Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

The regulation of cancer cell invasive behaviour by Chloride Interacellular Channel 3 (CLIC3) and Transglutaminasae 2

A study into the role of secreted CLIC3 in tumour cell invasion. The initiation and progression of cancers is thought to be linked to their relationship with a population of activated fibroblasts, which are associated with tumours. I have used an organotypic approach, in which plugs of collagen I are preconditioned with fibroblastic cells, to characterise the mechanisms through which carcinoma-associated fibroblasts (CAFs) influence the invasive behaviour of tumour cells. I have found that immortalised cancer-associated fibroblasts (iCAFs) support increased invasiveness of cancer cells, and that this is associated with the ability of CAFs to increase the fibrillar collagen content of the extracellular matrix (ECM). To gain mechanistic insight into this phenomenon, an in-depth SILAC-based mass proteomic analysis was conducted, which allowed quantitative comparison of the proteomes of iCAFs and immortalised normal fibroblast (iNFs) controls. Chloride Intracellular Channel Protein 3 (CLIC3) was one of the most significantly upregulated components of the iCAF proteome. Knockdown of CLIC3 in iCAFs reduced the ability of these cells to remodel the ECM and to support tumour cell invasion through organotypic plugs. A series of experiments, including proteomic analysis of cell culture medium that had been preconditioned by iCAFs, indicated that CLIC3 itself was a component of the iCAF secretome that was responsible for the ability of iCAFs to drive tumour cell invasiveness. Moreover, addition of soluble recombinant CLIC3 (rCLIC3) was sufficient to drive the extension of invasive pseudopods in cancer cell lines, and to promote disruption of the basement membrane in a 3D in vitro model of the ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition. My investigation into the mechanism through which extracellular CLIC3 drives tumour cell invasiveness led me to focus on the relationship between CLIC3 and the ECM modifying enzyme, transglutaminase-2 (TG2). Through this, I have found that TG2 physically associates with CLIC3 and that TG2 is necessary for CLIC3 to drive tumour cell invasiveness. These data identifying CLIC3 as a key pro-invasive factor, which is secreted by CAFs, provides an unprecedented mechanism through which the stroma may drive cancer progression.

Cell-cell communication in three dimensional microenvironments

Cellular behavior is dependent on a variety of extracellular cues required for normal tissue function, wound healing, and activation of the immune system. Removed from their in vivo microenvironment and cultured in vitro, cells lose many environmental cues and that may result in abberant behavior, making it difficult to study cellular processes. In order to mimic native tissue environments, optical tweezer and microfluidic technologies were used to place cells within defined areas of the culture environment. To provide three dimensional supports found in natural tissues, hydrogel scaffolds of poly (ethylene glycol) diacrylate and the basement membrane matrix Matrigel were used. Optical tweezer technology allowed precision placement and formation of homotypic and heterotypic arrays of human U937, HEK 293, and porcine mesenchymal stem cells. Alternatively, two microfluidic devices were designed to pattern Matrigel scaffolds. The first microfluidic device utilized laminar flow to spatially pattern multiple cell types within the device. Gradients of soluble molecules were then be formed and manipulated across the Matrigel scaffolds. Patterning Matrigel using laminar flow techniques require microfluidic expertise and do not produce consistent patterning conditions, limiting their use difficult in most cell culture laboratories. Thus, a buried Matrigel polydimethylsiloxane (PDMS) device was developed for spatial patterning of biological scaffolds. Matrigel is injected into micron sized channels of PDMS fabricated by soft lithography and allowed to thermally cure. Following curing, a second PDMS device was placed on top of the buried Matrigel channels to support media flow. In order to validate these systems, a cell-cell communication model system was developed utilizing LPS and TNFα signaling with fluorescent reporter systems to monitor communication in real time. We demonstrated the utility of microfluidic devices to support the cell-cell communication model system by co culturing three cell types within Matrigel scaffolds and monitoring signaling activity via fluorescent reporters.

Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures

In this study, Dicentrarchus labrax encephalitis virus (DIEV), which causes sea bass encephalitis, was propagated in cell culture, thus allowing study of its lytic cycle, DIEV infection of mammalian and fish cells induced different patterns of expression of capsid proteins, which were assembled as virus-like particles, accumulating in the cytoplasm either as diffuse masses or in vesicles, as shown by electron microscopy, These particles correspond to virions, as shown by their ability to induce Secondary infection, Fish cells proved to be more permissive for DIEV than mammalian cells, although virus yield remained low, RNA analysis of infected sea bass cells revealed DIEV RNA3, in addition to genomic RNA1 and RNA2, and the presence of the RNA;! minus strand, thus demonstrating the replication of the DIEV genome, In addition, DIEV RNA-dependent RNA polymerase was associated with mature virions even after purification by a CsCl gradient, but it was dissociated when capsids were destabilized, In addition to providing more information about the relatedness of DIEV to the members of the family Nodaviridae, this study shows that fish nodaviruses may not be able to infect as wide a variety of cells as insect nodaviruses can.

Low impact to fixed cell processing aiming transmission electron microscopy

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.

Development of 3D Cancer Cell Models to Test Metabolic Interventions as an Anticancer Strategy

In recent years, it has become evident that the role of mitochondria in the metabolic rewiring is essential for cancer development and progression. The metabolic profile during tumorigenesis has been performed mainly in traditional 2D cell models, including cell lines of various lineages and phenotypes. Although useful in many ways, their relevance can be often debatable, as they lack the interactions between different cells of the tumour microenvironment and/or interaction with the extracellular matrix 1,2. Improved models are now being developed using 3D cell culture technology, contributing with increased physiological relevance 3,4. In this work, we improved a method for the generation of 3D models from healthy and tumour colon tissue, based on organoid technology, and performed their molecular and biochemical characterization and validation. Further, in-plate cryopreservation was applied to these models, and optimal results were obtained in terms of cell viability and functionality of the cryopreserved models. We also cryopreserved colon fibroblasts with the aim to introduce them in a co-culture cryopreserved model with organoids. This technology allows the conversion of cell models into “plug and play” formats. Therefore, cryopreservation in-plate facilitates the accessibility of specialized cell models to cell-based research and application, in cases where otherwise such specialized models would be out of reach. Finally, we briefly explored the field of bioprinting, by testing a new matrix to support the growth of colon tumour organoids, which revealed promising preliminary results. To facilitate the reader, we organized this thesis into chapters, divided by the main points of work which include development, characterization and validation of the model, commercial output, and associated applications. Each chapter has a brief introduction, followed by results and discussion and a final conclusion. The thesis has also a general discussion and conclusion section in the end, which covers the main results obtained during this work.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Antioxidant And Antiproliferative Activities Of Methanolic Extract From A Neglected Agricultural Product: Corn Cobs.

Neglected agricultural products (NAPs) are defined as discarded material in agricultural production. Corn cobs are a major waste of agriculture maize. Here, a methanolic extract from corn cobs (MEC) was obtained. MEC contains phenolic compounds, protein, carbohydrates (1.4:0.001:0.001). We evaluated the in vitro and in vivo antioxidant potential of MEC. Furthermore, its antiproliferative property against tumor cells was assessed through MTT assays and proteins related to apoptosis in tumor cells were examined by western blot. MEC showed no hydroxyl radical scavenger capacity, but it showed antioxidant activity in Total Antioxidant Capacity and DPPH scavenger ability assays. MEC showed higher Reducing Power than ascorbic acid and exhibited high Superoxide Scavenging activity. In tumor cell culture, MEC increased catalase, metallothionein and superoxide dismutase expression in accordance with the antioxidant tests. In vivo antioxidant test, MEC restored SOD and CAT, decreased malondialdehyde activities and showed high Trolox Equivalent Antioxidant Capacity in animals treated with CCl4. Furthermore, MEC decreased HeLa cells viability by apoptosis due an increase of Bax/Bcl-2 ratio, caspase 3 active. Protein kinase C expression increased was also detected in treated tumor cells. Thus, our findings pointed out the biotechnological potential of corn cobs as a source of molecules with pharmacological activity.

Chitosan/tripolyphosphate Nanoparticles Loaded With Paraquat Herbicide: An Environmentally Safer Alternative For Weed Control.

Paraquat is a fast acting nonselective contact herbicide that is extensively used worldwide. However, the aqueous solubility and soil sorption of this compound can cause problems of toxicity in nontarget organisms. This work investigates the preparation and characterization of nanoparticles composed of chitosan and sodium tripolyphosphate (TPP) to produce an efficient herbicidal formulation that was less toxic and could be used for safer control of weeds in agriculture. The toxicities of the formulations were evaluated using cell culture viability assays and the Allium cepa chromosome aberration test. The herbicidal activity was investigated in cultivations of maize (Zea mays) and mustard (Brassica sp.), and soil sorption of the nanoencapsulated herbicide was measured. The efficiency association of paraquat with the nanoparticles was 62.6 ± 0.7%. Encapsulation of the herbicide resulted in changes in its diffusion and release as well as its sorption by soil. Cytotoxicity and genotoxicity assays showed that the nanoencapsulated herbicide was less toxic than the pure compound, indicating its potential to control weeds while at the same time reducing environmental impacts. Measurements of herbicidal activity showed that the effectiveness of paraquat was preserved after encapsulation. It was concluded that the encapsulation of paraquat in nanoparticles can provide a useful means of reducing adverse impacts on human health and the environment, and that the formulation therefore has potential for use in agriculture.

Elevated Micronucleus Frequency In Patients With Type 2 Diabetes, Dyslipidemia And Periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Evaluation of in vitro human gingival fibroblast seeding on acellular dermal matrix

The acellular dermal matrix (ADM) was introduced in periodontology as a substitute for the autogenous grafts, which became restricted because of the limited source of donor's tissue. The aim of this study was to investigate, in vitro, the distribution, proliferation and viability of human gingival fibroblasts seeded onto ADM. ADM was seeded with human gingival fibroblasts for up to 21 days. The following parameters were evaluated: cell distribution, proliferation and viability. Results revealed that, at day 7, fibroblasts were adherent and spread on ADM surface, and were unevenly distributed, forming a discontinuous single cell layer; at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. At 7 days, about to 90% of adherent cells on ADM surface were cycling while at 14 and 21 days this proportion was significantly reduced. A high proportion of viable cell was detected on AMD surface both on 14 and 21 days. The results suggest that fibroblast seeding onto ADM for 14 days can allow good conditions for cell adhesion and spreading on the matrix; however, migration inside the matrix was limited.

Array-CGH testing in spontaneous abortions with normal karyotypes

In about 50% of first trimester spontaneous abortion the cause remains undetermined after standard cytogenetic investigation. We evaluated the usefulness of array-CGH in diagnosing chromosome abnormalities in products of conception from first trimester spontaneous abortions. Cell culture was carried out in short- and long-term cultures of 54 specimens and cytogenetic analysis was successful in 49 of them. Cytogenetic abnormalities (numerical and structural) were detected in 22 (44.89%) specimens. Subsequent, array-CGH based on large insert clones spaced at ~1 Mb intervals over the whole genome was used in 17 cases with normal G-banding karyotype. This revealed chromosome aneuplodies in three additional cases, giving a final total of 51% cases in which an abnormal karyotype was detected. In keeping with other recently published works, this study shows that array-CGH detects abnormalities in a further ~10% of spontaneous abortion specimens considered to be normal using standard cytogenetic methods. As such, array-CGH technique may present a suitable complementary test to cytogenetic analysis in cases with a normal karyotype.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Pesquisa de Rickettsia spp em carrapatos Amblyomma cajennense e Amblyomma dubitatum no Estado de São Paulo

Foi pesquisada a presença de riquétsias em 3.545 carrapatos Amblyomma cajennense e 2.666 Amblyomma dubitatum. Através do teste de hemolinfa, reação em cadeia pela polimerase e isolamento de rickettsia em cultivo celular, todos os Amblyomma cajennense foram negativos, sendo que 634 (23,8%) Amblyomma dubitatum mostraram-se infectados com Rickettsia bellii.