Gemma Marston

Non‐invasive molecular imaging for preclinical cancer therapeutic development

Molecular and non‐invasive imaging are rapidly emerging fields in preclinical cancer drug discovery. This is driven by the need to develop more efficacious and safer treatments, the advent of molecular‐targeted therapeutics, and the requirements to reduce and refine current preclinical in vivo models. Such bioimaging strategies include MRI, PET, single positron emission computed tomography, ultrasound, and optical approaches such as bioluminescence and fluorescence imaging. These molecular imaging modalities have several advantages over traditional screening methods, not least the ability to quantitatively monitor pharmacodynamic changes at the cellular and molecular level in living animals non‐invasively in real time. This review aims to provide an overview of non‐invasive molecular imaging techniques, highlighting the strengths, limitations and versatility of these approaches in preclinical cancer drug discovery and development.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits mouse MC-26 colorectal cancer cell liver metastasis via inhibition of PGE2-dependent cell motility

The omega‐3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti‐CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E‐type PG synthesis.

CEA-targeted nanoparticles allow specific in vivo fluorescent imaging of colorectal cancer models

Fluorescent imaging of colorectal tumor cells would improve tumor localization and allow intra-operative staging, facilitating stratification of surgical resections thereby improving patient outcomes. We aimed to develop and test fluorescent nanoparticles capable of allowing this in vivo. Dye-doped silica nanoparticles were synthesized. Anti-CEA (carcinoembryonic antigen) or control IgGs were conjugated to nanoparticles using various chemical strategies. Binding of CEA-targeted or control nanoparticles to colorectal cancer cells was quantified in vitro, and in vivo after systemic-delivery to murine xenografts. CEA-targeted, polyamidoamine dendrimer-conjugated, nanoparticles, but not control nanoparticles, allowed strong tumor-specific imaging. We are the first to demonstrate live, specific, in vivo imaging of colorectal cancer cells using antibody-targeted fluorescent nanoparticles. These nanoparticles have potential to allow intra-operative fluorescent visualization of tumor cells.

Reduced type II interleukin-4 receptor signalling drives initiation, but not progression, of colorectal carcinogenesis: evidence from transgenic mouse models and human case–control epidemiological observations

We investigated the role of interleukin (IL)-4 receptor (IL-4R) signalling during mouse carcinogen-induced colorectal carcinogenesis and in a case–control genetic epidemiological study of IL-4Rα single nucleotide polymorphisms (SNPs). Azoxymethane-induced aberrant crypt focus (ACF; 6 weeks) and tumours (32 weeks) were analysed in wild-type (WT) BALB/c mice, as well as in IL-4Rα − /−, IL-13 −/− and ‘double-knockout’ (DKO) animals. Colorectal cancer (CRC) cases (1502) and controls (584) were genotyped for six coding IL-4Rα SNPs. The association with CRC risk and CRC-specific mortality was analysed by logistic regression. Lack of IL-4Rα expression was associated with increased ACFs [median 8.5 ACFs per mouse (IL-4Rα −/−) versus 3 (WT); P = 0.007], but no difference in the number of colorectal tumours [mean 1.4 per mouse (IL-4Rα −/−) versus 2 (WT)], which were smaller and demonstrated reduced nuclear/cytoplasmic β-catenin translocation compared with WT tumours. Tumour-bearing IL-4Rα −/− mice had fewer CD11b+/Gr1+ myeloid-derived suppressor splenocytes than WT animals. IL-13 −/− mice developed a similar number of ACFs to IL-4Rα −/− and DKO mice. There was a significant increase in CRC risk associated with the functional SNP Q576R [odds ratio 1.54 (95% confidence interval 0.94–2.54), P trend 0.03 for the minor G allele]. There was no effect of IL-4Rα genotype on either CRC-specific or all-cause mortality. These combined pre-clinical and human data together demonstrate that reduced IL-4R signalling has stage-specific effects on colorectal carcinogenesis (increased CRC initiation and risk but reduced tumour progression and no effect on CRC mortality). These results should prompt evaluation of the effect of pharmacological manipulation of IL-4R signalling on future CRC risk and for CRC treatment.

Changes in plasma chemokine C-C motif ligand 2 levels during treatment with eicosapentaenoic acid predict outcome in patients undergoing surgery for colorectal cancer liver metastasis.

The mechanism of the anti-colorectal cancer (CRC) activity of the omega-3 fatty acid eicosapentaenoic acid (EPA) is not understood. We tested the hypothesis that EPA reduces expression of chemokine C-C motif ligand 2 (CCL2), a pro-inflammatory chemokine with known roles in metastasis. We measured CCL2 in clinical samples from a randomized trial of EPA in patients undergoing liver surgery for CRC liver metastasis (LM) and preclinical models. Genome-wide transcriptional profiling of tumors from EPA-treated patients was performed. EPA decreased CCL2 synthesis by CRC cells in a dose-dependent manner. CCL2 was localized to malignant epithelial cells in human CRCLM. EPA did not reduce CCL2 content in human or mouse tumors compare to control. However, EPA treatment was associated with decreased plasma CCL2 levels compared with controls (P=0.04). Reduction in plasma CCL2 following EPA treatment predicted improved disease-free survival (HR 0.32; P=0.003). Lack of ‘CCL2 response’ was associated with a specific CRCLM gene expression signature. In conclusion, reduction in plasma CCL2 in patients with CRCLM treated with EPA predicts better clinical outcome and a specific tumor gene expression profile. Further work is needed to validate CCL2 as a therapeutic response biomarker for omega-3 fatty acid treatment of CRC patients.

High-frequency ultrasound for in vivo measurement of colon wall thickness in mice.

Mouse models are becoming increasingly important in the study of molecular mechanisms of colorectal disease and in the development of novel therapeutics. To enhance this phase of preclinical research, cost-effective, easy to use noninvasive imaging is required to detect and monitor changes in the colon wall associated with disease pathology. This study investigated the feasibility of using 40-MHz (high frequency) B-mode ultrasound (HF-US) to image the normal mouse colon and measure its thickness in vivo by establishing a robust imaging protocol and conducting a blinded comparison of colon wall thickness (CWT) measurement between and within operators. The in vivo and ex vivo appearance of mouse colon under HF-US revealed distinct patterns. Colon wall thickness was reproducibly and accurately measured using HF-US compared with histology measurement. The technique was more sensitive in detecting changes in CWT in distal than proximal colon as it showed the highest level of inter- and intraoperator reproducibility. Using the protocol described, it is possible to detect changes in thickness of 0.09 mm and 0.25 mm in distal and proximal colon, respectively. In conclusion, HF-US provides an easy to use and noninvasive method to perform anatomical investigations of mouse colon and to monitor changes in CWT.

The use of high-frequency ultrasound imaging and biofluorescence for in vivo evaluation of gene therapy vectors

BackgroundNon-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential.MethodsThis study assessed the utility of high-frequency ultrasound (HF-US) combined with biofluoresence imaging (BFI) to determine the longitudinal impact of a Herpesvirus saimiri amplicon on human colorectal cancer xenograft growth.ResultsHF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. The volumes correlated better with final ex vivo volume than mechanical callipers (R2 = 0.7993, p = 0.0002 vs. R2 = 0.7867, p = 0.0014). HF-US showed that the amplicon caused lobe formation. BFI demonstrated retention and expression of the amplicon in the xenografts and quantitation of the fluorescence levels also correlated with tumour volumes.ConclusionsThe use of multi-modal imaging provided useful and enhanced insights into the behaviour of gene therapy vectors in vivo in real-time. These relatively inexpensive technologies are easy to incorporate into pre-clinical studies.

Abstract 1572: Characterising the tumor vasculature in vivo in mouse models of colorectal cancer using contrast enhanced high-frequency ultrasound

Angiogenesis is essential for tumours to grow beyond a few millimetres in size and to metastasise, making it an attractive target for both cancer imaging and therapeutic intervention. Although microvessel density (MVD) is routinely used to assess tumour vascularity in fixed tissue, few techniques are currently available for imaging and quantitating tumour vasculature parameters in vivo. High-frequency ultrasound (HFUS) allows high resolution, non-invasive longitudinal imaging of tumours in vivo. Incorporation of microbubble contrast agent into imaging protocols allows visualisation of the vasculature, determination of relative blood flow and perfusion, and the ability to directly target and quantify molecular biomarkers of disease in vivo. We have been investigating in vivo tumour blood flow and vascularity in the growth of human colorectal cancer (CRC) cell xenografts. CD1 Nu/Nu mice implanted with SW480 cell xenografts were used to investigate the relationship between tumour growth and different vascular parameters obtained by contrast enhanced (CE) HFUS. Mice were imaged at day 21 and day 27 of tumour growth using the VisualSonics Vevo 770 system. A single bolus of microbubbles was injected via a syringe driver into the tail vein during 2D CE HFUS imaging, with the wash-in data loops captured prior to 3D CE HFUS imaging, and tissue samples collected for histology and immunohistochemistry. Time intensity curves were generated post-acquisition and used to determine perfusion kinetics and vascular data. A number of strongly significant correlations were observed relating in vivo and histological tumour characteristics with relative blood flow and total vascularity. These included the size of the tumour, tumour growth and the total vascular space. This study shows that CE HFUS is a powerful qualitative and quantitative tool for the analysis of tumour angiogenesis in vivo. It provides a relatively non-invasive method to monitor tumour blood flow and vascularity in longitudinal studies thus allowing effects on angiogenesis due to therapeutic intervention to be monitored. We are currently developing this technique for use with autochthonous colorectal cancer models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1572. doi:10.1158/1538-7445.AM2011-1572