The development of new drugs requires high throughput and cost-effective screening infrastructures associated to suitable cellular models1. Fourier Transform Infrared (FT-IR) spectroscopy and microscopy has the potential to detect small and early biochemical changes associated with cellular events, and in combination with pattern recognition/classification methods, it should be utilized as a complementary technique to perform high throughput measurements and screening in pre-clinical drug testing and for routine clinical diagnostic and prognostic analyses2. We explore the possibility to apply FT-IR microspectroscopy to characterize spectral signatures of activation and/or inhibition of intracellular signaling pathways in polymorphonuclear neutrophils (PMNs) incubated “ex vivo” with specific agonists and/or inhibitors of intracellular phosphonetworks3, a pre-requisite to develop new anti-inflammatory drugs, and to screen the efficacy of different drug formulations in inhibiting a key metabolic pathway relevant for the cell transformation process or to point out which is the most effective in relation to the considered cell model before performing clinical trials. To this scope two distinct clones of K562 cells4, a cell line model of human chronic myelogenous leukaemia (CML), respectively with low and rescued expression of receptor-type protein tyrosin phosphatase gamma (PTPRG) were incubated for 10 minutes with a selective inhibitor of BCR-ABL tyrosine kinase phosphorylation, or with a potent and selective inhibitor of the Src-family tyrosine kinase, respectively. In other experiements, the cystic fibrosis transmittance regulator (CFTR) deficient IB3-1 epithelial cell line and the corresponding isogenic CFTR rescued C38 cell line were incubated with the pro-inflammatory cytokine interleukin 8 (IL-8) or with the antibiotic azitromycin (AZT). At the end of perturbation, cells were fixed in 1% paraformaldehyde buffered solution in PBS, washed with distilled water and cell density was normalized to 5x106 cells/mL. A drop (10 µL) of each sample replicate was deposited on a 2 mm thick ZnSe infrared transparent window. Samples were dried in air and then stored in the vacuum and in the dark until the IR measurements. The acquisition of IR spectra was carried out at DA_NE light laboratory5 (LNF-INFN – Italy) using a Globar source and a Bruker Equinox55 FT-IR interferometer with a KBr beam splitter, connected to a Bruker Hyperion 3000 FT-IR microscope (Bruker Optik GmbH, Ettlingen, Germany) equipped with both 15x magnification objective and condenser. All measurements were performed in air and spectra were acquired in transmission modality from homogeneous zones of the sample containing a comparable number of cells in each selected sample area (typically from a 100x100 µm2 illuminated area, sometime reduced to 20x20 µm2 to obtain the spectrum of single cell). The transmitted IR light was collected by a single element 250x250 µm2 mercury-cadmium-telluride (MCT) liquid nitrogen-cooled detector for both the background and the sample. To obtain an acceptable S/N, 128 or 256 scans were averaged in the 700–4000 cm-1 spectral interval with a spectral resolution of 4 cm-1. Absorbance spectra were finally obtained by background spectrum subtraction from each sample spectrum. All spectral manipulations and calculations were performed with the use of OPUS™ 6.0 software. Atmosphere compensation was applied. The mean spectrum of replicate measurements of the different sample areas was calculated in the 4000–800 cm-1 frequency interval and the 1800–800 cm-1 spectral interval was selected and baseline corrected (Rubberband method with 64 points). To identify contribution in peaks, the second-order derivative was computed by using a generalized Savitzky-Golay algorithm with 9 smoothing points. Preliminary results that will be presented and discussed in the poster clearly indicate that FT-IR spectroscopy and microscopy can be utilized to identify signatures of phosphonetworks activation/inhibition and to compare the effects of different drugs on ex vivo cell models. We conclude that the application of this complementary and global analytical thechnique is very useful to biomedical research, diagnosis, monitoring and prognosis of diseases, and to drive therapeutic interventions. However, following relevant aspects must be considered to improve FT-IR microspectroscopy for basic science, pre-clinical and clinical applications. With a conventional thermal source and a FPA detector faster data collection at a spatial resolution roughly two times the diffraction limit can be obtained in a high number of single cells within larger sample area in a few minutes6; however, to obtain good quality IR spectra at the highest possible spatial resolution and S/N ratio the use of Synchrotron Radiation IR microscope equipped with a single-point or with a FPA detector seems more appropriate. The development and availability of microfluidic devices are necessary for the analysis on single cell in solution. High throughput measurememnts require the rapid elaboration of acquired data and the development of new data treatment methods for the quantitative estimation of the cell molecular contents or for determining the effects of stress on subcellular components. The availability of validated hyperspectral IR libraries of cells and cell events is necessary to easily identify spectral changes. These aspects, together with the possibility to obtain biochemical and structural hybrid images within the same analytical sample, for instance by cuncurrent IR and X-ray analysis should be considered in the future development of DASIM action, where synchrotrons should play a key role in supporting the development and validation of new intrumentations, devices and procedures for ruotine applications of FT-IR microspectroscopy. REFERENCES 1. R.D. Blumenthal and D.M. Goldenberg, Mol Biotechnol. 35, 185-198 (2007). 2. K.Z. Liu, M. Xu, and D.A. Scott. Br. J. Haematol. 136, 713-722 (2007). 3. C. Giagulli, et al. J. Immunol. 177, 604-611, (2006). 4. A. Mafficini, et al. Biomarker Insights 2, 217–224 (2007). 5. M. Cestelli Guidi, et al. J. Opt. Soc. Amer. A22, 2810 (2005). 6.°° L.M. Miller and R.J. Smith. Vibrational Spectroscopy 38, 237-240 (2005).

Development of FT-IR microspectroscopy for pre-clinical and clinical applications

BELLISOLA, GIUSEPPE;BERGAMINI, Corinna;BOLOMINI VITTORI, Matteo;DELLA PERUTA, Marco;GIAGULLI, Cinzia;LAUDANNA, Carlo;MAFFICINI, Andrea;MELOTTI, Paola Maria;SORIO, Claudio
2008

Abstract

The development of new drugs requires high throughput and cost-effective screening infrastructures associated to suitable cellular models1. Fourier Transform Infrared (FT-IR) spectroscopy and microscopy has the potential to detect small and early biochemical changes associated with cellular events, and in combination with pattern recognition/classification methods, it should be utilized as a complementary technique to perform high throughput measurements and screening in pre-clinical drug testing and for routine clinical diagnostic and prognostic analyses2. We explore the possibility to apply FT-IR microspectroscopy to characterize spectral signatures of activation and/or inhibition of intracellular signaling pathways in polymorphonuclear neutrophils (PMNs) incubated “ex vivo” with specific agonists and/or inhibitors of intracellular phosphonetworks3, a pre-requisite to develop new anti-inflammatory drugs, and to screen the efficacy of different drug formulations in inhibiting a key metabolic pathway relevant for the cell transformation process or to point out which is the most effective in relation to the considered cell model before performing clinical trials. To this scope two distinct clones of K562 cells4, a cell line model of human chronic myelogenous leukaemia (CML), respectively with low and rescued expression of receptor-type protein tyrosin phosphatase gamma (PTPRG) were incubated for 10 minutes with a selective inhibitor of BCR-ABL tyrosine kinase phosphorylation, or with a potent and selective inhibitor of the Src-family tyrosine kinase, respectively. In other experiements, the cystic fibrosis transmittance regulator (CFTR) deficient IB3-1 epithelial cell line and the corresponding isogenic CFTR rescued C38 cell line were incubated with the pro-inflammatory cytokine interleukin 8 (IL-8) or with the antibiotic azitromycin (AZT). At the end of perturbation, cells were fixed in 1% paraformaldehyde buffered solution in PBS, washed with distilled water and cell density was normalized to 5x106 cells/mL. A drop (10 µL) of each sample replicate was deposited on a 2 mm thick ZnSe infrared transparent window. Samples were dried in air and then stored in the vacuum and in the dark until the IR measurements. The acquisition of IR spectra was carried out at DA_NE light laboratory5 (LNF-INFN – Italy) using a Globar source and a Bruker Equinox55 FT-IR interferometer with a KBr beam splitter, connected to a Bruker Hyperion 3000 FT-IR microscope (Bruker Optik GmbH, Ettlingen, Germany) equipped with both 15x magnification objective and condenser. All measurements were performed in air and spectra were acquired in transmission modality from homogeneous zones of the sample containing a comparable number of cells in each selected sample area (typically from a 100x100 µm2 illuminated area, sometime reduced to 20x20 µm2 to obtain the spectrum of single cell). The transmitted IR light was collected by a single element 250x250 µm2 mercury-cadmium-telluride (MCT) liquid nitrogen-cooled detector for both the background and the sample. To obtain an acceptable S/N, 128 or 256 scans were averaged in the 700–4000 cm-1 spectral interval with a spectral resolution of 4 cm-1. Absorbance spectra were finally obtained by background spectrum subtraction from each sample spectrum. All spectral manipulations and calculations were performed with the use of OPUS™ 6.0 software. Atmosphere compensation was applied. The mean spectrum of replicate measurements of the different sample areas was calculated in the 4000–800 cm-1 frequency interval and the 1800–800 cm-1 spectral interval was selected and baseline corrected (Rubberband method with 64 points). To identify contribution in peaks, the second-order derivative was computed by using a generalized Savitzky-Golay algorithm with 9 smoothing points. Preliminary results that will be presented and discussed in the poster clearly indicate that FT-IR spectroscopy and microscopy can be utilized to identify signatures of phosphonetworks activation/inhibition and to compare the effects of different drugs on ex vivo cell models. We conclude that the application of this complementary and global analytical thechnique is very useful to biomedical research, diagnosis, monitoring and prognosis of diseases, and to drive therapeutic interventions. However, following relevant aspects must be considered to improve FT-IR microspectroscopy for basic science, pre-clinical and clinical applications. With a conventional thermal source and a FPA detector faster data collection at a spatial resolution roughly two times the diffraction limit can be obtained in a high number of single cells within larger sample area in a few minutes6; however, to obtain good quality IR spectra at the highest possible spatial resolution and S/N ratio the use of Synchrotron Radiation IR microscope equipped with a single-point or with a FPA detector seems more appropriate. The development and availability of microfluidic devices are necessary for the analysis on single cell in solution. High throughput measurememnts require the rapid elaboration of acquired data and the development of new data treatment methods for the quantitative estimation of the cell molecular contents or for determining the effects of stress on subcellular components. The availability of validated hyperspectral IR libraries of cells and cell events is necessary to easily identify spectral changes. These aspects, together with the possibility to obtain biochemical and structural hybrid images within the same analytical sample, for instance by cuncurrent IR and X-ray analysis should be considered in the future development of DASIM action, where synchrotrons should play a key role in supporting the development and validation of new intrumentations, devices and procedures for ruotine applications of FT-IR microspectroscopy. REFERENCES 1. R.D. Blumenthal and D.M. Goldenberg, Mol Biotechnol. 35, 185-198 (2007). 2. K.Z. Liu, M. Xu, and D.A. Scott. Br. J. Haematol. 136, 713-722 (2007). 3. C. Giagulli, et al. J. Immunol. 177, 604-611, (2006). 4. A. Mafficini, et al. Biomarker Insights 2, 217–224 (2007). 5. M. Cestelli Guidi, et al. J. Opt. Soc. Amer. A22, 2810 (2005). 6.°° L.M. Miller and R.J. Smith. Vibrational Spectroscopy 38, 237-240 (2005).
Fourier Transform Infrared spectroscopy; chronic myelogenous leukaemia; PTPRG
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/320265
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact