Normal Tissue Microenvironment Research Articles

PPARG and the PTEN-PI3K/AKT Signaling Axis May Cofunction in Promoting Chemosensitivity in Hypopharyngeal Squamous Cell Carcinoma.

It has been demonstrated that PPARG may interact with the PTEN-PI3K/AKT pathway, contributing to its involvement in the chemotherapy treatment of hypopharyngeal squamous cell carcinoma (HSCC). However, the underlying mechanism remains largely unknown. In this study, gene expression profiles of 17 HSCC patients, comprising 8 chemotherapy-sensitive patients (CSP) and 9 chemotherapy-nonsensitive patients (CNSP), were collected and analyzed to investigate expression patterns, correlations, influencing factors of the PPARG-PTEN-PI3K/AKT pathway, and its role in regulating chemosensitivity. The results revealed significantly increased expression (p < 0.04) of AKT1, AKT2, AKT3, PIK3CA, PPARG, and PTEN in the CSP group compared to the CNSP group. Specifically, AKT2 exhibited significant overexpression in tumor tissue (p = 0.01), while AKT2, AKT3, PPARG, and PTEN displayed significant increases in normal tissue (p ≤ 0.04). Positive correlations (R ∈ [0.43, 0.71], p < 0.014) were observed between PIK3CA, AKT1, AKT2, AKT3, and PTEN, with AKT2, AKT3, and PTEN also showing significant correlations with PPARG (R ∈ [0.35, 0.47], p < 0.04). Age, gender, and disease stage had no influence on PPARG, PIK3CA, and PTEN expression, but they may affect AKT expressions. Pathway analysis revealed that PPARG may interact with the PTEN-PI3K/AKT signaling pathway, playing a crucial role in regulating chemosensitivity in the normal tissue microenvironment. Our results suggest that AKT1 and PIK3CA may be associated with chemosensitivity in HSCC tumor cells, while PPARG and PTEN might exhibit a correlation with a specific segment of the PI3K/AKT pathway, potentially influencing chemosensitivity in the normal tissue microenvironment of HSCC patients.

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features.

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic-OA, or lipoic-LA), cross-linking of SH groups in the micelle's core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate-rhodamine (MUTMAC-R6G) and fluorescein isothiocyanate-rhodamine (FITC-R6G). While FITC-R6G has been described before as an FRET-producing pair, for MUTMAC-R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC-R6G and FITC-R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75-80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.

Non-pituitary GH regulation of the tissue microenvironment.

Non-pituitary growth hormone (npGH) expression is well established in extrapituitary tissues, but an understanding of the physiological role of npGH remains rather limited. Pro-tumorigenic npGH impacting the tumor microenvironment has been reviewed. We focus here on autocrine/paracrine npGH effects in non-tumorous tissues and discuss its mechanisms of action in the normal tissue microenvironment. We address the tissue-specific effects of npGH in regulating stem, endothelial, immune, and epithelial cells and highlight the related role of npGH-associated changes in tissue aging.

Definition of a novel breast tumor-specific classifier based on secretome analysis

BackgroundDuring cancer development, the normal tissue microenvironment is shaped by tumorigenic events. Inflammatory mediators and immune cells play a key role during this process. However, which molecular features most specifically characterize the malignant tissue remains poorly explored.MethodsWithin our institutional tumor microenvironment global analysis (T-MEGA) program, we set a prospective cohort of 422 untreated breast cancer patients. We established a dedicated pipeline to generate supernatants from tumor and juxta-tumor tissue explants and quantify 55 soluble molecules using Luminex or MSD. Those analytes belonged to five molecular families: chemokines, cytokines, growth factors, metalloproteinases, and adipokines.ResultsWhen looking at tissue specificity, our dataset revealed some breast tumor-specific characteristics, as IL-16, as well as some juxta-tumor-specific secreted molecules, as IL-33. Unsupervised clustering analysis identified groups of molecules that were specific to the breast tumor tissue and displayed a similar secretion behavior. We identified a tumor-specific cluster composed of nine molecules that were secreted fourteen times more in the tumor supernatants than the corresponding juxta-tumor supernatants. This cluster contained, among others, CCL17, CCL22, and CXCL9 and TGF-β1, 2, and 3. The systematic comparison of tumor and juxta-tumor secretome data allowed us to mathematically formalize a novel breast cancer signature composed of 14 molecules that segregated tumors from juxta-tumors, with a sensitivity of 96.8% and a specificity of 96%.ConclusionsOur study provides the first breast tumor-specific classifier computed on breast tissue-derived secretome data. Moreover, our T-MEGA cohort dataset is a freely accessible resource to the biomedical community to help advancing scientific knowledge on breast cancer.

Enhancing Proteasome-Inhibitory Activity and Specificity of Bortezomib By Endocytosis-Mediated Uptake of CD38 Targeted Nanoparticles in Multiple Myeloma

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. Due to its high expression on MM cells, CD38 is used as a marker for identification of MM cells, and there are several indications supporting the notion that CD38 plays significant roles in the progression of MM.We have fine-tuned the production of anti-CD38 chitosan NPs with a small size (50nm) to allow better penetration to tumors, and we showed that these NPs were stable at different storage conditions and periods. The chitosan NPs showed preferential drug release in MM tumor microenvironment compared to normal tissue microenvironment, and this release was pH dependent.The uptake of the anti-CD38 chitosan NPs to MM cells (cell lines and primary) was significantly higher than their uptake in normal cells (4-fold of BM MNCs, and 10-fold of PB MNCs) demonstrating that the anti-CD38 chitosan NPs are selective and specific to MM. BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest and apoptosis leading to cell death through inactivation of proliferative cell signaling pathways (MAPK, PI3K) and activation of pro-apoptotic pathways (PARP and Caspase-3). We also found that BTZ-loaded anti-CD38 NPs proved to be more efficacious than free BTZ and overcame CAM-DR and hypoxia-mediated drug resistance. The use of a 3DTEBM model, which recreate MM pathophysiology, corroborated the increase cytotoxicity of anti-CD38 chitosan NPs on MM cells relative to free drug and allowed to corroborated co-localization of the NPs in the MM cells.We found that BTZ-loaded NPs were significantly more potent in the proteasome activity inhibition than free BTZ and correlated with the boron content in MM cells treated with BTZ-loaded anti-CD38 NPs, which was 3-fold higher than in MM cells treated with BTZ as free drug. To confirm that the process involved in the internalization of the anti-CD38 chitosan NPs was endocytosis, we have shown that the NPs distribution in the cells co-localized with the distribution of endosomes and found that the anti-CD38 NPs were uptaken through clathrin-mediated and caveolae-mediated early endocytosis which progressed to late endocytosis and further delivery of the NPs content into the cells. Finally, we linked the internalization through endocytic pathways of anti-CD38 NPs to the enhanced proteasome activity inhibition of these NPs in MM cells. Treatment of MM cells with clathrin and caveolae-mediated early endocytosis, or late endocytosis inhibitors (but not macropinocytosis inhibitors) significantly reduced the proteasome inhibition of BTZ-loaded anti-CD38 chitosan NPs. When BTZ is administered as a free drug, it penetrated through passive diffusion into MM cells as well as in normal cells, and inhibition of the different endocytosis routes did not induce any change in its ability to inhibit the proteasome.In vivo studies showed that treatment with BTZ-loaded anti-CD38 targeted NPs (1mg/kg once a week) showed a markedly improved tumor growth inhibition, improved overall survival, and reduction of side effects compared to non-targeted NPs and free drug.In conclusion, anti-CD38 chitosan NPs showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through endocytosis, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. We are the first to report the enhancement of the efficacy and specificity of proteasome inhibitors due to endocytosis-driven delivery of NPs. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-Founder. Vij:Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Glycomimetics: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cellatrix LLC: Other: Founder and owner; Verastem: Research Funding; Cell Works: Research Funding; Abbvie: Research Funding; Tioma: Research Funding; Cleave Bioscience: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Discovery of Cell Aggregate-Inducing Peptides

Most cells within the human body interact with neighboring cells and extracellular matrix (ECM) components to establish a unique 3D organization. These cell–cell and cell–ECM interactions form a complex communication network of biochemical and mechanical signals critical for normal cell physiology. The behavior of cells in a 3D environment is fundamentally different from that of cells in monolayer culture. Aggregation can affect cell–cell interactions, being more representative of the normal tissue microenvironment. Therefore, 3D cell culture technologies have been developed. The general method for cell aggregate is a physical method; it is difficult to control the size and number of cell aggregates. In any case, no chemical method has been discovered yet, so a new method to solve these problems is needed. In this paper, we describe the induction of a cell aggregate of the newly discovered (Lys-Pro)12(KP24) peptide. Since it was revealed that KP24 had cell aggregate-inducing activity, its derivatives were molecularly designed to clarify the importance of the KP24 sequence. We report that cell aggregations were induced by KP24 to form aggregates of fibroblast cells. We evaluated KP24 derivative periodic peptides such as (Lys-Pro-Pro)8(KPP24) and (Lys-Lys-Pro)8(KKP24). The relationship between the structure of the peptide chain and the activity induced by the cell aggregations was investigated from the viewpoint of basic research and the biomedical engineering field.

Abstract 5549: Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments

Abstract LTX-315 is an oncolytic peptide that induces immunogenic cancer cell death and is currently evaluated in phase 1 clinical trials for its ability to trigger recruitment of tumor-specific T cells to tumors. Previous experimental studies identified LTX-315's ability to control cancer in mice grafted with various tumor cell lines. Here we extend our analyses to conditional genetic mouse models of both melanoma (driven by Braf and Pten alterations) and soft tissue sarcoma (driven by Kras and P53 alterations). In these genetic models, cancer cells are derived from somatic cells that are transformed in their normal tissue microenvironment and progress to high-grade tumors that are poorly infiltrated by T cells and typically resist prescribed chemo- and immunotherapeutic treatments. We report that LTX-315 not only delays tumor progression substantially in both models, but also profoundly alters the tumor microenvironment, most notably characterized with CD8+ T cell, NK cell and dendritic cell infiltration. Transition from a ‘cold' to a ‘hot' tumor microenvironment was also observed in patients with melanoma, sarcoma and breast cancer. Furthermore, CD8+ T cell depletion in mice abrogated long-term antitumor efficacy of LTX-315, indicating that these cells are required for drug-induced tumor control. Importantly, the ability to convert non-T-cell-infiltrated tumors into ones that display antitumor T cell immunity opens the possibility to prime and make unresponsive tumors sensitive for systemic immune treatments. Citation Format: Mikael J. Pittet, Hsin-Wei Liao, Baldur Sveinbjørnsson, Øystein Rekdal. Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5549.

Stress hormones concentrations in the normal microenvironment predict risk for chemically induced cancer in rats

Evidence show that stress hormones can influence cancer progression, but its role in carcinogenesis is poorly understood. In this study, we used a new method based on oral carcinogenesis model in rats to test the hypothesis that physiological levels of stress hormones in the normal tissue microenvironment would have significant predictive value for chemically induced cancer occurrence. Male Wistar rats were submitted to a tongue biopsy for measuring not-stress induced levels of norepinephrine, corticosterone, adrenocorticotropic hormone (ACTH) and brain-derived neurotrophic factor (BDNF) in the tissue before carcinogenic induction. Rats were treated with the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogen for twenty weeks and then euthanized for microscopic evaluation of the tongue lesions. Increased pre-carcinogen norepinephrine concentrations and reduced basal corticosterone levels in the normal tissue microenvironment were predictive for oral squamous cell carcinoma (OSCC) occurrence. Likewise, increased pre-carcinogen norepinephrine levels in the normal microenvironment were associated a lower expression of pCDKN2a-p16 in OSCCs. Post-carcinogen levels of corticosterone and BDNF in oral leukoplakia tissues (precursor lesion of OSCC) and post-carcinogen corticosterone concentrations in OSCCs were higher than basal levels in the normal mucosa. Increased norepinephrine concentrations in OSCCs were associated to a greater tumor volume and thickness. Furthermore, higher levels of norepinephrine, ACTH and BDNF in OSCCs were associated to a lesser intensity of the lymphoplasmocytic infiltrate. This study shows that pre-carcinogen stress hormones levels in the normal microenvironment may be predictive for chemically induced cancer in rats. Moreover, chemical carcinogenesis can promote stressor-like effects with hormonal changes in the tissue microenvironment, which may be associated to tumor progression.

Human Organ Culture: Updating the Approach to Bridge the Gap from In Vitro to In Vivo in Inflammation, Cancer, and Stem Cell Biology.

Human studies, critical for developing new diagnostics and therapeutics, are limited by ethical and logistical issues, and preclinical animal studies are often poor predictors of human responses. Standard human cell cultures can address some of these concerns but the absence of the normal tissue microenvironment can alter cellular responses. Three-dimensional cultures that position cells on synthetic matrices, or organoid or organ-on-a-chip cultures, in which different cell spontaneously organize contacts with other cells and natural matrix only partly overcome this limitation. Here, we review how human organ cultures (HOCs) can more faithfully preserve in vivo tissue architecture and can better represent disease-associated changes. We will specifically describe how HOCs can be combined with both traditional and more modern morphological techniques to reveal how anatomic location can alter cellular responses at a molecular level and permit comparisons among different cells and different cell types within the same tissue. Examples are provided involving use of HOCs to study inflammation, cancer, and stem cell biology.

Anti-CD38 Targeted Nanoparticles for Drug Delivery in Multiple Myeloma

INTRODUCTIONThe main limiting factor for the treatment with chemotherapies in multiple myeloma (MM) is the lack of specificity of the drugs. We propose the use of chitosan nanoparticles (cNPS) as specific and targeted drug delivery in MM using CD38 as targeting moiety. CD38 has been used as a therapeutic target in MM; anti-CD38 monoclonal antibodies are showing promising results of selective and efficient treatment of MM in preclinical studies and in early clinical trials. Moreover, we have recently shown that while the expression of several plasma cell markers such as CD138, CD56, and CD20 changed in different stages of the disease, CD38 is constantly expressed on all forms of MM cells. We hypothesize that the anti-CD38 targeted nanoparticles (CD38-NPs) will specifically deliver therapeutic agents to MM cells, thus improve therapeutic efficacy and reduce side effects.METHODS & RESULTS: cNPs were developed by ionic crosslinking and characterized by dynamic light scattering. A successful method to detect Bortezomib (Btz) by HPLC was developed and the encapsulation efficiency of Btz in cNPs was high (85%) after direct dispersion previous crosslinking. Stability (>30mV) and size (50nm) were not affected by drug loading and were constant for over a 2 month period at 4°C. The release kinetics of Btz from cNPs in different environments was investigated and found that our deliver system preferentially release drugs in tumor microenvironments; the tumor-like acidic pH (6.5-7.1) in MM conditioned media induced faster drug release (2.7-4.5 fold) than neutral pH representing blood and normal tissues. Then, the specificity and the kinetics of the binding of CD38-NPs to MM cells were investigated in vitro. The binding of CD38-NPs was significantly higher in MM cell lines and primary MM cells compared to normal mononuclear cells (MNCs) from peripheral blood and bone marrow. We further confirmed that CD38-NPs bind through CD38 within 2-3 hours, and the nanoparticles did not dissociate at least up to 24h after binding. Moreover, we tested the biodistribution and specificity of fluorescently labeled CD38-NPs in MM tumors models in vivo compared to the free dye and non-targeted cNPs; we found that free dye and non-targeted particles had random biodistribution in different normal tissues; while the CD38-NPs specifically bind the MM cells. Finally, the therapeutic efficacy of Btz-loaded CD38-NPs was assessed in vitro in the treatment of MM compared to free drug. Btz-loaded CD38-NPs induced significantly more killing and downregulation of pAKT in MM cell lines compared to free drug, while it did not affect MNCs. While 2.5nM free Btz induced almost no effect on MM cells, equivalent 2.5nM Btz-loaded CD38-NPs induced a profound killing of more than 50% of the MM cells. Btz-loaded CD38-NPs also led to the induction of a sub-G1 cell cycle arrest, which indicated accumulating apoptotic cells, decreased the percentage of cells in G2/M compared to free drug, and the effect was confirmed by specific signaling with reduced transition cell cycle proteins (pRb). We also found that Btz-loaded CD38-NPs significantly increased apoptosis and cell death (using Annexin/ PI assay) compared to free drug, which was confirmed by increased cleavage of caspase-3 and PARP. We also corroborated that empty (not drug loaded) CD38-NPs had no effect on survival, cell cycle, and apoptosis. Therefore, Btz-loaded CD38-NPs affected proliferation, cell cycle and apoptosis more profoundly compared to free Btz. Moreover, in vivo experiments to evaluate the efficacy and the toxicity of the Btz-loaded CD38-NPs compared with free Btz are ongoing. CONCLUSIONSCD38-NPs were prepared, characterized, and showed to be stable over at least 2 months after preparation. The particles had preferential Btz release in tumor-microenvironment compared to blood and normal tissue microenvironment, due to differences in acidity of the medium. CD38-NPs were shown to specifically bind to MM cells through surface CD38 receptors in vitro and in vivo. Moreover, Btz-loaded CD38-NPs reduced MM proliferation, inhibited cell cycle, and induced apoptosis more robustly than equivalent concentrations of free drug, but not in MNCs. These results confirmed that CD38-NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy. Therefore, CD38-NPs will reduce side effects and could be used as a new drug delivery approach in myeloma. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-founder. Vij:Shire: Consultancy; Takeda: Consultancy, Research Funding; Celgene: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Karyopharma: Consultancy; Bristol-Myers Squibb: Consultancy; Jazz: Consultancy; Amgen: Consultancy, Research Funding. Azab:Selexys: Research Funding; Vasculox: Research Funding; Cellatrix LLC: Other: Founder and owner; Karyopharm: Research Funding; Glycomimetics: Research Funding; Cleave Bioscience: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Cell Works: Research Funding.

Effect of genetic and environmental factors on the tumor microenvironment of tongue and buccal cancers.

e17500Background: A critical phase in the development of cancer is the conversion of growth-suppressive normal tissue microenvironment into a growth-promoting tumor microenvironment. This transform...

Regulation of prostate cancer progression by the tumor microenvironment

Prostate cancer remains the most frequently diagnosed cancer in men in North America, and despite recent advances in treatment patients with metastatic disease continue to have poor five-year survival rates. Recent studies in prostate cancer have revealed the critical role of the tumor microenvironment in the initiation and progression to advanced disease. Experimental data have uncovered a reciprocal relationship between the cells in the microenvironment and malignant tumor cells in which early changes in normal tissue microenvironment can promote tumorigenesis and in turn tumor cells can promote further pro-tumor changes in the microenvironment. In the tumor microenvironment, the presence of persistent immune infiltrates contributes to the recruitment and reprogramming of other non-immune stromal cells including cancer-associated fibroblasts and a unique recently identified population of metastasis-initiating cells (MICs). These MICs, which can also be found as part of the circulating tumor cell (CTC) population in PC patients, promote cancer cell transformation, enhance metastatic potential and confer therapeutic resistance. MICs act can on other cells within the tumor microenvironment in part by secreting exosomes that reprogram adjacent stromal cells to create a more favorable tumor microenvironment to support continued cancer growth and progression. We review here the current data on the intricate relationship between inflammation, reactive stroma, tumor cells and disease progression in prostate cancer.

Abstract 2370: Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition

Abstract During tumor formation, normal tissue microenvironment is transformed in an “altered” niche, composed of non-malignant supporting cells, which influence the homeostasis of cancer cells via paracrine regulators. Tumor-associated stromal cells (TASC) are the prominent stromal elements in most types of human carcinomas including colorectal cancer (CRC). The differentiation of TASC from other cell types, such as resident stromal cells or bone marrow-derived mesenchymal stem cells (BM-MSC) is mainly mediated by factors produced during the crosstalk with malignant cells. TASC produce various extracellular matrix proteins, chemokines, and other tumor-promoting factors which affect vascularization, proliferation, tumor cell invasiveness and survival. Emerging data suggests that they also play a critical role in determining response to therapy. For instance TASC-derived factors may contribute to the development of a protective milieu by influencing cell-cell/cell-matrix interactions, cancer cell survival, and suppression of anti-tumor immune responses. Moreover, physical contact between TASC and malignant cells supports tumor cell survival via activation of anti-apoptotic pathways or inducing epithelial-to-mesenchymal transition (EMT). We previously showed that BM-MSC are capable of triggering EMT in CRC cells, leading to increased tumor cell aggressiveness in vitro and in vivo. However, the role played by the stromal component and the processes induced on CRC cells remain to be fully elucidated. For this purpose we have addressed phenotypic and functional characterization of TASC in vitro and we have analyzed TASC-mediated effects on CRC development and progression in vivo. TASC were characterized for phenotype and differentiation capacity. Human CRC cells were cultured in the presence or absence of TASC for five days. After co-culture tumor cells were sorted by flow cytometry and evaluated for the expression of EMT-related genes by Real Time PCR and for in vitro invasiveness by chemoinvasion assay. Furthermore, their tumorigenicity was assessed upon injection in NOD/SCID mice and developing tumors were analyzed. Our results indicate that TASC resemble BM-MSC in morphology and phenotype. They comprise a multipotent subpopulation that is able to differentiate into adipogenic and osteogenic lineage. After co-culture with CRC cells they express membrane-bound TGF-β, through which they are capable to initiate EMT in tumor cells. Moreover CRC cells acquire a more invasive phenotype after co-culture with TASC. Upon subcutaneous injection in NOD/SCID mice, tumor cells co-cultured with TASC show a significantly faster growth kinetic and develop significantly larger tumor masses as compared to tumor cells alone. Thus our data show that the stromal component of CRC increases the tumor cells malignancy triggering EMT induction. Citation Format: Valentina Mele, Manuele Giuseppe Muraro, Raoul Droeser, Daniel Oertli, Markus Zuber, Raffaele Rosso, Ivan Martin, Michael Heberer, Giulio C. Spagnoli, Giandomenica Iezzi. Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2370. doi:10.1158/1538-7445.AM2015-2370

MicroRNA-21 expression and susceptibility to HPV-induced carcinogenesis - role of microenvironment in K14-HPV16 mice model.

Human papillomaviruses (HPVs) are responsible for several types of cancer. K14-HPV16 transgenic mice express the HPV16 early genes, developing multi-step carcinogenesis associated with marked inflammation, as observed in human patients. MicroRNAs (MiRNA) constitute a class of non-coding RNAs that regulate gene expression. In particular, miR-21 has been associated with carcinogenesis. However, little is known about this microRNA in the normal tissue microenvironment and its possible relationship with cancer predisposition. We hypothesized that miR-21 expression influences each tissue's susceptibility to HPV-induced carcinogenesis. In order to test this hypothesis, we evaluated miR-21 expression by RT-qPCR in ear and chest skin samples from 24–28 weeks old, female K14-HPV16 transgenic and wild-type mice. In wild-type mice (HPV −/−) miR-21 expression was lower in ear skin compared with that of chest skin (p = 0.036). Under the influence of HPV16 oncogenes, transgenic animals (HPV16 +/−), developed in situ carcinoma in all ear samples and epidermal hyperplasia in chest samples. These results are consistent with the hypothesis that microRNA expression in the microenvironment of normal tissues may influence HPV-associated carcinogenesis. Furthermore, among transgenic animals, miR-21 expression was lower in in situ carcinoma samples compared with hyperplasia (p = 0043). This suggests that, despite the well-known role of miR-21 as an oncogene, its anti-inflammatory and immunomodulatory properties may modulate HPV-induced carcinogenesis in a tissue-dependent manner. Further studies are warranted in order to explore the role of microRNAs in tissue susceptibility to carcinogenesis.

Biocompatible Porous Scaffolds from Derivatized PVA Composites

Introduction: Many polymer supports for cell seeding formulations based on chitosan are usually prepared by chemical cross-linking with glutaraldehyde, urea formaldehyde, epichlorohydrine, even if the chemical cross-linking agents possibly induce toxicity and other undesirable effects. Experimental: Derivatized poly (vinyl alcohol) was blended and foamed with chitosan to produce porous scaffolds with improved elastic and mechanical properties and also acceptable water solubility. Derivatized PVA (in example phosphorylated PVA) (P-PVA) also exhibited improved hydrophilicity, anionic properties and molecular electrostatic repulsion, and retained the bulk morphological features, compatible property, and solution stability. Results: Porous scaffolds formed by derivatized PVA crosslinked with chitosan proved a good cytocompatibility for human fibroblasts. Viability and apoptosis assays were performed indicating that this porous scaffold allow cell multiplication and scarce apoptotic induction. Conclusion: Tested porous scaffolds may represent an interesting support for three dimensional cell cultures destined to simulate tumor or normal tissue microenvironment.

Victory and defeat in the induction of a therapeutic response through vaccine therapy for human and canine brain tumors: a review of the state of the art.

Anti-tumor immunotherapy using tumor lysate-based vaccines has made great advances over recent decades. Cancer vaccines aim to elicit adaptive immune responses through various pathways by providing tumor and tumor-associated antigens with an immune stimulant or adjuvant. These anti-tumor vaccines are therefore developed as personalized treatments. Utilizing tumors as a source of vaccine antigens in immunotherapy has demonstrated promising results with minimal toxicity. However, to date, researchers have failed to overcome the overpowering immune suppressive effects within the tumor microenvironment. Immune suppression occurs naturally via multiple mechanisms. These mechanisms serve an important homeostatic role restoring a normal tissue microenvironment following an inflammatory response. Due to these suppressive mechanisms and the inherent heterogeneity of tumors, it is imperative to then elicit and maintain a specific tumoricidal response if vaccine therapy or some other combination of reagents is chosen. In this review, we focus on the historical use of tumors as a source of antigens to elicit a tumoricidal response and the limitations encountered that prevent greater success in immunotherapy. We describe the advantages and disadvantages of various vaccines and their ineffectiveness due to tumor-induced immune suppression.

Abstract PL04-02: Insights on cancer risk from normal breast tissue gene expression

Abstract Breast cancers evolve through interactions with the surrounding non-malignant tissue, with the non-malignant tissue providing a selective pressure during carcinogenesis. By conducting observational studies of non-malignant tissue, it is possible to identify pathways and phenotypes that contribute to cancer etiology and progression. Our gene expression profiling studies have demonstrated that cancer adjacent normal breast tissue is highly variable, with at least two distinct molecular subtypes. These subtypes are associated with histologic differences and distinct risk factor profiles, notably mammographic density. Given that mammographic density is the strongest, yet most poorly understood breast cancer risk factor, molecular studies to better understand this phenotype are important. We have also conducted studies of risk factor-associated gene expression in reduction mammoplasty tissue. Supervised analysis of gene expression in normal tissue is identifying novel biological phenotypes associated with established risk factors. For example, we have shown that aging is associated with specific gene expression changes in normal breast. Interestingly, the age-related pathways in non-diseased tissue are also dysregulated in the breast cancers that are more common in young women, providing a biological link between the aging process and tumor aggressiveness. Likewise, obesity causes alterations to the stromal milieu in normal breast tissue, with implications for the progression of basal-like breast cancers. Our results demonstrate that to study the evolution and outcome of breast cancers, it is important to consider the normal tissue microenvironment from which these cancers evolve. Citation Format: Melissa A. Troester. Insights on cancer risk from normal breast tissue gene expression. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PL04-02.

Anti-tumoral Properties of Endogenous Angiogenesis Inhibitors: A Case for Continued TIMP-2 Preclinical Development

Dr. Judah Folkman first introduced the concept of anti-angiogenic therapy almost four decades ago. This novel idea has subsequently been supported by extensive research in the multistep process of tumor-associated angiogenesis. As new blood vessels are formed, angiogenesis is switched on within the tumor microenvironment and the physiological balance between endogenous pro-angiogenic and anti-angiogenic factors is disrupted. Since then, angiogenesis inhibitors have been developed as drugs and administered in the clinic as part of anti-cancer therapy. Tissue inhibitor of metalloproteinase 2 (TIMP-2) is an endogenous inhibitor of angiogenesis that was initially discovered through the ability to inhibit matrix metalloproteinase (MMP) activity. Subsequently, TIMP-2 was shown to suppress endothelial cell proliferation and migration through MMP dependent and independent mechanisms. Moreover, recent studies indicate that TIMP-2- mediated inhibition of tumor growth may occur, at least in part, via mechanisms that are distinct from its ability to inhibit MMP activity. TIMP-2 is an essential element of the normal tissue microenvironment in the presence of low levels of MMP expression. However, in tumor tissue TIMP-2 levels are reduced. Recent experiments demonstrate that reconstitution of TIMP-2 expression in tumors not only inhibits tumor angiogenesis, but also acts directly on tumor cells to modulate interactions between the tumor cells and the microenvironment. These recent research findings support the idea that TIMP-2 is an excellent candidate for preclinical development as a novel biological agent for cancer therapy.

Cisplatin‐induced, coordinated transcriptional reprogramming of neurotrypsin and other proteases in cisplatin‐resistant ovarian cancer cells

The contribution of extracellular proteolysis to tumor growth, invasion, and metastasis has been recognized for many years. Deregulation of this process remodels normal tissue microenvironment, facilitating malignant transformation, cell migration and invasion, and sometimes resistance to chemotherapy. The mechanisms involved in these events have been extensively studied, but generally focused on individual proteases. We will show that cisplatin exposure, which is a common chemotherapy strategy, induces rapid and coordinated transcriptional reprogramming of 13 genes encoding proteolytic activities in cisplatin‐resistant ovarian cancer cells. These genes include 4 serine proteases, 4 kallikrein, and 5 matrix metallopepsidases. We will cover the transcriptional events involved in this deregulation, including the role of specific epigenetic modifiers at the promoters of these genes. We will also show cellular effects resulting from the suppression of these events, with emphasis on the activity of the serine protease neurotrypsin and its known substrate, the heparan sulfate proteoglycan agrin. Neurotrypsin cleaves agrin generating three fragments: N‐terminal agrin, agrin‐90, and agrin‐22. Together, we propose that cisplatin exposure induces coordinated deregulation of extracellular proteases contributing to escape drug toxicity in cisplatin‐resistant ovarian cancer cells.

Normal Hepatocyte Transplantation Delays the Emergence of Chemically Induced Preneoplastic Nodules in Rat Liver

Cancer often arises in a background of chronic tissue damage. It is also increasingly appreciated that such an injured tissue microenvironment might foster the selective emergence of altered cells, leading to neoplasia. Accordingly, reversal of chronic tissue damage could represent a potential strategy to counteract neoplastic disease. In these studies, we aim to investigate whether transplantation of normal cells in the context of an injured, neoplastic-prone microenvironment might impact on the evolution of the carcinogenic process. A rat model of chemically induced hepatocarcinogenesis was used. Animals were given a single dose of diethylnitrosamine (DENA), followed by two injections of retrorsine (RS), a pyrrolizidine alkaloid that imposes a persistent block on hepatocyte cell cycle. At the end of this protocol, rats were either given no further treatment or injected, via the portal circulation, with 4 million normal hepatocytes isolated from a syngenic donor. After 3 months, rats given DENA+RS alone displayed numerous discrete nodular lesions (up to 30 per liver), ranging 1 to 3 mm in size. On the other hand, in animals receiving DENA+RS and transplantation, donor hepatocytes were able to repopulate over 50% of the host liver, as expected. Most importantly, both the number and the size of hepatocyte nodules were greatly reduced in these animals (percent nodular area was 1.8 ± 0.3, down from a control value of 8.5 ± 2.8). The above data indicate that strategies aimed at reestablishing a normal tissue microenvironment might be relevant to the management of neoplastic disease.