Evaluation of fluorescence-guided surgery agents in a murine model of soft tissue fibrosarcoma.

BACKGROUND AND OBJECTIVES: Soft tissue sarcomas (STS) are mesenchymal malignancies. Treatment mainstay is surgical resection with negative margins ± adjuvant treatment. Fluorescence-guided surgical (FGS) resection can delineate intraoperative margins; FGS has improved oncologic outcomes in other malignancies. This novel strategy may minimize resection-associated morbidity while improving local tumor control. METHODS: We evaluate the tumor-targeting specificity and utility of fluorescence-imaging agents to provide disease-specific contrast. Mice with HT1080 fibrosarcoma tumors received one of five probes: cetuximab-IRDye800CW (anti-EGFR), DC101-IRDye800CW (anti-VEGFR-2), IgG-IRDye800CW, the cathepsin-activated probe Prosense750EX, or the small molecule probe IntegriSense750. Tumors were imaged daily using open- and closed-field fluorescence imaging systems. Tumor-to-background ratios (TBR) were evaluated. On peak TBR days, probe sensitivity was evaluated. Tumors were stained and imaged microscopically. RESULTS: At peak, closed-field imaging TBR of cetuximab-IRDye800CW (16.8) was significantly greater (P < 0.0001) than Integrisense750 (7.0), Prosense750EX (5.8), and DC101-IRDye800CW (3.7). All agents successfully localized as little as 1.0 mg of tumor tissue in the post-resection bed; cetuximab-IRDye800CW generated the greatest contrast (2.5). Cetuximab-IRDye800CW revealed strong tumor affinity microscopically; tumor fluorescence intensity was significantly greater (P < 0.0004) than 0.2 mm away from tumor border. CONCLUSION: This study demonstrates cetuximab-IRDye800CW superiority. FGS has the potential to improve post-resection morbidity and mortality by improving disease detection.

Targeting tumors with a killer-reporter adenovirus for curative fluorescence-guided surgery of soft-tissue sarcoma.

Fluorescence-guided surgery (FGS) of cancer is an area of intense interest. However, FGS of cancer has not yet been shown to be curative due to residual microscopic disease. Human fibrosarcoma HT1080 expressing red fluorescent protein (RFP) was implanted orthotopically in the quadriceps femoris muscle of nude mice. The tumor-bearing mice were injected with high and low-dose telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401, which labeled the tumor with GFP. Fluorescence-guided surgery (FGS) or bright light surgery (BLS) was then performed. OBP-401 could label soft-tissue sarcoma (STS) with GFP in situ, concordant with RFP. OBP-401-based FGS resulted in superior resection of STS in the orthotopic model of soft-tissue sarcoma, compared to BLS. High-dose administration of OBP-401 enabled FGS without residual sarcoma cells or local or metastatic recurrence, due to its dual effect of cancer-cell labeling with GFP and killing. High-dose OBP-401 based-FGS improved disease free survival (p = 0.00049) as well as preserved muscle function compared with BLS. High-dose OBP-401-based FGS could cure STS, a presently incurable disease. Since the parent virus of OBP-401, OBP-301, has been previously proven safe in a Phase I clinical trial, it is expected the OBP-401-FGS technology described in the present report should be translatable to the clinic in the near future.

Intraoperative detection and removal of microscopic residual sarcoma using wide-field imaging.

BACKGROUND: The goal of limb-sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization. METHODS: A primary mouse model of soft tissue sarcoma and a wide field-of-view imaging device were used to investigate a series of exogenously administered, near-infrared (NIR) fluorescent probes activated by cathepsin proteases for real-time intraoperative imaging. RESULTS: The authors demonstrated that exogenously administered cathepsin-activated probes can be used for image-guided surgery to identify microscopic residual NIR fluorescence in the tumor beds of mice. The presence of residual NIR fluorescence was correlated with microscopic residual sarcoma and local recurrence. The removal of residual NIR fluorescence improved local control. CONCLUSIONS: The authors concluded that their technique has the potential to be used for intraoperative image-guided surgery to identify microscopic residual disease in patients with cancer.

Compatibility of the linear-quadratic formalism and biologically effective dose concept to high-dose-per-fraction irradiation in a murine tumor.

PURPOSE: To evaluate the compliance of linear-quadratic (LQ) model calculations in the high-dose range as used in stereotactic irradiation in a murine tumor model. METHODS AND MATERIALS: Female 10-week-old Balb/c mice bearing 1-cm-diameter EMT6 tumors in the hind legs were used. Single doses of 10-25 Gy were compared with 2-5 fractions of 4-13 Gy given at 4-hour intervals. Cell survival after irradiation was determined by an in vivo-in vitro assay. Using an α/ß ratio determined for in vitro EMT6 cells and the LQ formalism, equivalent single doses for the hypofractionated doses were calculated. They were then compared with actually measured equivalent single doses for the hypofractionated doses. These fractionation schedules were also compared simultaneously to investigate the concordance/divergence of dose-survival curves plotted against actual radiation doses and biologically effective doses (BED). RESULTS: Equivalent single doses for hypofractionated doses calculated from LQ formalism were lower than actually measured doses by 21%-31% in the 2- or 3-fraction experiments and by 27%-42% in the 4- or 5-fraction experiments. The differences were all significant. When a higher α/ß ratio was assumed, the discrepancy became smaller. In direct comparison of the 2- to 5-fraction schedules, respective dose-response curves almost overlapped when cell survival was plotted against actual radiation doses. However, the curves tended to shift downward by increasing the fraction number when cell survival was plotted against BED calculated using an α/ß ratio of 3.5 Gy for in vitro EMT6 cells. CONCLUSION: Conversion of hypofractionated radiation doses to single doses using the LQ formalism underestimated the in vivo effect of hypofractionated radiation by approximately 20%-40%. The discrepancy appeared to be larger than that seen in the previous in vitro study and tended to increase with the fraction number. BED appeared to be an unreliable measure of tumor response.

[Intravenous photodynamic therapy: first experimental and clinical data].

We carried out the first investigation to establish rat tumor M-1 by laser irradiation of the large blood vessel zone in the inguinal triangle in conjunction with photosensitinogen growth inhibition. Relatively small-size metastases to the lymph nodes and liver (stage III-IV) were identified after the blood was exposed to laser radiation in combination with intravenous injection of photosensitinogen under clinical conditions.

A study of the immunological response to tumor ablation with irreversible electroporation.

Immune cell recruitment during the treatment of sarcoma tumors in mice with irreversible electroporation was studied by immunohistochemistry. Irreversible electroporation is a non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the membrane. Employing irreversible electroporation parameters known to completely ablate the tumors without thermal effects we did not find infiltration of immune cells probably because of the destruction of infiltration routes. We confirm here that immune response is not instrumental in irreversible electroporation efficacy, and we propose that irreversible electroporation may be, therefore, a treatment modality of interest to immunodepressed cancer patients.

Treatments for the activating macrophages that reduces surgical stress and postoperative mortalities from bacterial infections and tumor metastases.

BACKGROUND: Some of the mortalities caused by infectious diseases and/or distant metastases following surgery are thought to be due to immunological suppression. For this reason, techniques that reduce immunological suppression following surgery may reduce mortalities and/or incidences of micrometastases in distant organs. MATERIALS AND METHODS: Mice were anesthetized and their peritoneal cavities were opened for 30 min. Immunological suppression was estimated by the presence of tumor necrosis factor-a (TNF) after injection with OK-432 (dead bacterial bodies). The mice were administered with either Staphylococcus aureus or cancer cells of Meth A fibrosarcoma. Survival times and lung metastastic foci were then observed at 3 weeks. Results were compared for mice with or without treatment by OK432 or TNF prior to surgery. RESULTS: While significant suppression of TNF production was observed after laparotomy, administration of a macrophage-activating agent (TNF or OK-432) 3 h prior to laparotomy prevented immune suppression after the laparotomy. Laparotomy increased mortalities from bacterial infections and promoted the number of lung metastases. By contrast, administration of TNF or OK-432 3 h prior to the laparotomy decreased mortalities and metastases after the laparotomy. CONCLUSION: These results suggest that appropriate activation of macrophages prior to surgery is a method to reduce some of the detrimental effects caused by surgical operations.

Radiofrequency ablation of lung malignancies.

Radiofrequency ablation is a new technology that has been used successfully to treat hepatic tumors. Recently, an increasing number of reports have described the use of radiofrequency ablation for primary and metastatic lung tumors. Although such early experience appears promising, many questions regarding patient selection, radiofrequency ablation technique, effectiveness of ablation on lung tumors, radiographic follow-up, and survival remain unanswered. This article addresses these issues and provides the thoracic surgeon with a current review of the application of radiofrequency ablation to lung tumors.

Normalization of hypothalamic serotonin (5-HT 1B) receptor and NPY in cancer anorexia after tumor resection: an immunocytochemical study.

Tumor growth leads to anorexia and decreased food intake, the regulation of which is via the integrated hypothalamic peptidergic and monoaminergic system. Serotonin (5-HT), an anorectic monoamine acts primarily via 5-HT 1B-receptors in hypothalamic nuclei while neuropeptide Y (NPY) acts an orexigenic peptide. We previously reported that 5-HT 1B-receptors are up regulated while NPY is down regulated in tumor-bearing (TB)-related anorexia, contributing to food intake reduction. In anorectic TB rats we hypothesize that after tumor resection when food intake has reverted to normal, normalization of 5-HT 1B-receptor and NPY will occur. The aim of this study was to demonstrate normalization of these hypothalamic changes compared to Controls. In anorectic tumor-bearing rats after tumor resection (TB-R) and in sham-operated (Control) rats, distribution of 5-HT 1B-receptors and NPY in hypothalamic nuclei was analyzed using peroxidase antiperoxidase immunocytochemical methods. Image analysis of immunostaining was performed and the data were statistically analyzed. Immunostaining specificity was controlled by omission of primary or secondary antibodies and pre-absorption test. Our results show that after TB-R versus Controls a normalization of food intake, 5-H-1B-receptor and NPY expression in the hypothalamus occurs. These data, discussed in context with our previous studies, support the hypothesis that tumor resection results not only in normalization of food intake but also in reversible changes of anorectic and orexigenic hypothalamic modulators.

Combination radiofrequency ablation with intratumoral liposomal doxorubicin: effect on drug accumulation and coagulation in multiple tissues and tumor types in animals.

PURPOSE: To determine whether use of radiofrequency (RF) ablation combined with intravenously (IV) administered liposomal doxorubicin, as compared with use of RF ablation or doxorubicin alone, facilitates increased tissue coagulation and interstitial drug accumulation in animal models. MATERIALS AND METHODS: The institutional animal care and use committee approved this study. In experiment 1, multiple canine sarcomas were implanted in seven mildly immunosuppressed dogs and grown to a mean diameter of 4.8 cm. Tumors were assigned to three treatment groups: internally cooled RF ablation (12 minutes, 2000-mA pulsed technique) followed by IV liposomal doxorubicin (10 mg per animal) (n = 6), RF ablation alone (n = 6), and liposomal doxorubicin alone (n = 4). In experiment 2, the livers and kidneys of 10 rabbits and the thigh muscles of 10 rats were randomly assigned to one of two treatment groups: conventional RF ablation (90 degrees C +/- 2, 5 minutes) followed by IV liposomal doxorubicin (5 mg per rabbit, 1 mg per rat) or RF ablation alone (n = 5, each). Coagulation diameter and interstitial doxorubicin concentration (tissues were homogenized in acid alcohol, with doxorubicin extracted for 24 hours at 5 degrees C and quantified with fluorimetry) were measured 48 hours after treatment and compared. Multivariate analysis of variance and subsequent pairwise t tests (alpha = .05, two-tailed test) were performed. RESULTS: Data are means +/- standard errors of the mean. A larger diameter of tumor destruction was observed in canine sarcomas treated with RF ablation-liposomal doxorubicin (3.7 cm +/- 0.6) compared with that in tumors treated with RF ablation (2.3 cm +/- 0.1) or liposomal doxorubicin (0.0 cm +/- 0.0) alone (P < .01). A new finding was a completely necrotic red zone (1.6 cm +/- 0.7) surrounding the central RF ablation-induced white coagulation zone. Greater but nonuniform drug uptake was observed particularly in this red zone (77.0 ng/g +/- 18.2) compared with uptake in the central zone (15.1 ng/g +/- 3.2), peripheral area of untreated tumor (38.9 ng/g +/- 8.0), and tumors treated with liposomal doxorubicin alone (43.9 ng/g +/- 6.7 for all regions) (P < .01 for all individual comparisons). In experiment 2, use of combined therapy led to increased coagulation in all tissues (liver: 17.6 mm +/- 3.1, P = .03; kidney: 11.0 mm +/- 3.1, P = .03; muscle: 13.1 mm +/- 1.3, P < .01) compared with use of RF ablation alone (liver, 13.4 mm +/- 1.5; kidney, 7.9 mm +/- 0.7; muscle, 8.6 mm +/- 0.5). Combined therapy, as compared with liposomal doxorubicin therapy alone, was also associated with increased doxorubicin accumulation in liver, kidney, and muscle (1.56 microg/g +/- 0.34, 4.36 microg/g +/- 1.78, and 3.63 microg/g +/- 1.43, respectively, vs 1.00 microg/g +/- 0.18, 1.23 microg/g +/- 0.32, and 0.87 microg/g +/- 0.53, respectively) (P < or = .01 for all individual comparisons). CONCLUSION: Use of RF ablation combined with liposomal doxorubicin facilitates increased tissue coagulation and interstitial doxorubicin accumulation in multiple tissues and tumor types and may be useful for treatment of large tumors and achieving an ablative margin within the untreated tissue surrounding RF ablation-treated tumors.

Serum cartilage-derived retinoic acid-sensitive protein (CD-RAP) levels in swarm rat chondrosarcoma.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a new protein that was isolated from bovine articular chondrocytes and human melanoma cell lines (melanoma inhibitory protein or MIA). In normal tissue its expression is limited to cartilage, and in morbid tissue to melanoma, chondrosarcoma, and breast cancer. Serum levels of CD-RAP/MIA correlate with the progression of malignant melanoma, but there have been no reports on chondrosarcoma. Here, it was first demonstrated by RT-PCR and immunohistological methods that CD-RAP was expressed in tissue from a Swarm rat chondrosarcoma that was used as an experimental model. The course following tumor transplantation and changes in serum CD-RAP after tumor excision were then observed to investigate whether serum CD-RAP could be used as a marker of tumor activity. Consequently, serum CD-RAP in control rats tended to decrease as the animal grew, whereas it rose in proportion to tumor proliferation in rats that had received a tumor graft. Serum CD-RAP levels dropped rapidly following excision of the tumor in a group of tumor-excised rats. In those rats which had a recurrence following excision of the tumor, serum CD-RAP rose prior to the appearance of the tumor. Serum CD-RAP thus sensitively reflected tumor onset and proliferation, so that it appeared to be an effective marker of tumor activity for Swarm rat chondrosarcoma.

Antimetastatic effectiveness of amifostine therapy following surgical removal of Sa-NH tumors in mice.

The effects of dose per fraction on the ability of amifostine exposure to elevate angiostatin levels in the serum of mice and to inhibit spontaneous metastases formation using the well-characterized murine Sa-NH sarcoma were investigated. Amifostine was administered intraperitoneally at doses of 50, 100, or 200 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8 mm in diameter. Amifostine was again administered immediately following surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Nontumor-bearing control animals were treated using the same dosing and surgery schedules. The average number of pulmonary metastases per animal was determined for each experimental group. A significant reduction (P <.05) in the average number of pulmonary metastases was observed only in the group of animals exposed to a dose per fraction of 50 mg/kg. A dose of 100 mg/kg was less effective while 200 mg/kg had no effect on metastases formation in this study. The effects of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin were also determined using Western analysis. Correlating with the antimetastatic effect measured, exposure of animals to 50 mg/kg of amifostine resulted in a four-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in both tumor-bearing as well as nontumor-bearing animals. In contrast, a dose of 200-mg/kg amifostine administered intraperitoneally under these conditions had no measurable effect on angiostatin serum levels in this animal system. The enhanced ability of relatively low doses of amifostine to inhibit spontaneous metastases formation suggests that effective antimetastatic therapies with amifostine can be designed with minimal toxic side effects. While the dose responses for angiostatin production and metastases inhibition by amifostine are well correlated, the precise mechanism of action underlying these phenomena is unclear but is suggestive of a redox driven process(es).

Tumor removal enhances immunity induced by B7.1.

Costimulation of tumor T cells by B7.1 has been shown to be important for eliciting cell-mediated anti-tumor immunity. We constructed a stable B7.1 gene transfectant of a poorly immunogenic murine sarcoma, Moloney murine sarcoma virus-induced tumor cell line (MMSV). This transfectant, MMSV-B7.1, failed to produce any tumor development in syngeneic mouse models. When MMSV-B7.1 was simultaneously injected with wild-type MMSV, about half of the coinjected mice remained tumor free and displayed an increase in T cell population, upregulation of the mRNA level of various cytokines such as IL-4, IL-5, IL-10, IL-13, IL-15 and IFN-gamma, and complete rejection of reinjected MMSV. To investigate whether MMSV-B7.1 demonstrates any vaccinal effect, the transfectant was injected following the surgical removal of the primary tumor mass. Following a re-challenge with wild-type MMSV, all vaccinated mice maintained their tumor free status and displayed a rapid recovery of down-regulated cytokine levels. The results suggest that B7.1 vaccination after tumor removal might be useful for the prevention of tumor recurrence.

Resection of solid tumors reverses T cell defects and restores protective immunity.

We have previously reported that CTL were demonstrable early after inoculation of CMS5 fibrosarcoma cells, but that they disappeared within 3 wk. These mice were unable to reject a challenge with CMS5 tumor cells. Other studies demonstrated cell surface phenotype and signaling abnormalities of cells within the spleen. Since we assumed that such an environment would make it more difficult to elicit antitumor immune responses via immunotherapy, we asked whether resection of the tumor could reverse these abnormalities. Although early after tumor cell inoculation tumor resection leads to the development of immunity, the effect at late time points has not been studied critically. To test this, mice were inoculated s.c. with CMS5 cells and after 28 days the tumors were resected. We observed a gradual normalization of the cellular phenotype of the spleen. In particular, there was a decrease in the number of Mac1+/Gr1(high) cells and an increase in the number of CD3+ cells in the spleen within 24-48 h of tumor resection. By day 10, these values were normal. Levels of p56lck increased as well. The functional implications of these changes were illustrated by the reduced growth rate or the complete rejection of a challenge of tumor cells in the resected mice. Both CD4+ and CD8+ cells were involved in the restoration of tumor immunity. Our results suggested that tumor resection not only led to the reversal of immune suppression, but also unmasked a population of primed T cells able to mediate protective immunity.

Gamma-globulin inhibits tumor spread in mice.

Intravenous (i.v.) Ig is the human serum Ig fraction that is mainly composed of IgG prepared from plasma pools of over 15,000 healthy blood donors and is suitable for i.v. use. High-dose i.v. Ig is currently used to treat patients with diverse autoimmune conditions. Autoimmunity and malignancy co-exist frequently, and share etiological and pathological mechanisms. Since the two diseases are similarly treated, we studied the efficacy of i.v. Ig as a treatment for malignant conditions. The administration of i.v. Ig to mice inoculated i.v. with melanoma or sarcoma cells induced a statistically significant inhibition of metastatic lung foci and prolongation of survival time. Similar results were seen with SCID mice inoculated with SK-28 human melanoma cells. In a different model, melanoma was induced in the foot pad, followed by leg amputation, after the development of the tumor lesion. A lower number of melanoma recurrences and prolongation of survival time were demonstrated in the i.v. Ig-treated groups. In vitro studies revealed that i.v. Ig was found to stimulate the production of IL-12, an anti-tumor and anti-angiogenic cytokine. Moreover, it enhanced NK cell activity, thus explaining its beneficial effect in SCID mice (which lack B and T but possess NK cells). The results indicate that i.v. Ig acts as an anti-tumor agent. Since it has only minor side effects and is used extensively for other clinical conditions, i.v. Ig may be considered as a potential therapy for the prevention of tumor spread in humans.

Effect of a microwave coagulator on implanted liver neoplasms in rats.

An experimental study was conducted to determine the applicability of a microwave (MW) source for coagulation and dissection of metastatic tumour and liver parenchyma. Twenty PVG rats were studied after implantation of MC28 tumour cells into the liver. Eleven days later they underwent sham laparotomy (control) MW coagulation of the tumour deposit, local resection of the tumour deposit with 2 mm clearance or resection of the tumour with MW coagulation of the resection zone. The control animals all died with blood-stained ascites and heavy tumour burden in the liver. After MW coagulation, tumours disappeared in all but one rat. Local resection also led to tumour clearance in 4 of 5 rats. MW coagulation of the resection zone allowed rapid bloodless resection, and no tumour recurrence was observed after 40-89 days. We conclude that MW coagulation is a potentially useful tool for ablation of hepatic metastasis and as an adjunct to hepatic resection.

The effects of cisplatinum and vincristine on peripheral nerve regeneration.

Current treatment modalities for extremity sarcoma often include tumor extirpation plus neoadjuvant therapy. Limb-sparing surgery may require reconstruction of critical nerve defects. Neurotoxic side effects from adjuvant chemotherapy have been reported and raise concerns regarding the effects of chemotherapy on nerve regeneration. In an attempt to define the effects of adjuvant chemotherapy on peripheral nerve regeneration, cisplatin and vincristine were administered to rats following isografting of the posterior tibial nerve. Parameters used to assess peripheral nerve regeneration included walking track analysis and histomorphology. Sixty 250-g Sprague-Dawley rats were randomly allocated into one of three treatment groups. Each animal underwent a 15-mm reversed interposition nerve isograft from 30 donor rats into the right posterior tibial nerve. Ten animals served as control. The remaining animals were divided into two groups of 25 animals each. One group received cisplatin (75 mg/m2) and the other group received vincristine (1 mg/m2). Chemotherapy was administered at 4-week cycles for a total of six cycles (24 weeks). Walking track analysis was performed monthly. Nerve specimens were harvested from the grafted segment and the distal posterior tibial nerve for histomorphology. Walking track analysis demonstrated no statistical difference in print length between the control and chemotherapeutic groups at the conclusion of the study. The number of axons per square millimeter and nerve fiber density were not statistically different between control and chemotherapeutic groups. In the rodent posterior tibial nerve model, postoperative adjuvant therapy does not significantly alter functional outcome in peripheral nerve regeneration. The practice of immediate nerve grafting after tumor extirpation, despite planned postoperative chemotherapy, is supported.

Isolated lung perfusion with cisplatin in a rat lung solitary tumor nodule model.

BACKGROUND: The present study was conducted to evaluate the toxicity, pharmacokinetics and anti-tumor potency of isolated lung perfusion (ILP) with cisplatin in a visible lung tumor nodule model in rats. MATERIALS AND METHODS: A solitary tumor nodule was established by the injection of Methylcholanthrene-induced sarcoma cells into the left lung. Thirty rats were randomized to undergo ILP with either 0.1, 0.25, or 0.5 mg/mL cisplatin and buffered hespan (BHE), or with an intravenous injection of 1.0 or 2.5 mg cisplatin. RESULTS: The highest dose of cisplatin tolerated by the rats was 0.1 mg/mL for perfusion. A much higher platinum concentration in the tumor, of 6.67 +/- 1.64 vs. 2.51 +/- 0.60 micrograms/g tissue, but a significantly lower concentration in the serum and kidneys, was achieved by perfusion compared to that achieved by intravenous injection. A significantly lower tumor weight and 20% complete treatment response was achieved in rats given cisplatin than in those given BHE perfusion at 43.9 +/- 11.6 vs. 226.3 +/- 44.6 mg. CONCLUSION: ILP with cisplatin achieved superior results to intravenous injection according to the levels of toxicity and pharmacokinetic analysis, and it was effective against a visible tumor nodule model in rats.

Enhancement of experimental pulmonary metastasis and inhibition of subcutaneously transplanted tumor growth following cryosurgery.

We have previously reported that inhibition of anti-tumor immune responses and a corresponding enhancement of metastatic tumor growth occurred in rats following cryosurgery of 3-methylcholanthrene-induced WKA rat fibrosarcoma (KMT-17). In this study, to evaluate the enhancement of metastasis arising from the inhibition of anti-tumor immune responses following cryosurgery, we examined how cryosurgery affected experimental pulmonary metastasis and the growth of subcutaneously transplanted tumor. To reveal the effect of cryosurgery on pulmonary metastasis, rats received a subcutaneous inoculation of KMT-17 tumor in the right flank (1 x 10(6)) and i.v. injection (1 x 10(5)) on the same day or 4 days later. The right flank tumors were treated with cryosurgery 5 days after subcutaneous transplantation. The pulmonary metastasis of the rats, which were injected i.v. one day before treatment, was enhanced by cryosurgery as compared with surgical excision, though the pulmonary metastasis of rats, which were injected i.v. 5 days before treatment, was un-affected by cryosurgery. These observations suggest that cryosurgery may enhance the pulmonary metastasis in its early steps but has no effects in its later stages. To reveal the effect of cryosurgery on the growth of distant tumors, rats received subcutaneous inoculations of KMT-17 tumor in the right (1 x 10(6)) and left (1 x 10(4) approximately 10(5)) flanks. Tumors in the right flank were treated with cryosurgery 5 days after inoculation and the growth of untreated left flank tumors was observed. In this double grafted tumor system, however, cryosurgery significantly inhibited the growth of the untreated left flank tumors. Spleen cells obtained from rats which had undergone cryosurgery 4 or 10 days previously (cryo-spleen cells) were used for in vivo neutralizing Winn assay. Antitumor activity of cryo-spleen cells was decreased as compared with that of rats after surgical excision in both spleen cells from 4 and 10 days after treatment. These findings suggest that effector cells in the spleen may not participate in subcutaneous tumor regression and that the evaluation of antitumor effect using the double grafted tumor system needs caution.

A rat model of pancreatic ductal adenocarcinoma: targeting chemical carcinogens.

BACKGROUND: Current experimental models of pancreatic cancer either fail to reproduce the ductal phenotype or cause simultaneous cancers in other organs also. To develop an animal of pancreatic cancer that accurately mimics the human condition, we restricted carcinogenic exposure to the pancreas and specifically targeted ductal epithelial cells. Three different carcinogens were either implanted directly into the pancreas or infused into the pancreatic duct, with or without near-total pancreatectomy (as a means of inducing pancreatic ductal cell proliferation). METHODS: Groups of male Sprague-Dawley rats were exposed to varying doses of dimethylbenzanthracine (DMBA), methynitronitrosoguanidine, or ethylnitronitrosoguanidine either through direct implantation into the pancreas or infusion into the pancreatic duct. Near-total pancreatectomy was added in all groups except two DMBA implantation groups. Surviving rats were killed at 3, 6, 9, or 12 months, and the pancreata were evaluated histologically. RESULTS: All three carcinogens caused pancreatic inflammation, ductal hyperplasia, atypia, and dysplasia beginning by 3 months and becoming more prominent at later time points. Only DMBA caused frequent invasive pancreatic ductal adenocarcinoma, which was first evident by 6 months. The prevalence of pancreatic cancer among DMBA-treated rats evaluated after 10 months was 39% (19 of 49). The addition of pancreatic resection did not enhance pancreatic cancer development. CONCLUSIONS: Of the strategies tested, only direct implantation of DMBA into the rat pancreas frequently produces pancreatic cancer histologically similar to human ductal adenocarcinoma. The development of hyperplastic, atypical, and dysplastic changes preceding and accompanying carcinomas suggests that these lesions are preneoplastic. This model recapitulates the progression from normal to neoplastic epithelium and is likely to be useful for the study of morphologic and molecular mechanisms underlying the early stages of pancreatic carcinogenesis and for the investigation of novel diagnostic and therapeutic techniques.