PCaAnalyser: A 2D-Image Analysis Based Module for Effective Determination of Prostate Cancer Progression in 3D Culture

Three-dimensional (3D) in vitro cell based assays for Prostate Cancer (PCa) research are rapidly becoming the preferred alternative to that of conventional 2D monolayer cultures. 3D assays more precisely mimic the microenvironment found in vivo, and thus are ideally suited to evaluate compounds and their suitability for progression in the drug discovery pipeline. To achieve the desired high throughput needed for most screening programs, automated quantification of 3D cultures is required. Towards this end, this paper reports on the development of a prototype analysis module for an automated high-content-analysis (HCA) system, which allows for accurate and fast investigation of in vitro 3D cell culture models for PCa. The Java based program, which we have named PCaAnalyser, uses novel algorithms that allow accurate and rapid quantitation of protein expression in 3D cell culture. As currently configured, the PCaAnalyser can quantify a range of biological parameters including: nuclei-count, nuclei-spheroid membership prediction, various function based classification of peripheral and non-peripheral areas to measure expression of biomarkers and protein constituents known to be associated with PCa progression, as well as defining segregate cellular-objects effectively for a range of signal-to-noise ratios. In addition, PCaAnalyser architecture is highly flexible, operating as a single independent analysis, as well as in batch mode; essential for High-Throughput-Screening (HTS). Utilising the PCaAnalyser, accurate and rapid analysis in an automated high throughput manner is provided, and reproducible analysis of the distribution and intensity of well-established markers associated with PCa progression in a range of metastatic PCa cell-lines (DU145 and PC3) in a 3D model demonstrated

Induction of Reactive Bone Stromal Fibroblasts in 3D Models of Prostate Cancer Bone Metastases

A dynamic interplay between prostate cancer (PCa) cells and reactive bone stroma modulates the growth of metastases within the bone microenvironment. Of the stromal cells, metastasis-associated fibroblasts (MAFs) are known to contribute but are the least studied cell type in PCa tumour progression. It is the aim of the current study to establish a biologically relevant 3D in vitro model that mimics the cellular and molecular profiles of MAFs found in vivo. Using 3D in vitro cell culture models, the bone-derived fibroblast cell line, HS-5, was treated with conditioned media from metastatic-derived PCa cell lines, PC3 and MDA-PCa 2b, or mouse-derived fibroblasts 3T3. Two corresponding reactive cell lines were propagated: HS5-PC3 and HS5-MDA, and evaluated for alterations in morphology, phenotype, cellular behaviour, plus protein and genomic profiles. HS5-PC3 and HS5-MDA displayed distinct alterations in expression levels of N-Cadherin, non-functional E-Cadherin, alpha-smooth muscle actin (α-SMA), Tenascin C, and vimentin, along with transforming growth factor receptor expression (TGF β R1 and R2), consistent with subpopulations of MAFs reported in vivo. Transcriptomic analysis revealed a reversion of HS5-PC3 towards a metastatic phenotype with an upregulation in pathways known to regulate cancer invasion, proliferation, and angiogenesis. The exploitation of these engineered 3D models could help further unravel the novel biology regulating metastatic growth and the role fibroblasts play in the colonisation process

Chemokine receptor expression on integrin-mediated stellate projections of prostate cancer cells in 3D culture

The chemokine receptor CXCR7 has emerged as a regulator of prostate tumor growth and invasion, along with the well-established role of its closely related receptor, CXCR4, and their shared ligand, SDF-1α. Consequently, inhibition of the CXCR7/CXCR4/SDF-1α axis may assist in controlling prostate tumor growth and progression. To facilitate the development of potential therapeutics, further clarification of CXCR7 function is required, specifically in relation to CXCR4. In this study, we report that CXCR7 and CXCR4 were co-expressed in LNCaP, DU145 and PC3 cell lines in 2D culture. When cultured in 3D using Matrigel, a marked up-regulation of both receptors was observed in PC3 cells. Interestingly, both CXCR7 and CXCR4 co-localized within radiating cellular structures, termed stellate projections, which protruded outward into the matrix. The stellate projections were rich in the expression of pro-invasive integrin β1, β-laminin and MMP-11 proteins. The development of the stellate projections was mediated by integrin β1-mediated interactions with the ECM, which also regulated the expression of CXCR7 and CXCR4. Taken together, these results demonstrate that integrin-mediated cell-ECM interactions can modulate tumor cell morphology, and regulate the expression of chemokine receptors which are associated with the invasive phenotype and progression of PCa

Regulation of the chemokine receptors CXCR7 and CXCR4 in 3-D culture models of prostate cancer

CXCR7 was recently identified as the second member of the chemokine receptor family to bind stromal derived factor-1α (SDF-1α), a chemokine which is known to influence the establishment of cancer metastasis. Whilst expression of CXCR7 is highly restricted in non-malignant cells, it is widely expressed in many different tumor cell lines. In prostate cancer, a disease known to be highly regulated by the other SDF-1α -binding receptor CXCR4, CXCR7 has been found to regulate cell growth and invasion. In vivo prostate tumor biopsies show a pattern where CXCR7 expression increases with invasive grade, as previously reported for CXCR4. However, there is limited knowledge on the role of CXCR7 and its function in prostate cancer. In this study, we aim to more thoroughly characterize CXCR7 function across prostate cancer cell lines, in particular its regulation of cell growth and behavior. We will also assess its expression and function in response to its ligands and inhibitors, alongside CXCR4, in order to assess how these receptors are regulated in relation to each other. For this we employ western blotting expression studies, immunocytochemical visualization, and metabolic-based proliferation assays. Further, we will study the regulation of both CXCR7 and CXCR4 in 3D culture models of prostate cancer cell lines. Our preliminary data from 2D culture suggests that less invasive prostate cancer cell lines express higher levels of CXCR7 than more invasive cell lines, contrary to reports in vivo where CXCR7 expression was seen to increase with invasive grade. We have chosen to assess these aspects in 3D cultures of prostate cancer cell lines to determine whether culturing in 3D permits a phenotype more reflective of what has been reported for CXCR7 expression in prostate cancer in vivo. Further elucidation of CXCR7 function with respect to CXCR4 will shed light on how these receptors contribute to regulation of the metastatic process in prostate cancer – a process known to be heavily regulated by CXCR4. As CXCR4 has been established as a therapeutic target in prostate cancer, a more detailed knowledge of the CXCR7 receptor with which it shares a partial redundancy may indicate whether combinatorial therapies may be more effective in combating prostate cancer progression

In vivo biomarker expression patterns are preserved in 3D cultures of Prostate Cancer

Here we report that Prostate Cancer (PCa) cell-lines DU145, PC3, LNCaP and RWPE-1 grown in 3D matrices in contrast to conventional 2D monolayers, display distinct differences in cell morphology, proliferation and expression of important biomarker proteins associated with cancer progression. Consistent with in vivo growth rates, in 3D cultures, all PCa cell-lines were found to proliferate at significantly lower rates in comparison to their 2D counterparts. Moreover, when grown in a 3D matrix, metastatic PC3 cell-lines were found to mimic more precisely protein expression patterns of metastatic tumour formation as found in vivo. In comparison to the prostate epithelial cell-line RWPE-1, metastatic PC3 cell-lines exhibited a down-regulation of E-cadherin and α6 integrin expression and an up-regulation of N-cadherin, Vimentin and β1 integrin expression and re-expressed non-transcriptionally active AR. In comparison to the non-invasive LNCaP cell-lines, PC3 cells were found to have an up-regulation of chemokine receptor CXCR4, consistent with a metastatic phenotype. In 2D cultures, there was little distinction in protein expression between metastatic, non-invasive and epithelial cells. These results suggest that 3D cultures are more representative of in vivo morphology and may serve as a more biologically relevant model in the drug discovery pipeline

PCaAnalyser: a 2D-image analysis based module for effective determination of prostate cancer progression in 3D culture.

Three-dimensional (3D) in vitro cell based assays for Prostate Cancer (PCa) research are rapidly becoming the preferred alternative to that of conventional 2D monolayer cultures. 3D assays more precisely mimic the microenvironment found in vivo, and thus are ideally suited to evaluate compounds and their suitability for progression in the drug discovery pipeline. To achieve the desired high throughput needed for most screening programs, automated quantification of 3D cultures is required. Towards this end, this paper reports on the development of a prototype analysis module for an automated high-content-analysis (HCA) system, which allows for accurate and fast investigation of in vitro 3D cell culture models for PCa. The Java based program, which we have named PCaAnalyser, uses novel algorithms that allow accurate and rapid quantitation of protein expression in 3D cell culture. As currently configured, the PCaAnalyser can quantify a range of biological parameters including: nuclei-count, nuclei-spheroid membership prediction, various function based classification of peripheral and non-peripheral areas to measure expression of biomarkers and protein constituents known to be associated with PCa progression, as well as defining segregate cellular-objects effectively for a range of signal-to-noise ratios. In addition, PCaAnalyser architecture is highly flexible, operating as a single independent analysis, as well as in batch mode; essential for High-Throughput-Screening (HTS). Utilising the PCaAnalyser, accurate and rapid analysis in an automated high throughput manner is provided, and reproducible analysis of the distribution and intensity of well-established markers associated with PCa progression in a range of metastatic PCa cell-lines (DU145 and PC3) in a 3D model demonstrated

Advanced read-map generation.

<p>The major steps involved in generating the classified read-map via boundary-mask and threshold-mask. Images depict a DU145 spheroid grown in a 3D matrix following immuno-labelling for the α6 integrin subunit. Panels in this figure refer to the intensity of the antibody and distribution of the α6 integrin subunit. Labelling was present primarily in the peripheral region of the spheroid structure.</p

Tabbed-pane for defining parameters.

<p>One of the five tabbed sections related to the ‘mask-generation’ is visible.</p

Pseudo code of Algorithm 1.

<p>Major steps of the spheroid detection algorithm. Variables with sample values are placed within the angle brackets (i.e. < … >).</p

Pseudo code of Algorithm 3.

<p>The major steps in detecting spheroid-membership of a nucleus are shown.</p