Acquired resistance to EGFR tyrosine kinase inhibitors alters the metabolism of human head and neck squamous carcinoma cells and xenograft tumours.

BACKGROUND: Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management. METHODS: Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance. RESULTS: Comparison of NMR metabolite profiles obtained from control (CAL(S)) and EGFR TKI-resistant (CAL(R)) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CAL(R) relative to CAL(S) monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism. CONCLUSIONS: Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

Acute tumour response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) evaluated by non-invasive diffusion-weighted MRI.

BACKGROUND: Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts. METHODS: Nude mice bearing human BRAF(V600D) WM266.4 melanoma or BRAF(V600E) Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis. RESULTS: Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls. CONCLUSION: Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.

Size selectivity of hyaluronan molecular sieving by extracellular matrix in rabbit synovial joints.

In joint fluid the polymer hyaluronan (HA) confers viscous lubrication and greatly attenuates trans-synovial fluid loss (outflow buffering). Outflow buffering arises from the molecular sieving (reflection) and concentration polarization of HA at the synovial membrane surface. Outflow buffering declines if HA chain length is reduced, as in arthritis, and this has been attributed to reduced HA reflection. This was tested directly in the present study. Infused solutions of HA of approximately 2200 kDa (HA2000, 0.2 mg ml(-1)) or approximately 500 kDa (HA500, 0.2 mg ml(-1)) or approximately 140 kDa (HA140, 0.2-4.0 mg ml(-1)) were filtered across the synovial lining of the knee joint cavity of anaesthetized rabbits at a constant rate, along with a freely permeating reference solute, 20 kDa fluorescein-dextran (FD20). After a priming period the femoral lymph was sampled over 3 h. Mixed intra-articular (i.a.) fluid and subsynovial fluid were sampled at the end. Fluids were analysed by gel exclusion chromatography. The trans-synovial concentration profile was found to depend on polymer size. The i.a. concentration of HA2000 increased substantially relative to infusate and the subsynovial and lymph concentrations fell substantially. For HA500 and HA140 the trans-synovial concentration gradients were less pronounced, and absent for FD. The reflected fractions for HA2000, HA500 and HA140 across the cavity-to-lymph barrier were 0.65 +/- 0.05 (n = 10), 0.43 +/- 0.09 (n = 3) and 0.19 +/- 0.05 (n = 7), respectively, at matched filtration rates (P < 0.0001, analysis of variance). Reflected fractions calculated from HA i.a. accumulation or subsynovial dilution showed the same trend. The results demonstrate size-selective molecular sieving by the synovial extracellular matrix, equivalent to steric exclusion from cylindrical pores of radius 33-59 nm. The findings underpin the concentration polarization-outflow buffering theory and indicate that reduced HA chain length in arthritis exacerbates lubricant loss from a joint.