BACKGROUND
photodynamic therapy  is a promising modality in both the curative and palliative treatment against a variety of experimental and naturally occurring human cancers  <cit> .
essentially, pdt is a two-step process that begins with the administration of photosensitizer for selective uptake in the target tissue.
the second phase involves exposure to non-thermal light at a wavelength specific to the photosensitizer at the sensitized target tissue.
the activation of the photosensitizer by light is an oxygen-dependent process that results in the generation of highly cytotoxic species including singlet oxygen.
the release of these reactive molecules results in damage to both tumor cells and to the tumor microenvironment.
the significance of pdt is that there is a degree of treatment selectivity that allows tumor destruction with minimal involvement of healthy tissue.
this is achieved by a combination of selective accumulation of photosensitizer within the tumor and by control of the light geometry and illumination parameters  <cit> .
lung cancer became one of the first cancers to be considered for pdt and has been used as an adjuvant treatment over the last  <dig> years  <cit> .
currently, pdt is used either to treat microinvasive endobronchial non-small cell lung carcinoma  or to palliate patients with completely or partially obstructing endobronchial nsclc  <cit> .
despite the generally refractory nature of these type of tumors, central type of tumors with identifiable endobronchial lesions which could be easily accessed bronchoscopically for illumination have been successfully treated with pdt  <cit> .
pdt can preserve lung function, limiting surgical trauma and postoperative pain as well as used in combination with other therapeutic modalities such as chemotherapy  <cit> .
photosensitizer-induced fluorescence detection aimed at enhancing optical contrast to improve tumor visibility has been extensively investigated to develop 'tumor selective' imaging methods  <cit> .
the lack of tumor selectivity, complex pharmacokinetics and the fact that some photosensitizers may cause prolonged skin photosensitivity, make the clinical application of fluorescence detection and pdt more complex  <cit> .
these limitations have led to the development of second-generation photosensitizers, which usually produce shorter periods of photosensitivity, longer activation wavelengths, higher tumor-to-normal tissue concentration, excellent antitumor effect and higher quantum yields of 1o <dig>  <cit> .
studies showed that derivatives from chlorophylls/chlorins are potent photosensitizers  <cit> , of which mono-l-aspartyl chlorin e <dig>  is undergoing clinical trials in japan for the treatment of endobronchial lung cancer  <cit> .
this report investigates a new formulation that consists of a mixture of chlorin e <dig>  derived from the plant spirullina platensis and polyvinylpyrrolidone  .
pvp is a biocompatible hydrophilic polymer that has been used to improve dissolution of lipophilic drugs and to modify the biodistribution of the drug.
the mixture of ce <dig> and pvp has a mass fraction ratio of 1: <dig>  ce6-pvp absorbs light of wavelength above  <dig> nm and produces less long-term normal tissue phototoxicity than photofrin  <cit> .
in our previous studies, we have demonstrated that ce6-pvp selectively accumulated in the poorly differentiated human nasopharyngeal and human bladder carcinoma xenografts in animal models  <cit> .
we have also reported the potential application of ce6-pvp in photodynamic therapy in one angiosarcoma patient  <cit> .
the present work examines the fluorescence pharmacokinetic of ce6-pvp in nsclc and small cell lung carcinoma  xenografts on the chick chorioallantoic membrane  model.
we have applied the receiver operating characteristics  concept to compare the sensitivity and specificity of fluorescence imaging on nsclc and sclc using white light as the gold standard.
photosensitizing efficacy of ce6-pvp was also investigated between the two histology of lung carcinoma using murine xenografts model.
RESULTS
fluorescence bronchoscopy has been reported to enhance the diagnostic accuracy and definition of the intra-epithelial cancer within the bronchi  <cit> .
this technique has become more attractive for clinical use since more effective 2nd generations of photosensitizers have been clinically implemented and tested.
newer formulations of photosensitizers were intended to reduce common side effects such as skin photosensitivity, nausea, vomiting and transiently raised liver transaminase levels.
following the above rationale, we have investigated the use of pvp in combination with ce <dig> for application in lung cancers.
as it was important to establish if cellular localization of ce6-pvp was also exhibited in human lung tumor, the cam tumor xenograft was employed here.
we have demonstrated that this method of examining fluorescence uptake and retention in tissue explants on the cam model provides a reliable means for direct, comparative visualization in situ of human tumors  <cit> .
inoculation of human nsclc  and sclc  into highly vascularized cam led to the disseminated tumor growth on the surface of the cam .
typical fluorescence intensity image of nsclc and sclc are illustrated in fig.
2b and 2d, respectively.
intense red fluorescence was macroscopically visible in the tumor cells under blue light, as compared to non-malignant epithelium of the cam after  <dig> minutes post incubation with ce6-pvp.
the fluorescence retention by the lung tumor xenografts after topical administration was quantitatively evaluated using image-processing techniques and charted as a function of time .
high differential fluorescence intensity was observed between nsclc xenografts and its surrounding normal cam tissue compared to sclc xenografts.
the average of the red-to-blue intensity ratio of nsclc xenograft was higher than that of sclc xenograft.
the fluorescence intensity elimination rate constant for nsclc, sclc and normal cam was calculated to be  <dig> ,  <dig> , and  <dig>  min- <dig> respectively, suggesting that ce6-pvp is being retained longer in nsclc than sclc.
normal cam had a faster elimination rate of ce6-pvp.
we have applied roc curve analysis from  <dig>  to  <dig> h post administration of ce6-pvp to validate the ability of the photosensitizer to discriminate nsclc and sclc from normal cam membrane.
the area under the curve  were then compared in order to make a fair judgment of the effectiveness of ce6-pvp without being constricted to single values of sensitivity and specificity, which largely depend on the cut-off fluorescence intensity value chosen to distinguish normal from malignant region .
the following is a rough guide for classifying the accuracy of ce6-pvp based on the auc:  <dig> –  <dig>  = excellent;  <dig>  –  <dig>  = good;  <dig>  –  <dig>  = fair;  <dig>  –  <dig>  = poor; and  <dig>  –  <dig>  = fail.
the auc for nsclc were  <dig> ,  <dig> , and  <dig> , at  <dig>  h,  <dig> h and  <dig> h respectively  indicating that shorter exposure times resulted in lower accuracy.
the greatest auc was observed from  <dig> h post drug administration onwards: i.e.
<dig> ,  <dig>  and  <dig>  at  <dig> h,  <dig> h and  <dig> h respectively .
for sclc, the auc were  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> , and  <dig>  at  <dig>  h,  <dig> h,  <dig> h,  <dig> h,  <dig> h and  <dig> h respectively .
this result showed no improvement in fluorescence accuracy in demarcating sclc from the normal surrounding cam.
to evaluate the overall quality of fluorescence intensity discrimination between nsclc and sclc, a combined roc was generated from  <dig>  to  <dig> h post drug administration.
the sensitivity and the specificity were calculated using different threshold  values to distinguish healthy from malignant tissue .
for nsclc, the highest combined sensitivity and specificity were 90% and 78% , whereas for sclc it was 57% and 79% respectively , implying that fluorescence mediated ce6-pvp had distinctly higher rate of sensitivity for the detection of disseminated lesions of nsclc than with sclc.
an area of  <dig> represents a perfect discrimination of tumor from normal tissue; an area of  <dig>  represents no discrimination between normal and abnormal.
the p value indicates whether the area under the roc is significantly different from  <dig> .
*if the p value is <  <dig> , the area under the roc curve is significantly different .
for tumors, each point represents a mean of  <dig> eggs whereas for normal, each point represents a mean of  <dig> eggs.
to determine the efficacy of ce6-pvp mediated pdt, nude mice bearing nsclc and sclc tumors were administered with  <dig>  mg/kg of the photosensitizer.
pdt was performed on using light generated by a diode laser system  at the light dose of  <dig> j/cm <dig> and fluence rate of  <dig> mw/cm <dig>  the area of tumor necrosis was measured by evan's blue dye staining at  <dig> h post pdt.
strong heterogeneous staining was observed in the untreated controls  indicating occurrence of spontaneous, albeit limited necrosis, whereas in the pdt treated tumor, tissue damage was clearly evident as an unstained area .
nsclc tumors irradiated at  <dig> and  <dig> h drug-light interval exhibited extent of tumor necrosis of  <dig> ± 7% and  <dig> ± 4% respectively.
when pdt treatment was performed on sclc models using the same parameter, it was observed that irradiation at  <dig> h drug-light interval resulted in  <dig> ± 9% of tumor necrosis while irradiation at  <dig> h drug-light interval resulted in  <dig> ± 8% tumor necrosis.
thus, we conclude that sclc were only moderately sensitive to ce6-pvp mediated pdt.
almost all pdt studies were concerned with nsclc due to the referral patterns in the clinics  <cit> .
although pdt has also been shown to be effective in the clinical treatment of sclc  <cit> , little preclinical data exist comparing the efficacy or tendency for resistance toward photosensitization between these two tumor histologies.
in this study, we observed a certain degree of resistance to pdt in sclc xenografts that could be related to faster elimination rate of ce6-pvp, which resulted in lower cellular accumulation of the photosensitizer.
there are already a variety of molecular markers that have been implicated in the pathogenesis of sclc  <cit>  thus making it difficult to hypothesize the molecular basis of acquired resistance towards photosensitization in our experiments.
generally, in lung cancer four types of multidrug resistance have been identified, i.e., classical multidrug resistance , non-p-glycoprotein mdr , atypical mdr  and lung resistance-related protein  <cit> .
previous evidence indicates that sclc cell lines and tumors express multidrug resistance-associated protein, i.e.
mrp <dig>  atp binding cassette c <dig>  <cit>  and abcg <dig>   <cit> .
hence, one plausible reason to explain the lack of activity of ce6-pvp in sclc is the possible existence of abc transporters of chlorin-based  photosensitizers in this tumor histology.
the importance of human abcg <dig> in the transport of tetrapyrrole structure has been implicated  <cit> .
it was reported that cancer cell lines that expresses abcg <dig> was found to efflux some of the chlorophyll based photosensitizers and thus may confer resistance to this treatment modality  <cit> .
it has been suggested that by inhibiting abcg <dig> transport using tyrosine kinase inhibitors , it is likely to be a more successful approach to enhancing clinical pdt  <cit> .
CONCLUSIONS
photosensitization with ce6-pvp for  <dig> hours of exposure time appeared to be most effective in detecting nsclc in cam model.
furthermore, pdt at  <dig> h drug-light interval resulted in a better tumor necrosis in nsclc xenograft model.
sclc xenografts were found to manifest a certain degree of resistance to photosensitization with ce6-pvp.
despite the limited activity of ce6-pvp in the sclc xenografts, it is conceivable that the combined modality of fluorescence imaging and targeted photodynamic therapy using ce6-pvp may still have a potential role in sclc.
this warrants for additional studies on the molecular mechanisms of photosensitization resistance in sclc to overcome this important clinical problem.

 photodynamic therapy is a promising modality in both the curative and palliative treatment against a variety of experimental and naturally occurring human cancers. essentially, pdt is a two-step process that begins with the administration of photosensitizer for selective uptake in the target tissue. the second phase involves exposure to non-thermal light at a wavelength specific to the photosensitizer at the sensitized target tissue. the activation of the photosensitizer by light is an oxygen-dependent process that results in the generation of highly cytotoxic species including singlet oxygen. the release of these reactive molecules results in damage to both tumor cells and to the tumor microenvironment. the significance of pdt is that there is a degree of treatment selectivity that allows tumor destruction with minimal involvement of healthy tissue. this is achieved by a combination of selective accumulation of photosensitizer within the tumor and by control of the light geometry and illumination parameters. lung cancer became one of the first cancers to be considered for pdt and has been used as an adjuvant treatment over the last years. currently, pdt is used either to treat microinvasive endobronchial non-small cell lung carcinoma or to palliate patients with completely or partially obstructing endobronchial nsclc. despite the generally refractory nature of these type of tumors, central type of tumors with identifiable endobronchial lesions which could be easily accessed bronchoscopically for illumination have been successfully treated with pdt. pdt can preserve lung function, limiting surgical trauma and postoperative pain as well as used in combination with other therapeutic modalities such as chemotherapy. photosensitizer-induced fluorescence detection aimed at enhancing optical contrast to improve tumor visibility has been extensively investigated to develop'tumor selective' imaging methods. the lack of tumor selectivity, complex pharmacokinetics and the fact that some photosensitizers may cause prolonged skin photosensitivity, make the clinical application of fluorescence detection and pdt more complex. these limitations have led to the development of second-generation photosensitizers, which usually produce shorter periods of photosensitivity, longer activation wavelengths, higher tumor-to-normal tissue concentration, excellent antitumor effect and higher quantum yields of 1o. studies showed that derivatives from chlorophylls/chlorins are potent photosensitizers, of which mono-l-aspartyl chlorin e is undergoing clinical trials in japan for the treatment of endobronchial lung cancer. this report investigates a new formulation that consists of a mixture of chlorin e derived from the plant spirullina platensis and polyvinylpyrrolidone. pvp is a biocompatible hydrophilic polymer that has been used to improve dissolution of lipophilic drugs and to modify the biodistribution of the drug. the mixture of ce and pvp has a mass fraction ratio of 1: ce6-pvp absorbs light of wavelength above nm and produces less long-term normal tissue phototoxicity than photofrin. in our previous studies, we have demonstrated that ce6-pvp selectively accumulated in the poorly differentiated human nasopharyngeal and human bladder carcinoma xenografts in animal models. we have also reported the potential application of ce6-pvp in photodynamic therapy in one angiosarcoma patient. the present work examines the fluorescence pharmacokinetic of ce6-pvp in nsclc and small cell lung carcinoma xenografts on the chick chorioallantoic membrane model. we have applied the receiver operating characteristics concept to compare the sensitivity and specificity of fluorescence imaging on nsclc and sclc using white light as the gold standard. photosensitizing efficacy of ce6-pvp was also investigated between the two histology of lung carcinoma using murine xenografts model.  fluorescence bronchoscopy has been reported to enhance the diagnostic accuracy and definition of the intra-epithelial cancer within the bronchi. this technique has become more attractive for clinical use since more effective 2nd generations of photosensitizers have been clinically implemented and tested. newer formulations of photosensitizers were intended to reduce common side effects such as skin photosensitivity, nausea, vomiting and transiently raised liver transaminase levels. following the above rationale, we have investigated the use of pvp in combination with ce for application in lung cancers. as it was important to establish if cellular localization of ce6-pvp was also exhibited in human lung tumor, the cam tumor xenograft was employed here. we have demonstrated that this method of examining fluorescence uptake and retention in tissue explants on the cam model provides a reliable means for direct, comparative visualization in situ of human tumors. inoculation of human nsclc and sclc into highly vascularized cam led to the disseminated tumor growth on the surface of the cam. typical fluorescence intensity image of nsclc and sclc are illustrated in fig. 2b and 2d, respectively. intense red fluorescence was macroscopically visible in the tumor cells under blue light, as compared to non-malignant epithelium of the cam after minutes post incubation with ce6-pvp. the fluorescence retention by the lung tumor xenografts after topical administration was quantitatively evaluated using image-processing techniques and charted as a function of time. high differential fluorescence intensity was observed between nsclc xenografts and its surrounding normal cam tissue compared to sclc xenografts. the average of the red-to-blue intensity ratio of nsclc xenograft was higher than that of sclc xenograft. the fluorescence intensity elimination rate constant for nsclc, sclc and normal cam was calculated to be,, and min- respectively, suggesting that ce6-pvp is being retained longer in nsclc than sclc. normal cam had a faster elimination rate of ce6-pvp. we have applied roc curve analysis from to h post administration of ce6-pvp to validate the ability of the photosensitizer to discriminate nsclc and sclc from normal cam membrane. the area under the curve were then compared in order to make a fair judgment of the effectiveness of ce6-pvp without being constricted to single values of sensitivity and specificity, which largely depend on the cut-off fluorescence intensity value chosen to distinguish normal from malignant region. the following is a rough guide for classifying the accuracy of ce6-pvp based on the auc: – = excellent; – = good; – = fair; – = poor; and – = fail. the auc for nsclc were,, and, at h, h and h respectively indicating that shorter exposure times resulted in lower accuracy. the greatest auc was observed from h post drug administration onwards: i.e., and at h, h and h respectively. for sclc, the auc were,,,,, and at h, h, h, h, h and h respectively. this result showed no improvement in fluorescence accuracy in demarcating sclc from the normal surrounding cam. to evaluate the overall quality of fluorescence intensity discrimination between nsclc and sclc, a combined roc was generated from to h post drug administration. the sensitivity and the specificity were calculated using different threshold values to distinguish healthy from malignant tissue. for nsclc, the highest combined sensitivity and specificity were 90% and 78%, whereas for sclc it was 57% and 79% respectively, implying that fluorescence mediated ce6-pvp had distinctly higher rate of sensitivity for the detection of disseminated lesions of nsclc than with sclc. an area of represents a perfect discrimination of tumor from normal tissue; an area of represents no discrimination between normal and abnormal. the p value indicates whether the area under the roc is significantly different from. *if the p value is <, the area under the roc curve is significantly different. for tumors, each point represents a mean of eggs whereas for normal, each point represents a mean of eggs. to determine the efficacy of ce6-pvp mediated pdt, nude mice bearing nsclc and sclc tumors were administered with mg/kg of the photosensitizer. pdt was performed on using light generated by a diode laser system at the light dose of j/cm and fluence rate of mw/cm the area of tumor necrosis was measured by evan's blue dye staining at h post pdt. strong heterogeneous staining was observed in the untreated controls indicating occurrence of spontaneous, albeit limited necrosis, whereas in the pdt treated tumor, tissue damage was clearly evident as an unstained area. nsclc tumors irradiated at and h drug-light interval exhibited extent of tumor necrosis of ± 7% and ± 4% respectively. when pdt treatment was performed on sclc models using the same parameter, it was observed that irradiation at h drug-light interval resulted in ± 9% of tumor necrosis while irradiation at h drug-light interval resulted in ± 8% tumor necrosis. thus, we conclude that sclc were only moderately sensitive to ce6-pvp mediated pdt. almost all pdt studies were concerned with nsclc due to the referral patterns in the clinics. although pdt has also been shown to be effective in the clinical treatment of sclc, little preclinical data exist comparing the efficacy or tendency for resistance toward photosensitization between these two tumor histologies. in this study, we observed a certain degree of resistance to pdt in sclc xenografts that could be related to faster elimination rate of ce6-pvp, which resulted in lower cellular accumulation of the photosensitizer. there are already a variety of molecular markers that have been implicated in the pathogenesis of sclc thus making it difficult to hypothesize the molecular basis of acquired resistance towards photosensitization in our experiments. generally, in lung cancer four types of multidrug resistance have been identified, i.e., classical multidrug resistance, non-p-glycoprotein mdr, atypical mdr and lung resistance-related protein. previous evidence indicates that sclc cell lines and tumors express multidrug resistance-associated protein, i.e. mrp atp binding cassette c and abcg. hence, one plausible reason to explain the lack of activity of ce6-pvp in sclc is the possible existence of abc transporters of chlorin-based photosensitizers in this tumor histology. the importance of human abcg in the transport of tetrapyrrole structure has been implicated. it was reported that cancer cell lines that expresses abcg was found to efflux some of the chlorophyll based photosensitizers and thus may confer resistance to this treatment modality. it has been suggested that by inhibiting abcg transport using tyrosine kinase inhibitors, it is likely to be a more successful approach to enhancing clinical pdt.  photosensitization with ce6-pvp for hours of exposure time appeared to be most effective in detecting nsclc in cam model. furthermore, pdt at h drug-light interval resulted in a better tumor necrosis in nsclc xenograft model. sclc xenografts were found to manifest a certain degree of resistance to photosensitization with ce6-pvp. despite the limited activity of ce6-pvp in the sclc xenografts, it is conceivable that the combined modality of fluorescence imaging and targeted photodynamic therapy using ce6-pvp may still have a potential role in sclc. this warrants for additional studies on the molecular mechanisms of photosensitization resistance in sclc to overcome this important clinical problem. 
the present study evaluated the potential use of chlorin e – polyvinylpyrrolidone as a multimodality photosensitizer for fluorescence detection and photodynamic therapy on nsclc and sclc xenografts. little is known about the comparative efficacy of pdt in treating non-small cell lung carcinoma and small cell lung carcinoma, despite ongoing clinical trials treating lung cancers. human nsclc and sclc tumor cell lines were used to establish tumor xenografts in the chick chorioallantoic membrane model as well as in the balb/c nude mice. irradiation at h drug-light interval showed greater tumor necrosis against human nsclc xenografts in nude mice. photodynamic therapy is an effective local cancer treatment that involves light activation of a photosensitizer, resulting in oxygen-dependent, free radical-mediated cell death. sclc xenografts were observed to express resistance to photosensitization with ce6-pvp. in the cam model, ce6-pvp was applied topically and fluorescence intensity was charted at various time points.
 photodynamic therapy is an effective local cancer treatment that involves light activation of a photosensitizer, resulting in oxygen-dependent, free radical-mediated cell death. little is known about the comparative efficacy of pdt in treating non-small cell lung carcinoma and small cell lung carcinoma, despite ongoing clinical trials treating lung cancers. the present study evaluated the potential use of chlorin e – polyvinylpyrrolidone as a multimodality photosensitizer for fluorescence detection and photodynamic therapy on nsclc and sclc xenografts.  human nsclc and sclc tumor cell lines were used to establish tumor xenografts in the chick chorioallantoic membrane model as well as in the balb/c nude mice. in the cam model, ce6-pvp was applied topically and fluorescence intensity was charted at various time points. tumor-bearing mice were given intravenous administration of ce6-pvp and laser irradiation at nm. tumor response was evaluated at h post pdt. studies of temporal fluorescence pharmacokinetics in cam tumor xenografts showed that ce6-pvp has a selective localization and a good accuracy in demarcating nsclc compared to sclc from normal surrounding cam after h post drug administration. irradiation at h drug-light interval showed greater tumor necrosis against human nsclc xenografts in nude mice. sclc xenografts were observed to express resistance to photosensitization with ce6-pvp.  the formulation of ce6-pvp is distinctly advantageous as a diagnostic and therapeutic agent for fluorescence diagnosis and pdt of nsclc. 