We present mathematical models to study the mechanism of interaction of tumour infiltrating cytotoxic lymphocytes (TICLs) with tumour cells. We focus on the phase spaces of the systems and the nature of the solutions for the cell densities in the short and long term. The first model describes the production of offspring through cell proliferation, death and local kinetic interactions. The second model characterises the spatial distribution dynamics of the cell densities through reaction diffusion, which describes the random movement of the cells, and chemotaxis, which describes the immune cell movements towards the tumour cells. We then extend these models further to incorporate the effects of immunotherapy by developing two new models. In both situations, we analyse the phase spaces of the homogeneous models, investigate the presence of travelling wave solutions in our systems, and provide numerical simulations. Our analysis shows that cancer dormancy can be attributed to TICLs. Our study also shows that TICLs reduce the tumour cell density to a cancer dormant state but even with immunotherapy do not completely eliminate tumour cells from body tissue. Travelling wave solutions were confirmed to exist in the heterogeneous model, a linear stability analysis of the homogeneous models and numerical simulations show the existence of a stable tumour dormant state and a phase space analysis confirms that there are no limit cycles. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Erratum is pasted onto back endpaper. / Bibliography: leaves 189-219. / xxiii, 221 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Current understanding of metastatic processes and the clinical significance of these processes is discussed. The study of circulating solid tumour cells in peripheral blood is reviewed from an historical perspective. Newer molecular and cell biological techniques which may facilitate more reliable tumour evaluation are reviewed with special reference given to polymerase chair reaction (PCR) / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 1997?

Thesis (Ph. D. in Cell Biology)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Activin-A is a member of transforming growth factor-β (TGF-β) superfamily of cytokines which includes TGF-βs, Activins, Nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and anti-Mullerian hormone (AMH). TGF-β, Activin and Nodal are known to activate SMAD2/3, while BMPs and GDFs are known to activate SMAD1/5/8 signaling pathways. Activin-A binds to type II transmembrane serine threonine kinase receptor (ActRIIA or ActRIIB), which in turn activates type I receptor (ActRIB) leading to phosphorylation of SMAD2/SMAD3. Upon phosphorylation, SMAD2/3 forms a complex with SMAD4, which then translocates to nucleus. In the nucleus, SMAD2/3/4 complex, along with other co-factors regulates expression of a large number of genes. Unlike TGF-β, role of Activin in cancer is not well understood. Activin has been shown to be overexpressed in several cancers including metastatic prostate cancer, colorectal cancer, lung cancer, hepatocellular carcinoma and pancreatic cancer. Activin signaling has been shown to promote aggressiveness of esophageal squamous cell carcinoma and enhancing skin tumorigenesis and progression. Nodal, which binds to the same set of receptors, has also been shown to be overexpressed in several cancers. However, role of Activins in breast cancer progression is not well studied. Activin is expressed by normal breast epithelium and is known to play a role in mammary gland development. Earlier, a study had reported downregulation of Activin signaling in breast tumors. On the contrary, increased serum level of Activin has been reported in women with metastatic breast cancers. It is pertinent to mention here that TGF-β, which has been implicated in the progression and metastatic spread of breast cancers, also functions through the same set of downstream effectors- SMAD2 and SMAD3. Hence we wanted to evaluate the status of Activin signaling pathway in breast tumors and investigate its functional role in cancer progression. Gene expression profiling of 80 breast tumors and 20 normal samples was earlier performed in our laboratory revealed overexpression of INHBA in tumors compared to normal tissue samples. An independent set of 30 tumor and 15 normal samples were used to verify these results. Real-time PCR analysis revealed around 11.31 fold upregulation (p<0.001) of INHBA in breast tumors in comparison to normals. While no change in expression of INHA was observed, INHBB was found to be significantly downregulated in tumor samples. These results indicated upregulation of Activin-A in breast tumors. Further, a significant upregulation of ACVR2A and SMAD2 which act as signal transducers of Activin signaling pathway, was observed in breast tumors. Interestingly, while an increase in the expression of TGF-β1 was observed, TGFBR2 was found to be significantly downregulated in breast tumors. In addition, PCR analysis revealed significant downregulation of FST, β-glycan, IGSF1 and IGSF10, which act as negative regulators of Activin signaling pathway. Functional antagonism between TGF-β/Activin and BMP signaling pathway has been shown in both development and disease. Further analysis revealed that various BMPs including BMP2, BMP4 and BMP6 are downregulated in breast tumors compared to normal tissue samples. Various components and regulators of BMP signaling pathway were also found to be deregulated, indicating suppression of BMP signaling in breast tumors. To evaluate whether Activin signaling is active in breast tumor cells, immunohistochemistry with another set of 13 normal and 29 tumor samples was performed. Immunohistochemistry analysis revealed that most of the tumors have higher levels of Activin-A compared to normals tissues. Interestingly, no significant changes in expression of Activin-A was observed between normals and low grade tumors, suggesting that Activin-A may play an important role towards the late stages of the disease. In good correlation, breast tumors showed increased phospho SMAD2 and phospho SMAD3 levels compared to normal tissues. Also, in the same set of tumors, BMP2 staining showed a reduced expression pattern compared to normal tissues. Expression of inhibin in some normal and breast tumor samples revealed that most of the tumor samples have lower levels of inhibin compared to normal tissues. In order to understand the role of Activin-A in cancer progression, a panel of cell lines was selected. Treatment of cells with Activin-A resulted in activation of canonical SMAD as well as non-canonical Erk1/2 and PI3K signaling pathways. However, Activin-A treatment did not lead to activation of TAK1/p38 MAPK pathway. To begin with, it was important to evaluate effect of Activin-A on proliferation of various cell lines. Primarily, SMAD2/3 signaling pathway inhibits proliferation of normal epithelial cells, and hence, it is considered to have a tumor suppressive role. owever, this signaling pathway remains intact in most ( 98%) of the breast cancers. BrdU incorporation assay showed that Activin-A does not promote proliferation of cells under monolayer culture conditions. However, soft agar assay results showed that Activin signaling promotes anchorage independent growth of cancer cells. TGF-β is widely known as an inducer of epithelial mesenchymal transition (EMT). Also, EMT is considered to be a prerequisite for epithelial cells to undergo migration and invasion. During EMT, cells loose epithelial characteristics and acquire mesenchymal features along with cytoskeletal rearrangement. Treatment of cells with Activin-A resulted in downregulation of E-cadherin and upregulation of various mesenchymal markers. In addition, confocal microscopy imaging revealed a mesenchymal morphology of cells treated with Activin-A. Also, collagen gel contraction assay results indicated that Activin-A enhances the contractile property of HaCaT cells significantly. Cells undergone EMT are believed to acquire migratory and Invasive behaviour. In agreement with this, both scratch assay and trans-well migration assay showed that Activin-A enhances the migration of various cell lines. Further, Trans-well matrigel invasion assays were performed to assess how Activin affects invasion of various cancer cells. Matrigel invasion assay results showed that Activin-A enhances invasion of various cancer cell lines significantly. Also, RT-PCR, zymography and Luciferase assay results showed that Activin-A induces MMP2 expression. As described earlier, Activin-A activates both canonical as well as non canonical signaling pathways. In this direction, it was interesting to investigate the contribution of SMAD signaling pathway in pro-tumorigenic actions of Activin-A. Inhibiting SMAD3 activity either by its stable knockdown or by using a SMAD3 specific small molecule inhibitor revealed that Activin-A regulation of EMT markers is SMAD3 dependent. Further, it was observed that SMAD3 contributes significantly in mediating Activin-A induced migration and invasion. Hence, it is likely that SMADs may play an important role in breast tumor progression. Next, stable overexpression of Activin-A in MCF-7 or its knockdown in MDA-MB-231 and H460 cells was performed to assess the effect of Activin-A on the behaviour of these cells. BrdU assay indicated no change in proliferation of cells upon overexpression or knockdown of Activin-A. However, soft agar assay results showed that Activin-A expression affects anchorage independent growth of these cells. MCF-7 cells are generally considered to be less aggressive in their tumor forming ability. Activin-A overexpressing MCF7 cells and control cells were respectively injected into right and left flank of immunocompromised mice and followed till the tumors reached to a prominent size. Our results show that Activin-A overexpressing MCF-7 cells have better tumor forming ability in comparison to control cells. In contrast to MCF-7 cells, MDA-MB-231 cells are known to be aggressive in their tumorigenic potential. In order to understand the effect of Activin-A knockdown on the tumor forming ability in MDA-MB-231 cells, 0.5 million cells (optimal cell number generally used is 1-2 million) were injected subcutaneously in immunocompromised mice. The results showed that while control cells gave rise to a tumor in 7 out of 10 animals, Activin-A knockdown cells could form a tumor in only 3 out of 10 animals. Also, the tumors formed by control cells were significantly larger by weight as compared to tumors formed by knockdown cells. Further, immunohistochemistry showed that tumors formed by MCF-7 cells overexpressing Activin-A have higher Ki-67 percentage as compared to control tumors. One of the factors known to be important for tumor growth is VEGF, which leads to recruitment of blood vessels and hence providing nourishment to the tumor cells. Hence Activin-A regulation of VEGF expression was evaluated next. Activin-A treatment or its stable overexpression in MCF-7 cells resulted in increased VEGF expression in these cells. This was also confirmed by VEGF promoter activity assay. To assess if Activin-A can play a role in metastatic spread of cancer cells, tail vein injection of Activin-A overexpressing MCF-7 cells was performed in immunocompromised mice. Even though no significant difference was found in the number of nodules formed by control or Activin-A overexpressing cells, it was observed that Activin-A overexpressing cells formed much bigger nodules as compared to the control cells. This suggests that Activin-A may play an important part in the establishment of metastases from the disseminated cancer cells. Tumor forming ability of cancer cells and aggressiveness of various cancers has been associated with the presence of cells having stem-like phenotype. In this direction, CD44high and CD24low expression status was analysed upon overexpression and knockdown of Activin-A in MCF-7 and MDA-MB-231 cells respectively. FACS analysis of Activin-A overexpressing MCF-7 cells and Activin-A knockdown MDA-MB-231 cells shows that Activin-A expression leads to enrichment of breast cancer stem-like cells. In conclusion, this study highlights the importance of Activin-A signaling pathway in the progression of breast tumors. It is also important to note the role of SMAD signalling in the progression of breast cancers since these effectors are common between TGF-β, Activin and nodal factors, which have been shown to be involved in cancer progression in a context dependent manner.

Systems pathology attempts to introduce more holistic approaches towards pathology and attempts to integrate clinicopathological information with “-omics” technology. This doctorate researches two examples of a systems approach for pathology: (1) a personalized patient output prediction for ovarian cancer and (2) an analytical approach differentiates between individual and collective tumour invasion. During the personalized patient output prediction for ovarian cancer study, clinicopathological measurements and proteomic biomarkers are analysed with a set of newly engineered bioinformatic tools. These tools are based upon feature selection, survival analysis with Cox proportional hazards regression, and a novel Monte Carlo approach. Clinical and pathological data proves to have highly significant information content, as expected; however, molecular data has little information content alone, and is only significant when selected most-informative variables are placed in the context of the patient's clinical and pathological measures. Furthermore, classifiers based on support vector machines (SVMs) that predict one-year PFS and three-year OS with high accuracy, show how the addition of carefully selected molecular measures to clinical and pathological knowledge can enable personalized prognosis predictions. Finally, the high-performance of these classifiers are validated on an additional data set. A second study, an analytical approach differentiates between individual and collective tumour invasion, analyses a set of morphological measures. These morphological measurements are collected with a newly developed process using automated imaging analysis for data collection in combination with a Bayesian network analysis to probabilistically connect morphological variables with tumour invasion modes. Between an individual and collective invasion mode, cell-cell contact is the most discriminating morphological feature. Smaller invading groups were typified by smoother cellular surfaces than those invading collectively in larger groups. Interestingly, elongation was evident in all invading cell groups and was not a specific feature of single cell invasion as a surrogate of epithelialmesenchymal transition. In conclusion, the combination of automated imaging analysis and Bayesian network analysis provides an insight into morphological variables associated with transition of cancer cells between invasion modes. We show that only two morphologically distinct modes of invasion exist. The two studies performed in this thesis illustrate the potential of a systems approach for pathology and illustrate the need of quantitative approaches in order to reveal the system behind pathology.

Indiana University-Purdue University Indianapolis (IUPUI) / Cancer stem cells (CSCs) are proposed to play a major role in tumor progression, metastasis, and recurrence. The Human Epidermal growth factor Receptor 2 (HER2) gene is amplified and/or its protein product overexpressed in approximately 20% of breast cancers. HER2 overexpression is associated with increased CSCs, which may explain the aggressive phenotype and increased likelihood of recurrence for HER2+ breast cancers. Telomerase is reactivated in tumor cells, including CSCs, but has limited activity in normal tissues, providing support for the use of telomerase inhibition in anti-cancer therapy. Telomerase inhibition via an antagonistic oligonucleotide, imetelstat (GRN163L), has been shown to be effective in limiting cell growth in vitro and limiting tumor growth. Moreover, we have previously shown imetelstat can decrease metastases to the lungs, leading us to question if this is due to imetelstat targeting the CSC population. In this thesis, we investigated the effects of imetelstat on CSC and non-CSC populations of HER2+ breast cancer cell lines, as well as a triple negative breast cancer cell line, which lacks HER2 overexpression. Imetelstat inhibited telomerase activity in both CSC and non-CSC subpopulations. Moreover, imetelstat treatment alone and in combination with trastuzumab significantly reduced the CSC fraction and inhibited CSC functional ability, as shown by a significant decrease in mammosphere counts and invasive potential. Tumor growth rate was slower in combination treated mice compared to either drug alone. Additionally, there was a trend toward decreased CSC marker expression in imetelstat treated xenograft cells compared to vehicle control. The decrease in CSC marker expression we observed occurred prior to and after telomere shortening, suggesting imetelstat acts on the CSC subpopulation in telomere length dependent and independent mechanisms. Our study suggests addition of imetelstat to trastuzumab may enhance the effects of HER2 inhibition therapy.