Measurement of the acute metabolic response to hypoxia in rat tumours in vivo using magnetic resonance spectroscopy and hyperpolarised pyruvate.

PURPOSE: To estimate the rate constant for pyruvate to lactate conversion in tumours in response to a hypoxic challenge, using hyperpolarised (13)C1-pyruvate and magnetic resonance spectroscopy. METHODS AND MATERIALS: Hypoxic inspired gas was used to manipulate rat P22 fibrosarcoma oxygen tension (pO2), confirmed by luminescence decay of oxygen-sensitive probes. Hyperpolarised (13)C1-pyruvate was injected into the femoral vein of anaesthetised rats and slice-localised (13)C magnetic resonance (MR) spectra acquired. Spectral integral versus time curves for pyruvate and lactate were fitted to a precursor-product model to estimate the rate constant for tumour conversion of pyruvate to lactate (kpl). Mean arterial blood pressure (MABP) and oxygen tension (ArtpO2) were monitored. Pyruvate and lactate concentrations were measured in freeze-clamped tumours. RESULTS: MABP, ArtpO2 and tumour pO2 decreased significantly during hypoxia. kpl increased significantly (p<0.01) from 0.029±0.002s(-1) to 0.049±0.006s(-1) (mean±SEM) when animals breathing air were switched to hypoxic conditions, whereas pyruvate and lactate concentrations were minimally affected by hypoxia. Both ArtpO2 and MABP influenced the estimate of kpl, with a strong negative correlation between kpl and the product of ArtpO2 and MABP under hypoxia. CONCLUSION: The rate constant for pyruvate to lactate conversion, kpl, responds significantly to a rapid reduction in tumour oxygenation.

MALDI-MSI and label-free LC-ESI-MS/MS shotgun proteomics to investigate protein induction in a murine fibrosarcoma model following treatment with a vascular disrupting agent.

Tumour vasculature is notoriously sinusoidal and leaky, and is hence susceptible to vascular disruption. Microtubule destabilising drugs such as the combretastatins form the largest group of tumour vascular disrupting agents and cause selective shutdown of tumour blood flow within minutes to hours, leading to secondary tumour cell death. Targeting the tumour vasculature is a proven anticancer strategy but early treatment response biomarkers are required for personalising treatment planning. Protein induction following treatment with combretastatin A4-phosphate was examined in a mouse fibrosarcoma model (fs188), where tumour cells express only the matrix-bound isoform of vascular endothelial growth factor A (VEGF188). These tumours are relatively resistant to vascular disruption by combretastatin A4-phosphate and hence a study of protein induction following treatment could yield insights into resistance mechanisms. The distribution of a number of proteins induced following treatment were visualised by MALDI-mass spectrometry imaging. Responses identified were validated by LC-ESI-MS/MS and immunohistochemical staining. Significant changes in proteins connected with necrosis, cell structure, cell survival and stress-induced molecular chaperones were identified. Protein-protein interactions were identified using STRING 9.0 proteomic network software. These relationship pathways provided an insight into the activity of the active tumour milieu and a means of linking the identified proteins to their functional partners.

Tissue factor, angiogenesis and tumour progression.

Tissue factor, the primary initiator of the coagulation cascade, maintains vascular integrity in response to injury. It is now recognised that, in addition to the role as a procoagulant activator, tissue factor participates in many tumour-related processes that contribute to malignant disease progression. The present review details the recent evidence supporting a role for tissue factor in tumour haemostasis, angiogenesis, metastasis and malignant cell survival. Furthermore, future research directions are discussed that may enhance our understanding of the role and regulation of this protein, which could ultimately lead to the innovative design and development of new anticancer therapies.

Bone marrow-derived endothelial progenitor cells do not contribute significantly to new vessels during incisional wound healing.

OBJECTIVE: To assess the contribution of bone marrow (BM)-derived endothelial progenitor cells (EPCs) to the neovascularisation of cutaneous incisional wounds. METHODS: Lethally irradiated C57Bl/6 mice were transplanted with BM mononuclear cells from Tie2/lacZ mice, which constitutively overexpressed beta-galactosidase (beta-gal) in endothelial cells (ECs). Chimeras were wounded and the number of X-gal-stained (beta-gal(+)) BM-derived EPCs were calculated in histological wound sections. RESULTS: EPCs were measured in skin sections from unwounded BM transplant (BMT) mice, or at day 1 and 3 postwounding, at the level of 0.1 +/- 0.1 (mean +/- SEM) per skin/wound section. In day-5 to day-14 wounds, the number of EPCs increased gradually (1.3 +/- 0.5 at day 5 and 4.8 +/- 0.9 at day 10), peaking at day 14, when there was a significant increase in the number of EPCs per wound section (6.5 +/- 1.7) when compared to unwounded skin. Between days 14 and 18 postwounding, there was a rapid fall-off in the number of beta-gal(+) EPCs (0.8 +/- 0.5 at day 18) and numbers returned to baseline by day 21 (0.1 +/- 0.1). No evidence of vascular structures derived from BM-derived EPCs ("in situ" vasculogenesis) was observed and it was calculated that these cells contributed only 4.4% +/- 1.5% to total wound ECs at their peak. CONCLUSION: These findings indicate that the revascularization of dermal incisional wounds primarily occurs through angiogenesis because the low frequency and temporal expression of EPCs suggests that they do not make a significant contribution to the neovascularization process.

The microcirculation in acute murine cutaneous incisional wounds shows a spatial and temporal variation in the functionality of vessels.

A mouse perfusion model using fluorescently labeled dextran has been developed to investigate the functionality of blood vessels during cutaneous wound healing. By immunostaining cryostat sections of perfused wounds with antibodies that identify vessels, we were able to assess their functionality. There was an increase in the proportion of CD31(+)-perfused vessels in all wound regions with time, although the vessels of the wound margins and superficial granulation tissue (GT) took the longest to become perfused. More than 50% of the latter vessels were not perfused at 10 days postwounding. This is consistent with the growth of functional vessels from the wound base proceeding to the more superficial GT. The CD34 marker was expressed by a subpopulation of CD31(+) vessels. However, in contrast to CD31(+) vessels, the functionality of CD34(+) vessels did not change significantly with time and 50-75% of CD34(+) vessels in the GT and wound margins were nonfunctional. This might be explained either by apoptosis of the CD34(+) vessels or the loss of the marker with time. This study has important implications for assays of wound-healing angiogenesis based on histology and immunohistochemical markers for vessels, because vessel functionality differs both spatially and temporally during wound healing.