Although PDT for superficial, diffuse disease is a challenging application, it possesses substantial clinical potential for disseminated malignancies including those of the pleural and abdominal surfaces (42
). Similarly, PDT of diffuse disease in a small animal, especially within the thoracic cavity, is complex, thereby limiting the development of pre-clinical models for this application. We demonstrate that PDT can be delivered to the mouse thoracic cavity via a cylindrical diffusing fiber with therapeutic fluences and acceptable morbidity. Furthermore, we exploited the benefits of small animal imaging with a non-ionizing radiation modality, that being MRI, to quantify the spatial distribution of tumor burden and investigate PDT effects by location within the murine thoracic cavity. These data serve to establish this animal model, together with the treatment and monitoring approaches, as methods with significant potential value for the pre-clinical investigation of intrathoracic PDT for disseminated pleural malignancies.
The orthotopic model of the present report was adapted from that described by Onn et al (37
), who surveyed a panel of NSCLC cell lines and a small cell lung carcinoma cell line for their tumorigenicity and growth characteristics after intrathoracic injection. This method was chosen because it can be established without surgery, which limits morbidity prior to PDT, and it is associated with diffuse pleural spread, which is relevant to clinical PDT of pleural disease. In our observations of intrathoracic H460 proliferation, dissemination and pleural effusion were generally present within 9 days of the injection of 1 × 106
cells. Microscopic lesions within the lung itself were readily visible at histopathological evaluation. However, when compared to the cell lines studied by Onn et al (37
), we found that intrathoracic dissemination of H460 tumor cells progressed more rapidly and/or was aided by independent pleural seeding.
Few pre-clinical models of intrathoracic PDT of orthotopic lung disease currently exist. Among those established, most involve localized
light delivery to the diseased lung in conjunction with thoracotomy. For example, Opitz et al (44
) reported that focal illumination of localized subpleural mesothelioma nodules in rats resulted in 50-55% necrosis of tumor when treated with mTHPC and 80-88% when treated with verteporfin. The underlying aorta, bronchus and esophagus were spared from damage; however drug doses that produced the greatest amount of tumor necrosis also led to fibrosis in adjacent normal lung tissue. From these results, the authors concluded that focal intracavitary PDT in the rat was well tolerated, and a similar illumination approach was subsequently applied for PDT-enhanced uptake of the chemotherapy drug, Liporubicin, in sarcomas of the rat thoracic cavity (45
In contrast to the above results with focal lung illumination, the evaluation of pneumonectomy followed by spherical illumination to treat diffuse thoracic disease proved fatal to all animals studied (18
). Thus, the results of the present report are significant in that they establish that intracavitary light delivery to the murine thoracic cavity is feasible, and well-tolerated with minimal mortality. The improved mortality rate we experienced at therapeutic fluences is undoubtedly related to the lack of accompanying surgery, but nonetheless, control of tumor progression was still demonstrated. It is also important to note that different photosensitizers were used in these studies; our work utilized HPPH while Krueger et al (18
) used mTHPC, which could account for differences in outcome.
HPPH-PDT of the murine thoracic cavity can result in significant numbers of animal deaths as evidenced by a mortality rate of ≥50% at the highest fluences tested; however, a dose of 50 J/cm resulted in a more modest mortality rate of 11%. As noted in the Results, fluence-dependent mortality was considered separately from mortality related to placement of the light fiber itself. This distinction is important because fluence-dependent mortality is inherent to the study, and as suggested by histopathology, likely attributable to PDT effects on normal lung tissue (including pneumonia and vascular congestion, the severity of which can be expected to increase with increasing fluence). In contrast, technical mortality due to light fiber insertion or positioning is potentially addressable, for example through ultrasound guidance of fiber insertion or increasing operator experience with the technique.
Damage to the normal tissues, although undesirable, is inevitable in PDT of large surface areas with untargeted photosensitizers. The histopathologic changes associated with normal tissue injury in this study are consistent with that reported by others in thoracic PDT of tumor-free animals. Interstitial light delivery via a bare-tipped fiber in mTHPC-PDT of rat lung led to vascular damage, necrosis, RBC extravasation, edema, and fibrin deposition in the treated foci, as well as neutrophil infiltration into the surrounding areas (46
). Similar evidence of PDT-induced vascular disruption, alveolar congestion and hemorrhagic necrosis was noted in the lungs of mTHPC-treated pigs (47
) and Photofrin-treated rats (48
). Unlike that noted in the studies by Fielding et al (46
), however, our evaluation of interstitial HPPH-PDT of the mouse thoracic cavity did not create marked zones of necrosis, but rather scattered foci of necrotic cells. Treatment-induced pneumothorax, as has been detected in other studies (47
) could provide one explanation of this finding as it would alter the geometry of light exposure to the lung, while theoretically increasing diffuse dose to the pleural surfaces. Interestingly, diffuse light delivery via spherical illumination after pneumonectomy in the rat also did not produce identifiable focal zones of necrosis (18
), which reconciles well with the results of our study.
Local PDT damage to organs of the thoracic cavity manifested as an acute systemic inflammatory response, characterized by massive increases in circulating neutrophils and macrophages, decreases in blood lymphocytes, and thrombocytopenia. Such an inflammatory response is a well-documented characteristic of PDT, including applications that utilize HPPH or Photofrin as photosensitizers (49
). Furthermore, elevations in AST, ALT and CPK were also noted within 24 hours after thoracic PDT, all of which could be a consequence of acute damage to lung or muscle tissue (51
). Elevations in CPK have also been found after Photofrin- or HPPH-PDT of canine lung (52
) and returned to normal within a week of treatment (53
). Importantly, in the present study, no biochemical evidence of a systemic response was found after treatment with light-only despite its association with limited damage to the lung interstitium and vasculature, most likely attributable to physical or thermal damage of tissue in close contact with the light fiber. Similarly, light-only controls produced no effect on tumor weight or volume compared to untreated and/or HPPH-only controls. This further indicates that local histopathologic damage from light (alone) was too inconsequential to produce a more generalized effect.
MRI has been used to detect primary tumor as well as metastases to the rodent lung (54
) and provides the advantage of assessing treatment effect in the same animal. Although imaging the murine lung can prove challenging due to organ motion (57
), it is feasible, especially with the benefit of respiratory and cardiac gating (58
). Our initial studies establish that tumor volume measured by MRI is highly correlated with the mass of tumor collected and weighed at necropsy, and thereby provides a good representation of total tumor burden. Imaging was then applied to measure the intra-animal change in tumor burden between the day before (day −1) and five days after (day +5) PDT. Focus was placed on these time points as opposed to imaging more frequently because we had found that the repeated physiological stress of prolonged anesthesia (at least 1 hour from setup to acquisition of all three imaging planes within one mouse) on animals with lung disease led to their demise immediately after MRI. As an aside, a more slowly growing tumor model would enable the imaging to be scheduled less frequently and could perhaps facilitate multi-time point monitoring over a longer duration after PDT.
MRI results showed PDT to significantly slow tumor progression, with the PDT-treated tumors increasing in volume by 2.2-fold between day −1 and day +5, compared to a 3.4-fold increase in the control tumor volume. This increase in tumor volume in control mice is in good agreement with the tumor growth curve, which predicted an approximately four-fold increase in tumor burden between 11 days (corresponding to day −1) and 17 days (corresponding to day +5) after injection of the tumor cells. The presence of a significantly smaller increase in disease burden in the PDT-treated mice suggests that PDT reduced growth rate, which is supported by the histologic observation (data not shown) that fewer mitoses per high power field were present in PDT-treated tumors (5-9 mitoses) than in the light-treated controls (13 – 17 mitoses). Because this tumor model is highly aggressive and grows rapidly, evidence of a PDT-created reduction in tumor burden would best be detected within the first one or two days after treatment. That said, the presence of inflammation complicates interpretation of MR images within the first several days of treatment, so day +5 was instead used as the post-treatment imaging time point. At 24 h after PDT, necrosis is detectable via histologic assessment, but the presence of pre-existing necrosis invalidates any attempt to estimate treatment-induced tumor necrosis. Moreover, the presence of pre-existing necrosis suggests that these tumor nodules contain areas of hypoxia, which could reduce therapy-induced cell death by limiting delivery of photosensitizer and creation of the oxygen-dependent reactive species responsible for PDT-induced damage. Such hypoxic areas are, indeed, characteristic of clinical lung malignancies (61
). Continuing studies will more closely examine tumor hypoxia and any limitations it imposes on PDT-created cell death, as well as the introduction of approaches that augment direct cellular damage when combined with PDT.
A second advantage to MR imaging is the ability to provide information on the spatial distribution of tumor within the thoracic cavity. Our analyses discovered initial tumor growth to favor the right caudal ventral compartment of the thoracic cavity, which is most logically explained by injection of the tumor cells into the right side of the cavity and the effects of gravity as the tumor disseminates. PDT significantly reduced tumor progression, but it had no substantial impact on tumor burden distribution. This suggests that the treatment light was scattered throughout the cavity, and therefore no spatial compartment was completely spared by PDT. More generally, the results serve to prove this analytic approach as useful in quantification of heterogeneities in tumor distribution within the murine thoracic cavity. In clinical applications of PDT for disseminated pleural disease, it is necessary to overcome heterogeneities in the distribution of millimeter-sized nodules in order to control disease burden; this makes it both relevant and valuable to assess new PDT approaches in pre-clinical models for their ability to reduce not only overall disease burden but also the heterogeneities in its distribution.
PDT of disseminated thoracic malignancies is a potential adjuvant modality for better disease control and improved overall survival in the treatment of malignant pleural mesothelioma (1
) and NSCLC with pleural spread (3
). Due to the involvement of the pleural surfaces, treatment with even the most aggressive surgery or radiation therapy compatible with life cannot completely sterilize the region; therefore a local recurrence is inevitable (64
). PDT can potentially fill this need by providing an adjuvant approach to deliver superficial treatment to large surface areas with a single application (67
). It is an evolving application, and thus stands to be benefited by potential insights from pre-clinical studies that model PDT delivery to diffuse thoracic disease. In the present report, we describe an animal model of disseminated thoracic malignancy that is reproducible, well-tolerated, and feasible, together with a technically straightforward procedure to deliver intrathoracic PDT. HPPH-mediated interstitial PDT of the murine thoracic cavity slows progression of disseminated disease with acceptable damage to normal tissue consistent with mechanisms of HPPH action, including vascular disruption and necrotic death. Utilizing MR imaging, it is possible to study PDT effects on tumor burden and distribution, much in the same way that a patient would be evaluated by noninvasive imaging.