Niche inheritance: a cooperative pathway to enhance cancer cell fitness though ecosystem engineering

Cancer cells can be described as an invasive species that is able to establish itself in a new environment. The concept of niche construction can be utilized to describe the process by which cancer cells terraform their environment, thereby engineering an ecosystem that promotes the genetic fitness of the species. Ecological dispersion theory can then be utilized to describe and model the steps and barriers involved in a successful diaspora as the cancer cells leave the original host organ and migrate to new host organs to successfully establish a new metastatic community. These ecological concepts can be further utilized to define new diagnostic and therapeutic areas for lethal cancers.Comment: 8 pages, 1 Table, 4 Figure

Microenvironmental Variables Must Influence Intrinsic Phenotypic Parameters of Cancer Stem Cells to Affect Tumourigenicity

<div><p>Since the discovery of tumour initiating cells (TICs) in solid tumours, studies focussing on their role in cancer initiation and progression have abounded. The biological interrogation of these cells continues to yield volumes of information on their pro-tumourigenic behaviour, but actionable generalised conclusions have been scarce. Further, new information suggesting a dependence of tumour composition and growth on the microenvironment has yet to be studied theoretically. To address this point, we created a hybrid, discrete/continuous computational cellular automaton model of a generalised stem-cell driven tissue with a simple microenvironment. Using the model we explored the phenotypic traits inherent to the tumour initiating cells and the effect of the microenvironment on tissue growth. We identify the regions in phenotype parameter space where TICs are able to cause a disruption in homeostasis, leading to tissue overgrowth and tumour maintenance. As our parameters and model are non-specific, they could apply to any tissue TIC and do not assume specific genetic mutations. Targeting these phenotypic traits could represent a generalizable therapeutic strategy across cancer types. Further, we find that the microenvironmental variable does not strongly affect the outcomes, suggesting a need for direct feedback from the microenvironment onto stem-cell behaviour in future modelling endeavours.</p></div

Analysis of glioma cell heterogeneity by lineage-tracing in murine and human model systems

Intra-tumour heterogeneity and plasticity are key factors in treatment resistance and the recurrence of Glioblastoma (GBM), which is invariably fatal. Genetics, epigenetics, cell metabolism and plastic cancer stem cell (CSC) hierarchies interact with volatile micro-environmental forces to promote and shape cell identity. Improved understanding of these factors will inform more precise and effective GBM therapies. Here, we aim to develop a fluorescent tracking approach for patient derived GBM cells to investigate the relationship between clones, environment and CSC marker expression. Using a murine GBM model combined with Rosa26-confetti fluorescent labelling, we trialled suitable techniques for detection of labelled tumour clones and concluded fluorescent imaging and flow cytometry were the most effective. For patient-derived cells, we modified LeGO-vector fluorescent labelling with the aim of tracking a greater number of clones. We further optimised this technique for simultaneous flow cytometry detection of clones and their CSC marker expression. In the final chapter, we address the hypothesis that whole population CSC surface marker plasticity is a result of emergent clonal predominance. In two patient derived GBM lines, under steady-state environmental conditions, serial passaging and assessment of clonal marker expression detected distinct marker expression patterns between clones in the same culture dish. For both cell lines, transfer and culture of clonal mixtures to Matrigel® spheroids produced an expected plastic transition in population marker expression but also considerable predominance of certain clones. While the clonalsurface marker dynamics of the two cell lines were markedly distinct, divergent surface marker plasticity between clones of the same cell line was a consistent observation. Taken together these results supported our hypothesis that population marker plasticity is in part a result of emergent clonal predominance. We propose our developed techniques are suitable for rapid and economic characterisation of patient specific gene disruption, therapeutic vulnerabilities and resistance mechanisms

LONGITUDINAL CLONAL LINEAGE DYNAMICS AND FUNCTIONAL CHARACTERIZATION OF PANCREATIC CANCER CHEMO-RESISTANCE AND METASTASIZATION

In recent years, technological advancements, such as next-generation sequencing and single-cell interrogation techniques, have enriched our understanding in tumor heterogeneity. By dissecting tumors and characterizing clonal lineages, we are better understanding the intricacies of tumor evolution. Tumors are represented by the presence of and dynamic interactions amongst clonal lineages. Each lineage and each cell contributes to tumor dynamics through intrinsic and extrinsic mechanisms, and the variable responses of clones to perturbations in the environment, especially therapeutics, underlie disease progression and relapse. Thus, there exists a pressing need to understand the molecular mechanisms that determine the functional heterogeneity of tumor sub-clones to improve clinical outcomes. Clonal replica tumors (CRTs) is an in vivo platform created specifically to enable robust tracing and functional study of clones within a tumor. The establishment of CRTs is built upon our current concept of tumor heterogeneity, intrinsic cancer cell hierarchy and clonal self-renewal properties. The model allows researchers to create large cohorts of tumors in different animals that are identical in their clonal lineage composition (clonal correlation amongst tumors \u3e0.99). CRTs allow simultaneously tracking of tens of thousands of clonal lineages in different animals to provide a high level of resolution and biological reproducibility. CRTs are comprised of barcoded cells that can be identified and quantified. A critical feature is that we have developed a systematic method to isolate and expand essentially any of the clonal lineages present within a CRT in their naïve state; that is, we can characterize each sub-clonal lineage at the molecular and functional levels and correlate these findings with the behavior of the same lineage in vivo and in response to drugs. Here, based on the CRT model and its concept, we studied differential chemo-resistance among clones, where we identified pre-existing upregulation in DNA repair as a mechanism for chemo-resistance. Furthermore, through stringent statistical testing, we demonstrated orthotopic CRTs to be a powerful and robust model to quantitatively track clonal evolution. Specifically, we longitudinally tracked clones in models of pancreatic ductal adenocarcinoma (PDAC) from primary tumor expansion through metastasization, where we captured unexpected clonal dynamics and “alternating clonal dominance” naturally occurring in unperturbed tumors. Moreover, by characterizing pro- and none-metastasizing clones, we were able to identified key clonal intrinsic factors that determined the nature of tumor metastases. Finally, I will discuss distinct clonal evolution patterns that emerged under different environmental pressures, leading to the hypothesis of “tumor clonal fingerprint”, where the characteristic of a tumor could be defined by actively maintained ratio of different tumor lineages, which could provide measurable insights to how we approach treatments

Cross talk between CD271 expressing tumour subpopulations and autophagy in the drug resistance, invasion and survival of cutaneous metastatic melanoma

Phd ThesisCutaneous malignant melanoma remains an increasing world health problem. Although targeted therapy to hyper-activated MAPK signalling has significantly increased survival for patients with BRAF/NRAS mutant melanomas, the development of acquired resistance is inevitable and associated with the emergence of chemo-resistant subpopulations expressing the neurotrophin receptor CD271. The relationship between drug-induced CD271 expressing subpopulations and autophagy however, remains undefined. The central aim of the present study was to define the relationship between CD271 (both constitutive and drug-induced) expression and autophagy, and the impact of CD271 inhibition or autophagy modulation on the potential re-sensitisation of BRAF mutant metastatic melanoma cells to MEK inhibition. Semi-quantitative immuno-histochemical analysis (IHC) of CD271 expression in primary cutaneous melanomas of different AJCC stage, revealed a significant stepwise increase in expression in advanced, stage III melanomas, paralleled with biphasic expression of p62 and consistent with the paradoxical role of autophagy in cancer. Additionally, chemical inhibition of autophagy with chloroquine or a specific Vps34 inhibitor, selectively inhibited cell viability CD271 positive but not negative subpopulations, isolated from BRAF mutant melanoma cell line, collectively suggesting constitutive CD271 expression is associated with an increase in basal pro-survival autophagy. Prolonged exposure of BRAFV600E mutant melanoma cells to the MEK 1/2 specific inhibitor trametinib resulted in increased CD271 expression, LC3-II accumulation and reduced p62 expression, again highlighting the relationship between CD271 expression and autophagy activation. Genetic or chemical inhibition of CD271 in trametinib-induced drug-resistant BRAF mutant melanoma subpopulations resulted in significant inhibition of cell viability and resensitisation to trametinib-induced apoptosis. Furthermore, Inhibition of autophagy with chloroquine/ Vps34 inhibitor also resulted in reduced colony forming capacity, invasion and the re-sensitization of CD271 expressing BRAF mutant subpopulations to the cytotoxic effects of trametinib in vitro, with Vps34 inhibition additionally promoting trametinib-induced death and the prevention of tumour invasion and dissemination of trametinib-induced drug resistant subpopulations in a zebra fish xenograft model. iv Attempts to harness cytotoxic autophagy to overcome drug-induced resistance to trametinib additionally revealed treatment of drug-induced resistant CD271 expressing BRAF mutant melanoma subpopulations with 9-tetrahydrocannabinol (THC) resulted in significant inhibition of cell viability. Collectively these data underpin the intimate relationship between trametinib-induced CD271 drug-resistant subpopulations and pro-survival autophagy and suggest the targeting of CD271 or indirect clinical modulation of autophagy (either through selective inhibition or the harnessing of its cytotoxic effects induced by THC) may provide valuable therapeutic strategies through which to overcome the resistance of BRAF/NRAS mutant metastatic melanoma to MEK inhibition.Cancer Research UK, The North Eastern Research Skin Fund and the National Institute for Cancer Research