Esha T. Shah

Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOM™ assay data using the Fisher-Kolmogorov model

BackgroundStandard methods for quantifying IncuCyte ZOOM™ assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time. Unfortunately, these measurements give no insight into the details of the cellular-level mechanisms acting to close the initially-vacant area. We provide an alternative method enabling us to quantify the role of cell motility and cell proliferation separately. To achieve this we calibrate standard data available from IncuCyte ZOOM™ images to the solution of the Fisher-Kolmogorov model.ResultsThe Fisher-Kolmogorov model is a reaction-diffusion equation that has been used to describe collective cell spreading driven by cell migration, characterised by a cell diffusivity, D, and carrying capacity limited proliferation with proliferation rate, λ, and carrying capacity density, K. By analysing temporal changes in cell density in several subregions located well-behind the initial position of the leading edge we estimate λ and K. Given these estimates, we then apply automatic leading edge detection algorithms to the images produced by the IncuCyte ZOOM™ assay and match this data with a numerical solution of the Fisher-Kolmogorov equation to provide an estimate of D. We demonstrate this method by applying it to interpret a suite of IncuCyte ZOOM™ assays using PC-3 prostate cancer cells and obtain estimates of D, λ and K. Comparing estimates of D, λ and K for a control assay with estimates of D, λ and K for assays where epidermal growth factor (EGF) is applied in varying concentrations confirms that EGF enhances the rate of scratch closure and that this stimulation is driven by an increase in D and λ, whereas K is relatively unaffected by EGF.ConclusionsOur approach for estimating D, λ and K from an IncuCyte ZOOM™ assay provides more detail about cellular-level behaviour than standard methods for analysing these assays. In particular, our approach can be used to quantify the balance of cell migration and cell proliferation and, as we demonstrate, allow us to quantify how the addition of growth factors affects these processes individually.

Reproducibility of scratch assays is affected by the initial degree of confluence: Experiments, modelling and model selection.

Scratch assays are difficult to reproduce. Here we identify a previously overlooked source of variability which could partially explain this difficulty. We analyse a suite of scratch assays in which we vary the initial degree of confluence (initial cell density). Our results indicate that the rate of re-colonisation is very sensitive to the initial density. To quantify the relative roles of cell migration and proliferation, we calibrate the solution of the Fisher-Kolmogorov model to cell density profiles to provide estimates of the cell diffusivity, D, and the cell proliferation rate, λ. This procedure indicates that the estimates of D and λ are very sensitive to the initial density. This dependence suggests that the Fisher-Kolmogorov model does not accurately represent the details of the collective cell spreading process, since this model assumes that D and λ are constants that ought to be independent of the initial density. Since higher initial cell density leads to enhanced spreading, we also calibrate the solution of the Porous-Fisher model to the data as this model assumes that the cell flux is an increasing function of the cell density. Estimates of D and λ associated with the Porous-Fisher model are less sensitive to the initial density, suggesting that the Porous-Fisher model provides a better description of the experiments.

Logistic Proliferation of Cells in Scratch Assays is Delayed

Scratch assays are used to study how a population of cells re-colonises a vacant region on a two-dimensional substrate after a cell monolayer is scratched. These experiments are used in many applications including drug design for the treatment of cancer and chronic wounds. To provide insights into the mechanisms that drive scratch assays, solutions of continuum reaction–diffusion models have been calibrated to data from scratch assays. These models typically include a logistic source term to describe carrying capacity-limited proliferation; however, the choice of using a logistic source term is often made without examining whether it is valid. Here we study the proliferation of PC-3 prostate cancer cells in a scratch assay. All experimental results for the scratch assay are compared with equivalent results from a proliferation assay where the cell monolayer is not scratched. Visual inspection of the time evolution of the cell density away from the location of the scratch reveals a series of sigmoid curves that could be naively calibrated to the solution of the logistic growth model. However, careful analysis of the per capita growth rate as a function of density reveals several key differences between the proliferation of cells in scratch and proliferation assays. Our findings suggest that the logistic growth model is valid for the entire duration of the proliferation assay. On the other hand, guided by data, we suggest that there are two phases of proliferation in a scratch assay; at short time, we have a disturbance phase where proliferation is not logistic, and this is followed by a growth phase where proliferation appears to be logistic. These two phases are observed across a large number of experiments performed at different initial cell densities. Overall our study shows that simply calibrating the solution of a continuum model to a scratch assay might produce misleading parameter estimates, and this issue can be resolved by making a distinction between the disturbance and growth phases. Repeating our procedure for other scratch assays will provide insight into the roles of the disturbance and growth phases for different cell lines and scratch assays performed on different substrates.

Repositioning “old” drugs for new causes: identifying new inhibitors of prostate cancer cell migration and invasion

The majority of prostate cancer (PCa) deaths occur due to the metastatic spread of tumor cells to distant organs. Currently, there is a lack of effective therapies once tumor cells have spread outside the prostate. It is therefore imperative to rapidly develop therapeutics to inhibit the metastatic spread of tumor cells. Gain of cell motility and invasive properties is the first step of metastasis and by inhibiting motility one can potentially inhibit metastasis. Using the drug repositioning strategy, we developed a cell-based multi-parameter primary screening assay to identify drugs that inhibit the migratory and invasive properties of metastatic PC-3 PCa cells. Following the completion of the primary screening assay, 33 drugs were identified from an FDA approved drug library that either inhibited migration or were cytotoxic to the PC-3 cells. Based on the data obtained from the subsequent validation studies, mitoxantrone hydrochloride, simvastatin, fluvastatin and vandetanib were identified as strong candidates that can inhibit both the migration and invasion of PC-3 cells without significantly affecting cell viability. By employing the drug repositioning strategy instead of a de novo drug discovery and development strategy, the identified drug candidates have the potential to be rapidly translated into the clinic for the management of men with aggressive forms of PCa.

MUC13 overexpression in renal cell carcinoma plays a central role in tumor progression and drug resistance

Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti‐apoptotic proteins, BCL‐xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi‐kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1−/−) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1−/−and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1−/− mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 ± 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 ± 0.12 ng/ml and 2.91 ± 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1−/− HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1−/− HFD-fed mice, but not in NOD/SCID mice. In Rag1−/− HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of long-term Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1−/− mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia.

Mechanistic and experimental models of cell migration reveal the importance of intercellular interactions in cell invasion

Moving fronts of cells are essential for development, repair and disease progression. Therefore, understanding and quantifying the details of the mechanisms that drive the movement of cell fronts is of wide interest. Quantitatively identifying the role of intercellular interactions, and in particular the role of cell pushing, remains an open question. Indeed, perhaps the most common continuum mathematical idealization of moving cell fronts is to treat the population of cells, either implicitly or explicitly, as a population of point particles undergoing a random walk that neglects intercellular interactions. In this work, we report a combined experimental-modelling approach showing that intercellular interactions contribute significantly to the spatial spreading of a population of cells. We use a novel experimental data set with PC-3 prostate cancer cells that have been pretreated with Mitomycin-C to suppress proliferation. This allows us to experimentally separate the effects of cell migration from cell proliferation, thereby enabling us to focus on the migration process in detail as the population of cells recolonizes an initially-vacant region in a series of two-dimensional experiments. We quantitatively model the experiments using a stochastic modelling framework, based on Langevin dynamics, which explicitly incorporates random motility and various intercellular forces including: (i) long range attraction (adhesion); and (ii) finite size effects that drive short range repulsion (pushing). Quantitatively comparing the ability of this model to describe the experimentally observed population-level behaviour provides us with quantitative insight into the roles of random motility and intercellular interactions. To quantify the mechanisms at play, we calibrate the stochastic model to match experimental cell density profiles to obtain estimates of cell diffusivity, D, and the amplitude of intercellular forces, f0. Our analysis shows that taking a standard modelling approach which ignores intercellular forces provides a poor match to the experimental data whereas incorporating intercellular forces, including short-range pushing and longer range attraction, leads to a faithful representation of the experimental observations. These results demonstrate a significant role for intercellular interactions in cell invasion. Author summary Moving cell fronts are routinely observed in various physiological processes, such as wound healing, malignant invasion and embryonic morphogenesis. We explore the effects of a previously overlooked mechanism that contributes to population-level front movement: pushing. Our framework is flexible and incorporates range of reasonable biological phenomena, such as random motility, cell-to-cell adhesion, and pushing. We find that neglecting finite size effects and intercellular forces, such as cell pushing, reduces our ability to mimic and predict our experimental observations.

No effect of administration of unacylated ghrelin on subcutaneous PC3 xenograft growth in a Rag1-/- mouse model of metabolic dysfunction

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1−/− mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 μg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1−/− mice on a high-fat diet, but did not significantly improve other metabolic parameters. We hypothesise that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome. Conflict of interest The authors declare no conflict of interest.

Abstract 3491: A role for the long non-coding RNAGHRLOSin cancer

Long non-coding RNA (lncRNA) genes are abundant in the human genome, and many are now recognized as oncogenes and/or tumor suppressors. We previously characterized the structure of GHRLOS, an antisense gene on the opposite strand of the multifunctional ghrelin gene (GHRL), however, its expression and function in disease has not been described. To this end, using The Cancer Genome Atlas (TCGA) data set, we revealed that GHRLOS is differentially expressed in a number of cancers. In particular, expression was elevated in endometrial cancer (1.2 - fold; P = 7.1 x 10 -3 Welch9s two-sample t-test; n = 24 vs. n = 175) and prostate cancer (1.2-fold; P = 3.7 x 10 -6 ; n = 52 vs. n = 498) compared to normal tissues. Using qRT-PCR (and commercial cDNA panels) we confirmed that GHRLOS expression was upregulated in endometrial cancer (1.96-fold, P = 0.005 Welch’s two-sample t-test, n = 5 vs. n = 17) and prostate cancer (2.46-fold, P = 0.0045 Welch’s two-sample t-test, n = 5 vs. n = 21) compared to normal tissues. Initial studies, using siRNA designed to silence endogenous GHRLOS expression, significantly reduced cell migration in the PC3 prostate cancer cell line (0.47-fold change, P = 0.042 Kruskal-Wallis test, n = 2) conversely, preliminary data using forced GHRLOS overexpression increased migration and proliferation. Taken together, we show that the long non-coding RNA GHRLOS is differentially expressed in tumor tissue and regulates cell migration and proliferation; possibly by modulating alternative splicing of the overlapping, multifunctional ghrelin gene locus. Targeting GHRLOS could provide a valuable and novel way to target the ghrelin axis in disease. Ongoing studies aim to validate in vitro functional results in complementary mouse xenograft models and identify genes and pathways regulated by this novel lncRNA. Citation Format: Esha T. Shah, Penny Jeffery, Patrick Thomas, Inge Seim, Lisa Chopin. A role for the long non-coding RNA GHRLOS in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2017-3491