HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

Gene expression down-regulation in CD90(+) prostate tumor-associated stromal cells involves potential organ-specific genes

Background: The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THYI. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment. Methods: Prostate CD90(+) stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder. Results: The tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes. Conclusion: CD90(+) prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Importance of cone beam computed tomography for diagnosis of calcifying cystic odontogenic tumor associated to odontoma. Report of a case

The calcifying cystic odontogenic tumour (CCOT) is a rare benign cystic neoplasm not infrequently associated with odontoma. This report documents a case of CCOT associated with compound odontoma arising in the anterior maxilla in a 25-year-old woman. Conventional radiographs showed a large calcified mass with poorly visualized radiolucent margins. The extent and condition of the internal structure of the CCOT associated with odontoma was able to be determined based on radiographic findings from cone beam computed tomography. This advanced image technique proved to be extremely useful in the radiographic assessment of this particular neoplasm of the jawbones.

Interleukin-10 production by tumor infiltrating macrophages plays a role in Human Papillomavirus 16 tumor growth

Background: Human Papillomavirus, HPV, is the main etiological factor for cervical cancer. Different studies show that in women infected with HPV there is a positive correlation between lesion grade and number of infiltrating macrophages, as well as with IL-10 higher expression. Using a HPV16 associated tumor model in mice, TC-1, our laboratory has demonstrated that tumor infiltrating macrophages are M2-like, induce T cell regulatory phenotype and play an important role in tumor growth. M2 macrophages secrete several cytokines, among them IL-10, which has been shown to play a role in T cell suppression by tumor macrophages in other tumor models. In this work, we sought to establish if IL-10 is part of the mechanism by which HPV tumor associated macrophages induce T cell regulatory phenotype, inhibiting anti-tumor activity and facilitating tumor growth. Results: TC-1 tumor cells do not express or respond to IL-10, but recruit leukocytes which, within the tumor environment, produce this cytokine. Using IL-10 deficient mice or blocking IL-10 signaling with neutralizing antibodies, we observed a significant reduction in tumor growth, an increase in tumor infiltration by HPV16 E7 specific CD8 lymphocytes, including a population positive for Granzyme B and Perforin expression, and a decrease in the percentage of HPV specific regulatory T cells in the lymph nodes. Conclusions: Our data shows that in the HPV16 TC-1 tumor mouse model, IL-10 produced by tumor macrophages induce regulatory phenotype on T cells, an immune escape mechanism that facilitates tumor growth. Our results point to a possible mechanism behind the epidemiologic data that correlates higher IL-10 expression with risk of cervical cancer development in HPV infected women.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)(3) selectively potentiates its photodynamic activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)(3) into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)(3) and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

NFAT1 transcription factor is central in the regulation of tissue microenvironment for tumor metastasis

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-beta in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

Sialylation of beta 1 Integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

In previous studies, we determined that beta 1 integrins from human colon tumors have elevated levels of alpha 2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta 1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha 2-6 sialylation of the beta 1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha 2-6 sialylation exhibit beta 1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha 2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha 2-6-sialylated, beta 1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes ( unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta 1 subunit, suggesting that beta 1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.

The prognostic value of CD147/EMMPRIN is associated with monocarboxylate transporter 1 co-expression in gastric cancer

The aim of the present work was to assess the role of monocarboxylate transporters (MCTs), namely MCT1 and MCT4 as well as MCT/CD147 co-expression in gastric tissues and evaluate their clinico-pathological significance in gastric carcinoma. For that, we analysed the immunohistochemical expression of MCT1, MCT4 and CD147, in a large series of gastric samples, including non-neoplastic, tumour and metastatic tissues. A significant decrease in MCT4 plasma membrane expression was observed from non-neoplastic to gastric primary malignant tissues and to lymph-node metastasis and both MCT1 and MCT4 correlated with CD147. Importantly, both MCT4 and CD147 were more frequently expressed in Lauren`s intestinal-type tumours and MCT1/CD147 co-expression was associated with advanced gastric carcinoma, Lauren`s intestinal type, TNM staging and lymph-node metastasis. Our results showed that the prognostic value of CD147 was associated with MCTI co-expression in gastric cancer cells, supporting the view that CD147 plasma membrane activity is dependent on MCT co-expression. (C) 2009 Elsevier Ltd. All rights reserved.

Bioinformatics construction of the human cell surfaceome

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

HOXB5 expression in oral squamous cell carcinoma

Human HOX genes encode transcription factors that act as master regulators of embryonic development. They are important in several processes such as cellular morphogenesis and differentiation. The HOXB5 gene in particular has been reported in some types of neoplasm, but not in oral cancer. OBJECTIVE: The present study investigated the expression of HOXB5 in oral squamous cell carcinoma (SCC) and in non-tumoral adjacent tissues, focusing on verifying its possible role as a broad tumor-associated gene and its association with histopathological and clinical (TNM) characteristics. MATERIAL AND METHODS: RT-PCR was performed to amplify HOXB5 mRNA in 15 OSCCs and adjacent non-tumoral epithelium. A possible association with TNM and histopathologic data was verifed by the chi-square and post-hoc t-test. RESULTS: HOXB5 was amplifed in 60% non-tumoral epithelium and in 93.3% carcinomas. No statistically signifcant differences were found regarding the HOXB5 mRNA expression and TNM or histological grade. CONCLUSION: HOXB5 is expressed in OSCCs and its role in cancer progression should be further investigated.

Cross talk Initiated by Endothelial Cells Enhances Migration and Inhibits Anoikis of Squamous Cell Carcinoma Cells through STAT3/Akt/ERK Signaling

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL-6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

Surgical correction of Tessier number 0 cleft

The no. 0-14 cleft involves the midline of the face and cranium. It may include both a true and a false median cleft lip, with or without associated hypotelorism or hypertelorism. The no. 0 cleft is the most common of the craniofacial clefts. The objective of this study was to review the functional outcome and aesthetic results of the different techniques applied for each case. We have conducted a retrospective analysis of our series consisting of 32 cases of Tessier no. 0 cleft, in the period between 1997 and 2007. The patients were divided into 2 groups: those with the true median cleft and those with the false median cleft. The clinical findings, lip malformation, alveolar cleft, nasal appearance, septal involvement, associated deformities, and surgical procedures, were all reviewed. Holoprosencephaly was present in 9 cases, with a false median cleft upper lip and an absence of the premaxilla, septum, and columella (only 1 patient underwent lip and columella reconstruction at 2 years of age). Nine patients had an incomplete median cleft lip. Seven of these cases had associated median alveolar cleft, and 1 had an intranasal tumor, associated with lipoma of corpus callosum, characteristic of the Pai syndrome. Six cases of a bifid nose were seen, 2 of which were associated with an alveolar median cleft and hypertelorism. An isolated median alveolar cleft was present in 7 cases, 2 of them associated with a no. 30 cleft. This article presents a large series of Tessier no. 0 cleft, describing the differences between the false and the true median cleft. The surgical procedures may vary in relation to the type of involvement.

HLA polymorphisms as incidence factor in the progression to end-stage renal disease in Brazilian patients awaiting kidney transplant

Chronic renal failure (CRF) leads in the majority of instances to end-stage renal disease (ESRD) requiring renal replacement therapy. Age, gender, genetics, race, hypertension, and smoking among others are factors associated with ESRD. Our interest was to evaluate the possible associations of class I and II HLA antigens with ESRD renal disease independent of other factors, among patients with CRF, having various diagnoses in the Brazilian population of the Sao Paulo state. So 21 HLA-A, 31 HLA-B, and 13 HLA-DR were detected in 105 patients who were compared with 160 healthy controls of both sexes who were not related to the patients evaluated until 2005. We calculated allelic frequencies, haplotypes frequencies, etiological fractions (EF), preventive fractions, and relative risks (RR). We compared demographic data of patients and controls. The antigens positively associated with ESRD were: HLA-A78 (RR = 30.31 and EF = 0.96) and HLA-DR11 (RR = 18.87 and EF = 0.65). The antigens HLAB14 (RR = 29.90 and EF = 0.75) was present at a significantly lower frequency among patients compared with controls. In contrast, no haplotype frequency showed statically significant associations. Further molecular studies may clarify types and subtypes of alleles involved with ESRD progression.

Clear cell odontogenic carcinoma: case report with immunohistochemical findings adding support to the challenging diagnosis

Clear cell odontogenic carcinoma (CCOC) is a rare odontogenic tumor associated with aggressive clinical behavior, metastasis, and low survival. We report a case of CCOC affecting the mandible of a 39-year-old man. The tumor presented a biphasic pattern composed of clear cell nests intermingled with eosinophilic cells and separated by collagenous stroma. Immunoreactivity to cytokeratin (CK), specifically AE1/AE3 and CK 8, 14, 18, and 19 was found, as well as to epithelial membrane antigen (EMA). The tumor cells were negative for S100 protein, CK 13, vimentin, smooth muscle actin, laminin and type IV collagen. Low labeling indices for the proliferation markers Ki-67 and proliferating cell nuclear antigen and to p53 protein might predict a favorable prognosis for the lesion. A surgical resection was performed, followed by adjuvant radiotherapy. A 2-year follow-up has shown no signs of recurrence. The significance of histochemical and immunohistochemical resources in the correct diagnosis of CCOC is analyzed.