Lung cancer patients are diagnosed at late stages when curative treatments are no longer possible; thus, molecular biomarkers for noninvasive detection are urgently needed. In this sense, we previously identified and validated an epigenetic 4-gene signature that yielded a high diagnostic performance in tissue and invasive pulmonary fluids. We analyzed DNA methylation levels using the ultrasensitive digital droplet PCR in noninvasive samples in a cohort of 83 patients. We demonstrated that BCAT1 is the candidate that achieves high diagnostic efficacy in circulating DNA derived from plasma (area under the curve: 0.85). Impact of potentially confounding variables was also explored.

There is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.

Introduction: SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis.Methods: Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell-and patient -derived xenografts and metastasis. YES1 levels were eval-uated in mouse and patient plasma-derived exosomes.Results: Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell-and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/ monitoring.Conclusions: Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.

Simple Summary Apart from genetic changes, cancer is characterized by epigenetic alterations, which indicate modifications in the DNA (such as DNA methylation) and histones (such as methylation and acetylation), as well as gene expression regulation by non-coding (nc)RNAs. These changes can be used in biological fluids (liquid biopsies) for diagnosis, prognosis and prediction of cancer drug response. Although these alterations are not widely used as biomarkers in the clinical practice yet, increasing number of commercial kits and clinical trials are expected to prove that epigenetic changes are able to offer valuable information for cancer patients. Early alterations in cancer include the deregulation of epigenetic events such as changes in DNA methylation and abnormal levels of non-coding (nc)RNAs. Although these changes can be identified in tumors, alternative sources of samples may offer advantages over tissue biopsies. Because tumors shed DNA, RNA, and proteins, biological fluids containing these molecules can accurately reflect alterations found in cancer cells, not only coming from the primary tumor, but also from metastasis and from the tumor microenvironment (TME). Depending on the type of cancer, biological fluids encompass blood, urine, cerebrospinal fluid, and saliva, among others. Such samples are named with the general term "liquid biopsy" (LB).

We have studied blood levels of cytokines/chemokines in patients with metastatic renal cell carcinoma treated with sunitinib or pazopanib, with the goal of identifying biomarkers that can predict efficacy and survival. We have found that high levels of CXCL10, CXCL11, HGF and IL-6 before treatment associate with poor prognosis in these patients. Moreover, these factors are correlated in patients with renal carcinoma, suggesting a coordinated expression and secretion. We have developed a prognostic signature including these factors that predicts very accurately prognosis. Our results may help defining better the group of renal cell carcinoma patients who may benefit from sunitinib/pazopanib.

Our goal was to assess the correlation of immune parameters with the response to induction chemotherapy (ICT) in head and neck squamous cell carcinoma (HNSCC) patients. Pretreatment biopsies from 64 patients with HNSCC that received ICT were assessed for PD-L1 protein expression and density of CD8+ and FOXP3+ tumor infiltrating lymphocytes (TIL). In addition, the neutrophil-to-lymphocyte ratio (NLR) was calculated from pretreatment whole blood counts. In total, 55% of cases exhibited PD-L1 combined proportion score (CPS) positivity (¿1% stained cells). PD-L1 CPS positivity correlated with a high density of both CD8+ (p = 0.01) and FOXP3+ (p < 0.001) TILs. There was no correlation between PD-L1 expression or TIL density and NLR values. In univariate analyses, the absence of PD-L1 CPS expression (p = 0.042) and a high NLR (p = 0.034) were significantly correlated with response to ICT. Neither CD8+ TIL (p = 0.99) nor FOXP3+ TIL densities (p = 0.71) were associated with response to ICT. In multivariate analysis, only a high NLR was associated with response to ICT (HR = 4.06, 95% CI = 1.06¿15.5, p = 0.04). In addition, a high NLR was also independently associated with lower disease-specific (p = 0.03) and overall survival rates (p = 0.04), particularly in the subset of patients who received definitive surgical treatment. These results suggest that NLR could emerge as a predictive biomarker of response to ICT.

Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10(+) DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8(+) T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.

Background Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. Methods TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. Results Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro.

Low dose computed tomography (LDCT) screening, together with the recent advances in targeted and immunotherapies, have shown to improve non-small cell lung cancer (NSCLC) survival. Furthermore, screening has increased the number of early stage-detected tumors, allowing for surgical resection and multimodality treatments when needed. The need for improved sensitivity and specificity of NSCLC screening has led to increased interest in combining clinical and radiological data with molecular data. The development of biomarkers is poised to refine inclusion criteria for LDCT screening programs. Biomarkers may also be useful to better characterize the risk of indeterminate nodules found in the course of screening or to refine prognosis and help in the management of screening detected tumors. The clinical implications of these biomarkers are still being investigated and whether or not biomarkers will be included in further decision-making algorithms in the context of screening and early lung cancer management still needs to be determined. However, it seems clear that there is much room for improvement even in early stage lung cancer disease-free survival (DFS) rates; thus, biomarkers may be the key to refine risk-stratification and treatment of these patients. Clinicians' capacity to register, integrate, and analyze all the available data in both high risk individuals and early stage NSCLC patients will lead to a better understanding of the disease's mechanisms, and will have a dire

PURPOSE This study aimed to assess the efficacy of the combination of nivolumab (nivo) plus ipilimumab (ipi) as a first-line therapy with respect to the 12-month overall survival (OS) in patients with metastatic uveal melanoma (MUM) who are not eligible for liver resection. METHODS This was a single-arm, phase II trial led by the Spanish Multidisciplinary Melanoma Group (GEM) on nivo plus ipi for systemic treatment-naive patients of age. 18 years, with histologically confirmed MUM, Eastern Cooperative Oncology Group-PS 0/1, and confirmed progressive metastatic disease (M1). Nivo (1 mg/kg once every 3 weeks) and ipi (3 mg/kg once every 3 weeks) were administered during four inductions, followed by nivo (3 mg/kg once every 2 weeks) until progressive disease, toxicity, or withdrawal. The primary end point was 12-month OS. OS, progression-free survival (PFS), and overall response rate were evaluated every 6 weeks using RECIST (v1.1). Safety was also evaluated. Logistic regression and Cox proportional hazard models comprising relevant clinical factors were used to evaluate the potential association with response to treatment and survival. Cytokines were quantified in serum samples for their putative role in immune modulation/angiogenesis and/or earlier evidence of involvement in immunotherapy. RESULTS A total of 52 patients with a median age of 59 years (range, 26-84 years) were enrolled. Overall, 78.8%, 56%, and 32% of patients had liver M1, extra-liver M1, and elevated lactate dehydrogenase. Stable disease was the most common outcome (51.9%). The primary end point was 12-month OS, which was 51.9% (95% CI, 38.3 to 65.5). The median OS and PFS were 12.7 months and 3.0 months, respectively. PFS was influenced by higher LDH values. CONCLUSIONS Nivo plus ipi in the first-line setting for MUM showed a modest improvement in OS over historical benchmarks of chemotherapy, with a manageable toxicity profile. (C) 2021 by American Society of Clinical Oncology

Pazopanib is an oral angiogenesis tyrosine kinase inhibitor (TKI) recommended in metastatic renal cell carcinoma (mRCC) for treatment-naive patients or those experiencing cytokine failure. We conducted a phase 2, open-label, single-arm study in ten Spanish centres among mRCC patients whose disease progressed on first-line TKI. Patients received pazopanib until disease progression, death, or unacceptable toxicity. Twenty-seven patients were included (median age 62 yr, 51.9% male). The objective overall response rate was 14.8% (95% confidence interval [CI] 1.4-28.2%). Median progression-free survival was 6.7 mo (95% CI 3.7-11.2) and median overall survival was 20.6 mo (95% CI 12.6-27.4). Lower circulating levels of IL-10 (p = 0.002) were observed in responding patients at 8 wk after treatment. The median pazopanib treatment duration was 6.0 mo (range 1.0-47.0). Most patients (48.1%) had mild or moderate adverse events (AEs), while 44.4% had severe AEs. Pazopanib was clinically active and well tolerated as a second-line treatment in mRCC patients after TKI failure, and circulating IL-10 levels could predict response. Patient summary: Pazopanib could be used as a second-line therapy for the treatment of metastatic renal cell carcinoma after failure of tyrosine kinase inhibitor (TKI) therapy when drugs such as nivolumab and cabozantinib are not available.

Introduction The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. Methods A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. Results Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. Conclusions YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.

Bevacizumab (as other monoclonal antibodies) has now become a mainstay in the treatment of several cancers in spite of some limitations, including poor tumour penetration and the development of resistance mechanisms. Its nanoencapsulation may be an adequate strategy to minimize these problems. The aim of this work was to evaluate the efficacy of bevacizumab-loaded nanoparticles (B-NP-PEG) on a xenograft model of human colorectal cancer. For this purpose, human serum albumin nanoparticles were prepared by coacervation, then coated with poly(ethylene glycol) and freeze-dried. B-NP-PEG displayed a mean size of about 300 nm and a bevacizumab loading of approximately 145 ¿g/mg. An in vivo study was conducted in the HT-29 xenograft model of colorectal cancer. Both, free and nanoencapsulated bevacizumab, induced a similar reduction in the tumour growth rate of about 50%, when compared to controls. By microPET imaging analysis, B-NP-PEG was found to be a more effective treatment in decreasing the glycolysis and metabolic tumour volume than free bevacizumab, suggesting higher efficacy. These results correlated well with the capability of B-NP-PEG to increase about fourfold the levels of intratumour bevacizumab, compared with the conventional formulation. In parallel, B-NP-PEG displayed six-times lower amounts of bevacizumab in blood than the aqueous formulation of the antibody, suggesting a lower incidence of potential undesirable side effects. In summary, albumin-based nanoparticles may be adequate carriers to promote the delivery of monoclonal antibodies (i.e. bevacizumab) to tumour tissues.

Due to improvements in systemic therapies and longer survivals, cancer patients frequently present with recurrent brain metastases (BM). The optimal therapeutic strategies for limited brain relapse remain undefined. We analyzed tumor control and survival in patients treated with salvage focal radiotherapy in our center. Thirty-three patients with 112 BM received salvage stereotactic radiosurgery (SRS) or fractionated stereotactic radiotherapy (FSRT) for local or regional recurrences. Local progression was observed in 11 BM (9.8 %). After 1 year, 72 % of patients were free of distant brain failure, and the 2-year overall survival (OS) was 37.7 %. No increase in toxicity or neurologically related deaths were observed. The 2- and 3-year whole brain radiation therapy free survival (WFS) rates were 92.9 % and 77.4 %, respectively. Hence, focal radiotherapy is a feasible salvage of recurrent BM in selected group of patients with limited brain disease, achieving a maintained intracranial control and less neurological toxicity.

Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1¿programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.

The present work proposes a brazing procedure to join a self-passivating tungsten alloy (W-10Cr-0.5Y) with Eurofer. The results indicated the achievement of high continuity W-Eurofer joints using 80Cu-20Ti filler material, which involves operative brazeability. The resulting microstructure of the braze changes considerably compared to the brazing attempts with pure tungsten, which is associated to the reactive character of chromium. A high interaction between molten filler and W base material close to the braze was detected, with preferential grain boundary penetration of Cu-Ti into W alloy. It gave rise to a loss of hardness at the W base material near the joint. Regarding to the strength of the joints, shear strength of similar to 90 MPa was obtained.

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (similar to 40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Background Programmed death-ligand 1 (PD-L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy. The current study evaluated the feasibility and efficacy of PD-L1 immunostaining and quantitation on direct Papanicolaou-stained cytological smears compared with formalin-fixed paraffin-embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona). Methods PD-L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC). Results In 57 sets of samples, both PD-L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin-fixed paraffin-embedded samples was observed in 97.3% of the cases. Conclusions The quantification of PD-L1 expression on direct Papanicolaou-stained cytology smears is feasible and reliable for both PD-L1 assays.

Relapse rates in surgically resected non-small-cell lung cancer (NSCLC) patients are between 30% and 45% within five years of diagnosis, which shows the clinical need to identify those patients at high risk of recurrence. The eighth TNM staging system recently refined the classification of NSCLC patients and their associated prognosis, but molecular biomarkers could improve the heterogeneous outcomes found within each stage. Here, using two independent cohorts (MDA and CIMA-CUN) and the eighth TNM classification, we show that TMPRSS4 protein expression is an independent prognostic factor in NSCLC, particularly for patients at stage I: relapse-free survival (RFS) HR, 2.42 (95% CI, 1.47-3.99), p < 0.001; overall survival (OS) HR, 1.99 (95% CI, 1.25-3.16), p = 0.004). In stage IA, high levels of this protein remained associated with worse prognosis (p = 0.002 for RFS and p = 0.001 for OS). As TMPRSS4 expression is epigenetically regulated, methylation status could be used in circulating tumor DNA from liquid biopsies to monitor patients. We developed a digital droplet PCR (ddPCR) method to quantify absolute copy numbers of methylated and unmethylated CpGs within the TMPRSS4 and SHOX2 (as control) promoters in plasma and bronchoalveolar lavage (BAL) samples. In case-control studies, we demonstrated that TMPRSS4 hypomethylation can be used as a diagnostic tool in early stages, with an AUROC of 0.72 (p = 0.008; 91% specificity and 52% sensitivity) for BAL and 0.73 (p = 0.015; 65% specificity and 90% sensitivity) for plasma, in early stages. In conclusion, TMPRSS4 protein expression can be used to stratify patients at high risk of relapse/death in very early stages NSCLC patients. Moreover, analysis of TMPRSS4 methylation status by ddPCR in blood and BAL is feasible and could serve as a non-invasive biomarker to monitor surgically resected patients.

High mortality rates caused by NSCLC show the need for the identification of novel therapeutic targets. In this study we have investigated the biological effects and molecular mechanisms elicited by TMPRSS4 in NSCLC. Overexpression of TMPRSS4 in LKR13¿cells increased malignancy, subcutaneous tumor growth and multiorganic metastasis. In conditional knock-down (KD) experiments, abrogation of TMPRSS4 in H358 and H2170¿cells altered proliferation, clonogenicity, tumor engraftment and tumor growth. Reduction in S and G2/M phases of the cell cycle, decreased BrdU incorporation and increased apoptosis was also found. Transcriptomic analysis in KD cells revealed downregulation of genes involved in DNA replication, such as MCM6, TYMS and CDKN1A (p21). In patients, expression of a signature of MCM6/TYMS/TMPRSS4 genes was highly associated with poor prognosis. Downregulation of TMPRSS4 significantly increased sensitivity to chemotherapy agents. In experiments using cisplatin, apoptosis and expression of the DNA-damage marker ¿-H2A was higher in cells lacking TMPRSS4. Moreover, in vivo assays demonstrated that tumors with no TMPRSS4 were significantly more sensitive to cisplatin than controls. These results show that TMPRSS4 can be considered as a novel target in NSCLC, whose inhibition increases chemosensitivity.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.

Several circulating biomarkers and single nucleotide polymorphisms (SNPs) have been correlated with efficacy and tolerability to antiangiogenic agents. These associations remain unexplored in well-differentiated, metastatic pancreatic neuroendocrine tumors treated with the multitargeted tyrosine kinase inhibitor sunitinib. We have assessed the effect on tumor response at 6 months, overall survival, progression-free survival and safety of 14 SNPs, and 6 soluble proteins. Forty-three patients were recruited. Two SNPs in the vascular endothelial growth factor receptor 3 (VEGFR-3) gene predicted lower overall survival: rs307826 with hazard ratio (HR) 3.67 (confidence interval [CI] 95%, 1.35-10.00) and rs307821 with HR 3.84 (CI 95%, 1.47-10.0). Interleukin-6 was associated with increased mortality: HR 1.06 (CI 95%, 1.01-1.12), and osteopontin was associated with shorter PFS: HR 1.087 (1.01-1.16), independently of Ki-67. Furthermore, levels of osteopontin remained higher at the end of the study in patients considered non-responders: 38.5 ng/mL vs. responders: 18.7 ng/mL, p-value=0.039. Dynamic upward variations were also observed with respect to IL-8 levels in sunitinib-refractory individuals: 28.5 pg/mL at baseline vs. 38.3 pg/mL at 3 months, p-value=0.024. In conclusion, two VEGFR-3 SNPs as well as various serum biomarkers were associated with diverse clinical outcomes in patients with well-differentiated pancreatic neuroendocrine tumors treated with sunitinib.

CONTEXT: - The rapid advances in targeted therapies in non-small cell lung cancer (NSCLC) make the optimization and implementation of cytology specimens for molecular testing a priority. Up to 70% of patients with NSCLC are diagnosed at advanced stages and tissue biopsies often cannot be taken. Although cytology samples provide high-quality material for molecular testing, molecular cytopathology is not yet well known or widely used. OBJECTIVE: - To report the many advances in molecular cytopathology and the suitability and utility of cytology samples in molecular and genetic testing of NSCLC. DATA SOURCES: - Data sources comprised published peer-reviewed literature and personal experience of the authors. CONCLUSIONS: - Molecular testing can be performed on cytologic specimens, especially on direct smears. Rapid on-site evaluation by cytopathologists has improved the adequacy and the management of cytology samples for molecular testing. Mutational profiling of NSCLC using next-generation sequencing can be performed on cytology samples from very small amounts of DNA. Fluorescence in situ hybridization assays on cytology specimens, including stained direct smear, offer some distinct advantages over their histologic counterpart, and are used to detect ALK and ROS1 rearrangements in NSCLC. Cytology specimens allow assessment of the entire tumor cell nucleus, avoiding signal loss from truncation artifacts. The use of cytology samples for assessing programmed death ligand-1 protein expression is currently being developed. Protocols for bisulfite conversion and DNA droplet digital polymerase chain reaction assays have been optimized for cytology smear to investigate aberrant DNA methylation of several NSCLC-related genes

A major complication of colorectal cancer (CRC), one of the most frequent and deadly types of cancer, is disease progression via liver metastases. At this stage, very few treatment options are available for patients, and the disease remains incurable. Herein, we used a well-established mouse model of CRC liver metastasis (CLM) to identify new regulators of this process. Using serial transplantation of murine MC38 adenocarcinoma cells, we obtained liver metastatic variants that displayed extremely strong colonization abilities. Using these newly established cell lines, we performed gene expression arrays and microRNA (miR) profiling. Comparative and predictive analyses between the two arrays showed higher expression of c-met and concomitant reduction of miR-146a in the mestastatic variants. In CRC patients, expression levels of both c-met and miR-146a were similar between primary tumors and liver metastases. Interestingly, we identified c-met as a new target for miR-146a, as miR-146a was able to impede c-met translation. Of relevance, overexpression of miR-146a in metastatic clones showed reduced in vitro malignancy and abolished the development of primary tumor and liver metastases. Our results document a new mechanism for c-met regulation in CLM and highlight the crucial role of miR-146a in suppressing tumorigenesis.

Colorectal cancer (CRC) is one of the most frequent tumor types in Western countries. Approximately 20 % of patients show metastasis at the time of diagnosis, with the liver being one of the most affected organs. Transforming growth factor-beta (TGF-ß) plays a regulatory role not only in the physiology of the normal colon but also in the development of CRC and its metastatic process. In this review, we analyze the molecular mechanisms leading to TGF-ß dysregulation in tumor and stroma cells and the modification of the microenvironment that fosters CRC metastasis. Recent genomic studies have identified a CRC subtype with a mesenchymal and aggressive phenotype having TGF-ß as a hub gene of this signature. Consistent with these findings, the inhibition of TGF-ß signaling has been shown to impair experimental CRC metastasis to the liver. Based on these and other results conducted in various tumor types, the pharmaceutical industry has developed a variety of strategies to target TGF-ß. We provide up-to-date information of these therapies, which are currently in preclinical or clinical trials.

Despite a continuing debate about the existence of cancer stem cells (CSCs), recent discoveries have provided further support for their existence and their roles in drug resistance, cancer recurrence and metastasis. CSC characteristics, such as self-renewal and tumour initiation, and supporting cellular processes, particularly the epithelial-to-mesenchymal transition, are attracting a great deal of attention from cancer researchers as they offer opportunities for discovering novel therapeutic targets for future drug development. However, the identification of potential CSC targets presents clear obstacles due to a lack of truly specific CSC markers and the reality of CSC plasticity, making this task a significant challenge. Agents that target developmental signalling pathways, such as Notch, Wnt and Hedgehog, are now in clinical trials whilst alternative approaches including immune-based therapies and microRNA-mediated pathway inhibitors are producing promising pre-clinical results. Here, we discuss the contribution of CSCs to cancer metastasis and the scope of opportunities for therapeutic intervention. In particular, we consider CSC-targeting agents for which there is experimental evidence of anti-metastatic properties and which may have potential to eventually limit relapse and impede metastasis in patients.

Id1 promotes carcinogenesis and metastasis, and predicts prognosis of non-small cell lung cancer (NSCLC)-adenocarcionoma patients. We hypothesized that Id1 may play a critical role in lung cancer colonization of the liver by affecting both tumor cells and the microenvironment. Depleted levels of Id1 in LLC (Lewis lung carcinoma cells, LLC shId1) significantly reduced cell proliferation and migration in vitro. Genetic loss of Id1 in the host tissue (Id1(-/-) mice) impaired liver colonization and increased survival of Id1 animals. Histologically, the presence of Idl in tumor cells of liver metastasis was responsible for liver colonization. Microarray analysis comparing liver tumor nodules from Id1(+/+) mice and Idl(-/-) mice injected with LLC control cells revealed that Id1 loss reduces the levels of EMT-related proteins, such as vimentin. In tissue microarrays containing 532 NSCLC patients' samples, we found that Idl significantly correlated with vimentin and other EMT-related proteins. Idl loss decreased the levels of vimentin, integrin beta 1, TGF beta 1 and snail, both in vitro and in vivo. Therefore, Id1 enables both LLC and the host microenvironment for an effective liver colonization, and may represent a novel therapeutic target to avoid NSCLC liver metastasis. (C) 2017 Elsevier B.V. All rights reserved.

Metastatic spread is responsible for the majority of cancer deaths and identification of metastasis-related therapeutic targets is compulsory. TMPRSS4 is a pro-metastatic druggable transmembrane type II serine protease whose expression has been associated with the development of several cancer types and poor prognosis. To study the role and expression of this protease in cancer, we have developed molecular tools (active recombinant proteins and a polyclonal antibody) that can be used for diagnostic purposes and for testing anti-TMPRSS4 drugs. In addition, we have evaluated TMPRSS4 protein expression in several cancer tissue microarrays (TMAs). Full length and truncated TMPRSS4 recombinant proteins maintained the catalytic activity in two different expression systems (baculovirus and E. coli). Sensitivity of the rabbit polyclonal antisera against TMPRSS4 (ING-pAb) outperformed the antibody most commonly used in clinical settings. Analysis by immunohistochemistry in the different TMAs identified a subset of adenocarcinomas, squamous carcinomas, large cell carcinomas and carcinoids of the lung, which may define aggressive tumors. In conclusion, our biological tools will help the characterization of TMPRSS4 activity and protein expression, as well as the evaluation of anti-TMRSS4 drugs. Future studies should determine the clinical value of assessing TMPRSS4 levels in different types of lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines. In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped.

Metastasis involves a series of changes in cancer cells that promote their escape from the primary tumor and colonization to a new organ. This process is related to the transition from an epithelial to a mesenchymal phenotype (EMT). Recently, some authors have shown that migratory cells with an EMT phenotype share properties of cancer stem cells (CSCs), which allow them to form a new tumor mass. The type II transmembrane serine protease TMPRSS4 is highly expressed in some solid tumors, promotes metastasis and confers EMT features to cancer cells. We hypothesized that TMPRSS4 could also provide CSC properties. Overexpression of TMPRSS4 reduces E-cadherin and induces N-cadherin and vimentin in A549 lung cancer cells, supporting an EMT phenotype. These changes are accompanied by enhanced migration, invasion and tumorigenicity in vivo. TMPRSS4 expression was highly increased in a panel of lung cancer cells cultured as tumorspheres (a typical assay to enrich for CSCs). H358 and H441 cells with knocked-down TMPRSS4 levels were significantly less able to form primary and secondary tumorspheres than control cells. Moreover, they showed a lower proportion of ALDH+ cells (examined by FACS analysis) and lower expression of some CSC markers than controls. A549 cells overexpressing TMPRSS4 conferred the opposite phenotype and were also more sensitive to the CSC-targeted drug salinomycin than control cells, but were more resistant to regular chemotherapeutic drugs (cisplatin, gemcitabine and 5-fluorouracil). Analysis of 70 NSCLC samples from patients revealed a very significant correlation between TMPRSS4 expression and CSC markers ALDH (p¿=¿0.0018) and OCT4 (p¿=¿0.0004), suggesting that TMPRSS4 is associated with a CSC phenotype in patients' tumors. These results show that TMPRSS4, in addition to inducing EMT, can also promote CSC features in lung cancer; therefore, CSC-targeting drugs could be an appropriate treatment for TMPRSS4+ tumors.

1 in CRC cell¿CAFs attachment and its impact on liver metastasis. CAFs were obtained after xenotransplantation of Mc38 cells into EGFP-C57BL/6 mice. Attachment experiments with CRC cells and CAFs (with or without TGFß1 and the inhibitory peptide P17) were carried out, as well as in vivo liver metastasis assays. TGFß1 induced adhesion of CRC cells to CAFs, whereas exposure to P17 abrogated this effect. Co-injection of Mc38 cells with CAFs intrasplenically increased liver metastasis, as compared to injection of tumor cells alone. Pretreatment of Mc38 cells with TGFß1 enhanced the metastatic burden, in comparison to untreated Mc38 + CAFs. TGFß1-pretreated Mc38 cells co-metastatized with CAFs to the liver in a highly efficient way. Importantly, the metastatic burden was significantly reduced (p < 0.001) when P17 was administered in mice. The number of PCNA+ and CD-31+ cells was also reduced by P17 in these animals, indicating a decrease in proliferation and angiogenesis upon TGFß1 signaling blockade. Through microarray analysis, we identified potential TGFß1-regulated genes that may mediate cancer cell¿stroma interactions to increase metastasis. In conclusion, TGFß1 promotes co-travelling of CRC cells and CAFs to the liver to enhance metastasis. Our results strongly support the use of TGFß1 targeted drugs as a novel strategy to reduce liver metastasis in CRC patients.

The generation of new antileishmanial drugs has become a priority. Selenium and its derivatives stand out as having promising leishmanicidal activity. In fact, some parasites express selenoproteins and metabolize selenium. Recently, selenium derivatives have shown the potential to reduce parasitemia, clinical manifestations, and mortality in parasite-infected mice. In this paper, after selecting four candidates according to drug similarity parameters, we observed that two of them, called compounds 2b [methyl-N,N¿-di(thien-2-ylcarbonyl)-imidoselenocarbamate] and 4b [methyl-N,N¿-di(5-nitrothien-3-ylcarbonyl)-imidoselenocarbamate], exhibit low 50% inhibitory concentrations (IC50s) (<3 ¿M) and good selectivity indexes (SIs) (>5) in Leishmania major promastigotes and lack toxicity on macrophages. In addition, in analysis of their therapeutic potential against L. major in vitro infection, both compounds display a dramatic reduction of amastigote burden (~80%) with sublethal concentrations. Furthermore, in macrophages, these selenocompounds induce nitric oxide production, which has been described to be critical for defense against intracellular pathogens. Compounds 2b and 4b were demonstrated to cause cell cycle arrest in G1 . Interestingly, evaluation of expression of genes related to proliferation (PCNA), treatment resistance (ABC transporter and alpha-tubulin), and virulence (quinonoid dihydropteridine reductase [QDPR]) showed several alterations in gene expression profiling. All these results prompt us to propose both compounds as candidates to treat leishmanial infections.

The spread of lung cancer cells to distant sites represents a common event associated with poor prognosis. A fraction of tumor cells named cancer stem cells (CSCs) have the ability to overcome therapeutic stress and remain quiescent. However, whether these CSCs have also the capacity to initiate and sustain metastasis remains unclear. Here, we used tumor sphere cultures (TSC) isolated from mouse and human lung cancer models to enrich for CSCs, and assessed their metastatic potential as compared to non-CSCs. As expected, TSC overexpressed a variety of stem cell markers and displayed chemoresistance. The CSC phenotype of TSC was confirmed by their higher growth ability in soft agar and tumorigenic potential in vivo, despite their reduced in vitro cell growth kinetics. Surprisingly, the appearance of spontaneous lung metastases was strongly delayed in mice injected with TSC as compared to non-TSC cells. Similarly, this finding was confirmed in several other models of metastasis, an effect associated with a retarded colonization activity. Interestingly, such delay correlated with a quiescent phenotype whose underlined mechanisms included an increase in p27 protein and lower phospho-ERK1/2 levels. Thus, these data suggest that cells enriched for CSC properties display an impaired metastatic activity, a finding with potential clinical implications.

The biological activity of thyroid hormones (TH) is regulated by selenoenzymes of the iodothyronine deiodinase (DIO) family catalysing TH activating and inactivating reactions. Besides TH metabolism, several studies indicate an important role of DIO isoenzymes in tumorigenesis and cancer growth. It is therefore of therapeutic importance to identify modulators of DIO expression. We have synthesized and studied a series of selenocompounds containing a methyl- or benzyl-imidoselenocarbamate backbone. One of these novel compounds had chemotherapeutic activities in a murine xenograft tumour model by an unknown mechanism. Therefore, we tested their effects on DIO expression in vitro. In HepG2 hepatocarcinoma cells, DIO1 activity was strongly (up to 10-fold) increased by the methyl-but not by the corresponding benzyl-imidoselenocarbamates. Steady-state mRNA levels remained unaltered under these conditions indicating a post-transcriptional mode of action. The effects were further characterized in HEK293 cells stably expressing DIO1, DIO2 or DIO3. Even within the artificial genetic context of the expression vectors, all three DIO isoenzymes were up-regulated by the methyl-and to a lesser extent by the benzyl-imidoselenocarbamates. Consistent stimulating effects were observed with methyl-N, N'-di(quinolin-3-ylcarbonyl)-imidoselenocarbamate (EI201), a selenocompound known for its anti-tumour activity. DIO inducing effects were unrelated to the intracellular accumulation of selenium, yet the precise mode of action remains elusive. Collectively, our data highlight that these selenocompounds may constitute interesting pharmacological compounds for modifying DIO expression potentially affecting the balance between cell differentiation and proliferation.

INTRODUCTIONThe need for new treatments for advanced prostate cancer has fostered the experimental use of targeted therapies. Sunitinib is a multi-tyrosine kinase inhibitor that mainly targets membrane-bound receptors of cells within the tumor microenvironment, such as endothelial cells and pericytes. However, recent studies suggest a direct effect on tumor cells. In the present study, we have evaluated both direct and indirect effects of Sunitinib in prostate cancer and how this drug regulates hypoxia, using in vitro and in vivo models. METHODSWe have used both in vitro (PC-3, DU145, and LNCaP cells) and in vivo (PC-3 xenografts) models to study the effect of Sunitinib in prostate cancer. Analysis of hypoxia based on HIF-1 expression and FMISO uptake was conducted. ALDH activity was used to analyze cancer stem cells (CSC). RESULTSSunitinib strongly reduced proliferation of PC-3 and DU-145 cells in a dose dependent manner, and decreased levels of p-Akt, p-Erk1/2, and Id-1, compared to untreated cells. A 3-fold reduction in tumor growth was also observed (P<0.001 with respect to controls). Depletion of Hif-1 levels in vitro and a decrease in FMISO uptake in vivo showed that Sunitinib inhibits tumor hypoxia. When combined with radiotherapy, this drug enhanced cell death in vitro and in vivo, and significantly decreased CD-31, PDGFR, Hif-1, Id1, and PCNA protein levels (whereas apoptosis was increased) in tumors as compared to controls or single-therapy treated mice. Moreover, Sunitinib reduced the number of ALDH+ cancer stem-like cells and sensitized these cells to radiation-mediated loss of clonogenicity. DISCUSIONOur results support the use of Sunitinib in prostate cancer and shows that both hypoxia and cancer stem cells are involved in the effect elicited by this drug. Combination of Sunitinib with radiotherapy warrants further consideration to reduce prostate cancer burden.

Id1 has been shown to play a critical role in tumorigenesis and angiogenesis. Moreover, recent reports have involved Id1 in the maintenance of cancer stem cell features in some tumor types. The Id1 gene generates two isoforms through alternative splicing: Id1a and Id1b. We have investigated the role of each isoform in cancer development. Using lentiviral systems we modified the endogenous expression of each of these isoforms in cancer cells and analyzed their biological effect both in vitro and in vivo. Overexpression of Id1b in murine CT26 and 3LL cells caused a G0/G1 cell cycle arrest and reduced proliferation, clonogenicity and phospho-ERK1/2 levels, while increasing p27 levels. High levels of Id1a had an opposite effect and the proportion of cells in the S phase increased significantly. In vivo models confirmed the inhibitory role of Id1b in primary tumor growth and metastasis. Through microarray analysis we found that the cancer stem cell (CSC) markers ALDH1A1 and Notch-1 were up-regulated specifically in Id1b-overexpressing cells. By using qPCR we also found overexpression of Sca-1, Tert, Sox-2 and Oct-4 in these cells. Increased levels of Id1b promoted self-renewal and CSC-like properties, as shown by their high capacity for developing secondary tumorspheres and retaining the PKH26 dye. The acquisition of CSC phenotype was confirmed in human PC-3 cells that overexpressed Id1b. Our results show that Id1b maintains cells in a quiescent state and promotes self-renewal and

Inhibitor of differentiation-1 (Id1) might constitute a novel prognostic factor able to differentiate indolent from aggressive prostate tumors. In this study, 2 cohorts of 52 and 79 prostate cancer patients were selected for Id1 expression analysis. Higher levels of Id1 protein in advanced poor-prognosis patients and a correlation of higher Id1 mRNA expression levels with a lower survival in stage I to III patients were observed. Background: In the prostate-specific antigen era, potentially indolent prostate tumors are radically treated, causing overtreatment. Molecular prognostic factors might differentiate indolent from aggressive tumors, allowing avoidance of unnecessary treatment. Patients and Methods: Fifty-two prostate cancer patients (20 organ-confined and 32 metastatic) were selected. All formalin-fixed and paraffin-embedded primary biopsies and matched metastases of 15 of them were evaluated for tumor and endothelial cell Id1 protein expression. Seventy-nine additional patients with organ-confined prostate cancer were selected for Id1 mRNA in silico analysis. Results: Among metastatic cancer subjects, 48% of primary tumors and 38% of metastases showed Id1 tumor cell expression, and 79% of primary tumors and 81% of metastases showed endothelial immunoreactivity. In the organ-confined group none of them showed Id1 protein tumor cell expression and 50% displayed endothelial expression. In the metastatic patients group, lower levels of Id1 protein predicted a nonsignificant longer overall survival (13 months vs. 7 months; P = .79). In the in silico analysis, however, lower levels of Id1 mRNA predicted a longer disease-free survival (61 months vs. not-reached; P = .018) and the hazard ratio for progression was 0.451 (P = .022) in favor of patients showing lower levels. Conclusion: In our cohort, it seems to be a differential epithelial expression of Id1 protein according to the prognostic features (metastatic/poor prognosis vs. organ-confined/good prognosis). In localized tumors treated with radical prostatectomy, higher Id1 mRNA expression levels might predict a higher hazard ratio for progression and a shorter disease-free survival. Further validation of these results in larger prospective series is warranted.

Id-1 is a member of the helix-loop-helix family of proteins that regulates the activity of transcription factors to suppress cellular differentiation and to promote cell growth. Overexpression of Id-1 in tumor cells correlates with increased malignancy and resistance to chemotherapy and radiotherapy. Id-1B is an isoform generated by alternative splicing that differs from the classical Id-1 in the 13-C-terminal amino acids, whose function is at present unknown. We have studied the role of Id-1B in cancer and its expression in healthy/malignant lung tissues. Overexpression of Id-1B in A549 lung and PC3 prostate cancer cells reduced anchorage-dependent and independent proliferation and clonogenic potential. Moreover, it increased the proportion of cells in the G0/G1 phase of the cell cycle and p27 levels, while reduced phospho-Erk and cyclin A levels. Through microarray analysis, we identified genes involved in cell growth and proliferation that are specifically deregulated as a consequence of Id-1B overexpression, including IGF2, BMP4, Id2, GATA3, EREG and AREG. Id-1B overexpressing cells that were treated with 4Gy irradiation dose were significantly less resistant to cell death. In vivo assays demonstrated that tumors with high Id-1B levels exhibited less growth (p< 0.01), metabolic activity (glucose uptake) and angiogenesis (p< 0.05) compared to tumors with low Id-1B expression; mice survival was significantly extended (p< 0.05). Quantification by qRT-PCR revealed that expression of Id-1B was significantly lower (p< 0.01) in human lung tumors compared to their matched nonmalignant counterparts. In conclusion, our results demonstrate that Id-1B decreases the malignancy of lung and prostate cancer cells, sensitizes them to radiotherapy-induced cell death, and counteracts the pro-tumorigenic role of the classical form of Id-1.

Purpose: To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell-carcinoma (MRCC) and to evaluate in vitro their mechanism of action in sunitinib resistance. Methods: We screened 673 microRNAs using TaqMan Low-density-Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n = 41). The most relevant differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenotypes selected from an independent cohort (n = 101). In vitro experiments were conducted to study the role of miR-942 in sunitinib resistance. Results: TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p = 0.0074). High expression of miR-942, miR-628-5p, miR-133a, and miR-484 was significantly associated with decreased time to progression and overall survival. These microRNAs were also overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive cell line. MiR-942 overexpression in Caki-2 up-regulates MMP-9 and VEGF secretion which, in turn, promote HBMEC endothelial migration and sunitinib resistance. Conclusions: We identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity or resistance to sunitinib. MiR-942 was the best predictor of efficacy. We describe a novel paracrine mechanism through which high miR-942 levels in MRCC cells up-regulates MMP-9 and VEGF secretion to enhance endothelial migration and sunitinib resistance. Our results support further validation of these miRNA in clinical confirmatory studies.

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition), accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.

A series of 31 new selenoesters were synthesized and their cytotoxic activity was evaluated against a prostate cancer cell line (PC-3). The most active compounds were also tested against three tumoural cell lines (MCF-7, A-549 and HT-29) and one non-tumour prostate cell line (RWPE-1). Thirteen compounds showed significant activity towards all tumour cells investigated, and some of them were even more potent than etoposide and cisplatin, which were used as reference drugs. Because of their pronounced potency and/or selectivity, four analogues (5, 21, 28 and 30), were selected in order to assess their redox properties related to a possible redox modulating activity. The glutathione peroxidase (GPx) assay showed slight activity for compound 30 and the 2,2-diphenyl-1-picrylhydrazyl-(DPPH) assay showed a weak activity for compounds 5 and 28. The present results revealed that analogues 5, 21, 28 and 30 might serve as a useful starting point for the design of improved anti-tumour agents.

BACKGROUND: TMPRSS4 is a membrane-anchored protease involved in cell migration and invasion in different cancer types including lung cancer. TMPRSS4 expression is increased in NSCLC and its inhibition through shRNA reduces lung metastasis. However, molecular mechanisms leading to the protumorigenic regulation of TMPRSS4 in lung cancer are unknown. METHODS: miR-205 was identified as an overexpressed gene upon TMPRSS4 downregulation through microarray analysis. Cell migration and invasion assays and in vivo lung primary tumour and metastasis models were used for functional analysis of miR-205 overexpression in H2170 and H441 cell lines. Luciferase assays were used to identify a new miR-205 direct target in NSCLC. RESULTS: miR-205 overexpression promoted an epithelial phenotype with increased E-cadherin and reduced fibronectin. Furthermore, miR-205 expression caused a G0/G1 cell cycle arrest and inhibition of cell growth, migration, attachment to fibronectin, primary tumour growth and metastasis formation in vivo. Integrin ¿5 (a proinvasive protein) was identified as a new miR-205 direct target in NSCLC. Integrin ¿5 downregulation in lung cancer cells resulted in complete abrogation of cell migration, a decreased capacity to adhere to fibronectin and reduced in vivo tumour growth, compared with control cells. TMPRSS4 silencing resulted in a concomitant reduction of integrin ¿5 levels. CONCLUSION: We have demonstrated for the first time a new molecular pathway that connects TMPRSS4 and integrin ¿5 through miR-205 to regulate cancer cell invasion and metastasis. Our results will help designing new therapeutic strategies to inhibit this novel pathway in NSCLC.

Altered expression and activity of proteases is a key event in cancer, particularly in relation to invasion, modification of the extracellular matrix and metastasis. The transmembrane protease, serine 4 (TMPRSS4) is closely related to other cancer-associated proteases, such as hepsin, TMPRSS2 and matriptase. We review in this study up-to-date information about expression, role, regulation and clinical relevance of TMPRSS4 in cancer. Increased expression of this protease is associated with acquisition of epithelial to mesenchymal transition, invasion and metastasis in vivo. Signalling in cancer cells involves upregulation of integrin-¿5 (ITG-¿5) and urokinase-type plasminogen activator (uPA), downregulation of E-cadherin and activation of uPA enzymatic activity at the plasma membrane, as well as phosphorylation of FAK, Src, Akt and ERK1/2 intracellularly. Upregulation of miR-205 hinders the protumorigenic effects elicited by TMPRSS4 through restoration of E-cadherin levels and direct targeting of ITG-¿5. High levels of TMPRSS4 have been found in several types of solid tumours in patients, and association with poor prognosis has been consistently described. On the basis of this information and the structural characteristics of this druggable protease, we suggest that TMPRSS4 could be a novel potential therapeutic target in solid tumours.

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation and recurrence after chemotherapy. Targeting CSCs and non-CSCs with specific compounds may be an effective approach to reduce lung cancer growth and metastasis. The aim of this study was to investigate the effect of salinomycin, a selective inhibitor of CSCs, with or without combination with paclitaxel, in a metastatic model. To evaluate the effect of these drugs in metastasis and tumor microenvironment we took advantage of the immunocompetent and highly metastatic LLC mouse model. Aldefluor assays were used to analyze the ALDH+/- populations in murine LLC and human H460 and H1299 lung cancer cells. Salinomycin reduced the proportion of ALDH+ CSCs in LLC cells, whereas paclitaxel increased such population. The same effect was observed for the H460 and H1299 cell lines. Salinomycin reduced the tumorsphere formation capacity of LLC by more than 7-fold, but paclitaxel showed no effect. In in vivo experiments, paclitaxel reduced primary tumor volume but increased the number of metastatic nodules (p<0.05), whereas salinomycin had no effect on primary tumors but reduced lung metastasis (p<0.05). Combination of both drugs did not improve the effect of single therapies. ALDH1A1, SOX2, CXCR4 and SDF-1 mRNA levels were higher in metastatic lesions than in primary tumors, and were significantly elevated in both locations by paclitaxel treatment. On the contrary, such levels were reduced (or in some cases did not change) when mice were administered with salinomycin. The number of F4/80+ and CD11b+ cells was also reduced upon administration of both drugs, but particularly in metastasis. These results show that salinomycin targets ALDH+ lung CSCs, which has important therapeutic effects in vivo by reducing metastatic lesions. In contrast, paclitaxel (although reducing primary tumor growth) promotes the selection of ALDH+ cells that likely modify the lung microenvironment to foster metastasis.

Colorectal cancer (CRC) frequently metastasizes to the liver, a phenomenon that involves the participation of transforming-growth-factor-beta(1) (TGF beta(1)). Blockade of the protumorigenic effects elicited by TGF beta(1) in advanced CRC could attenuate liver metastasis. We aimed in the present study to assess the antimetastatic effect of TGF beta(1)-blocking peptides P17 and P144, and to study mechanisms responsible for this activity in a mouse model. Colon adenocarcinoma cells expressing luciferase were pretreated with TGF beta(1) (Mc38-luc(TGF beta 1) cells), injected into the spleen of mice and monitored for tumor development. TGF beta(1) increased primary tumor growth and liver metastasis, whereas systemic treatment of mice with either P17 or P144 significantly reduced tumor burden (p < 0.01). In metastatic nodules, mitotic/apoptotic ratio, mesenchymal traits and angiogenesis (evaluated by CD-31, as well as circulating endothelial and progenitor cells) induced by TGF beta(1) were consistently reduced following injection of peptides. In vitro experiments revealed a direct effect of TGF beta(1) in Mc38 cells, which resulted in activation of Smad2, Smad3 and Smad1/5/8, and increased invasion and transendothelial migration, whereas blockade of TGF beta(1)-signaling reverted these features. Because TGF beta(1)-mediated epithelial -mesenchymal transition (EMT) has been suggested to induce a cancer stem cell (CSC) phenotype, we analyzed the ability of this cytokine to induce tumorsphere formation and the expression of CSC markers. In TGF beta(1)-treated cells, tumorspheres were enriched in CD44 and SOX2, which were diminished in the presence of P17. Our data provide a preclinical rationale to evaluate P17 and P144 as potential therapeutic options for the treatment of metastatic CRC. (c) 2012 Elsevier Inc. All rights reserved.

Background: Inhibitor of DNA binding 1 (Id1) and 3 (Id3) genes have been related with the inhibition of cell differentiation, cell growth promotion and tumor metastasis. Recently, Id1 has been identified as an independent prognostic factor in patients with lung adenocarcinoma, regardless of the stage. Furthermore, Id1 may confer resistance to treatment (both, radiotherapy and chemotherapy). Methods: We have studied, using monoclonal antibodies for immunohistochemistry, the Id1 and Id3 tumor epithelial expression in 17 patients with stage III-N2 non-small cell lung cancer (NSCLC) treated with definitive chemoradiotherapy. Results: Id1 expression is observed in 82.4% of the tumors, whereas Id3 expression is present in 41.2% of the samples. Interestingly, Id1 and Id3 expression are mutually correlated (R = 0.579, p = 0.015). In a subgroup analysis of patients with the most locally advanced disease (T4N2 stage), co-expression of Id1 and Id3 showed to be related with a worse overall survival (45 vs 6 months, p = 0.002). A trend towards significance for a worse progression free survival (30 vs 1 months, p = 0.219) and a lower response rate to the treatment (RR = 50% vs 87.5%, p = 0.07) were also observed. Conclusions: A correlation between Id1 and Id3 protein expression is observed. Id1 and Id3 co-expression seems associated with a poor clinical outcome in patients with locally advanced NSCLC treated with definitive chemoradiotherapy.

The immobilization of cells within alginate-poly-L-lysine-alginate (APA) microcapsules has been demonstrated to be an effective technology design for long term delivery of therapeutic products. Despite promising advances, biosafety aspects still remain to be improved. Here, we describe a complete characterization of the strategy based on TGL triple-fusion reporter gene - which codifies for Herpes Simplex virus type 1 thymidine-kinase (HSV1-TK), green fluorescent protein (GFP) and Firefly Luciferase - (SFG(NES)TGL) to inactivate encapsulated cells and their therapeutic effects. Myoblasts genetically engineered to secrete erythropoietin (EPO) were retroviraly transduced with the SFG(NES)TGL plasmid to further characterize their ganciclovir (GCV)-mediated inactivation process. GCV sensitivity of encapsulated cells was 100-fold lower when compared to cells plated onto 2D surfaces. However, the number of cells per capsule and EPO secretion decayed to less than 15% at the same time that proliferation was arrested after 14 days of GCV treatment in vitro. In vivo, ten days of GCV treatment was enough to restore the increased hematocrit levels of mice implanted with encapsulated TGL-expressing and EPO-secreting cells. Altogether, these results show that TGL triple-fusion reporter gene may be a good starting point in the search of a suitable biosafety strategy to inactivate encapsulated cells and control their therapeutic effects. (C) 2012 Elsevier Ltd. All rights reserved.

Since circulating tumor cells (CTCs) have metastatic potential, their genetic and phenotypic characteristics could provide crucial information to establish the most effective therapy. We assessed the clinical utility of a methodology that allows the simultaneous analysis of CTC phenotype and genotype in colon cancer patients and, in addition, whether this methodology could provide complementary information to that obtained by the primary tumor biopsy. Thirty-three non-metastatic (stages 0-III) colon cancer patients and 9 healthy donor samples were evaluated. All peripheral blood samples (10 ml) were analyzed by cytokeratin immunomagnetic enrichment. Eight samples were analyzed by immunocytochemistry and 25 samples were analyzed by FICTION technique for simultaneous cytokeratin expression and chromosome 17 and ERBB2 gene status. A further study was carried out in one patient who showed CTC heterogeneity in chromosomal abnormalities. We analyzed HER2 protein expression on CTCs and FISH and HER2 protein expression in primary tumor of this patient. Our results show that 9.09% of patients had cytokeratin-positive CTCs (CK+/CTCs in peripheral blood). One of the patients showed heterogeneity in chromosomal 17 abnormalities and two different CK expression patterns on CTCs: one CK+/CTCs and one CK-/CTCs. Furthermore, 63.33% of these CTCs overexpressed HER2 protein while the primary tumor of this patient was diploid and did not express HER2 protein. We describe a methodology that allows the simultaneous genetic and phenotypic analysis of CTCs in colon cancer patients, which may provide essential information to select patients who might benefit from specific therapy.

Inhibitor of differentiation-1 (Id1) plays a role in cell proliferation, acquisition of epithelial to mesenchymal transition (EMT) features and angiogenesis. Id1 was shown to be expressed in some tumor types, mainly in advanced dedifferentiated stages. However, recent studies using a validated and highly specific monoclonal antibody against Id1 have challenged many of the results obtained by immunohistochemistry. The goal of our work was to perform a thorough analysis of Id1 expression in mouse embryos and adult tissues, as well as healthy and malignant mouse and human samples using this validated antibody (Perk et al., 2006). Our results show that Id1 was highly expressed in the oropharyngeal cavity, lung, cartilage and skin of E14 and E15 mouse embryos, but expression was progressively reduced in more developed embryos. Immunostaining only remained in epithelial cells of the gut and uterus of adult mice. Mammary MMTV-Myc and MMTV-Myc/VEGF transgenic mouse tumors, and squamous cell carcinomas of the lung induced by N-nitroso-tris-chloroethylurea (NTCU) were highly positive for Id1, unlike their respective healthy counterparts. Id1 immunostaining in a human tissue microarray (TMA) revealed strong expression in cancers of the oral cavity, bladder and cervix. Some tumor specimens of esophagus, thyroid and breast were also strongly positive. Our results suggest that Id1 is an oncofetal protein highly expressed in particular tumor types that should be reanalyzed in future studies using large cohorts of patients to reassess its diagnostic/prognostic value. Moreover, MMTV-Myc- and NTCU-induced tumors could serve as appropriate mouse models to study Id1 functions in breast and lung cancer, respectively.

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively its applicability. Producing reduced size microcapsules involves broadening the possibilities to employ this technology in the treatment of many disorders. Nervous system diseases (NSD) represent a clear example of that. This work describes the feasibility of reducing the size of alginate-poly-L-lysine-alginate (APA) microcapsules up to 100 ¿m in a highly monodisperse way using the novel Flow Focusing technique. C(2)C(12) myoblasts genetically engineered to express the triple reporter gene thymidine kinase-green fluorescent protein-luciferase (TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might be affected due to the physicochemical changes arising from such dramatic size reduction. Not only were negative effects at this level not noticed in terms of cell viability, cell proliferation and KDR secretion, but once again the suitability of APA microcapsules was also reinforced against other microcapsule designs. Furthermore, the fully viable and functional biosystems were successfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3 weeks.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related deaths. Currently available chemotherapeutic options are not curative due in part to tumor resistance to conventional therapies. We generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2r¿ null mice by injection of human alpha-feto protein (hAFP)- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. In this model, low-dose metronomic administration of cyclophosphamide (LDM-CTX) caused complete regression of the tumor mass. A significant increase in survival (P<0.0001), reduced aberrant angiogenesis and hyperproliferation, and decrease in the number of circulating tumor cells were found in LDM-CTX-treated animals, in comparison with untreated mice. Co-administration of LDM-CTX with anti-VEGF therapy further improved the therapeutic efficacy. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, discontinuation of therapy resulted in tumor regrowth. Moreover, in-vitro LDM-CTX treatment reduced hepatosphere formation in both number and size, and the resulting spheres were enriched in CD13+ cells indicating that these cells were particularly resistant to therapy. Co-treatment of the CD13-targeting drug, bestatin, with LDM-CTX leads to slower tumor growth and a decreased tumor volume. Therefore, combining a CD13 inhibitor, which targets the CSC-like population, with LDM-CTX chemotherapy may be used to eradicate minimal residual disease and improve the treatment of liver cancer.

Methylimidoselenocarbamates have previously proven to display potent antitumor activities. In the present study we show that these compounds act as multikinase inhibitors. We found that the most effective compound, quinoline imidoselenocarbamate EI201, inhibits the PI3K/AKT/mTOR pathway, which is persistently activated and contributes to malignant progression in various cancers. EI201 blocked the phosphorylation of AKT, mTOR and several of its downstream regulators (p70(S6K) and 4E-BP1) and ERK1/2 in PC-3, HT-29 and MCF-7 cells in vitro, inducing both autophagy and apoptosis. EI201 also contributes to the loss of maintenance of the self-renewal and tumorigenic capacity of cancer stem cells (CSCs). 0.1 mu mol/L EI201 triggered a reduction in size and number of tumorspheres in PC-3, HT-29 and MCF-7 cells and 4 mu mol/L induced the elimination of almost all the tumorspheres in the three studied cell lines. In addition, EI201 suppressed almost 80% prostate tumor growth in vivo (p < 0.01) compared to controls at a relatively low dose (10 mg/kg) in a mouse xenograft model. There was a significant decrease in the subcutaneous primary tumor [18F]-FDG uptake (76.5% reduction, p < 0.05) and in the total tumor burden (76.8% reduction, p < 0.05) after EI201 treatment compared to vehicle control, without causing toxicity in mice. Taken together, our results support further development of EI201 as a novel multi-kinase inhibitor that may be useful against cancers with aberrant upregulation of PI3K/AKT and MAPK signaling pathways.

Introduction: Lung cancer is the leading cause of cancer deaths worldwide. Although standard treatment regimens have produced promising results with neoadjuvant and adjuvant strategies, outcomes for patients with lung cancer are still considered disappointing. Recent data provide evidence that the tumor-stromal environment is a leading player in carcinogenesis, not just a supporting tumor compartment. Areas covered: This article reviews the current understanding of the lung cancer microenvironment and the complex bidirectional interplay between the tumor and lung microenvironment in non-small cell lung cancer (NSCLC). In particular, this review emphasizes the role of fibroblasts, mesenchymal stem cells and myeloid cells as well as mediators and molecular pathways regulated by these cells within the tumor microenvironment (TME) that contribute to lung tumor initiation, progression and metastasis. In addition, this review also summarizes the therapeutic strategies currently being applied in preclinical and clinical trials. Expert opinion: Considering the recent advances in understanding lung tumor stroma, lung cancer progression could be effectively hampered by combining cytotoxic strategies with therapies that target the TME. However, more preclinical and clinical research is needed to prove the efficacy of this strategy for treating NSCLC.

Background: Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. Results: The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. Conclusions: We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

Bone microenvironment and cell-cell interactions are crucial for the initiation and development of metastasis. By means of a pharmacologic approach, using the multitargeted tyrosine kinase inhibitor sunitinib, we tested the relevance of the platelet-derived growth factor receptor (PDGFR) axis in the bone marrow (BM) stromal compartment for the initiation and development of lung cancer metastasis to bone. PDGFR beta was found to be the main tyrosine kinase target of sunitinib expressed in BM stromal ST-2 and MC3T3-E1 preosteoblastic cells. In contrast, no expression of sunitinib-targeted receptors was found in A549M1 and low levels in H460M5 lung cancer metastatic cells. Incubation of ST-2 and human BM endothelial cells with sunitinib led to potent cell growth inhibition and induction of apoptosis in a dose-dependent manner. Similarly, sunitinib induced a robust proapoptotic effect in vivo on BM stromal PDGFR beta(+) cells and produced extensive disruption of tissue architecture and vessel leakage in the BM cavity. Pretreatment of ST-2 cells with sunitinib also hindered heterotypic adhesion to lung cancer cell lines. These effects were correlated with changes in cell-cell and cell-matrix molecules in both stromal and tumor cells. Pretreatment of mice with sunitinib before intracardiac inoculation of A549M1 or H460M5 cells caused marked inhibition of tumor cells homing to bone, whereas no effect was found when tumor cells were pretreated before inoculation. Treatment with sunit

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-alpha, TNF-alpha, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-gamma-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.

Purpose: Blastic natural killer (NK) cell lymphoma/blastic plasmacytoid dendritic cell neoplasm (BNKL) is a rare and aggressive neoplasia characterized by infiltration of blast CD4(+)/CD56(+) cells in the skin, the bone marrow, and peripheral blood. Currently, more efforts are required to better define molecular and biological mechanisms associated with this pathology. To the best of our knowledge, no mouse model recapitulated human BNKL so far. Experimental Design: Primary bone marrow cells from a BNKL patient were injected in nonobese diabetes/severe combined immunodeficient interleukin (IL) 2r gamma(-/-) mice with the intent to generate the first BNKL orthotopic mouse model. Moreover, because of the lack of efficient treatments for BNKL, we treated mice with lenalidomide, an immunomodulatory and antiangiogenic drug. Results: We generated in mice a fatal disease resembling human BNKL. After lenalidomide treatment, we observed a significant reduction in the number of peripheral blood, bone marrow, and spleen BNKL cells. Tumor reduction parallels with a significant decrease in the number of circulating endothelial and progenitor cells and CD31(+) murine endothelial cells. In mice treated with lenalidomide, BNKL levels of active caspase-3 were significantly augmented, thus showing proapoptotic and cytotoxic effects of this drug in vivo. An opposite result was found for proliferating cell nuclear antigen, a proliferation marker. Conclusions: Our BNKL model might better define the cellular and molecular mechanisms involved in this disease, and lenalidomide might be considered for the future therapy of BNKL patients.

Cell microencapsulation may represent a breakthrough to overcome problems associated with cell therapy. Advances in material biocompatibility and production protocols have put this field close to its clinical application. However, issues such as the possibility of tracking cell-containing microcapsules, monitoring cell viability, and discontinuation of the therapeutic activity when necessary, still remain unsolved. We demonstrate here simultaneous monitoring and pharmacological control of myoblasts-containing alginate microcapsules, injected in immunocompetent mice after transduction with the SFG(NES)TGL triple reporter retroviral vector, which contains green fluorescence protein (GFP), firefly luciferase and herpes simplex virus type 1 thymidine-kinase (HSV1-TK). Naked (as controls) or microencapsulated cells were subcutaneously injected in C57BL/6J mice and followed up by luminometry. Signal for naked cells disappeared 2 weeks after cell injection, whereas signal for microencapsulated cells remained strong for 8 months, thus demonstrating the presence of living cells. Treatment of mice with the thymidine-kinase substrate ganciclovir caused death of microencapsulated myoblasts, as seen by a drastic decay in the light emission and histological analysis. Hence, we conclude that incorporation of the SFG(NES)TGL vector into microencapsulated cells represents an accurate tool for controlling cell location and viability in a non-invasive way. Moreover, cell death can be induced by administration of ganciclovir, in case therapy needs to be interrupted. This system may represent a step forward in the control and biosafety of cell- and gene- therapy-based microencapsulation protocols.

BACKGROUND: Prostate cancer (PrCa) has a high incidence in Western countries and at present, there is no cure for hormone refractory prostate cancer. Transgenic mouse models have proven useful for understanding mechanisms of prostate carcinogenesis. The characterization of genetically modified mouse PrCa models using high-throughput genomic analyses provides important information to guide appropriate experiment applications for such model. METHODS: We have analyzed the transcriptome of the hormone refractory and highly metastatic Fetal Globin-SV40/T-antigen (Ggamma-globin-Tag) transgenic mouse model for PrCa compared to normal mouse prostate tissue. Gene expression patterns found in Ggamma-globin-Tag mouse prostate tumors were compared with publicly available human localized and metastatic prostate tumors (GEO accession # GSE3325) through hierarchical cluster analysis, Pearson's rank correlation coefficient, and Self Organizing Feature Maps (SOM) analyses. RESULTS: Ggamma-globin-Tag tumors clustered closely with human metastatic tumors and gene expression patterns had a significant correlation (P < 0.01), unlike human localized primary tumors (P > 0.6). Bioinformatic analyses identified deregulated genetic pathways and networks in Ggamma-globin-Tag tumors, which displayed similarities to alterations in human PrCa. Changes in the expression of genes involved in DNA replication and repair (Rb1, p53, Myc, PCNA, DNMT3A) and growth factor signaling pathways (TGFbeta2, ERK1/2, NRas, and Notch1) are deregulated in the Ggamma-globin-Tag tumors, suggesting their key role in the oncogenic process. Identification of an enrichment of putative binding sites for transcription factors revealed eight transcription factors that may be important in Ggamma-globin-Tag carcinogenesis, including SP1, NF-Y, CREB, Elk1, and E2F. Novel genes related to microtubule regulation were also identified in Ggamma-globin-Tag tumors as potentially important candidate targets for PrCa. Overexpression of stathmin-1, whose expression was increased in human metastatic prostate tumors, was validated in Ggamma-globin-Tag tumors by immunohistochemistry. This protein belongs to the SV40/T-antigen cancer signature identified in previous studies in prostate, breast, and lung cancer mouse models. CONCLUSIONS: Our results show that the Ggamma-globin-Tag model for hormone refractory PrCa shares important features with aggressive, metastatic human PrCa. Given the role of stathmin-1 in the destabilization of microtubles and taxane resistance, the Ggamma-globin-Tag model and other SV40/T-antigen driven transgenic models may be useful for testing potential therapies directed at stathmin-1 in human prostate tumors.

Prostate cancer is a long latency type of tumor that usually develops in men older than 50 years of age. Prostate epithelial neoplasia (PIN), the initial malignant lesion, progresses to invasive carcinoma over the course of years. Because of the particular features of prostate carcinogenesis, this type of tumor may represent a paradigm for cancer prevention. Several clinical trials have evaluated the effect of different compounds on prostate tumor development, including finasteride, selenium, vitamin E, and carotenes. Although some results are promising, no conclusive data have been achieved as to recommend any of these compounds as preventive agents. Results from some trials, such as SELECT, where supplementation of selenium and/or vitamin-E was used, have been rather disappointing. However, many novel chemopreventive agents that target different cancer-related pathways are being developed lately. Appropriate animal models (in particular, genetically modified mice) are being used to assess the efficacy of these novel compounds. The transgenic adenocarcinoma of the mouse prostate (TRAMP) model has been validated as an accurate model to test a variety of preventive agents. Genistein, alpha-difluoromethylornithine, toremifene, R-flurbiprofen, celecoxib, and green tea polyphenols have been shown to prevent prostate cancer development in TRAMP mice. In conclusion, new chemopreventive compounds which are effective in animal models are likely to be tested soon in clinical trials, with the final goal of reducing prostate cancer incidence in men.

Vascular endothelial growth factor (VEGF) is overexpressed during the transition from prostate intraepithelial neoplasia (PIN) to invasive carcinoma. We have mimicked such a process in vitro using the PIN-like C3(1)/Tag-derived Pr-111 cell line, which expresses low levels of VEGF and exhibits very low tumorigenicity in vivo. Elevated expression of VEGF164 in Pr-111 cells led to a significant increase in tumorigenicity, invasiveness, proliferation rates and angiogenesis. Moreover, VEGF164 induced strong changes in cell morphology and cell transcriptome through an autocrine mechanism, with changes in TGF-beta1- and cytoskeleton-related pathways, among others. Further analysis of VEGF-overexpressing Pr-111 cells or following exogenous addition of recombinant VEGF shows acquisition of epithelial-mesenchymal transition (EMT) features, with an increased expression of mesenchymal markers, such as N-cadherin, Snail1, Snail2 (Slug) and vimentin, and a decrease in E-cadherin. Administration of VEGF led to changes in TGF-beta1 signaling, including reduction of Smad7 (TGF-beta inhibitory Smad), increase in TGF-betaR-II, and translocation of phospho-Smad3 to the nucleus. Our results suggest that increased expression of VEGF in malignant cells during the transition from PIN to invasive carcinoma leads to EMT through an autocrine loop, which would promote tumor cell invasion and motility. Therapeutic blockade of VEGF/TGF-beta1 in PIN lesions might impair not only tumor angiogenesis, but also the early dissemination of malignant cells outside the epithelial layer.

Background: Different isoforms of VEGF-A (mainly VEGF(121), VEGF(165) and VEGF(189)) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF(121/165)b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results: Recombinant VEGF(121/165)b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF(165). Furthermore, treatment of endothelial cells with VEGF(121/165)b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF(121/165)b isoforms. A549 and PC 3 cells overexpressing VEGF(121)b or VEGF(165)b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found. Conclusions: Our results demonstrate that VEGF(121/165)b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF(121/165)b-based therapies in patients.