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The use of tissue-engineered 3D models of cancer has grown in popularity with recent advances in the field of cancer research. 3D models are inherently more biomimetic compared to 2D cell monolayers cultured on tissue-culture plastic. Nevertheless 3D models still lack the cellular and matrix complexity of native tissues. This review explores different 3D models currently used, outlining their benefits and limitations. Specifically, this review focuses on stiffness and collagen density, compartmentalization, tumor-stroma cell population and extracellular matrix composition. Furthermore, this review explores the methods utilized in different models to directly measure cancer invasion and growth. Of the models evaluated, with PDX and in vivo as a relative “gold standard”, tumoroids were deemed as comparable 3D cancer models with a high degree of biomimicry, in terms of stiffness, collagen density and the ability to compartmentalize the tumor and stroma. Future 3D models for different cancer types are proposed in order to improve the biomimicry of cancer models used for studying disease progression.

Increased levels and diversity of human endogenous retrovirus (HERV) transcription characterize most cancer types and are linked with disease outcomes. However, the underlying processes are incompletely understood. Here, we show that elevated transcription of HERVH proviruses predicted survival of lung squamous cell carcinoma (LUSC) and identified an isoform of CALB1, encoding calbindin, ectopically driven by an upstream HERVH provirus under the control of KLF5, as the mediator of this effect. HERVH-CALB1 expression was initiated in preinvasive lesions and associated with their progression. Calbindin loss in LUSC cell lines impaired in vitro and in vivo growth and triggered senescence, consistent with a protumor effect. However, calbindin also directly controlled the senescence-associated secretory phenotype (SASP), marked by secretion of CXCL8 and other neutrophil chemoattractants. In established carcinomas, CALB1-negative cancer cells became the dominant source of CXCL8, correlating with neutrophil infiltration and worse prognosis. Thus, HERVH-CALB1 expression in LUSC may display antagonistic pleiotropy, whereby the benefits of escaping senescence early during cancer initiation and clonal competition were offset by the prevention of SASP and protumor inflammation at later stages.

Background Transcriptional activation of otherwise repressed retrotransposable elements (RTEs) is a hallmark of cancer, shaping tumour progression and immunogenicity by multifaceted, yet incompletely understood, mechanisms. Methods We used an extended pan-cancer transcriptome assembly to identify potential effects of RTEs on the genes within which they have integrated or those in proximity. These were subsequently verified in test cases by further analysis of transcriptional profiles in cancer patient data, and by in vitro studies involving restoration of gene activity, and proliferation and migration assays in cancer cell lines. Results We report that cancer-specific transcriptional activation of RTEs causes frequent reduction or loss of gene function. Exonisation and alternative splicing of RTEs creates non-functional RNA and protein isoforms and derepressed RTE promoter activity initiates antisense transcription, both at the expense of the canonical isoforms. Contrary to theoretical expectation, transcriptionally activated RTEs affect genes with established tumour-promoting functions, including the common essential RNGTT and the lung cancer-promoting CHRNA5 genes. Furthermore, the disruptive effect of RTE activation on adjacent tumour-promoting genes is associated with slower disease progression in clinical data, whereas experimental restoration of gene activity enhances tumour cell growth and invasiveness in vitro. Conclusions These findings underscore the gene-disruptive potential of seemingly innocuous germline RTE integrations, unleashed only by their transcriptional utilisation in cancer. They further suggest that such metastable RTE integrations are co-opted as sensors of the epigenetic and transcriptional changes occurring during cellular transformation and as executors that disrupt the function of tumour-promoting genes.

Ameloblastoma is a benign, epithelial cancer of the jawbone, which causes bone resorption and disfigurement to patients affected. The interaction of ameloblastoma with its tumour stroma drives invasion and progression. We used stiff collagen matrices to engineer active bone forming stroma, to probe the interaction of ameloblastoma with its native tumour bone microenvironment. This bone-stroma was assessed by nano-CT, transmission electron microscopy (TEM), Raman spectroscopy and gene analysis. Furthermore, we investigated gene correlation between bone forming 3D bone stroma and ameloblastoma introduced 3D bone stroma. Ameloblastoma cells increased expression of MMP-2 and -9 and RANK temporally in 3D compared to 2D. Our 3D biomimetic model formed bone nodules of an average surface area of 0.1 mm 2 and average height of 92.37 ± 7.96 μm over 21 days. We demonstrate a woven bone phenotype with distinct mineral and matrix components and increased expression of bone formation genes in our engineered bone. Introducing ameloblastoma to the bone stroma, completely inhibited bone formation, in a spatially specific manner. Multivariate gene analysis showed that ameloblastoma cells downregulate bone formation genes such as RUNX2 . Through the development of a comprehensive bone stroma, we show that an ameloblastoma tumour mass prevents osteoblasts from forming new bone nodules and severely restricted the growth of existing bone nodules. We have identified potential pathways for this inhibition. More critically, we present novel findings on the interaction of stromal osteoblasts with ameloblastoma.

Background Cancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling. Methods We utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a “healthy” or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling. Results CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin). Conclusions These results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.

Background Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. Results In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T ( FT ) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A - 1 ( PROTEIN PHOSPHATASE 2A - 1 ), LsPP2AA3 ( PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3 ) and LsTIP41 ( TAP42 - INTERACTING PROTEIN OF 41   kDa ), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A - 1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A - 1 , LsPP2AA3 and LsTIP41 , would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. Conclusions Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments.

The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis.

Epithelial to mesenchymal transition (EMT) in cancer is the process described where cancer epithelial cells acquire mesenchymal properties which can lead to enhanced invasiveness. Three-dimensional cancer models often lack the relevant and biomimetic microenvironment parameters appropriate to the native tumour microenvironment thought to drive EMT. In this study, HT-29 epithelial colorectal cells were cultivated in different oxygen and collagen concentrations to investigate how these biophysical parameters influenced invasion patterns and EMT. Colorectal HT-29 cells were grown in physiological hypoxia (5% O2) and normoxia (21% O2) in 2D, 3D soft (60 Pa), and 3D stiff (4 kPa) collagen matrices. Physiological hypoxia was sufficient to trigger expression of markers of EMT in the HT-29 cells in 2D by day 7. This is in contrast to a control breast cancer cell line, MDA-MB-231, which expresses a mesenchymal phenotype regardless of the oxygen concentration. In 3D, HT-29 cells invaded more extensively in a stiff matrix environment with corresponding increases in the invasive genes MMP2 and RAE1. This demonstrates that the physiological environment can directly impact HT-29 cells in terms of EMT marker expression and invasion, compared to an established cell line, MDA-MB-231, which has already undergone EMT. This study highlights the importance of the biophysical microenvironment to cancer epithelial cells and how these factors can direct cell behaviour. In particular, that stiffness of the 3D matrix drives greater invasion in HT-29 cells regardless of hypoxia. It is also pertinent that some cell lines (already having undergone EMT) are not as sensitive to the biophysical features of their microenvironment.

Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer (“tumouroid”) model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In terms of drug response, the IC50 for paclitaxel for MIA Paca-2 cells increased from 0.819 nM in 2D to 3.02 nM in ACMs and to 5.87 nM and 3.803 nM in simple and complex tumouroids respectively, indicating that drug penetration may be significantly reduced in the latter. The results demonstrate the need for biomimetic models during initial drug testing and evaluation.

Ameloblastoma is a benign, locally invasive epithelial odontogenic neoplasm of the jaw. Treatment of choice is jaw resection, often resulting in significant morbidity. The aim of this study was to recapitulate ameloblastoma in a completely humanised 3D disease model containing ameloblastoma cells, osteoblasts and activated osteoclasts to investigate the RANKL pathway within the ameloblastoma stromal environment and its response to the RANKL antibody denosumab. In vitro bone was engineered by culturing human osteoblasts (hOB) in a biomimetic, dense collagen type I matrix, resulting in extensive mineral deposits by day 21 forming alizarin red positive bone like nodules throughout the 3D model. Activated TRAP + human osteoclasts were confirmed through the differentiation of human CD14+ monocytes after 10 days within the model. Lastly, the ameloblastoma cell lines AM-1 and AM-3 were incorporated into the 3D model. RANKL release was validated through TACE/ADAM17 activation chemically or through hOB co-culture. Denosumab treatment resulted in decreased osteoclast activation in the presence of hOB and ameloblastoma cells. These findings stress the importance of accurately modelling tumour and stromal populations as a preclinical testing platform.