1.

Introduction

The inflammatory response elicited by photodynamic therapy (PDT) represents a complex and dynamic process characterized by elements of the innate immune system.^1,2 A hallmark of this host response is the massive recruitment of leukocytes, a significant fraction of which are neutrophils, to the treated tumor site.^3,4 This phenomenon has been observed with several different photosensitizers, including the US FDA approved Photofrin.⁵ In our own work using in vivo imaging, we have recently demonstrated that PDT with the phthalocyanine photosensitizer, Pc4, or with 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH), induces a significant increase in ${\mathrm{Gr}1}^{+}$ cells in irradiated versus control EMT6 mouse mammary carcinoma tumors.^6,7 Several animal studies have also investigated the importance of intratumor neutrophil accumulation to the long-term curative outcome of PDT using selective depletion or inactivation of neutrophils and have demonstrated that the depletion of these effector cells results in a decrease of the PDT-mediated tumor cure rate.^3,8,9

Neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G are three serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. These proteases are externalized in an active form during neutrophil activation at inflammatory sites, and recent studies have started to elucidate their contribution to the immune response. In this context, NE has received particular attention.^10,11 Although NE has been implicated in a variety of diseases due to its capacity to degrade the extracellular matrix, new findings provide compelling evidence that NE contributes considerably to the regulation of host functions.¹² A recent study reported that NE is involved in polymorphonuclear leukocyte-mediated host protection in a mouse model of Pseudomonas aeruginosa-induced pneumonia.¹³ The study also demonstrated that extracellularly active NE has the capacity to induce mRNA expression of the proinflammatory cytokines tumor necrosis factor $\alpha$ (TNF-$\alpha$), macrophage inflammatory protein-2, and interleukin-6 (IL-6).

The growing recognition of the role of proteinases and proteolytic cascades in both the growth and metastasis of tumors has led to the development not only of therapeutic strategies using proteinase inhibitors but also of methods to detect and image tumors in vivo via tumor-associated proteolytic activities.^14–16 These selective protease probes belong to a family of imaging agents that are optically invisible or at least display reduced fluorescence when administered intravenously until activated by enzymatic cleavage at the disease site. This allows localization of fluorescence signal in areas of enzyme activity with low background in tissue regions that do not activate the probes. Thus, increased signal levels report enhanced proteolytic activity. Recently, several of these activatable imaging probes have been developed. For example, commercially available protease activatable probes targeting matrix metalloproteinases (MMPs) (MMPSense®, Perkin Elmer, Hopkinton, MA) have been widely used to illustrate enzyme activity in tumors and in other non-neoplastic diseases, such as stroke and arthritis.^17–19 In light of recent findings that NE mediates the upregulation of proinflammatory cytokines¹³ and based on the knowledge that the local inflammatory response to PDT is also characterized by increased expression of several cytokines, including IL-1$\beta$, TNF-$\alpha$ and IL-6,²⁰ we performed an imaging study to characterize the NE enzyme activity in PDT-treated tumors using a newly developed NE-selective near infrared activatable optical probe, neutrophil elastase 680 FAST (NE680). We evaluated if PDT conditions that induce significant neutrophil accumulation in treated tumor sites alter local NE protease activity. We report that in vivo imaging results are consistent with significantly increased NE activity in PDT-treated tumors, and the extent of probe accumulation correlates with the magnitude of intratumor neutrophil influx associated with different treatment protocols.

2.

Materials and methods

3.

Results and Discussion

In a study reported by Sun et al.,⁵ the authors examined the activity of neutrophils in SCC VII tumors subjected to Photofrin-PDT by assaying for tumor levels of neutrophilic myeloperoxidase (MPO). MPO, a neutrophil-specific enzyme, is released extracellularly upon cell activation and is considered an indicator of neutrophil-mediated cytotoxicity during inflammatory responses. The treatment protocol involved an intravenous administration of $10\mathrm{mg}{\mathrm{kg}}^{-1}$ Photofrin followed by illumination with $120\mathrm{mW}{\mathrm{cm}}^{-2}$ at 630 nm after a drug-light interval of 24 h. This treatment regimen resulted in a significant and sustained release of MPO into the tumor interstitium, with increased levels detectable as early as 2 h and reaching maximum enhancement at 8- to 13-h postirradiation. The findings from this report not only provided a rationale to investigate NE, another member of the neutrophil-associated enzyme family, but also informed the choice for the Photofrin-PDT protocol and the time point for NE imaging adopted for this current study.

In vivo whole-mouse fluorescence imaging using the IVIS Spectrum imager showed that the NE680 fluorescence was greatly enhanced in SCC VII tumors subjected to Photofrin-PDT [Fig. 1(c)] relative to controls [Fig. 1(a) and 1(b)]. As described in Sec. 2, the NE680 probe was injected in treated mice immediately following irradiation, and imaging was performed 5 h later. Thus, assuming that the cleaved probe does not clear on this time scale, NE680 levels imaged in PDT-treated tumors reflect cumulative elastase enzyme activity during the initial 5-h postirradiation. A quantitative comparison among the different groups was performed by measuring the fluorescence signal from a region of interest drawn over tumor regions of the mouse [Fig. 2(a)]. The NE680 fluorescence measured from Photofrin-PDT-treated SCC VII tumors was approximately threefold to fourfold higher than that measured in control tumors ($n=3$ for each group). The difference in fluorescence intensity between the untreated and drug-only control tumors was not statistically significant. In these controls, the source of observed NE680 fluorescence may be attributed to constitutive levels of NE present in a tumor. The differences observed in vivo in tumoral NE680 probe activation were further investigated by measurements in excised tumor samples [Fig. 2(b)], which are not influenced by possible treatment-induced changes in overlying skin. Interestingly, the ex vivo NE levels in treated SCC VII tumors ($n=2$) were found to be approximately 2.5-fold greater than those in controls. This slightly lower level of enzyme activity in excised tumors may suggest a significant activation of neutrophils in the skin immediately adjacent to the tumor in vivo.

Fig. 1

Whole-mouse fluorescence images of neutrophil elastase (NE) activity in SCC VII tumors established on the right hind leg of C3H mice. (a) Control [$(-)$ drug $(-)$ light], (b) Unirradiated drug-only control [$(+)$ drug $(-)$ light] and (c) Photofrin-PDT treated [$(+)$ drug $(+)$ light)]. $10\mathrm{mg}{\mathrm{kg}}^{-1}$ Photofrin was injected intravenously, and irradiation was performed 24 h later with a fluence of $150\mathrm{J}{\mathrm{cm}}^{-2}$ at an irradiance of $100\mathrm{mW}{\mathrm{cm}}^{-2}$. Immediately after irradiation, 100 μl of NE680 probe was administered via tail vein injection. Imaging was performed 5 h later on an IVIS Spectrum system. NE680 was excited and detected with a combination of $675/30$ and $720/20\mathrm{nm}$ filters, respectively. The color bar represents radiance efficiency.

Fig. 2

(a) Quantitative image analysis of in vivo NE680 fluorescence levels measured from region of interests assigned over tumor sites on the mice ($n=3$ for each group). (b) Ex vivo NE680 levels measured in excised tumor tissue ($n=2$) using the IVIS system. Data are normalized to intensities in control tumor samples. The representative images in the inset illustrate the intensities on the same color scale for representative control (left), unirradiated drug-only control (center), and Photofrin-PDT-treated (right) tumor specimens.

Figures 3 and 4 illustrate NE activity in EMT6 tumors established in BALB/c mice. Fluorescence measured from any activatable probe at a target site includes contributions from a combination of probe delivery and activation. Therefore, measurement of NE680 intensity in an unirradiated tumor in the contralateral leg offers an important internal control in that it is not influenced by variations in intravenous probe administration. Consequently, any fluorescence enhancement observed in treated versus unirradiated tumors is most likely due to PDT-induced changes in elastase activity. Figure 3(a) shows a representative image of NE680 fluorescence acquired from an EMT6 tumor subjected to Photofrin-PDT. The tumor on the contralateral leg was not irradiated and exhibits fluorescence intensity that is below the lower threshold of the display scale. The data from unirradiated and irradiated EMT6 tumors ($n=3$ for each group) are summarized in Fig. 3(b) and show that Photofrin-PDT induces an enhancement of NE activity by at least threefold at 5-h postirradiation. These results obtained from EMT6 tumors are remarkably similar to those observed in SCC VII tumors (Fig. 2).

Fig. 3

(a) Representative whole-mouse fluorescence image of NE activity in EMT6 tumors established in both hind legs of a BALB/c mouse. The tumor on the right leg was subjected to illumination, whereas the tumor on the left leg was shielded from any light exposure, thereby providing a Photofrin-sensitized, unirradiated control. $10\mathrm{mg}{\mathrm{kg}}^{-1}$ Photofrin was injected intravenously, and irradiation on the right leg tumor was performed 24 h later with a fluence of $150\mathrm{J}{\mathrm{cm}}^{-2}$ at an irradiance of $100\mathrm{mW}{\mathrm{cm}}^{-2}$. Immediately after irradiation, 100 μl of NE680 probe was administered, and imaging on the IVIS system was done 5 h later. (b) Quantitative comparison of NE680 fluorescence in irradiated versus unirradiated control tumors ($n=3$ for each group). Data are normalized to intensities in unirradiated drug-only control tumor samples. Error bars represent standard deviation.

Fig. 4

(a) NE680 fluorescence image acquired from a BALB/c mouse with bilateral EMT6 tumors. The tumor on the right leg was subjected to illumination, whereas the tumor on the left leg was shielded from light exposure and served as an HPPH-sensitized, unirradiated control. HPPH (1 $1\mu \mathrm{m}\mathrm{o}\mathrm{l}{\mathrm{kg}}^{-1}$) was injected intravenously, and irradiation was performed 24 h later with a fluence of $100\mathrm{J}{\mathrm{cm}}^{-2}$ at an irradiance of $75\mathrm{mW}{\mathrm{cm}}^{-2}$. Five hours following irradiation and NE680 administration, whole-mouse imaging was performed. (b) Quantitative comparison of NE680 fluorescence in irradiated versus unirradiated control tumors ($n=3$ for each group). Data are normalized to intensities in unirradiated drug-only control tumor samples. Error bars represent standard deviation.

Figure 4 exhibits data from EMT6 tumors ($n=3$) subjected to HPPH-PDT. In the whole-mouse image of Fig. 4(a), in addition to an increased level of NE680 induced by irradiation, there is also an evidence of some upregulation of elastase activity in the unirradiated tumor. This suggests the possibility that intratumoral HPPH drug accumulation stimulates a modest increase in neutrophil activation and consequent NE release. The summary data plot in Fig. 4(b) shows that NE680 fluorescence is approximately twofold higher in treated versus unirradiated tumors, which is significantly lower than the threefold difference observed with Photofrin-PDT.

In order to verify that the enhanced levels of NE680 fluorescence measured in irradiated tumors relative to unirradiated controls were caused by increased elastase activity and cleavage of the probe, we performed in vivo experiments to examine the attenuation of NE function in irradiated tumors with the introduction of an established NE inhibitor. Sivelestat is a selective NE inhibitor and has been shown to be effective clinically in mitigating the effect of systemic inflammatory response in patients with acute lung injury.²⁵ PDT-treated tumors that received Sivelestat via intratumor injection were assayed for NE680 fluorescence, and the levels were compared to those obtained from unirradiated and irradiated tumors in the absence of Sivelestat. Figure 5(a) shows significant inhibition of NE activity upon administration of Sivelestat into SCC VII tumors subjected to Photofrin-PDT. The introduction of the inhibitor resulted in decreased NE680 fluorescence to intensities observed in unirradiated drug-only control tumors. The presence of Sivelestat in Photofrin-PDT-treated EMT6 tumors also diminished the NE680 fluorescence from their levels observed in irradiated tumors [Fig. 5(b)]. The NE680 levels between unirradiated drug-only control and irradiated plus Sivelestat groups for both SCC VII and EMT6 tumors were not different at the $P=0.1$ significance level. Kossodo et al.²² performed a number of corroborating studies to extensively characterize the NE680 probe and verified that the dominant protease involved in probe cleavage is NE, and no other neutrophil- or nonneutrophil-associated proteases. Our own findings and that of Kossodo et al., therefore, strongly suggest that measured changes in NE680 probe fluorescence is a readout of corresponding variations in intratumoral NE activity.

Fig. 5

Comparison of NE680 fluorescence levels in (a) SCC VII and (b) EMT6 tumors among three groups: Photofrin-sensitized but unirradiated (drug control), subjected to irradiation (PDT treated) and irradiated plus administered the NE inhibitor, Sivelestat (PDT+Sivelestat). Sivelestat was delivered via direct intratumor injection 15 min prior to NE680 administration in the mice. Data are normalized to intensities in unirradiated drug-only control tumor samples. Error bars represent standard deviation.

In order to interpret the difference in the extent of NE680 enhancement observed with Photofrin- versus HPPH-PDT treatment regimens, we evaluated neutrophil influx into PDT-treated tumors sensitized with Photofrin versus HPPH. We have recently reported on the use of confocal imaging to visualize populations of ${\mathrm{Gr}1}^{+}$ cells and ${\mathrm{MHC}\text{-}\mathrm{II}}^{+}$ antigen presenting cells in tumors subjected to HPPH-PDT.⁷ The HPPH-PDT treatment conditions were identical to those used in this study. We found that HPPH-PDT results in 1.5- and 2.5-fold increase in intratumoral accumulation of ${\mathrm{Gr}1}^{+}$ cells at 5-h and 24-h postirradiation, respectively. To assay for Photofrin-PDT-induced influx of ${\mathrm{Gr}1}^{+}$ cells, we used whole-mount labeling of ex vivo tissues obtained from control and treated tumors. Figure 6(a) and 6(b) show fluorophore-labeled infiltrating ${\mathrm{Gr}1}^{+}$ cells imaged in an unirradiated control and PDT-treated tumor specimen, respectively. The tissue fragment illustrated in Fig. 6(b) was excised from a treated tumor at 5-h postirradiation. Each image is comprised of twenty-four $600\times 600{\mu \text{m}}^2$ individual fields, which were stitched to create a montage with a FOV of $3.6\times 2.4{\mathrm{mm}}^2$, as described in Sec. 2. Analysis of the images revealed that the influx of ${\mathrm{Gr}1}^{+}$ cells into Photofrin-PDT-treated EMT6 tumors was at least 10 times greater than that measured in unirradiated drug-only control tumors ($n=2$ for each group). The cell densities were found to be similar in Photofrin-PDT-treated tumor tissues excised at 5-h and 24-h postirradiation (data not shown). These results are qualitatively consistent with those reported by Sun et al.,⁵ who observed a 10-fold increase in accumulated neutrophils in Photofrin-PDT treated versus untreated SCC VII tumors, as measured indirectly via MPO assay. Gollnick et al.²⁶ reported that the number of infiltrating neutrophils were considerably fewer following HPPH- versus Photofrin-PDT. The authors attributed the results to the lower degree of inflammation associated with HPPH-PDT (Ref. 27) and to the kinetics of tumor destruction, with tumor regression occurring within 24 h after Photofrin-PDT versus a prolonged period of 72 to 96 h with HPPH-PDT. As NE is an enzyme released from neutrophil granules upon activation, it is therefore likely that the stronger inflammatory response triggered by Photofrin-PDT contributes to increased elastase activity imaged in irradiated tumors sensitized with Photofrin versus HPPH.

Fig. 6

Confocal fluorescence images of Alexa488-conjugated anti-${\mathrm{Gr}1}^{+}$ labeled cells in tissue fragments excised from EMT6 tumors in BALB/c mice. (a) Control tumor that received Photofrin but was not irradiated, and (b) tumor that was PDT treated and excised 5-h postirradiation. The FOV imaged in both of these tumors is $3.6\times 2.4{\mathrm{mm}}^2$, constructed from a montage of 24 individual $600\times 600{\mu \text{m}}^2$ fields. The ${\mathrm{Gr}1}^{+}$ cell counts in Photofrin-PDT-treated tumor tissue are at least 10-fold greater than that in the unirradiated drug-only control tissue.

In conclusion, this study was motivated by recent reports that highlight the extended functions of neutrophils and their downstream effects in mounting both innate and adaptive host response.¹⁰ Serine proteases released from activated neutrophils have emerged as a major player in the regulation of inflammatory response, such as specifically altering the function of cytokines and chemokines.¹¹ Mittendorf et al.²⁸ recently showed that a novel breast cancer antigen, Cyclin E, is cleaved after specific uptake of NE by breast cancer cells, and this increases the susceptibility of the cancer cells to lysis by cytotoxic T lymphocytes-specific for the antigen. This novel observation along with other recent findings therefore demonstrates that extracellularly released NE has physiologic functions that contribute to host defense, and hence the conventional view that assigns NE as pathogenic for tissue-destructive diseases, such as acute or chronic pulmonary diseases, warrants careful reassessment.^13,28 Further, as PDT induces increased accumulation of neutrophils in treated tumors and NE is released from activated neutrophils and stimulates proinflammatory cytokines, our characterization of significant NE increase in PDT-treated tumors establishes a rationale to further evaluate a previously undescribed role of NE in linking the elements of innate and adaptive host response associated with PDT.