presence of molecular crosstalk between cancer cells and TME components. Collectively, these results highlighted the emerging need of new therapies targeting this crosstalk between the cancer cells and TME components in MT cancer.

Difference between total and intact assays for N-terminal propeptide of type I procollagen reflects degradation of pN-collagen rather than denaturation of intact propeptide.

BACKGROUND: The concentration of N-terminal propeptide of type I procollagen (PINP) in the serum reflects the rate of type I collagen formation. Intact PINP assay measures the trimeric propeptide while total P1NP assay measures both trimeric and monomeric forms. In this study we compared these two assays emphasizing the possible differences. METHODS: Intact and total PINP were measured from serum in healthy Finnish blood donors (n = 34) and in the patients with chronic renal failure before and after haemodialysis (n = 39). In addition, the serum of a normal man, pooled hospital serum samples and the serum of a patient with haemodialysis treatment were fractioned by gel filtration and trimeric and monomeric forms were located. Fractions were lyophilized and intact and total PINP were measured in each fraction. Samples from bedridden geriatric patients (n = 173) were also measured using intact and total PINP assays and a degradation marker of type I collagen (ICTP). RESULTS: The correlation between intact and total PINP in controls was 0.89 and their PINP concentrations were similar. In haemodialysis or bedridden geriatric patients, the PINP methods gave significantly different results. In gel filtration studies, intact PINP hardly measured monomeric form even if its concentration was disproportionately increased in haemodialysis patients. In bedridden geriatric patients, the difference of total and intact PINP correlated significantly to degradation marker ICTP. CONCLUSIONS: Difference between total and intact assays for PINP seem to reflect degradation of pN-collagen rather than denaturation of intact propeptide.

A novel organotypic model mimics the tumor microenvironment.

Carcinoma cell invasion is traditionally studied in three-dimensional organotypic models composed of type I collagen and fibroblasts. However, carcinoma cell behavior is affected by the various cell types and the extracellular matrix (ECM) in the tumor microenvironment. In this study, a novel organotypic model based on human uterine leiomyoma tissue was established and characterized to create a more authentic environment for carcinoma cells. Human tongue squamous cell carcinoma cells (HSC-3) were cultured on top of either collagen or myoma. Organotypic sections were examined by immunohistochemistry and in situ hybridization. The maximal invasion depth of HSC-3 cells was markedly increased in myomas compared with collagen. In myomas, various cell types and ECM components were present, and the HSC-3 cells only expressed ECM molecules in the myoma model. Organotypic media were analyzed by radioimmunoassay, zymography, or Western blotting. During carcinoma cell invasion, matrix metalloprotease-9 production and collagen degradation were enhanced particularly in the myoma model. To evaluate the general applicability of the myoma model, several oral carcinoma, breast carcinoma, and melanoma cell lines were cultured on myomas and found to invade in highly distinct patterns. We conclude that myoma tissue mimics the native tumor microenvironment better than previous organotypic models and possibly enhances epithelial-to-mesenchymal transition. Thus, the myoma model provides a promising tool for analyzing the behavior of carcinoma cells.

The enamel matrix derivative (Emdogain) enhances human tongue carcinoma cells gelatinase production, migration and metastasis formation.

Enamel matrix derivative Emdogain (EMD) is widely used in periodontal treatment to regenerate lost connective tissue and to improve the attachment of the teeth. Gelatinases (MMP-2 and -9) have an essential role in the promotion and progression of oral cancer growth and metastasis formation. We studied the effects of EMD on human tongue squamous cell carcinoma (HSC-3) cells in vitro and in vivo. In vitro, EMD (100 microg/ml and 200 microg/ml) remarkably induced the MMP-2 and -9 production from HSC-3 cells analysed by zymography and enzyme-linked immunosorbent assay. EMD also slightly induced the MMP-2 and -9 production from benign human mucosal keratinocytes (HMK). Furthermore, EMD clearly induced the transmigration of HSC-3 cells but had no effect on the HMK migration in transwell assays. The in vitro wound closure of HSC-3 cells was notably accelerated by EMD, whereas it had only minor effect on the wound closure of HMKs. The migration of both cell lines was inhibited by a selective cyclic anti-gelatinolytic peptide CTT-2. EMD had no effect on HSC-3 cell proliferation or apoptosis and only a limited effect on cell attachment to various extracellular matrix components. The in vivo mice experiment revealed that EMD substantially induced HSC-3 xenograft metastasis formation. Our results suggest that the use of EMD for patients with oral mucosal carcinomas or premalignant lesions should be carefully considered, possibly avoided.