Thesis advisor: Thomas N. Seyfried / Mycoplasmas are the smallest, self-replicating free-living prokaryotes, and have been associated with carcinogenesis. Mycoplasmas can be detected in a high percentage of a wide variety of primary human cancers. Some mycoplasma species such as M. fermentans and M. hyorhinis can transform normal murine and human cell lines into tumorigenic cells. Mycoplasma infection can activate oncogenes as well as inactivate tumor suppressor genes. These observations suggest that mycoplasmas can be both carcinogenic and or onco-modulatory. I found that the metastatic macrophage VM-M3 cell line (referred to as M3+) was infected with mycoplasmas. Mycoplasmal16S rDNA sequencing showed M3+ cells were infected by the mycoplasma species M. arginini. Antibiotic was used to eradicate M. arginini from M3+ cells (referred to as M3- cells). The energetics of the infected M3+ cells and the non-infected M3- cells was studied by measuring respiration (oxygen consumption) and fermentation (lactate production). Respiration was enhanced and fermentation was reduced in the M3- cells compared to the M3+ cells. Glucose enhanced the fermentation and reduced the respiration of both the M3+ and the M3- cells. The M3+ cells produced higher quantities of metabolites indicative of immunological activation (itaconic acid, succinate, and citrulline) compared to M3- cells. In addition, in-vitro proliferation was higher in the M3+ cells than in the M3- cells at high cell densities. Primary subcutaneous tumor growth and metastasis was less in mice inoculated with the M3- cells than with the M3+ cells. The survival of a VM mouse was longer when inoculated with the M3- cells compared to the M3+ cells. Altogether these data indicates that M. arginini is an onco-modulator associated with activation, deregulated energetics and enhanced tumor progression of VM-M3 metastatic macrophage cells. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Thomas N. Seyfried / It has long been posited that all cancer cells are dependent on glucose for energy, termed the "Warburg Effect". As a result of an irreversible injury to the mitochondria, cancer cells are less efficient in aerobic respiration. Therefore, calorie restriction was thought to be a natural way to attenuate tumor growth. Calorie restriction lowers blood glucose, while increasing the circulation of ketone bodies. Ketone bodies are metabolized via oxidative phosphorylation in the mitochondria. Only cells that are metabolically capable of aerobic respiration will thus be able to acquire energy from ketone bodies. To date, calorie restriction has been shown to greatly reduce tumor growth and angiogenesis in the murine CT2A, EPEN, and human U87 brain tumor models. Using the novel VM-M3 model for invasive brain cancer and systemic metastatic cancer, I found that though calorie restriction had some efficacy in reducing brain tumor invasion and primary tumor size, metastatic spread was unaffected. Using a bioluminescent-based ATP assay, I determined the viability of metastatic mouse VM-M3 tumor cells grown in vitro in serum free medium in the presence of glucose alone (25 mM), glutamine alone (4 mM), or in glucose + glutamine. The VM-M3 cells could not survive on glucose alone, but could survive in glutamine alone indicating an absolute requirement for glutamine in these metastatic tumor cells. Glutamine could also maintain viability in the absence of glucose and in the presence of the F1 ATPase inhibitor oligomycin. Glutamine could not maintain viability in the presence of the Krebs (TCA) cycle enzyme inhibitor, 3-nitropropionic acid. The data indicate that glutamine can provide ATP for viability in the metastatic VM-M3 cells through Krebs cycle substrate level phosphorylation in the absence of energy from either glycolysis or oxidative phosphorylation. I therefore developed a metabolic therapy that targeted both glucose and glutamine metabolism using calorie restriction and 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog. Primary tumor growth was about 20-fold less in DON treated mice than in untreated control mice. I also found that DON treatment administered alone or in combination with CR inhibited metastasis to liver, lung, and kidney as detected by bioluminescence imaging and histology. Although DON treatment alone did not reduce the incidence of tumor metastasis to spleen compared to the controls, DON administered together with CR significantly reduced the incidence of metastasis to the spleen, indicating a diet/drug synergy. In addition, the phagocytic capabilities of the VM-M3 tumor cells were enhanced during times of energy stress. This allowed for the digestion of engulfed material to be used in energy production. My data provide proof of concept that metabolic therapies targeting both glucose and glutamine metabolism can manage systemic metastatic cancer. Additionally, due to the phagocytic properties of the VM-M3 cell line also seen in a number of human metastatic cancers, I suggest that a unique therapy targeting metabolism and phagocytosis will be required for effective management of metastatic cancer. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Thomas N. Seyfried / Metastasis is the process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distant organs, and is the primary cause of morbidity and mortality for cancer patients. Most conventional cancer therapies are ineffective in managing tumor metastasis. This has been due in large part to the absence of in vivo metastatic models that represent the full spectrum of metastatic disease. Here I identify three new spontaneously arising tumors in the inbred VM mouse strain, which has a relatively high incidence of CNS tumors. Two of the tumors (VM-M2 and VM-M3) reliably expressed all of the major biological processes of metastasis to include local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Metastasis was assessed through visual organ inspection, histology, immunohistochemistry, and bioluminescence imaging. The metastatic VM tumor cells also expressed multiple properties of macrophages including morphological appearance, surface adhesion, phagocytosis, gene expression (CD11b, Iba1, F4/80, CD68, CD45, and CXCR4) and total lipid composition (glycosphingolipids and phospholipids). The third tumor (VM-NM1) grew rapidly and expressed properties of neural stem/progenitor cells, but was neither invasive nor metastatic. This thesis research also examined the influence of a genelinked up-regulation of the simple ganglioside GM3 in the metastatic VM-M3 tumor. Ganglioside GM3 has been shown to have anti-invasive effects through its ability to modulate integrins and matrix metalloproteases. Additionally, GM3 was previously shown to be elevated in resting macrophages when compared to activated macrophages. The bioluminescent VM-M3 cells (M3/Fluc) contain mostly GM2, GM1, and GD1a with undetectable levels of GM3. Additionally, the M3/Fluc cells express GalNAc-T, a key enzyme for the synthesis of complex gangliosides from GM3, the precursor used for complex ganglioside biosynthesis. Stable transduction of the M3/Fluc tumor with a lentiviral vector containing a cDNA sequence targeting the GalNAc-T gene (Fluc-TNG), resulted in a knock-down of GalNAc-T expression and an up-regulation of GM3 compared to the control (Fluc-csh) transduced M3/Fluc tumor cells. In vivo, the Fluc-TNG cells were significantly less invasive when implanted in the brain and less metastatic when implanted in the flank when compared to the control Fluc-csh tumors. My data indicate that spontaneous brain tumors can arise from different cell types in VM mice and that the ganglioside GM3 can inhibit invasion and metastasis in metastatic cancer cells with macrophage properties. The new VM tumor model will be useful for defining the biological processes of cancer metastasis and for evaluating potential therapies for tumor management. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Student No. 9404705H MDent Research Report School of Oral Sciences / Expression of the E-cadherin/β-catenin complex in oral squamous cell carcinoma and its correlation with histomorphology and metastasis. The immunohistochemical expression of E-cadherin and β-catenin was examined in 30 primary oral squamous cell carcinomas in patients with (n=19) and without (n=11) nodal metastasis, as confirmed on histopathological examination of the resected regional lymph nodes. The corresponding primary and nodal metastases tissue samples were available for 17 patients. The 30 primary carcinomas were histologically graded according to the invasive tumour front grading system and by conventional Broders’ criteria. None of the 30 primary carcinomas showed homogenous, membranous E-cadherin and β-catenin expression when compared to the normal oral squamous epithelium. Staining was heterogeneous in 73% (22/30) and in 77% (23/30) of the primary carcinomas stained for E-cadherin and β-catenin respectively. There was a highly significant reduction (P<0,001) of both E-cadherin and β- catenin expression with progression from the well-differentiated areas to the less differentiated tumour cells at the invasive tumour front. At the invasive tumour front, however, irrespective of the nodal status and invasive tumour front grading score, 28/30 (93%) tumours showed loss of E-cadherin expression. Loss of β-catenin expression was recorded in 22/30 (73%) cases. These findings indicate that E-cadherin and β-catenin play a key role in the loss of differentiation of tumour cells in oral squamous cell carcinoma and while they may be permissive for metastasis, in isolation, E-cadherin and β-catenin are probably not predictive of metastatic potential in oral squamous cell carcinoma.

No description available.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the five leading causes of cancer-related deaths in the West, and this, largely, is due to metastatic disease. In order to better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. Whilst such tissue is in routine diagnostic use, the cross-linkages induced by fixation have, in the past, precluded proteomic investigation for research purposes. Recent technological advances have, however, overcome this technical limitation. Using laser capture microdissection (P.A.L.M system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum and urine resulted in a less than 30 % overlap, indicating that our study has expanded the current database of proteins expressed in this malignancy substantially. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) 4 in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. We chose to investigate further the roles of S100P in lymphatic invasion in vitro and in vivo. By co-culturing a Panc1 S100P-overexpressing clone (S5L), or a vector control clone (V3L), with human dermal lymphatic endothelial cells (HDLEC), we were able to show that different receptors mediate S5L adhesion to resting and activated HDLEC as opposed to V3L; and that the presence of S5L cells in these co-cultures significantly increased permeability at one (p = 0.02), four (p = 0.002) and eight (p = 0.007) hours post-seeding, and significantly increased translymphatic endothelial migration at 72 hours (p = 0.006). Using the V3L and S5L cell lines, which were transduced to express luciferase, we also created an orthotopic mouse model of PDAC, as well as experimental metastatic mouse models, in CD1 nude mice. These models were used to evaluate the effects of S100P on primary tumour growth, metastasis and site-specific growth. S100P was only found to significantly increase primary tumour growth in this model (n = 10 animals/group), both by bioluminescence (p = 0.002) and tumour weight (p = 0.01). No metastases (spontaneous and/or experimental) were seen however. Thus, this model can be used to evaluate the anti-tumour efficacy of novel therapies to S100P in the future.

Metastasis is the leading cause of poor prognosis for individuals diagnosed with cancer. Breast cancer is particularly prevalent with 1 in 10 women receiving a breast cancer diagnosis in her lifetime. There are various types of breast cancers that are distinguished by molecular subtype as defined by specific biomarker expression profiles: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). The subtypes defined by the varying expression of these receptors respond differently to cancer treatments. For example, luminal A ([ER/PR+] HER2- KI67-) responds well to endocrine therapy and patients generally have a good prognosis, whereas triple negative breast cancer (TNBC) ([ER/PR-] HER2- basal marker+) has no specific targeted treatment available and the prognosis is usually poor. Patients with the HER2 subtype often develop resistance to the treatment specific to the breast cancer molecular subtype. Since 90% of all cancer-related deaths are due to metastatic disease, effectively treating all of these types of breast cancers before metastasis is an important factor in achieving a more positive outcome, In order for metastasis to occur, a tumor cell must have the ability to mobilize, intravasate into the vasculature, and then extravasate and proliferate into a tumor at a distant site. Numerous biological and environmental factors must facilitate each of these steps in order for metastasis to occur. One biomarker of metastasis is a tumor microenvironment of metastasis (TMEM). A TMEM is the physical apposition of a Mena-expressing tumor cell, a macrophage (a type of white blood cell), and an endothelial cell (a blood vessel cell). Each TMEM component plays a key role in breast cancer biology. The automated clinical assay MetaSite BreastTM was developed by MetaStat, Inc. to quantify TMEMs. The MetaSiteTM score directly correlates with risk of developing metastasis. The Mena protein is involved in cell motility and expressed isoforms can either promote metastasis (for example, MenaINV), or protect and prevent metastasis (for example, Mena11a). These isoforms are not expressed in a binary manner and studies have shown that the ratio of MenaINV to Mena11a can give insight into the pro-metastatic/anti-metastatic biology of the cell. To indirectly measure the amount of MenaINV, the Z-score of Mena11a is subtracted from the Z-score of pan-Mena (all Mena isoforms), yielding a theoretical maximum amount of MenaINV, called Menacalc. This process is performed by quantitative analysis of multiplexed immunofluorescence staining through the MenaCalcTM assay developed by MetaStat, Inc. The results of this study demonstrated that MenaCalcTM is a high-performing, high-throughput assay that was clinically validated under CLIA-approved protocol in January 2016. The assay surpassed all benchmark goals for precision and performance. For both day-to-day and run-to-run operations, precision and reproducibility were analyzed using Pearson’s R and slope. The day-to-day reproducibility yielded Pearson’s R values of 0.879 and 0.853 comparing Day 1 vs. Day 2 and Day 2 vs. Day 3, respectively. The slopes for the same comparisons were 0.985 and 0.982, respectively. The analysis of run-to-run precision had Pearson’s R values of 0.999 and 0.994 comparing Day 1 vs. Day 2 and Day 2 vs. Day 3, respectively. The slopes were 0.999 for both comparisons. The development of such an assay brings new elements of precision and reproducibility to the current market of breast cancer biomarker tests. Statistical analysis revealed a wide range of MenaCalcTM scores that were independent of total Mena expression. Individual images showed a range of MenaCalcTM values from a low of only 2.9% of cells with a high MenaCalcTM score to a high of 97.4% of cells with a high MenaCalcTM score. Regions of high MenaCalcTM scores correlated with areas of invasive tumor. Preliminary data assessing the synergistic use of both the MetaSite BreastTM and the MenaCalcTM assays were promising. These data suggests that both physical MetaSiteTM structures and protein expression levels can be used to more thoroughly understand the biology of breast cancer and the path to metastasis. Three clusters of combined MetaSiteTM/MenaCalcTM scores were observed: MetaSiteTM low/MenaCalcTM low, MetaSiteTM low/MenaCalcTM high, MetaSiteTM high/MenaCalcTM high. Because a MetaSiteTM High/MenaCalcTM Low score combination was not observed, a high MenaCalcTM score may be necessary for TMEM formation. Studies are ongoing to further evaluate the synergy of the MetaSite BreastTM and the MenaCalcTM in order to bring more power to the assessment of metastatic risk. / 2016-12-16T00:00:00Z

Les métastases représentent l’étape ultime de la progression tumorale. Les traitements inhibiteurs de l’angiogenèse offrent de nouvelles perspectives thérapeutiques, notamment pour les stades invasifs, mais cependant ils n’apportent qu’une prolongation modeste de la survie des patients. La faible réponse de certains types de cancers aux traitements ciblant l’angiogenèse tumorale, comme le carcinome de la prostate chez l’homme ou du sein chez la femme, est due au développement de résistances tumorales d’une part, et à l’accélération du processus métastatique mise en évidence par certaines études précliniques et cliniques d’autre part. L’objectif de ce travail de thèse a été d’étudier le rôle d’une protéine anti-angiogénique endogène, la thrombospondine 1 (TSP1), dans l’invasion et la dissémination métastatique dans le carcinome de la prostate et du sein. Notre laboratoire a montré que la densité microvasculaire est inversement corrélée à l’expression de la TSP1 dans les stades précoces de ces carcinomes. Cependant, cette corrélation est perdue aux stades avancés où l’expression de la TSP1 devient associée à un mauvais pronostic. Les travaux de ma thèse ont permis de montrer que la TSP1 stimule un ensemble de processus intervenant dans la progression tumorale, dont la migration et l’invasion cellulaires, l’extravasation et la dissémination métastatique. Nous avons aussi pu mettre en évidence que l’hypoxie induite par l’inhibition de l’angiogenèse est un activateur majeur de l’invasion et de la formation de métastases, que cette hypoxie soit produite par des molécules endogènes comme la TSP1 ou par des inhibiteurs pharmacologiques de ce processus. De plus, nous démontrons que la TSP1 est un facteur de mauvais pronostic puisque son expression est corrélée avec l’augmentation de l’invasion et de la rechute des patients atteints de cancers de la prostate androgéno-résistants. En conclusion, nos résultats suggèrent que l’expression de la TSP1 pourrait être un facteur prédictif de l’invasion et de l’occurrence de métastases, et que le ciblage de la TSP1 présente un fort potentiel thérapeutique pour bloquer à terme ces processus. / Prognosis is poor once tumors developed metastasis, and overall survival has been only modestly improved by the development of drugs inhibiting angiogenesis. The poor response of certain types of cancer to therapies that target tumor angiogenesis, including prostate and breast carcinomas, is due to the development of an evasion and to the acceleration of the metastatic process noted in some preclinical and clinical studies. The objective of this thesis was to study the role of an endogenous anti-angiogenic factor, thrombospondin 1 (TSP1), in the process of invasion and metastasis of prostate and breast carcinomas. Our laboratory has previously shown that microvessel density is inversely correlated with the expression of TSP1 in primary stages of breast and prostate carcinomas. However, this correlation is lost in advanced cancers, where TSP1 expression becomes associated with poor prognosis. We show that TSP1 stimulates a set of processes involved in tumor progression, including cell migration and invasion, extravasation and metastatic dissemination. We also demonstrate that hypoxia induced by an inhibition of angiogenesis resulting from the activity of endogenous or pharmacological molecules is a major activator of invasion and metastasis. In addition, we demonstrate that TSP1 is a poor prognostic factor since its expression is correlated with increased invasion and relapse in patients with androgen-resistant prostate cancer. In conclusion, our results suggest that the expression of TSP1 could be a predictor of invasion and metastasis occurrence, and that targeting TSP1 could be a great therapeutic potential to block these processes.

O osteossarcoma é um tumor ósseo maligno comum em cães, com preferência por raças de grande porte, tem alto potencial metastático e ocorre frequentemente no esqueleto apendicular. O diagnóstico é baseado na história clínica, exame físico, achados radiológicos e histopatológicos. O tratamento consiste na ressecção cirúrgica do tumor e o protocolo de tratamento de escolha é a amputação associada à quimioterapia. O osteossarcoma apresenta semelhanças em cães e humanos, portanto o osteossarcoma canino pode ser um modelo útil para estudar esta doença em humanos. O objetivo deste trabalho foi estabelecer e caracterizar a linhagem celular primária de osteossarcoma canino. Os fragmentos tumorais foram obtidos por meio de biópsias e excereses cirúrgicas do osteosarcoma, a confirmação diagnóstica realizada pelo exame histopatológico. Os fragmentos foram cultivados em meios de cultura DMEM-H, DMEM-Low, RPMI-1640 e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida, microscopia eletrônica de transmissão e varredura. A caracterização dos marcadores de superfície, citoplasmáticos e nucleares, análise das fases do ciclo celular e potencial elétrico mitocondrial foram realizados por citometria de fluxo. Foram obtidos 6 tumores, sendo 5 osteossarcomas e um condrossarcoma, dos quais foram obtidas 3 linhagens OST-1, OST-3, e CDS1. Os meios de cultivo DMEM-H e MEM foram eficientes para obtenção e manutenção das linhagens. Morfologicamente as células OST-1, apresentaram aspecto fusiforme enquanto as células OST-3 apresentaram pleomorfismo celular. Os marcadores de superfície, citoplasmáticos e nucleares nas células de osteossarcoma canino OST-3 apresentaram-se diferencialmente expressos, como os marcadores de origem mesenquimal, o marcador de reparo de DNA, P53, não foi expresso como também é encontrado em inúmeras linhagens de osteossarcoma. / Osteosarcoma is a common malignant bone tumor in dogs, with a preference for large breeds, has a high metastatic potential and occurs often in the appendicular skeleton. Diagnosis is based on clinical history, physical examination, radiological and histopatological findings. The treatment consists in surgical tumor resection, and treatment protocol choice is amputation associated with chemotherapy. Osteosarcoma has many similarities in dogs and humans, thus the canine osteosarcoma can be a usefull model to study this disease in humans. The aim of this work was to establish and characterize the canine osteosarcoma primary cell line. Tumor samples were obtained by surgical biopsy and the osteosarcoma confirmation was made by histopathological examination. Tumor fragments were cultured in DMEM-H, DMEM- LOW, RPMI-1640 and MEM culture media to establish the cell lines. Cell morphology analysis was carried out by photodocumentation in inverted microscopy and in transmission electron microscopy. The characterization of surface, cytoplasmic and nuclear markers and cell cycle phases, and mitochondrial electric potential analysis were performed by flow cytometry and analyzed with the Win MDI 2.8. Five osteosarcomas, 1 chondrosarcoma, and three cancer cell lines, OST-1, OST-3, and CDS1, were obtained. DMEM-H and MEM culture medium were efficient in cell lines establishing and maintenance. OST-1 cell line showed spindle shaped and OST-3 cell line showed pleomorphism. Surface, cytoplasmic and nuclear markers in the OST-3 canine osteosarcoma cell line were differentially expressed as mesenchimal origin markers, the DNA repair marker, P53, it was not expressed, it is also found in other osteossarcoma lines.

Cancer cells exhibit an abnormal metabolic phenotype characterized by glycolysis and lactate fermentation in the presence of oxygen, a phenomenon known as the Warburg effect. This dysregulated metabolism plays an important role in every aspect of cancer progression, from tumorigenesis to invasion and metastasis. The Warburg effect is a common phenotype shared by most, if not all, cancer types. It is especially prominent in metastatic tumors, which are notoriously resistant to treatment and responsible for the majority of cancer-related deaths. Thus, metabolic therapies which target the Warburg effect could offer novel therapeutic options for most cancer patients, including those with aggressive or late-stage cancers. The ketogenic diet is a high fat, low carbohydrate diet that induces a physiological state of nutritional ketosis - decreased blood glucose and elevated blood ketones. It has been investigated as a cancer therapy for its potential to exploit the Warburg effect by restricting glucose availability to glycolysis-dependent tumors, and has been reported to slow cancer progression in some animal models as well as in anecdotal reports and small clinical studies in humans. Interestingly, there is some evidence that the elevation in blood ketones induced by the ketogenic diet contributes to its anti-cancer effects, suggesting that ketone supplementation could possibly inhibit cancer progression on its own. Rapid growth outstrips a tumor's ability to adequately perfuse its tissue, creating regions of tumor hypoxia which exacerbate the Warburg effect and promote a malignant phenotype. Hyperbaric oxygen therapy is the administration of 100% oxygen at elevated barometric pressure. It supersaturates the blood with oxygen, increasing its diffusion distance into the tissues, and can therefore be used to increase intratumoral pO2 and reverse tumor hypoxia. Here we present evidence that the ketogenic diet, ketone supplementation, and hyperbaric oxygen therapy work individually and in combination to slow progression and extend survival in the VM-M3 model of metastatic cancer. This study strongly suggests that these cost effective, non-toxic metabolic therapies should be further evaluated in animal and human studies to determine their potential clinical use.