A TRACER culture invasion assay to probe the impact of cancer associated fibroblasts on head and neck squamous cell carcinoma cell invasiveness.

Cancer associated fibroblasts (CAFs) are a major cellular component of the tumour stroma and have been shown to promote tumour cell invasion and disease progression. CAF-cancer cell interactions are bi-directional and occur via both soluble factor dependent and extracellular matrix (ECM) remodelling mechanisms, which are incompletely understood. Previously we developed the Tissue Roll for Analysis of Cellular Environment and Response (TRACER), a novel stacked paper tumour model in which cells embedded in a hydrogel are infiltrated into a porous cellulose scaffold that is then rolled around an aluminum core to generate a multi-layered 3D tissue. Here, we use the TRACER platform to explore the impact of CAFs derived from three different patients on the invasion of two head and neck squamous cell carcinoma (HNSCC) cell lines (CAL33 and FaDu). We find that co-culture with CAFs enhances HNSCC tumour cell invasion into an acellular collagen layer in TRACER and this enhanced migration occurs independently of proliferation. We show that CAF-enhanced invasion of CAL33 cells is driven by a soluble factor independent mechanism, likely involving CAF mediated ECM remodelling via matrix metalloprotenases (MMPs). Furthermore, we find that CAF-enhanced tumour cell invasion is dependent on the spatial pattern of collagen density within the culture. Our results highlight the utility of the co-culture TRACER platform to explore soluble factor independent interactions between CAFs and tumour cells that drive increased tumour cell invasion.

A three-dimensional engineered heterogeneous tumor model for assessing cellular environment and response.

This protocol describes how to build and implement a three-dimensional (3D) cell culture system, TRACER (tissue roll for analysis of cellular environment and response), that enables analysis of cellular behavior and phenotype in hypoxic gradients. TRACER consists of infiltrating cells encapsulated in a hydrogel extracellular matrix (ECM) within a thin strip of porous cellulose scaffolding that is then rolled around an oxygen-impermeable mandrel for assembly of thick and layered 3D tissue constructs that develop cell-defined oxygen gradients. TRACER differs from other stacked-paper cell culture models because it is assembled from a single-piece scaffold, which facilitates rapid disassembly for analysis of different cell populations and metabolites. The protocol describes how to fabricate TRACER components, cell seeding in the scaffold, and scaffold assembly and disassembly. Furthermore, it provides methods to quantify live, dead, or proliferating cells, as well as gradients of oxygen using the nitroimidazole derivative EF5, in a layer-by-layer analysis with confocal microscopy or by flow cytometry of cells isolated from the TRACER scaffold. Additional methods to isolate live cells from TRACER layers for dose-response analysis with a clonogenic assay, as well as steps to extract RNA or fast-changing metabolites from TRACER layers, are also presented. Finally, we provide alternative steps to establish TRACER co-cultures for assessment of tumor cell invasion and metastasis, in this case in the absence of a hypoxic gradient. Although analysis time varies according to the assay chosen, scaffold fabrication and seeding typically take 2 h, and TRACER assembly takes 20 min on the day following scaffold seeding. The TRACER platform is designed for use by researchers and students who have basic tissue culture experience.

The Current Landscape of 3D In Vitro Tumor Models: What Cancer Hallmarks Are Accessible for Drug Discovery?

Cancer prognosis remains a lottery dependent on cancer type, disease stage at diagnosis, and personal genetics. While investment in research is at an all-time high, new drugs are more likely to fail in clinical trials today than in the 1970s. In this review, a summary of current survival statistics in North America is provided, followed by an overview of the modern drug discovery process, classes of models used throughout different stages, and challenges associated with drug development efficiency are highlighted. Then, an overview of the cancer hallmarks that drive clinical progression is provided, and the range of available clinical therapies within the context of these hallmarks is categorized. Specifically, it is found that historically, the development of therapies is limited to a subset of possible targets. This provides evidence for the opportunities offered by novel disease-relevant in vitro models that enable identification of novel targets that facilitate interactions between the tumor cells and their surrounding microenvironment. Next, an overview of the models currently reported in literature is provided, and the cancer biology they have been used to explore is highlighted. Finally, four priority areas are suggested for the field to accelerate adoption of in vitro tumour models for cancer drug discovery.

A TRACER 3D Co-Culture tumour model for head and neck cancer.

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment and have been shown to play an important role in the progression of cancer. To probe these tumour-stroma interactions, we incorporated CAFs derived from head and neck cancer patients and squamous carcinoma cells of the hypopharynx (FaDu) into the Tissue Roll for the Analysis of Cellular Environment and Response (TRACER) platform to establish a co-culture platform that simulates the CAF-tumour microenvironmental interactions in head and neck tumours. TRACER culture involves infiltrating cells into a thin fibrous scaffold and then rolling the resulting biocomposite around a mandrel to generate a 3D and layered structure. Patterning the fibrous scaffold biocomposite during fabrication enables control over the specific location of different cell populations in the rolled configuration. Here, we optimized the seeding densities and configurations of the CAF and FaDu cell tissue sections to enable a robust 3D co-culture system under normoxic conditions. Co-culture of CAFs with FaDu cells produced negligible effects on radiation resistance, but did produce increases in proliferation rate and invasive cell migration at 24 and 48 h of culture. Our study provides the basis for use of our in vitro co-culture TRACER model to investigate the tumour-stroma interactions, and to bridge the translational gap between preclinical and clinical studies.

No effect of menstrual cycle phase on lactate threshold.

No previous exercise studies in women have assessed the effects of the normal menstrual cycle on the lactate threshold (LT) measured during a graded, maximal exercise test. This is relevant to our understanding of exercise training and metabolism in eumenorrheic women. The present study, therefore, examined the effect of menstrual cycle phase on the LT. Eight moderately active, eumenorrheic women performed three maximal exercise tests with simultaneous determination of LT. Tests were performed in the early follicular (low estrogen and progesterone), midfollicular (elevated estrogen and low progesterone), and midluteal (elevated estrogen and progesterone) phases of the menstrual cycle. No significant differences were observed in LT measured across phases of the menstrual cycle whether data were expressed in absolute terms (1299 +/- 70, 1364 +/- 80, and 1382 +/- 71 ml O(2)/min, respectively) or relative to maximal oxygen uptake (V(o2 max); 52.1 +/- 1.7, 54.7 +/- 1.7, and 55.7 +/- 1.6%, respectively). In addition, there were no significant cycle phase differences in V(o2 max), maximal heart rate, heart rate at LT, or final lactate concentration. With data combined across all phases of the menstrual cycle, there was a significant correlation between the LT and the epinephrine breakpoint (r = 0.91, P < 0.0002) and norepinephrine breakpoint (r = 0.94, P < 0.0001). For epinephrine only, there was close correspondence between the epinephrine breakpoint (ml O(2)/min) and the LT. In conclusion, LT as well as V(o2 max) and other measures of cardiorespiratory fitness are not significantly affected by the changing sex steroid levels observed across the normal menstrual cycle. Data suggest that the onset of the steep increase in epinephrine determines the LT during graded exercise.

Disparities in reproductive health-related visits to the emergency department in Maryland by age and race, 1999-2005.

OBJECTIVE: To describe reproductive health-related visits to Maryland emergency departments (EDs) among women aged 15-44 years from 1999 to 2005. METHODS: We obtained data from the Healthcare Cost and Utilization Project State Emergency Department Database and State Inpatient Database. ICD-9-CM diagnosis codes were used to classify reproductive health-related visits. We calculated the annual rate of reproductive health visits to Maryland EDs from 1999 to 2005 for women aged 15-44 years and tested time trends using linear regression. Admission rates were defined as the percentage of ED visits that resulted in inpatient admission. We calculated age-specific and race-specific rate ratios for diagnoses using Poisson regression and admission rate ratios using Cochran-Mantel-Haenszel statistics. RESULTS: From 1999 to 2005, the rate of ED visits in Maryland increased 50%, from 28.0 to 42.1 visits per 1000 women. Lower genital tract infections were the most common diagnosis (21.4%). The rates were higher for women aged 15-24 than for women aged 25-44 (rate ratio 1.18, 95% confidence interval [CI] 1.17-1.18) and nearly three times higher for black women than white women (rate ratio 2.94, 95% CI 2.92-2.96). Admission rates were lower for women aged 15-24 than for women aged 25-44 (rate ratio 0.34, 95% CI 0.33-0.35) and were higher among black than white women (rate ratio 1.16, 95% CI 1.14-1.18). CONCLUSIONS: Disparities by age and race are evident for reproductive health-related ED visits in Maryland, and many of these ED visits are for conditions that are amenable to preventive measures.