Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer

The major limiting factor in the successful application of adjuvant therapy for metastatic disease is the lack of adjuvant specificity that leads to severe side effects. Reasoning that T cells of the immune system are highly specific, we generated tumor-specific T cells by genetic modification of mouse primary T cells with a chimeric receptor reactive with the human breast cancer-associated Ag erbB-2. These T cells killed breast cancer cells and secreted IFN-gamma in an Ag-specific manner in vitro. We investigated their use against metastatic breast cancer in mice in an adjuvant setting, and compared their effectiveness with the commonly applied adjuvants doxorubicin, 5-fluorouracil, and herceptin. Mice were inoculated orthotopically with the human erbB-2-expressing spontaneously metastatic mouse breast cancer 4T1.2 in mammary tissue, and the primary tumor was surgically removed 8 days later., Significant metastatic disease was demonstrated in lung and liver at the time of surgery on day 8 with increased tumor burden at later time points. T cell adjuvant treatment of day 8 metastatic disease resulted in dramatic increases in survival of mice, and this survival was significantly greater than that afforded by either doxorubicin, 5-fluorouracil, or herceptin.

Bisphenol A at the reference level counteracts doxorubicin transcriptional effects on cancer related genes in HT29 cells

Human exposure to Bisphenol A (BPA) results mainly from ingestion of food and beverages. Information regarding BPA effects on colon cancer, one of the major causes of death in developed countries, is still scarce. Likewise, little is known about BPA drug interactions although its potential role in doxorubicin (DOX) chemoresistance has been suggested. This study aims to assess potential interactions between BPA and DOX on HT29 colon cancer cells. HT29 cell response was evaluated after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis of several cancer-associated genes (c-fos, AURKA, p21, bcl-xl and CLU) shows that BPA exposure induces slight up-regulation exclusively of bcl-xl without affecting cell viability. On the other hand, a sub-therapeutic DOX concentration (40nM) results in highly altered c-fos, bcl-xl, and CLU transcript levels, and this is not affected by co-exposure with BPA. Conversely, DOX at a therapeutic concentration (4μM) results in distinct and very severe transcriptional alterations of c-fos, AURKA, p21 and CLU that are counteracted by co-exposure with BPA resulting in transcript levels similar to those of control. Co-exposure with BPA slightly decreases apoptosis in relation to DOX 4μM alone without affecting DOX-induced loss of cell viability. These results suggest that BPA exposure can influence chemotherapy outcomes and therefore emphasize the necessity of a better understanding of BPA interactions with chemotherapeutic agents in the context of risk assessment.

Bcl-xL is overexpressed in hormone-resistant prostate cancer and promotes survival of LNCaP cells via interaction with proapoptotic Bak.

Androgen-sensitive prostate cancer cells turn androgen resistant through complex mechanisms that involve dysregulation of apoptosis. We investigated the role of antiapoptotic Bcl-xL in the progression of prostate cancer as well as the interactions of Bcl-xL with proapoptotic Bax and Bak in androgen-dependent and -independent prostate cancer cells. Immunohistochemical analysis was used to study the expression of Bcl-xL in a series of 139 prostate carcinomas and its association with Gleason grade and time to hormone resistance. Expression of Bcl-xL was more abundant in prostate carcinomas of higher Gleason grades and significantly associated with the onset of hormone-refractory disease. In vivo interactions of Bcl-xL with Bax or Bak in untreated and camptothecin-treated LNCaP and PC3 cells were investigated by means of coimmunoprecipitation. In the absence of any stimuli, Bcl-xL interacts with Bax and Bak in androgen-independent PC3 cells but only with Bak in androgen-dependent LNCaP cells. Interactions of Bcl-xL with Bax and Bak were also evidenced in lysates from high-grade prostate cancer tissues. In LNCaP cells treated with camptothecin, an inhibitor of topoisomerase I, the interaction between Bcl-xL and Bak was absent after 36 h, Bcl-xL decreased gradually and Bak increased coincidentally with the progress of apoptosis. These results support a model in which Bcl-xL would exert an inhibitory effect over Bak via heterodimerization. We propose that these interactions may provide mechanisms for suppressing the activity of proapoptotic Bax and Bak in prostate cancer cells and that Bcl-xL expression contributes to androgen resistance and progression of prostate cancer.

Expression and role of BORIS/CTCFL in human cancer stem cells

Le cancer est défini comme la croissance incontrôlée des cellules dans le corps. Il est responsable de 20 % des décès en Europe. Plusieurs expériences montrent que les tumeurs sont issues et se développent grâce à un petit nombre de cellules, que l'on appelle cellules souches cancéreuses (CSC). Ces CSC sont également responsables de l'apparition de métastases et de la résistance aux médicaments anticancéreux. De ce fait, l'identification des gènes qui contribuent aux propriétés de ces CSC (comme la survie des tumeurs, les métastases et la résistance aux médicaments) est nécessaire pour mieux comprendre la biologie des cancers et d'améliorer la qualité des soins des patients avec un cancer. A ce jour, de nombreux marqueurs ont été proposés ainsi que de nouvelles thérapies ciblées contre les CSC. Toutefois, et malgré les énormes efforts de la recherche dans ce domaine, la quasi-totalité des marqueurs de CSC connus à ce jour sont aussi exprimés dans les cellules saines. Ce projet de recherche visait à trouver un nouveau candidat spécifique des CSC. Le gène BORIS (pour Brother of Regulator of Imprinted Sites), nommé aussi CTCFL (CTCF-like), semble avoir certaines caractéristiques de CSC et pourrait donc devenir une cible prometteuse pour le traitement du cancer. BORIS/CTCFL est une protéine nucléaire qui se lie à l'ADN, qui est exprimée dans les tissus normaux uniquement dans les cellules germinales et qui est réactivée dans un grand nombre de tumeurs. BORIS est impliqué dans la reprogrammation épigénétique au cours du développement et dans la tumorigenèse. En outre, des études récentes ont montré une association entre l'expression de BORIS et un mauvais pronostic chez des patients atteints de différents types de cancers. Nous avons développé une nouvelle technologie basée sur les Molecular Beacon pour cibler l'ARNm de BORIS et cela dans les cellules vivantes. Grâce à ce système expérimental, nous avons montré que seule une toute petite sous-population (0,02 à 5%) de cellules tumorales exprimait fortement BORIS. Les cellules exprimant BORIS ont pu être isolées et elles présentaient les caractéristiques de CSC, telles qu'une forte expression de hTERT et des gènes spécifiques des cellules souches (NANOG, SOX2 et OCT4). En outre, une expression élevée de BORIS a été mise en évidence dans des populations enrichies en CSC ('side population' et sphères). Ces résultats suggèrent que BORIS pourrait devenir un nouveau et important marqueur de CSC. Dans des études fonctionnelles sur des cellules de cancer du côlon et du sein, nous avons montré que le blocage de l'expression de BORIS altère largement la capacité de ces cellules à former des sphères, démontrant ainsi un rôle essentiel de BORIS dans l'auto- renouvellement des tumeurs. Nos expériences montrent aussi que BORIS est un facteur important qui régule l'expression de gènes jouant un rôle clé dans le développement et la progression tumorale, tels le gène hTERT et ceux impliqués dans les cellules souches, les CSC et la transition épithélio-mésenchymateuse (EMT). BORIS pourrait affecter la régulation de la transcription de ces gènes par des modifications épigénétiques et de manière différente en fonction du type cellulaire. En résumé, nos résultats fournissent la preuve que BORIS peut être classé comme un gène marqueur de cellules souches cancéreuse et révèlent un nouveau mécanisme dans lequel BORIS jouerait un rôle important dans la carcinogénèse. Cette étude ouvre de nouvelles voies pour mieux comprendre la biologie de la progression tumorale et offre la possibilité de développement de nouvelles thérapies anti-tumorales et anti-CSC avec BORIS comme molécule cible. - Cancer is defined as the uncontrolled growth of cells in the body. It causes 20% of deaths in the European region. Current evidences suggest that tumors originate and are maintained thanks to a small subset of cells, named cancer stems cells (CSCs). These CSCs are also responsible for the appearance of metastasis and therapeutic resistance. Consequently, the identification of genes that contribute to the CSC properties (tumor survival, metastasis and therapeutic resistance) is necessary to better understand the biology of malignant diseases and to improve care management. To date, numerous markers have been proposed to use as new CSC- targeted therapies. Despite the enormous efforts in research, almost all of the known CSCs markers are also expressed in normal cells. This project aimed to find a new CSC-specific candidate. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA binding protein involves in epigenetic reprogramming in normal development and in tumorigenesis. Recent studies have shown an association of BORIS expression with a poor prognosis in different types of cancer patients. Therefore, BORIS seems to have the same characteristics of CSCs markers and it could be a promising target for cancer therapy. BORIS is normally expressed only in germinal cells and it is re-expressed in a wide variety of tumors. We developed a new molecular beacon-based technology to target BORIS mRNA expressing cells. Using this system, we showed that the BORIS expressing cells are only a small subpopulation (0.02-5%) of tumor cells. The isolated BORIS expressing cells exhibited the characteristics of CSCs, with high expression of hTERT and stem cell genes (NANOG, SOX2 and OCT4). Furthermore, high BORIS expression was observed in the CSC-enriched populations (side population and spheres). These results suggest that BORIS might be a novel and powerful CSCs marker. In functional studies, we observed that BORIS knockdown significantly impairs the capacity to form spheres in colon and breast cancer cells, thus demonstrating a critical role of BORIS in the self-renewal of tumors. The results showed in the functional analysis indicate that BORIS is an important factor that regulates the expression of key-target genes for tumor development and progression, such as hTERT, stem cells, CSCs markers and EMT (epithelial mesenchymal transition)-related marker genes. BORIS could affect the transcriptional regulation of these genes by epigenetic modification and in a cell type dependent manner. In summary, our results support the evidence that BORIS can be classified as a cancer stem cell marker gene and reveal a novel mechanism in which BORIS would play a critical role in tumorigenesis. This study opens new prospective to understand the biology of tumor development and provides opportunities for potential anti-tumor drugs.

Interactions between cancer stem cells and their niche govern metastatic colonization.

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycinruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycinruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycinruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycinruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitroand the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Parabens can enable hallmarks and characteristics of cancer in human breast epithelial cells: a review of the literature with reference to new exposure data and regulatory status

A framework for understanding the complexity of cancer development was established by Hanahan and Weinberg in their definition of the hallmarks of cancer. In this review, we consider the evidence that parabens can enable development in human breast epithelial cells of 4/6 of the basic hallmarks, 1/2 of the emerging hallmarks and 1/2 of the enabling characteristics. Hallmark 1: parabens have been measured as present in 99% of human breast tissue samples, possess oestrogenic activity and can stimulate sustained proliferation of human breast cancer cells at concentrations measurable in the breast. Hallmark 2: parabens can inhibit the suppression of breast cancer cell growth by hydroxytamoxifen, and through binding to the oestrogen-related receptor gamma (ERR) may prevent its deactivation by growth inhibitors. Hallmark 3: in the 10nM to 1M range, parabens give a dose-dependent evasion of apoptosis in high-risk donor breast epithelial cells. Hallmark 4: long-term exposure (>20weeks) to parabens leads to increased migratory and invasive activity in human breast cancer cells, properties which are linked to the metastatic process. Emerging hallmark: methylparaben has been shown in human breast epithelial cells to increase mTOR, a key regulator of energy metabolism. Enabling characteristic: parabens can cause DNA damage at high concentrations in the short term but more work is needed to investigate long-term low-doses of mixtures. The ability of parabens to enable multiple cancer hallmarks in human breast epithelial cells provides grounds for regulatory review of the implications of the presence of parabens in human breast tissue.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Towards antibody-mediated targeting of dendritic cells for cancer immunotherapy with multivalent polymer-antigen conjugates

Der Fokus dieser Arbeit lag auf der definierten Synthese multifunktioneller Polymer-Konjugate zur Anwendung in der Krebs-Immunotherapie. Durch gezielte Variation der Kon-jugationsbedingungen wurde Zusammensetzung, Größe und Aggregationsverhalten in Zell-medium sowie in humanem Serum untersucht. Nach definierter physikalisch-chemischer Charakterisierung wurde dann die induzierte Antigen-Präsentation zur Aktivierung der T-Zellproliferation analysiert.rnDafür wurden zwei verschiedene polymere Carrier-Systeme gewählt, lineares Poly-L-lysin und eine Polylysinbürste (PLL-Bürste). Es wird vermutet, dass die PLL-Bürste aufgrund der anisotropen Form eine bessere Verteilung im Körper und eine verlängerte Zirkulationsdauer zeigen wird. Die zu konjugierenden biologisch aktiven Komponenten waren der antiDEC205-Antikörper (aDEC205) für die gezielte Adressierung CD8-positiver dendritischer Zellen (DC), ein Ovalbumin (OVA)-spezifisches Antigen mit der Kernsequenz SIINFEKL für die Spezifität der Immunantwort gegen Krebszellen, die dieses Antigen tragen, und ein immunaktivieren-der TLR9-Ligand, CpG1826. Die Effizienz dieses Konjugates dendritische Zellen zu aktivieren, welche wiederum eine Immunantwort gegen OVA-exprimierende Krebszellen induzieren, wurde durch die Konjugation aller Komponenten am identischen Trägermolekül deutlich höher erwartet.rnLineares Poly-L-lysin diente als Modellsystem um die Konjugationschemie zu etablieren und dann auf die zylindrische Polylysinbürste zu übertragen. Anhand dieser polymeren Träger wurde das Verhalten der verschiedenen Topologien des Knäuels und der Bürste im Hinblick auf den Einfluss struktureller Unterschiede sowohl auf Konjugationsreaktionen als auch auf das in situ und in vitro Verhalten untersucht.rnFluoreszenzmarkiertes Antigen und der CpG Aktivator konnten jeweils aufgrund einer Thiol-Modifizierung an die Thiol-reaktive Maleimidgruppe des heterobifunktionellen Linkers Sulfo-SMCC an PLL-AlexaFluor48 konjugiert werden. Anschließend wurde aDEC205-AlexaFluor647 an PLL gekoppelt, entweder durch Schiff Base-Reaktion des oxidierten Antikörpers mit PLL und anschließender Reduzierung oder durch Click-Reaktion des PEG-Azids modifizierten An-tikörpers mit Dicyclobenzylcyclooctin (DIBO)-funktionalisiertem PLL. Die Konjugation der biologisch aktiven Komponenten wurde mit Durchflusszytometrie (FACS) und konfokaler Laser Scanning Mikroskopie (CLSM) untersucht und die Zusammensetzung des Konjugatesrnmittels UV/Vis-Spektroskopie bestimmt. Die PLL-Bürste alleine zeigte eine hohe Zytotoxizität bei HeLa und JAWS II Zelllinien, wohingegen lineares PLL und PLL-Konjugate sowie die PLL Bürsten-Konjugate keine ausgeprägte Zytotoxizität aufwiesen. Die Polymer-Konjugate wie-sen keine Aggregation in Zellmedium oder humanem Serum auf, was mittels winkelabhängi-ger dynamischer Lichtstreuung bestimmt wurde. CLSM Aufnahmen zeigten Kolokalisation der an die einzelnen Komponenten gebundenen Fluoreszenzfarbstoffe in dendritischen Zel-len, was die erfolgreiche Konjugation und Internalisierung der Konjugate in die Zellen bele-gen konnte. FACS Messungen ergaben eine geringfügig erhöhte Aufnahme des adressierten PLL-Antigen-Antikörper-Konjugates verglichen mit dem PLL-Antigen-Konjugat. Experimente mit dem „Specific Hybridization Internalization Sensor“ (SHIP) zeigten jedoch nur Aufnahme der PLL-Konjugate in CD8+ unreife DC, nicht in reife DC, die nicht mehr unspezifisch, sondern nur noch über Rezeptoren internalisieren. Dies bewies die unspezifische Aufnahme des Kon-jugates, da Antikörper-Konjugation keine Rezeptor-vermittelte Endozytose in reife DC indu-zieren konnte. T-Zell-Proliferationsassays ergaben eine Aktivierung von CD8+ T-Zellen indu-ziert durch Antigen-tragende Konjugate, wohingegen Konjugate ohne Antigen als Negativ-kontrollen dienten und keine T-Zell-Proliferation erzielten. Es konnte jedoch kein Unter-schied zwischen adressierten und nicht adressierten Konjugaten aufgrund der unspezifischen Aufnahme durch das Polymer beobachtet werden. Lösliches SIINFEKL alleine bewirkte schon bei geringeren Konzentrationen eine T-Zell-Proliferation.rnEs war somit möglich, drei biologischen Komponenten an einen polymeren Träger zu konju-gieren und diese Konjugate im Hinblick auf Zusammensetzung, Größe, Internalisierung in dendritische Zellen und Aktivierung der T-Zell-Proliferation zu untersuchen. Außerdem wur-de die Konjugationschemie erfolgreich von dem Modellsystem des linearen PLL auf die PLL-Bürste übertragen. Die Polymer-Konjugate werde unspezifisch in DC aufgenommen und in-duzieren T-Zellproliferation, die mit Antigen-Präsentationsassays nachgewiesen wird. Es konnte jedoch durch Konjugation des Antikörpers keine Rezeptor-vermittelte Aufnahme in CD8+ DC erzielt werden.rnDiese Studien stellen einen erfolgsversprechenden ersten Schritt zur Entwicklung neuer Na-nomaterialien für die Anwendung in Krebs-Immuntherapie dar.

High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer

Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing, CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, pre-existing SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SC-like BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (AR(low)) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The AR(low) seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.