Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer ther... more Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.
Following a radiological incident the rapid identification of those individuals exposed to critic... more Following a radiological incident the rapid identification of those individuals exposed to critically high radiation doses is important for initial triage and medical treatment. It has been previously demonstrated that scoring of radiation-induced foci of the phosphorylated histone γ-H2AX, which form at the sites of DNA double-strand breaks, may be used to determine radiation exposure levels from blood samples. Although faster than the ‘gold standard’ dicentric assay, foci scoring is still impractical in a field situation where large numbers of people may need to be screened. To deal with such a situation, an inexpensive portable device with high throughput capacity is desirable. Here we describe a portable microfluidic fluorescence spectrometer device which passes a suspension of γ-H2AX immunofluorescence-stained lymphocytes through a focused 488 nm laser beam in a microfluidic chamber and records emission spectra over the range 495–725 nm. The recorded emission spectra are spectrally unmixed into their constituent parts from which radiation exposure levels are determined. Proof of principle is demonstrated using cultured lymphoblastoid cells, exposed to X-ray doses between 0 and 8 Gy. With the current prototype setup it takes approximately 6 min to acquire and analyse 10,000 spectra. Further effort is required to fully develop this approach into a portable triage tool that could be used to help classify people into appropriate treatment categories based on radiation exposure levels.
We present details of the development of a optical biochip, with integrated on-chip laser excitat... more We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength corrugations fabricated on a thin gold film. We have made
The expression level of the HER family is unreliable as a predictive marker for targeted therapie... more The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.
The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singl... more The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singly counted ions. A duo-plasmatron ion source and a 2 MV Tandem™ accelerator supply a range of ions from protons to calcium for this beamline and microscope endstation, with energy ranges from 0.5 to 12 MeV. A magnetic quadrupole triplet lens is used to focus the beam of ions. We present the design of this beamline, and early results showing the capability to count single ions with 98% certainty on CR-39 track etch. We also show that the beam targeting accuracy is within 5 μm and selectively target human fibroblasts with a <5 μm carbon beam, using γ-H2AX immunofluorescence to demonstrate which cell nuclei were irradiated. We discuss future commissioning steps necessary to achieve submicron targeting accuracy with this beamline.
Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical i... more Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical importance in efficient multiphoton excitation microscopy. Since measurement of the pulse front distortion due to a lens is not trivial we have developed an imaging interferometric cross-correlator which allows us to measure temporal delays and pulse-widths across the spatial profile of the beam. The instrument consists of a modified Michelson interferometer with a reference arm containing a voice-coil delay stage and an arm which contains the optics under test. The pulse replicas are recombined and incident on a 22×22 lenslet array. The beamlets are focused in a 0.5 mm thick BBO crystal (cut for Type I second harmonic generation), filtered to remove the IR component of the beam and imaged using a 500 fps camera. The GVD and pulse front distortion are extracted from the temporal stack of beamlet images to produce a low resolution spatio-temporal map.
Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer ther... more Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.
Following a radiological incident the rapid identification of those individuals exposed to critic... more Following a radiological incident the rapid identification of those individuals exposed to critically high radiation doses is important for initial triage and medical treatment. It has been previously demonstrated that scoring of radiation-induced foci of the phosphorylated histone γ-H2AX, which form at the sites of DNA double-strand breaks, may be used to determine radiation exposure levels from blood samples. Although faster than the ‘gold standard’ dicentric assay, foci scoring is still impractical in a field situation where large numbers of people may need to be screened. To deal with such a situation, an inexpensive portable device with high throughput capacity is desirable. Here we describe a portable microfluidic fluorescence spectrometer device which passes a suspension of γ-H2AX immunofluorescence-stained lymphocytes through a focused 488 nm laser beam in a microfluidic chamber and records emission spectra over the range 495–725 nm. The recorded emission spectra are spectrally unmixed into their constituent parts from which radiation exposure levels are determined. Proof of principle is demonstrated using cultured lymphoblastoid cells, exposed to X-ray doses between 0 and 8 Gy. With the current prototype setup it takes approximately 6 min to acquire and analyse 10,000 spectra. Further effort is required to fully develop this approach into a portable triage tool that could be used to help classify people into appropriate treatment categories based on radiation exposure levels.
We present details of the development of a optical biochip, with integrated on-chip laser excitat... more We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength corrugations fabricated on a thin gold film. We have made
The expression level of the HER family is unreliable as a predictive marker for targeted therapie... more The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.
The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singl... more The Surrey vertical beam is a new facility for targeted irradiation of cells in medium with singly counted ions. A duo-plasmatron ion source and a 2 MV Tandem™ accelerator supply a range of ions from protons to calcium for this beamline and microscope endstation, with energy ranges from 0.5 to 12 MeV. A magnetic quadrupole triplet lens is used to focus the beam of ions. We present the design of this beamline, and early results showing the capability to count single ions with 98% certainty on CR-39 track etch. We also show that the beam targeting accuracy is within 5 μm and selectively target human fibroblasts with a <5 μm carbon beam, using γ-H2AX immunofluorescence to demonstrate which cell nuclei were irradiated. We discuss future commissioning steps necessary to achieve submicron targeting accuracy with this beamline.
Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical i... more Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical importance in efficient multiphoton excitation microscopy. Since measurement of the pulse front distortion due to a lens is not trivial we have developed an imaging interferometric cross-correlator which allows us to measure temporal delays and pulse-widths across the spatial profile of the beam. The instrument consists of a modified Michelson interferometer with a reference arm containing a voice-coil delay stage and an arm which contains the optics under test. The pulse replicas are recombined and incident on a 22×22 lenslet array. The beamlets are focused in a 0.5 mm thick BBO crystal (cut for Type I second harmonic generation), filtered to remove the IR component of the beam and imaged using a 500 fps camera. The GVD and pulse front distortion are extracted from the temporal stack of beamlet images to produce a low resolution spatio-temporal map.
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Papers by Paul Barber