Imaginando el futuro / Looking to the future

Presentamos la visión futurista que de su especialidad tienen 7 líderes de opinión estrechamente comprometidos con la patología mamaria. Las especialidades incluidas fueron radiología, patología, cirugía, cirugía plástica, medicina nuclear, oncología médica y oncología radioterápica. Los autores plasman, en este artículo, sus opiniones y criterios respecto a los avances que vislumbran en su futuro profesional.Conceptos clave como sistemas de cribado sin radiación, transcriptómica clínica, diagnóstico funcional del tumor, inteligencia artificial, navegación intraoperatoria, biopsia líquida, ADN tumoral circulante, reconstrucción con técnicas microquirúrgicas avanzadas, hipofraccionamiento extremo o teragnosis, son algunos de los conceptos presentados y discutidos.Los autores justifican sus puntos de vista, abriendo líneas de trabajo a tener en cuenta para optimizar esfuerzos y el conocimiento futuro. (AU)

We present the futuristic vision of their specialty of seven opinion leaders closely involved in breast pathology. The specialties were radiology, pathology, surgery, plastic surgery, nuclear medicine, medical oncology, and radiation oncology. In this article, the authors express their opinions and criteria regarding the advances they foresee for their professional future.Key concepts such as radiation-free screening systems, clinical transcriptomics, functional tumor diagnosis, artificial intelligence, intraoperative navigation, liquid biopsy, circulating tumor DNA, reconstruction with advanced microsurgical techniques, extreme hypofractionation or theragnosis are some of the concepts presented and discussed.The authors justify their points of view, suggesting lines of work to optimize efforts and future knowledge. (AU)

Histopathological growth patterns of liver metastasis: updated consensus guidelines for pattern scoring, perspectives and recent mechanistic insights.

The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

Lipopolysaccharide-regulated secretion of soluble and vesicle-based proteins from a panel of colorectal cancer cell lines.

PURPOSE: To mimic the perioperative microenvironment where bacterial products get in contact with colorectal cancer (CRC) cells and study its impact on protein release, we exposed six CRC cell lines to lipopolysaccharide (LPS) and investigated the effect on the secretome using in-depth mass spectrometry-based proteomics. EXPERIMENTAL DESIGN: Cancer cell secretome was harvested in bio-duplicate after LPS treatment, and separated in EV and soluble secretome (SS) fractions. Gel-fractionated proteins were analysed by label-free nano-liquid chromatography coupled to tandem mass spectrometry. NF-κB activation, triggered upon LPS treatment, was evaluated. RESULTS: We report a CRC secretome dataset of 5601 proteins. Comparison of all LPS-treated cells with controls revealed 37 proteins with altered abundance in the SS, including RPS25; and 13 in EVs, including HMGB1. Comparing controls and LPS-treated samples per cell line, revealed 564 significant differential proteins with fold-change >3. The LPS-induced release of RPS25 was validated by western blot. CONCLUSIONS AND CLINICAL RELEVANCE: Bacterial endotoxin has minor impact on the global CRC cell line secretome, yet it may alter protein release in a cell line-specific manner. This modulation might play a role in orchestrating the development of a permissive environment for CRC liver metastasis, especially through EV-communication.

Liver prometastatic reaction: Stimulating factors and responsive cancer phenotypes.

Cancer is first a localized tissue disorder, whose soluble and exosomal molecules and invasive cells induce a host response providing the stromal components of the primary tumor microenvironment (TME). Once the TME is developed, cancer-derived molecules and cells can more efficiently spread out and a whole-body response takes place, whose pathophysiological changes may result in a paraneoplastic syndrome. Remote organ-specific prometastatic reactions may also occur at this time, facilitating metastatic activities of circulating tumor cells (CTCs) through premetastatic niche development at targeted organs. However, additional signaling factors from the inter-organ communication network involved in the pathophysiology and comorbidities of cancer patients may also regulate prometastatic reaction-stimulating effects of cancer and non-cancer tissue factors. This article provides a conceptual overview of our ongoing clinical research on the liver prometastatic reaction (LPR) of patients with colorectal cancer (CRC), their portal vein- and hepatic artery-driven LPR-Stimulating Factors (LPR-SF), and their resulting LPR-derived Metastasis-Stimulating Factors (LPR-MSF) acting on liver-invading CRC cells. In addition, we also provide new insights on the molecular subtyping of LPR-responsive cancer phenotypes in patients with CRC and melanoma; and on how to investigate and interpret the prometastatic infrastructure in the real pathophysiological context of patients with cancer undergoing surgical procedures and receiving pharmacological treatments with multiple side effects, including those affecting the LPR, its stimulating factors and responsive cancer phenotypes.

Soluble ICAM 1 and VCAM 1 Blood Levels Alert on Subclinical Atherosclerosis in Non Smokers with Asymptomatic Metabolic Syndrome.

BACKGROUND: Metabolic syndrome (MetS) is a heterogeneous clinical entity associated with insulin resistance, low-grade proinflammatory balance and impaired endothelial function, accelerating atherosclerosis. Atherosclerotic lesions worsen with age, smoking and co-morbidities, making it difficult to accurately diagnose the cardiovascular disease (CVD) risk. AIM: We investigate the association between subclinical atherosclerosis and the presence of blood parameters related to adipocyte and vascular endothelial cell dysfunction, in non-smokers with MetS, under 60 and without previous CVD events. METHODS: Seventy-eight asymptomatic individuals (average 46.5 years, 69% male; 59 MetS and 19 controls) were studied prospectively. Subclinical CVD was defined by the presence of carotid plaque and/or carotid intima-media thickness (CIMT) > 0.9 in 2/3D ultrasound-studies, left ventricular hypertrophy (LVH) or high coronary calcium score (CCS). Multiplex immunoassay by Luminex xMAP was performed to measure plasma levels of adipokines and endothelial cell-derived molecules. RESULTS: Compared with controls, MetS patients had higher prevalence of carotid plaque (25 vs. 0%, p = 0.01), CIMT>0.9 (73 vs. 26%, p = 0.001) and higher CCS (69 vs. 5, p = 0.01), which were associated with a remarkable decrease in plasma Omentin levels and increase in sICAM-1, sVCAM-1 and PAI-1 (p <0.05). There was a statistically significant association between CIMT and sICAM-1 (OR: 14.57, 95% CI: 2.56-82.73, p <0.001), sVCAM-1 (OR:7.33, 95% CI: 1,58-33.96, p = 0.007) and PAI-1 (OR:7.80, 95% CI: 1.04-22.10, p = 0.036) in patients with carotid plaque and/or CIMT>0.9. Positive correlation between plaque volume and sICAM-1 levels was also detected (r = 0.40, p = 0.045). CONCLUSIONS: We demonstrated that the increase of sICAM-1, sVCAM-1 and PAI-1, together with decrease of omentin-1 led to a proinflammatory imbalance pointing to the presence of subclinical atherosclerosis, and improving CVD risk stratification in non-smoking patients at early stage MetS beyond traditional scores.

International consensus guidelines for scoring the histopathological growth patterns of liver metastasis.

BACKGROUND: Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs. METHODS: Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined. RESULTS: Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006). CONCLUSIONS: The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies.

Role of TGF-ß in metastatic colon cancer: it is finally time for targeted therapy.

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.

The inhibitor of differentiation-1 (Id1) enables lung cancer liver colonization through activation of an EMT program in tumor cells and establishment of the pre-metastatic niche.

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 Id1 in tumor cells of liver metastasis was responsible for liver colonization. Microarray analysis comparing liver tumor nodules from Id1+/+ mice and Id1-/- 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 Id1 significantly correlated with vimentin and other EMT-related proteins. Id1 loss decreased the levels of vimentin, integrinß1, TGFß1 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.

Opposite Effects of Mechanical Action of Fluid Flow on Proangiogenic Factor Secretion From Human Adipose-Derived Stem Cells With and Without Oxidative Stress.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

Distinct Osteomimetic Response of Androgen-Dependent and Independent Human Prostate Cancer Cells to Mechanical Action of Fluid Flow: Prometastatic Implications.

BACKGROUND AND METHODS: Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. RESULTS: We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFß, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBß1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFß secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFß-stimulating effect on mechanically stimulated LnCap cells. CONCLUSIONS: MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321-333, 2017. © 2016 Wiley Periodicals, Inc.

Towards the introduction of the 'Immunoscore' in the classification of malignant tumours.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

IL-18 regulates melanoma VLA-4 integrin activation through a Hierarchized sequence of inflammatory factors.

Very late antigen-4 (VLA-4) is frequently overexpressed on melanoma cells contributing to inflammation-dependent metastasis. Melanoma cell adhesion to endothelium via VLA-4-vascular cell adhesion molecule-1 (VCAM-1) interaction was used to study VLA-4 activation during melanoma cell response to inflammation. Cooperation among major inflammatory mediators was analyzed in melanoma cells exposed to single inflammatory factors in the presence of inhibitors for other assayed mediators. A stepwise cascade of hierarchized molecules heterogeneously made and used during melanoma response to IL-18, induced hydrogen peroxide (H2O2), in turn activating VLA-4 and melanoma cell adhesion to endothelium. The cascade involved prostaglandin E2 (PGE2) production from melanoma induced by IL-18-dependent tumor necrosis factor-α (TNFα); next, PGE2-induced IL-1ß via vascular endothelial growth factor (VEGF) secretion, which in turn induced VLA-4 activation via cyclooxygenase 2-dependent H2O2. This sequence operated in IL-18R/VLA-4/VEGF-expressing murine (B16) and human (A375 and 883) melanomas, but not in those without this phenotype. Separation of active VLA-4-expressing B16 melanoma cells through immobilized VCAM-1 verified their higher IL-18R/TNFR1/VEGFR2 expression and metastatic growth than inactive VLA-4-expressing cells. However, cooperation among melanoma cell sub-populations with heterogeneous cytokine receptor levels may occur through VLA-4-stimulating factors, leading to intratumoral amplification of metastatic potential. Therefore, expression of the VLA-4-stimulating factor sequence may help to predict melanoma prometastatic risk, and offers therapeutic targets for metastatic melanoma deactivation through VLA-4 activation blockade.

Future perspectives in melanoma research. Meeting report from the "Melanoma Bridge. Napoli, December 2nd-4th 2012".

Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma. After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma. The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma. The third "Melanoma Research: "A bridge from Naples to the World" meeting, shortened as "Bridge Melanoma Meeting" took place in Naples, December 2 to 4th, 2012. The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma. This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients.

α2,3-Sialyltransferase ST3Gal IV promotes migration and metastasis in pancreatic adenocarcinoma cells and tends to be highly expressed in pancreatic adenocarcinoma tissues.

Sialyltransferases have received much attention recently as they are frequently up-regulated in cancer cells. However, the role played by each sialyltransferase in tumour progression is still unknown. α2,3-Sialyltransferases ST3Gal III and ST3Gal IV are involved in sialyl-Lewis(x) (SLe(x)) synthesis. Given that the role of ST3Gal III in pancreatic adenocarcinoma cells has been previously reported, in this study we have focused on investigating the role of ST3Gal IV in the acquisition of adhesive, migratory and metastatic capabilities and, secondly, in analyzing the expression of ST3Gal III and ST3Gal IV in pancreatic adenocarcinoma tissues versus control tissues. ST3Gal IV overexpressing pancreatic adenocarcinoma MDAPanc-28 cell lines were generated. They showed a heterogeneous increase in SLe(x), and enhanced E-selectin adhesion and migration. Furthermore, when injected into nude mice, increased metastasis and decreased survival were found in comparison with controls. The behaviour of MDAPanc-28 ST3Gal IV overexpressing cells in these processes was similar to the already reported MDAPanc-28 ST3Gal III overexpressing cells. Furthermore, pancreatic adenocarcinoma tissues tended to express high levels of ST3Gal III and ST3Gal IV together with other fucosyltransferase genes FUT3 and FUT6, all involved in the last steps of sialyl-Lewis(x) biosynthesis. In conclusion, both α2,3-sialyltransferases are involved in key steps of pancreatic tumour progression processes and are highly expressed in most pancreatic adenocarcinoma tissues.

Metastatic lesions with and without interleukin-18-dependent genes in advanced-stage melanoma patients.

IL-18 is an immune-stimulating cytokine that promotes experimental melanoma metastasis via vascular endothelial growth factor (VEGF)-induced very late antigen (VLA)-4. We studied genes associated with the ability of melanoma cells to allow metastasis under IL-18 effects, and we verified their expression in metastatic lesions from patients with melanoma. Human melanoma cell lines with and without the IL-18 receptor (IL-18R)/VEGF/VLA-4-expressing phenotype were identified, and their metastatic potential was studied in nude mice. RNA from untreated and IL-18-treated melanoma phenotypes was hybridized to a cDNA microarray, and their signature genes were studied. RNA from primary and metastatic lesions from patients with melanoma was hybridized to a cDNA microarray to identify lesions with the transcript patterns of melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype. IL-18R/VEGF/VLA-4-expressing A375 and 1182 melanoma cells produced a higher metastasis number than 526 and 624.28 melanoma cells, not using this prometastatic pathway. Melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype had distinct transcript patterns. However, the type I transcriptional cluster, including cutaneous and lymph node metastases, but not the type II cluster, not including cutaneous metastases, had signature genes from IL-18-treated melanoma cells with, but not without, the IL-18R/VEGF/VLA-4 phenotype. Metastatic melanoma lesions with and without IL-18-dependent genes were identified, suggesting that melanoma metastasis developed via inflammation-dependent and inflammation-independent mechanisms. Signature genes from melanomas with and without the IL-18R/VEGF/VLA-4 phenotype may serve as diagnostic biomarkers of melanoma predisposition to prometastatic effects of IL-18.

Identification of hepatic microvascular adhesion-related genes of human colon cancer cells using random homozygous gene perturbation.

Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT-29 human colorectal cancer cells to create a HT-29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT-29 adhesion to LSEC were altered. Next, HT-29 RHGP cell library fractions with upregulated or silenced LSEC adhesion-related genes were isolated. Around 160 clones having altered expression in LSEC adhesion-related genes were obtained, and nine relevant protein-coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT-29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT-29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT-29 cells (ITPKC gene) and their silenced status in adherent HT-29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT-29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics.

Pazopanib inhibits the activation of PDGFRß-expressing astrocytes in the brain metastatic microenvironment of breast cancer cells.

Brain metastases occur in more than one-third of metastatic breast cancer patients whose tumors overexpress HER2 or are triple negative. Brain colonization of cancer cells occurs in a unique environment, containing microglia, oligodendrocytes, astrocytes, and neurons. Although a neuroinflammatory response has been documented in brain metastasis, its contribution to cancer progression and therapy remains poorly understood. Using an experimental brain metastasis model, we characterized the brain metastatic microenvironment of brain tropic, HER2-transfected MDA-MB-231 human breast carcinoma cells (231-BR-HER2). A previously unidentified subpopulation of metastasis-associated astrocytes expressing phosphorylated platelet-derived growth factor receptor ß (at tyrosine 751; p751-PDGFRß) was identified around perivascular brain micrometastases. p751-PDGFRß(+) astrocytes were also identified in human brain metastases from eight craniotomy specimens and in primary cultures of astrocyte-enriched glial cells. Previously, we reported that pazopanib, a multispecific tyrosine kinase inhibitor, prevented the outgrowth of 231-BR-HER2 large brain metastases by 73%. Here, we evaluated the effect of pazopanib on the brain neuroinflammatory microenvironment. Pazopanib treatment resulted in 70% (P = 0.023) decrease of the p751-PDGFRß(+) astrocyte population, at the lowest dose of 30 mg/kg, twice daily. Collectively, the data identify a subpopulation of activated astrocytes in the subclinical perivascular stage of brain metastases and show that they are inhibitable by pazopanib, suggesting its potential to prevent the development of brain micrometastases in breast cancer patients.

Design, synthesis, and functional evaluation of leukocyte function associated antigen-1 antagonists in early and late stages of cancer development.

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its α(L)-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled.

Epithelial to mesenchymal transition and cancer stem cell phenotypes leading to liver metastasis are abrogated by the novel TGFß1-targeting peptides P17 and P144.

Colorectal cancer (CRC) frequently metastasizes to the liver, a phenomenon that involves the participation of transforming-growth-factor-ß(1) (TGFß(1)). Blockade of the protumorigenic effects elicited by TGFß(1) in advanced CRC could attenuate liver metastasis. We aimed in the present study to assess the antimetastatic effect of TGFß(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 TGFß(1) (Mc38-luc(TGFß1) cells), injected into the spleen of mice and monitored for tumor development. TGFß(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 TGFß(1) were consistently reduced following injection of peptides. In vitro experiments revealed a direct effect of TGFß(1) in Mc38 cells, which resulted in activation of Smad2, Smad3 and Smad1/5/8, and increased invasion and transendothelial migration, whereas blockade of TGFß(1)-signaling reverted these features. Because TGFß(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 TGFß(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.