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Research Articles
TNF-
–based accentuation in cryoinjury—dose, delivery, and response
Departments of 1 Biomedical Engineering, 2 Mechanical Engineering and 3 Urologic Surgery, University of Minnesota, Minneapolis, Minnesota; 4 Department of Pathology, West Virginia University, Morgantown, West Virginia; and 5 CytImmune Sciences, Rockville, Maryland
Requests for reprints: John C. Bischof, Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN 55455. Phone: 612-625-5513. E-mail: bischof{at}umn.edu
Abstract
Cryosurgery is a minimally invasive cancer treatment using cryogenic temperatures. Intraoperative monitoring of iceball growth is an advantage of the treatment. However, whereas the iceball can be easily visualized, destruction within the iceball is incomplete and the means to monitor the "kill zone" are urgently needed. Recently, we have shown the ability of tumor necrosis factor-
(TNF-) to enhance destruction within an iceball. To avoid systemic toxicity, we delivered TNF- selectively to the tumor by a gold nanoparticle of 30-nm diameter (CYT-6091) tagged with TNF- and thiol-derivatized polyethylene glycol. Using a dorsal skin fold chamber (DSFC) in a nude mouse, both normal skin and human prostate carcinoma (LNCaP Pro 5) were pretreated with soluble TNF- (topically or i.v.) or CYT-6091 (i.v.) and frozen after 4 h. The cryolesion was assessed after 3 days by comparing histologic necrosis with perfusion defects. Hind limb tumors were also treated by visibly encompassing the tumor with an iceball and assessing gross changes over time. A 5-µg dose of soluble TNF- or CYT-6091 increased the temperature threshold of necrosis in the tumor in the DSFC from –14.0 ± 1.6°C (n = 6) to 0.9 ± 1.5°C (n = 6) and –1.5 ± 3.7°C (n = 6), respectively. In hind limb tumors, the same dose resulted in significant tumor shrinkage and remission in 2 of 8 (for soluble TNF-) and in 3 of 8 (for CYT-6091). The nanoparticle alone group without TNF- increased the temperature threshold of necrosis to –7.0 ± 2.3°C in the tumor in the DSFC and more shrinkage of the tumor in the hind limb when compared with cryo alone treatment. Systemic toxicity was noted in all soluble TNF- groups but none with CYT-6091. These results suggest that it is possible to destroy all of a tumor within an iceball by preincubation with TNF- and systemic toxicity can be avoided by CYT-6091. [Mol Cancer Ther 2007;6(7):2039–47]
Introduction
Cryosurgery is currently used for the treatment of prostate and liver cancers and is also recommended for retinoblastoma, early-stage skin cancers, and breast and colon cancers. With recent advancements in the cryosurgical apparatus and improved image guidance, the procedure is increasingly safe and controlled (1). Despite advantages including ease of operation, low morbidity, and low cost, the efficacy is limited by the incomplete destruction of the tissue inside the iceball. This is reflected in the clinical guideline of achieving –40°C to ensure complete cell death and the reports of local recurrence after freezing within the prostate (2–4). Thus, whereas the iceball can be visualized by ultrasound, computed tomography, or magnetic resonance imaging, the thermal efficacy of the iceball is questionable, and the means to monitor the "kill zone" within it are urgently needed. The present work centers on the use of a nanoparticle-based drug carrier both as an adjuvant to increase the cryosensitivity within the periphery of the iceball (0 to –40°C), which would otherwise remain viable, and as a delivery agent to limit the systemic dose while producing an effective local dose at the tumor site. Specifically, the goal is to extend the edge of necrosis (i.e., kill zone) up to the edge of the iceball while simultaneously sparing surrounding normal tissue.
In recent years, various adjunctive therapies have been proposed, which, when supplemented with cryoablation, enhance overall destruction. These adjunctive therapies can be grouped into two categories: (a) combination with other forms of cancer therapies such as radiation, hyperthermia, chemotherapeutics, etc. (5–7); and (b) the use of adjuvants to accentuate established mechanisms (direct cell injury, vascular injury) of cryoinjury (8–10). The search for adjunctive therapies that can destroy all of the frozen tissue still remains a challenge. Our approach has been to augment mechanisms already present in cryoinjury.
Inflammation occurring either during or subsequent to the process of freezing is thought to play a critical role in tissue necrosis during cryotherapy. Damage to the endothelium and the ensuing inflammation have long been observed in frostbite injury and are considered critical in defining the edge of the lesion (11–13). Thus, one possible means of augmenting the efficacy of cryotherapy may be the induction of the localized inflammatory response within the solid tumor before freezing. Indeed, we observed that the topical administration of the proinflammatory cytokine tumor necrosis factor- (TNF-) at a low dose 4 h before cryosurgery augmented vascular injury in the cryolesion in vivo (10). This work was the first to show the ability to destroy all of a tissue within the iceball using an adjuvant approach.
Although TNF- is well known for its role in inflammation and immunity and for its antitumor properties, systemic toxicity associated with it has limited its use in cancer therapy to isolated limb perfusion (14–16). Thus, targeting of TNF- to the tumor could be a challenge in achieving the cryosurgical adjuvant efficacy. Recently, Paciotti et al. (17) described a colloidal gold nanoparticle drug delivery system that targets the delivery of TNF- to solid tumors. The nanodrug, termed CYT-6091, consists of a suspension of colloidal gold nanoparticles (average diameter, 30 nm) to which TNF- and thiol-derivatized polyethylene glycol are covalently bonded (17). We have recently used this nanodrug to augment hyperthermic injury in a hind limb model (18).
The purpose of this study was to establish the in vivo efficacy of TNF- on the temperature threshold (defined as the minimum temperature reached at the edge of a moderate freeze-thaw injury; ref. 19) with a localized administration of TNF- in both normal and cancer tissues and also to evaluate the feasibility of systemic application of both soluble and nanoparticle-bound TNF-. A specific goal was to show the possibility of complete destruction of tumor within an iceball with an appropriate systemically delivered TNF- adjuvant while minimizing TNF-–related toxicity. The model chosen was LNCaP Pro 5 human prostate cancer grown in a dorsal skin fold chamber (DSFC) and a hind limb tumor in a nude mouse preparation. The DSFC allowed a reproducible temperature history to be applied and monitored continuously (20). A thin microvascular preparation was created, where the LNCaP Pro 5 tumor could be propagated and visualized by intravital microscopy (21). A controlled cryotreatment protocol was applied and injury was assessed by intravital perfusion defect and postmortem histology. In the hind limb tumor model system, i.v. injections of both soluble TNF- and CYT-6091 were administered and tumor destruction was compared with and without cryosurgery. We observed, at the doses used in the study, that both soluble TNF- and CYT-6091 were capable of destroying the entire tumor within the iceball but only CYT-6091 accomplished this without systemic toxicity.
Materials and Methods
Animals
All animal protocols were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee. Male athymic nude mice weighing 20 to 25 g were purchased from the National Cancer Institute and housed according to the University-approved standard operating procedures. Animals with DSFCs were housed at slightly higher humidity and temperature to maintain the microvascular preparation. When appropriate, animals were anesthetized by an i.p. injection of ketamine and xylazine at 100 and 10 mg/kg, respectively.
Cell Culture
LNCaP Pro 5 human prostate cancer cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum, antibiotics, and 10–9 mol/L dihydrotestosterone as previously described (10, 22).
Transfection
LNCaP Pro 5 cells were transfected with DsRed-express fluorescent protein DNA to monitor the growth of tumor in the DSFC under fluorescence. One million cells mixed with 5 µg of DsRed-express plasmid vector (Clontech Laboratories) were suspended in 400-µL medium and placed in a 2-mm electroporation cuvette. Transfection was done with an ECM-600 (BTX, MA) electroporator at 100 V and 3,150 µF. Stable clones were identified and isolated by culturing in G-418 antibiotic selection medium.
DSFC Design and Implantation
The DSFC allows intravital two-dimensional, controlled growth and visualization of tumors as previously described (10, 13, 23). In brief, the dorsal skin of each mouse was sandwiched between two anodized aluminum frames with 10-mm-diameter viewing windows, separated by a distance of 450 µm and maintained by spacers on the screws. The epidermis was removed from the viewing side along with excess fascia to permit better visualization of the microvasculature (10). Windows milled from quartz glass microslides were used to cover the vascular area.
Tumor Cell Implantation
DSFC. LNCaP Pro 5 cells were suspended in 30 µL of Matrigel matrix (Matrigel diluted 3:1 in serum-free medium; BD Biosciences). One to two million cells were inoculated into the DSFC chamber window on both day 0 and day 4 of implantation. The reapplication was done to ensure complete growth throughout the chamber. The tumor growth was monitored by fluorescence microscopy and experiments were done on day 12 following DSFC implantation, when the tumor covered the entire chamber.
Hind Limb. One to two million LNCaP Pro 5 tumor cells, prepared as described above, were s.c. injected in the hind limb of mice. Experiments were done after 5 to 7 weeks when a tumor diameter of 8 to 9 mm was obtained. Animals forming round and symmetrical tumors were randomized into the various groups for experiments.
TNF-–Mediated Inflammation
On the day of the study, soluble TNF- or CYT-6091 (CytImmune Sciences) was administered either topically (DSFC) or i.v. (DSFC and hind limb). For topical administration in the DSFC, the glass window was removed and soluble TNF- (2 and 200 ng) dissolved in 30-µL saline was directly applied. The glass window was replaced after 15 min and cryosurgical treatment was done 4 h later. I.v. injections were done in the tail vein with 5 µg of soluble TNF- or CYT-6091 suspended in 100-µL saline in both the DSFC and hind limb tumors, and cryosurgery was done after 4 h. The TNF- dose was based on our previous work with TNF- and CYT-6091 in cryosurgery and hyperthermia (10, 18). The 4-h time interval was chosen between TNF- application and cryosurgery because up-regulation of cell adhesion molecules such as intercellular adhesion molecule and vascular cell adhesion molecule and reduction in blood perfusion within the tissue have been shown to take place within this time period (18, 24).
Groups
After randomization, the following groups were assigned in the DSFC (both normal skin and tumor) and hind limb: control (no treatment), sham treatment (cryoprobe not activated), cryo alone, Pt-cAu (gold nanoparticles with thiol-derivatized polyethylene glycol but no TNF-), Pt-cAu followed by cryosurgery, CYT-6091 alone (5 µg), CYT-6091 followed by cryosurgery (5 µg), soluble TNF- alone (5 µg), and soluble TNF- followed by cryosurgery (5 µg). The following groups were assigned only in the DSFC model system: topical TNF- alone (2 and 200 ng) and topical TNF- followed by cryosurgery (2 and 200 ng). The number of animals per group is summarized in Table 1
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Before cryosurgery, the glass window covering the chamber was replaced with a Lexan disc, which had a hole of 1-mm diameter in the center (for insertion of cryoprobe tip) and 0.5-mm diameter holes (for thermocouple placement) at radii of 2, 3, and 4 mm (Fig. 1A ), as previously reported (10). A hole was then made at the center of the tissue using a 21-gauge needle. A 1-mm-diameter brass fin fitted to a 5-mm cryoprobe (Endocare) was inserted from the top into the hole. A type T thermocouple with a bead diameter of 0.5 mm was soldered on the tip of the fin and other thermocouples were placed in the remaining holes to monitor temperature at the center and specific locations, respectively (Fig. 1A). The temperature profile throughout the chamber was simultaneously monitored by IR thermography. An IR camera (FLIR) was focused onto the skin of the mouse from the side of the chamber opposite the probe (Fig. 1A). The camera was calibrated from –20°C to 30°C in the field of view.
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4 mm and was then allowed to thaw passively at room temperature for 15 min. IR images were taken throughout the procedure. Temperature at a desired location inside the chamber was calculated with Thermagram IR image software (Thermoteknix). The temperature data from thermocouples were recorded using a Hydra Data Logger Series 2 (Fluke). For sham cryosurgery, the above described procedure was repeated, except the cryoprobe was not activated. At the end of above procedure, the Lexan window was replaced by glass and animals were returned to their cages for housing. Hind Limb. A small incision was made in the center of the tumor with a 21-gauge needle and the tip of the 1-mm probe (same as one used in DSFC) was inserted slightly through the slit as shown in Fig. 1B. Care was taken that the probe did not exert pressure to deform the tumor shape. The cryomachine was then activated with the tip temperature set at –120°C. The tumor was frozen under visual guidance until the edge of the iceball reached the periphery of the tumor (Fig. 1B). Occasionally, grossly asymmetrical probe placement and iceball growth occurred and these data were discarded. The time of freezing varied between 2 and 3 min, depending on the size of the tumor. The tumor was allowed to thaw passively at room temperature.
Temperature Measurement in the DSFC
To compensate for the condensation induced artifacts in temperature measurements, the IR camera was calibrated by controlled freezing studies using normal tissue. Tissue pieces (bologna beef slices) were frozen in vitro on a controlled directional stage, as previously described (25). The stage, with tissue pieces on the top, was set at fixed temperatures between –20°C and 30°C and allowed to equilibrate. Actual tissue temperatures on the upper surface were obtained from T-type thermocouple with a bead diameter of 0.1 mm. The temperature of this surface was then measured by IR camera. Thermocouple-measured temperature and IR-measured temperature were plotted and the equation obtained was used to correct the IR temperature measurement for use with the DSFC. A quasi-steady thermal model as previously described for the DSFC further validated these measurements (10). In brief, the system can be modeled as a one-dimensional cylindrical system and solved to obtain temperature as a function of radius (10, 20).
 | (1) |
Here a, b, c, and d are known constants dependent on probe radius, thermal conductivity of tissue, phase change temperature, and the temperature of the probe (10). Rpr is the probe tip radius (0.5 mm).
Injury Measurement
DSFC. The effects of TNF- and/or cryosurgery on vascular damage were visualized with 10 mg/mL of 70-kDa FITC–labeled dextran at 3 days posttreatment as previously described (10, 13). The animals were placed on a mouse stage specifically designed to image the vasculature through a Nikon inverted microscope. Stasis was defined as the lack of blood flow (i.e., perfusion defects) as evidenced by the lack of fluorescence signal within a vessel. The chamber was traversed radially in the four perpendicular directions (north, south, east, west) and radial locations of stasis (i.e., perfusion defects) noted using a micrometer scale fixed on the stage with the chamber center as the origin (19). The vasculature was simultaneously imaged using a Silicon Intensified Transmission camera (Hamamatsu, North Central Instruments) and videos were recorded and processed as previously described (10). In brief, the individual images were extracted and tiled to recreate the entire vasculature and obtain the edge of stasis (Fig. 2A
). Four such radial measurements were obtained from each animal. Temperatures at the edge of all the measured radii of stasis were then extrapolated from the respective IR image taken after cryosurgery and averaged to obtain the temperature threshold of injury for the particular animal (Fig. 2B). Animals were sacrificed after vascular imaging and the entire chamber fixed in 10% neutral-buffered formalin and processed for histology. After sectioning and staining with H&E, the radii of necrosis were measured on the slides and compared with the corresponding radii of stasis for each animal.
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Hind Limb. Tumor volumes were measured every 3 to 4 days for up to 30 days after the treatment. Tumor dimensions were measured using calipers and volumes were calculated as width x length x height x 0.53 (27).
Statistics
One-way ANOVA followed by Bonferroni's multiple comparison test was conducted to determine statistical significance between different groups using a statistical software (SPSS); P < 0.05 was considered significant.
Results
Growth and Temperature Profiles of LNCaP Pro 5 Tumors Inside DSFC
LNCaP Pro 5 tumors growing inside the DSFC were clearly visible throughout the chamber as shown by the red fluorescence of the DsRed protein (Fig. 2A). The tumors were clearly differentiated from normal skin, which only exhibited weak autofluorescence. This was important because, occasionally, the tumors did not grow or were localized in small regions in the chamber. Fluorescence microscopy thus ensured the presence of tumor in the entire chamber before carrying out experiments.
A linear calibration fit was obtained between the thermocouple-measured temperature and IR-measured temperature in the controlled directional stage experiments.
 | (2) |
This equation was used to obtain corrected temperatures from the IR images recorded during cryosurgery. The temperature profile in the DSFC varied radially, with temperatures lowest at the center and increasing outwardly (Fig. 2B). The thermocouple measurements have been validated with the thermal model for the DSFC previously used by Hoffmann and Bischof (20). The IR measurements at specific locations also correlated well with the thermocouple measurements and fell within its SD (Fig. 2C). The individual temperature profile at the end of treatment in the DSFC varied somewhat between animals, possibly due to differences in the tissue composition, blood perfusion, and variable contact between the probe and the tissue. These variations were accounted for by using individual thermal histories to calculate the temperature threshold of necrosis for each animal used in the experiments. In conclusion, we used IR for all animals where stasis occurred at T > -20°C and thermocouples with model verification where stasis occurred at T < -20°C.
Characterization of Cryotherapy in LNCaP Pro 5 Tumors in DSFC
Injury was characterized by intravital fluorescence of perfusion and postmortem histology. All control unfrozen tumors showed blood flow throughout the chamber as visualized by FITC-dextran fluorescence. Sham treatment displayed patent vasculature beyond the probe insertion site at 0.5-mm radius. The chambers frozen with or without TNF- intervention displayed a central static region surrounded by perfused tissue in the rest of the chamber. There was increased permeability (not quantified) at the edge of the injury evident as blurriness due to the leakage of dye as previously noted (10, 13). Aggregates of RBC were also seen occasionally on the tissue surface, primarily close to the injury edge, suggesting hemorrhage. WBC rolling and adhesion, indicative of enhanced inflammation, was observed just before cryotreatment in all animals pretreated with TNF- as previously noted (21). Posttreatment analysis of histology showed a centrally necrotic region surrounded by a transition region, which was composed of both viable and dead cells, followed by normal tissue morphology. The boundary for blood perfusion, as marked by DiOC7 injection, superimposes with the edge of necrosis on H&E-stained slides as shown in Fig. 3
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pretreatment were found as shown in Table 2
. The average radii of stasis (measured by FITC-Dextran) and radii of necrosis (measured by histology) on both normal skin and tumor showed an increase in all TNF- pretreatments. Injury, as measured by perfusion defect and histologic necrosis, was expected to match within 0.5 mm as previously reported for the transition width of the cryolesion (25). As can be seen, the average radii measured by these two methods in all groups were within 0.1 to 0.4 mm, which corresponds to roughly 1°C to 5°C temperature offset in the DSFC at a radial location between 3 and 4 mm. To further verify the colocalization of perfusion defect and histologic necrosis at precise microscopic locations, the dye DiOC7 was used to visualize stasis directly within the area of necrosis and the regions were found to match within 0.5 mm (Fig. 3).
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–Induced Accentuation in Cryoinjury in DSFC in normal skin increased the average radius of stasis from 2.4 ± 0.3 mm for freeze only to 3.0 ± 0.1 and 3.8 ± 0.4 mm for pretreatment with 2 and 200 ng of TNF-, respectively (Table 2). The corresponding temperature of necrosis was raised from –27.7 ± 5.0°C for freeze only to –13.0 ± 2.5°C and –2.6 ± 2.7°C for pretreatment with 2 and 200 ng of TNF-, respectively (Fig. 4A). Systemic injections of 5 µg of Pt-cAu, soluble TNF-, and CYT-6091 preparations showed an augmentation of injury to –11.5 ± 3.1°C, –11.0 ± 2.6°C, and –10.1 ± 4.8°C, respectively. Two of the seven normal skin animals pretreated with 5-µg soluble TNF- died a few hours after cryotreatment, which is likely due to systemic toxicity. No animals died after CYT-6091 injection and cryotreatment.
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and CYT-6091 also shifted the edge of necrosis closer to the iceball edge to temperatures of 0.9 ± 1.5°C and –1.6 ± 3.7°C, respectively, as shown in Fig. 4A. Systemic injection of 5 µg of Pt-cAu followed by cryosurgery produced a smaller augmentation to a temperature of –7.0 ± 2.3°C. Two of the eight tumor-bearing animals pretreated with 5 µg of soluble TNF- died a few hours after cryosurgery, again likely due to systemic toxicity. All the chambers pretreated with TNF- had a distinct eschar formation at day 3 when observed from the external (skin) side of the chamber (Fig. 3B). This was especially evident at the higher doses of TNF- (200 ng topical dose, 5 µg of soluble TNF- and CYT-6091), suggesting extensive tissue damage within the treated tissue.
TNF-–Induced Accentuation in Cryoinjury in Hind Limb
Tumors treated with either soluble TNF- or CYT-6091 before cryosurgery exhibited a drastic reduction in tumor volume already at day 3 as seen in Fig. 5A
. The volume was reduced to 80.7 ± 12.7% of the initial volume at day 3 by cryo alone. This compares to a greater reduction of 45.8 ± 14.6% and 50.4 ± 18.9% of the initial volume for cryosurgery done after pretreatment with 5 µg of soluble TNF- or CYT-6091, respectively. The effects of TNF- alone or CYT-6091 alone on tumors without cryotreatment as noted in Table 1 resulted in no significant reduction in tumor volume as compared with control tumor growth at the doses used in this study and tracked similar to control tumors (data not shown). The control tumor showed significant growth as compared with all groups. There was significant darkening and eschar formation at day 3 in both the soluble TNF-– and CYT-6091–treated groups as seen in Fig. 5C, which resolved between days 10 and 20 posttreatment in all animals. Some of the tumor animals in both these groups (2 of 8 in soluble TNF- and 3 of 8 in CYT-6091) collapsed completely over time (days 3–15) and were not palpable or measurable (day 30 shown in Fig. 5C). The remaining tumors in both these groups showed a significant growth delay. Only one of eight tumors in the CYT-6091 group and none in the soluble TNF- group reached original volume by day 30 (Fig. 5B). The differential response (i.e., reduction versus regression) was probably due to the variation in freezing of the tumors because total freeze time depended on the visual information of the iceball. Tumors treated with cryo alone showed significantly less growth delay, reaching original size within 10 days and continuing growth thereafter (Fig. 5A). The Pt-cAu group with cryo also showed a greater reduction in tumor volume at day 3 to 67.8 ± 11.0% and a significant growth delay when compared with cryo alone treatment as shown in Fig. 5A. Histologic analysis at day 30 in representative animals in each group showed no edema and only mild inflammation.
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–Induced Toxicity and CYT-6091 augmented the efficacy, the treatments were significantly different with regard to systemic toxicity. In hind limb tumors, two of eight animals pretreated with soluble TNF- died a few hours after cryotreatment. Such toxicity was also observed in both normal skin (2 of 7) and tumor (2 of 8) DSFCs pretreated with TNF- as mentioned above. No such toxicity was observed in animals pretreated with CYT-6091 followed by cryosurgery. Discussion
The data presented here provide compelling evidence for the use of localized TNF- as an adjuvant in cryosurgical procedures. The augmentation of injury observed in both DSFC and hind limb tumors suggests that tumor destruction is possible throughout a cryosurgical iceball with appropriately dosed and delivered TNF- adjuvant. The temperature threshold of necrosis increased with the addition of TNF- in a dose-dependent manner for all tissues in the DSFC as shown in Fig. 4A. In the hind limb, visually guided iceball growth led to significant reduction and, in some cases, complete remission of the tumor. In clinical situations where progression of the iceball is monitored and can be directly compared with the tumor volume by ultrasound, computed tomography, or magnetic resonance imaging, a closer match between freezing and regression is expected.
The vascular events following cryosurgery are thought to be critical in governing the kill zone at the periphery of an iceball, where temperatures are not low enough for direct cell death (19). Various in vivo studies have shown endothelial sloughing, invasion by neutrophils, free radical–mediated injury, and formation of thrombus after freezing insult (11, 12). In vitro results on microvascular endothelial cells in our lab found them to be more cryosensitive than numerous tumor cells with a total loss of viability in suspensions below –20°C. This suggests endothelium as a critical target in cryosurgical injury (19, 28). Hence, our approach has focused on endothelial activation by addition of an inflammatory agent (10).
We have predominantly worked with the proinflammatory cytokine TNF-, which is closely associated with vascular events and injury as well as blood flow. High local administration has been shown to selectively destroy tumor vasculature and cause direct apoptosis and necrosis of endothelial cells (29–31). In this study, we used low doses of TNF-, which did not cause significant direct necrosis. The main reported actions of TNF- on endothelium are that of increasing procoagulant activity, decreasing anticoagulant activity, increasing leukocyte adherence, and production of other cytokines (32–34). The presence of TNF- recruits neutrophils, which release toxic enzymes and free radicals, and this may be a further mechanism of enhanced cryosensitivity (21). Another important effect of TNF- is the reduction in blood flow, which would otherwise interfere with the expansion of the iceball during cryosurgery. Rubidium uptake studies done in a breast carcinoma show a 75% reduction in blood flow in hind limb tumors after TNF- addition at 4 h followed by a gradual return of perfusion (18). In summary, both preinflammation and reduction in blood flow within the tumor are suggested mechanisms of TNF-–enhanced cryosurgery (35).
An increase in the thermal threshold of cryoinjury with TNF- addition in both normal and tumor tissue is shown in Fig. 4A. The thermal threshold for tumor was found to be higher than for normal skin for all the treatments done in the DSFC. Tumor vasculature is disorganized and not fully developed compared with normal skin. In addition, whereas augmentation occurs in both tissues, it is greater in tumor with systemic delivery of TNF-, possibly due to greater accumulation of the drug (Fig. 4A). In clinical situations, selective inflammation of only the tumor tissue and not the surrounding normal tissue before cryotreatment would increase the difference in temperature threshold of injury between normal and tumor tissue up to 20°C to 25°C (Fig. 4A). This is important specifically at the edge of the tumor where overfreezing into surrounding normal tissue can cause various complications, especially in the prostate. Thus, differential action and preferential delivery of TNF- to the tumor can help in destroying the entire tumor within an iceball.
Systemic toxicity associated with TNF- also makes local delivery important for adjuvant use (31). Interestingly, reperfusion of large cryosurgical iceballs releases various cytokines, including TNF-, acutely into the blood system and can be associated with "cryoshock," which can lead to organ failure and death (36). This additive effect may explain the death of the animals in this study, which were systemically injected with soluble TNF- before cryosurgery. To avoid this toxicity, gold nanoparticles coated with TNF- were used to selectively deliver the TNF- to the tumor. A 100% survival was observed with the CYT-6091 injection followed by cryotreatment. These nanoparticles are proposed to preferentially extravasate into the tumor. The tumor uptake mechanism is passive due to the pore size in leaky vasculature and active by receptor binding of TNF- to both endothelial and tumor cells.
The augmentation observed with the pretreatment of Pt-cAu (Figs. 4 and 5) suggests that these particles alone may possibly trigger some inflammation or local host-mediated response, which augments cryoinjury. There has been a recent discussion of the potential effects of nanosized particles including inflammation on various tissues and organs (37, 38). Additionally, gold nanoparticles were shown to possess antiangiogenic properties in vivo whereas carbon nanotubes have been shown to cause lung inflammation (39–41). There is limited and conflicting information about interaction of nanosized particles with biological systems. The possible outcomes and applications of using specific nanosized particles as determined by their size, coating, and targeting continue to be an active area of research (42, 43). Our control with only gold particles (Fig. 4A) shows accentuation with cryo but less than that for TNF-–coated particles, suggesting that both size and TNF- are important in the CYT-6091 enhancement of cryo treatment.
Cryosurgical injury results in a complex interplay of direct cell injury to the tumor and endothelial cells, as well as vascular events including ischemia/reperfusion injury, thrombus formation, and leukocyte-mediated injury. We have shown that TNF- injection 4 h before cryosurgery augments cryoinjury in both tumor and normal tissue. Further, the appropriate dose of TNF- was shown for the first time to destroy all tumor tissues within an iceball. Whereas both soluble TNF- and CYT-6091 were effective in achieving destruction inside the entire iceball, only CYT-6091 accomplished this without systemic toxicity. Future work will be needed to clearly establish the effect of nanoparticle alone (Pt-cAu) and TNF- on the augmentation of injury. We believe that mechanisms at the level of the endothelium and microvasculature are responsible for the enhanced injury. The TNF-– and/or Pt-cAu–induced inflammatory and apoptotic pathways in cryoinjury in tumor and endothelial cells are being characterized in separate studies to assess their respective importance in the reported enhancement of cryoinjury.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11/ 1/06; revised 4/18/07; accepted 5/25/07.
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  Article on TNF-{alpha} based accentuation in cryoinjury Mol. Cancer Ther., August 1, 2007; 6(8): 2383 - 2383. [Full Text] [PDF]
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