Proton beams in radiation therapy.

The rationale for study of proton radiation therapy is that, for some anatomic sites and tumors, the treatment volume is smaller; i.e., there is less irradiation of nontarget tissue while the target is included in three dimensions at each treatment session. As a result, the dose to the target can be raised. The consequence is that the tumor control probability improves and the frequency and severity of treatment-related morbidity decrease. These results come about from the physical fact that the proton range in tissue is finite; in comparison, absorption of photons is an exponential function and, hence, some dose is received for the full-beam path through the body. Accordingly, the dose deep to the target for proton treatments can be zero for each beam path. This situation provides a virtually certain means of improving the treatment outcome for selected categories of patients. Experience to date with proton radiation therapy has been quite limited. As of June 1991, the total number of proton radiation-treated patients was 11,763 from the various centers. Of that number, approximately 46% and 32% have been treated for small benign intracranial lesions (principally pituitary adenomas and arteriovenous malformations) and for tumors of the eye, respectively. Thus, only some 2500 patients have been treated for all other tumor types. The results from three centers and approximately 2800 patients with uveal melanoma are that the local control rate was 96% (for failures in-field, marginal, and in other parts of the eye). The local control results for chondrosarcomas and chordomas of the skull base are 91% and 65%, respectively. These percentages compare with some 35% achieved with conventional treatment. Experience with arteriovenous malformations indicates that control of bleeding and disappearance of the lesion are comparable to those achieved by other procedures. The developments from the proton therapy programs have contributed greatly to radiation treatment planning, e.g., the first three-dimensional treatment planning system put into regular clinical use (uveal melanoma), beam's eye view, digital-reconstructed radiograph, dose-volume histograms, and definitions of the uncertainty in dose around any defined point. The potential for clinical gains is high. In May 1991, the Proton Radiation Oncology Group was formed to design, supervise, and coordinate clinical trials and to assist in data analysis. The efficacy of proton radiation therapy will be compared with that of photon therapy of the very highest technology.

Hyperbaric oxygen and radiobiology of a C3H mouse mammary carcinoma.

A series of radiation dose-tumor control response assays was performed on the first-to-fourth generation isotransplants of a C3H/J mouse mammary carcinoma for radiation administered under four different conditions: hypoxia, a clamp across the root of the tumor-bearing part for 1 minute before treatment; air, animal respiring air at one atmosphere of pressure; O2 30 or 44 psi, animal respiring pure oxygen at 30 or 44 psi for 15 minutes before and during local tumor irradiation. When radiation was administered as a single dose (v = 1) to 250 mm3 tumors, high pressure oxygen was only modestly effective in reducing the tumor control dose (TCD50); TCD50 hypoxia/O2 30 psi and TCD50 hypoxia/O2 44 psi were only 1.2 and 1.3, respectively. However, for similar treatment of 0.6 mm3 tumors and of even smaller "microcolonies," the ratios TCD50 hypoxia/O2 44 psi were 2.5 and 3.2, respectively. These results are discussed with respect to simple tumor models so as to estimate the proportion of hypoxic cells present in tumors under normal conditions and the influence of oxygen breathed at increased pressure on that proportion. High pressure oxygen was found much more effective when combined with fractionated irradiation (v = 10) than with single-dose therapy. Thus TCD50 hypoxia/O2 30 psi was 2.2 (v = 10) instead of 1.2 (v = 1). Finally, the ratio TCD50 hypoxia = 10/v = 1 was 2.15. These results are reviewed and inferences drawn as to the extent of tumor cell proliferation between treatments, repair of sublethal radiation injury by hypoxic cells, and the movement of cells from the hypoxic compartment to the aerobic compartment during fractionated irradiation (v = 10) under normal conditions.

Toxicity, radiation sensitivity modification, and combined drug effects of ascorbic acid with misonidazole in vivo on FSaII murine fibrosarcomas.

Ascorbic acid (ASC) has been shown to radioprotect nonmalignant tissue and to enhance the effects of misonidazole (MISO) on hypoxic cells in vitro. Since ASC is minimally toxic, it is an interesting candidate for improving the radiotherapeutic gain factor in vivo. The in vivo radiomodifying effects of ASC on a C3H/fSed murine fibrosarcoma (FSaII), on normal skin, and on bone marrow were determined with the use of the tumor growth delay and TCD50 (i.e., radiation dose required to control 50% of treated tumors for a minimum of 120 days) assays and the RD50 (i.e., dose required to cause a peak skin reaction of 2 in 50% of treated hind limbs) and LD50 (i.e., whole-body radiation lethal dose) assays, respectively. ASC was buffered to pH 7.35 and delivered ip at a dose of 4.5 mg g-1 body weight. ASC did not modify tumor growth delay induced by radiation or the TCD50 [87.1 Gy (control) vs. 85.6 Gy (ASC)]. Normal tissues, however, were radioprotected by ASC. RD50 values for 2+ acute skin reactions were 46.4 and 55.7 Gy for control and ASC-treated subjects, respectively; LD50 (30 days) values were 7.2 and 8.5 Gy. The enhancement ratios for skin and bone marrow were 1.20 and 1.18, and 95% confidence limits were (1.07 ... 1.34) and (1.14 ... 1.23), respectively. The therapeutic gain factor was 1.22 calculated as the ratio of the TCD50 and the reference normal tissue (RD50 or LD50). When ASC and MISO were combined, ASC reduced the in vivo radiosensitizing effects of MISO.

Impact of stromal sensitivity on radiation response of tumors.

BACKGROUND: Irradiation of tumors causes the death of both parenchymal tumor cells as well as normal tissue stromal cells (e.g., endothelium, connective tissue). However, it has been difficult to distinguish the contributions to overall tumor response after irradiation from the two compartments. The development of the severe combined immunodeficient (SCID) mouse provides a model in which the contribution of stromal cell responses to ionizing radiation to overall tumor response can be defined, because its normal tissue cells are extremely radiosensitive. Therefore, the results of irradiation of tumors in radiation-sensitive (SCID) and radiation-resistant hosts can be compared, and the contribution of the normal tissue stroma clarified. PURPOSE: Our purpose was to investigate the effects of radiation-induced stromal cell damage on tumor cell death, using tumor growth delay (GD) and local control (complete and permanent regression of the irradiated tumor) as end points. METHODS: Tumor GD and local control experiments were performed in SCID, athymic, and C3H mice. Sixty SCID and 60 nude mice for each of three human tumor cell lines (HGL9, HSTS26, HCT15) and for each of five murine cell lines (FSC1, FSC2, FSM1, FSM2, E01) and 60 SCID and 60 C3H mice for the FSa2 spontaneous C3H sarcoma were studied. Neoplasms were produced by injection of 10(6) cells from in vitro tissue cultures into the flanks of donor mice; after tumors had grown, experimental neoplasms were produced by transplanting 2- to 3-mm fragments into recipient mice. Animals were randomly assigned to various groups when tumors reached average volumes of 120 mm3. Graded, single-dose x irradiations (15-115 Gy, dose rate about 7 Gy/min) were given under acutely hypoxic conditions. Tumors were scored one to two times per week until recurrence. RESULTS: The x-ray doses needed to achieve local control in 50% of the animals (tumor control doses, TCD50) ranged from 45.1 to 58.0 Gy for human tumors and from 36.3 to 114.0 Gy for murine tumors. On average, the TCD50 values in SCID mice were only about 3.5% lower than values in nude or C3H mice. The amount of GD defined at 66% of the TCD50 for the various groups was, however, 27% longer in the SCID mice (P = .004). CONCLUSIONS: While the three-fold higher radiation sensitivity of the normal tissue stromal cells in the SCID mice did not alter the percentage of tumors controlled by x irradiation in the SCID mouse hosts as compared with other hosts, there appear to be significant differences in GD. Radiation-induced stromal cell damage does not significantly contribute to tumor cell death; however, it can prolong the interval of tumor regression.

Time-temperature relationship th hyperthermic treatment of malignant and normal tissue in vivo.

The effect of hyperthermia on normal and tumor tissue was studied following water bath heating of a methylcholanthrene-induced fibrosarcoma (FSaI) isotransplanted into the feet of C3H mice. The time-temperature relation for the 50% tumor control dose over the temperature range of 41.5--45.5 degrees showed a log linear relationship which followed a biphasically modified Arrhenius plot. At temperatures above 43 degrees, there was a 50% reduction in heating time to obtain the 50% tumor control dose for each 1 degree increase in temperature, corresponding to an activation energy of 140 kcal/mol. At temperatures below 43 degrees, the curve was steeper, with a tendency to double the treatment time for each 0.5 degree reduction in temperature (activation energy, approximately 230kcal/mol). Normal tissue damage in the tumor-bearing foot was estimated at two levels with a 50% response dose assay. Severe normal tissue damage showed a time-temperature relationship similar to the tumor response, thus indicating no variation in therapeutic ratio at different temperatures. However, for slight tissue damage, the therapeutic ratio increased with decreasing temperatures, yielding a better therapeutic ratio at lower temperatures. The time-temperature relationship obtained in the FSaI fibrosarcoma is supported by other studies and points to a general time-temperature relationship for hyperthermic tumor destruction.

Quantitative transplantation assays of spontaneous tumors of the C3H mouse as allografts in athymic NCr/Sed-nu/nu nude mice and isografts in C3Hf/Sed mice.

Three spontaneous tumors of the C3H mouse have been used in a comparison of their transplantability and radiation response (local control) in syngeneic C3Hf/Sed mice and in allogeneic athymic NCr/Sed-nu/nu nu nude mice. The tumors were: MCaIV, a moderately well-differentiated mammary carcinoma; FSaII, a poorly differentiated fibrosarcoma; and SCCVII, a moderately well differentiated squamous cell carcinoma. The tumors were studied as fourth to seventh generation transplants. Assays to determine the number of tumor cells that, on the average, transplant the tumor to half of the recipients or transplant sites (TD50) demonstrated that these 3 tumors transplanted into the s.c. tissue of the NCr/Sed-nu/nu as readily as of C3Hf/Sed mice. The TD50 for MCaIV was slightly but significantly lower in 4-week-old NCr/Sed-nu/nu mice which had received 6 Gy whole body irradiation (WBI) 24 h before transplantation, namely, 5.8 x 10(4) (95% confidence limits, 4.5-7.6) versus 7.8 x 10(4) (6.0-10.0). The 6-Gy WBI did not affect the TD50 for 8- to 10-week-old mice. Similarly, the TD50 for SCCVII was lower in 6-Gy WBI NCr/Sed-nu/nu recipients (1.5 x 10(4) versus 3.9 x 10(4)). The TD50 for FSaII was not affected by 6-Gy WBI. Further, the TD50 for FSaII following i.v. injection of tumor cells (transplant to lung) was the same for C3Hf/Sed and NCr/Sed-nu/nu mice (this obtained for normal or 6-Gy WBI-treated subjects). The radiation doses which on the average achieve control of half of the MCaIV, FSaII, and SCCVII tumors were lower, higher, and the same in NCr/Sed-nu/nu than in C3Hf/Sed mice, respectively. The radiation doses which achieve control of half of the MCaIV and SCCVII tumors were not affected by 6-Gy WBI before transplantation.

Comparative morphometric study of tumor vasculature in human squamous cell carcinomas and their xenotransplants in athymic nude mice.

When human tumor xenotransplants into nude mice are used as experimental models, it is important to know whether their microvascular anatomy is rather host or tumor specific. Therefore a morphometric comparison of the vascular network in human squamous cell carcinomas and their xenotransplants was carried out. Biopsies were taken from surgical specimens of three squamous cell carcinomas of the oral cavity. Part of the material was processed for histology and the rest was cut into 1-mm3 cubes and transplanted s.c. into the lateral thorax of athymic nude mice [NCr/Sed(nu/nu)]. The microvascular architecture of original tumors and of three first, one second, and one late generation xenografts was compared. Capillaries were identified in original human tumors by anti-factor VIII staining and in xenografts with antilaminin staining. The median distances between interphase tumor cells and blood vessels were determined and were found to be much longer in original human tumors than in xenografts, ranging from 81 microns to 99 microns and 53 microns to 65 microns, respectively. However, the characteristic qualitative histology of tumors appeared to be preserved in xenotransplants. Analysis of the topographic distribution of mitotic figures revealed that in both original tumors and xenotransplants proliferation of tumor cells was concentrated around blood vessels. Again, vascular distances in original tumors were significantly longer than in xenotransplants. In addition, xenotransplants into nude mice from a long passaged cell line from a human pharyngeal squamous cell carcinoma, FaDu, was investigated. FaDu showed a rare-fication of the capillary network with increasing tumor volume, but a constant median distance of mitotic figures from blood vessels. In conclusion, the pattern of spatial distribution of proliferating tumor cells as well as differentiation characteristics appear to be retained in xenograft tumors, but the density of the vascular system is host specific. This has to be taken into account when physiological parameters of blood supply are studied in xenotransplanted tumors.

Cell proliferation kinetics in human tumor xenografts measured with iododeoxyuridine labeling and flow cytometry: a study of heterogeneity and a comparison between different methods of calculation and other proliferation measurements.

The influence of overall treatment time in the results of fractionated radiation treatment was initially established in experimental tumors and, subsequently, in the clinic. The availability of techniques (antibodies against halogenated thymidine analogues and flow cytometry) which permit determinations of the duration of the synthesis phase, the labeling index, and the tumor potential doubling time (Tpot) in a short period of time and requiring only a small biopsy of tumor tissue, has expanded interest in the relationship between tumor cell proliferation and response to irradiation. A valuable tool in the study of this relationship are human tumor xenografts. Previous studies have shown a substantial intratumoral heterogeneity in the determinations of Tpot. Different methods of calculation of the kinetic parameters have been published. We have conducted a heterogeneity analysis and an evaluation of the different calculation methods in order to define the validity of Tpot as a proliferation rate measurement in human tumor xenografts. Results show the intertumoral variability in Tpot [between different types of human tumor xenografts systems (coefficient of variation = 88.2%)] to be greater than mean intratumoral variation (coefficient of variation = 30.8%); this suggests that this variation is potentially adequate to serve as a predictor of response. The diverse calculation methods provided significantly different absolute values but not different tumor ranking, probably because the time interval between labeling and sampling was maintained, for all the samples, between 6 and 8 h. Our study has found significant differences between the labeling index and the S-phase fraction determined with the DNA profile in 9 out of 10 tumor types. No correlation was found between the DNA index of the tumors in this series and their proliferation rate.

Enhancement by Corynebacterium parvum of the normal and tumor tissue response to hyperthermia.

The effect of Corynebacterium parvum treatment on the response of tumor and normal tissue to hyperthermia (43.5 degrees) was studied. Animals were C3Hf/Sed mice from our defined flora mouse colony. The time at hyperthermia that achieved control of one-half of methylcholanthrene-induced fibrosarcomas and the foot reaction were examined after treatment. C. parvum, if given 3 to 32 days before hyperthermia, enhanced the reaction to local hyperthermia of normal tissue. No enhancement was observed if C. parvum was given after hyperthermia. This enhancement was more dramatic for tumor response resulting in a therapeutic gain factor of congruent to 2.3 (3.7/1.6). Comparative studies on combined Corynebacterium and radiation failed to demonstrate the enhancement to normal tissue.

Enhancement of the thermal response of animal tumors by Corynebacterium parvum.

The effect of Corynecbacterium parvum treatment on the thermal response of animal tumors was studied. Tumors were methylcholanthrene-induced (FSa-II) and spontaneous (FSa-I) fibrosarcomas in C3Hf/Sed mice. C. parvum was given i.v. and was followed by local hyperthermia at 43.5 degrees 3 days later. Cell survival determined by lung colony assays showed that preadministration of C. parvum insignificantly enhanced the thermal response of both tumors. Studies of delay of tumor growth for FSa-II demonstrated that the enhancement ratio decreased with increasing time of treatment and reached a minimum of approximately equal to 1.7. The enhancement ratio for the time at hyperthermia which achieved tumor control in one-half of the treated tumors was 1.7. Together with our previous results on normal tissue responses, the therapeutic gain factor for obtaining 50% tumor control was found to be 1.1 (1.7/1.55) for the weekly immunogenic FSa-II tumor, while it was 2.3 for moderately immunogenic FSa-I as reported previously.

Effect of whole-body hyperthermia on cell survival, metastasis frequency, and host immunity in moderately and weakly immunogenic murine tumors.

The effects of whole-body hyperthermia (WBH) on animal tumors and on metastasis frequency were studied. The tumors were a chemically-induced fibrosarcoma, FSa-I, which is moderately immunogenic and a spontaneously arisen fibrosarcoma, FSa-II, which is very weakly immunogenic. The WBH was given at 42.5 degrees in an incubator which had an auxiliary heater for accurate temperature control. Animal core temperature reached 41.5 degrees in 30 min. The lung colony assay revealed that the WBH for 60 min given at 24 hr after i.v. injection of single cells gave no lethal damage to either FSa-I or -II tumor cells. A significant inhibition of tumor growth was found when large tumors were given three daily WBH treatments. The frequency of lung metastasis was enhanced when large weakly immunogenic FSa-II tumors were treated by WBH, although no increase in the frequency was observed for FSa-I tumors of any size. Local hyperthermia did not significantly increase the metastasis rate of both tumors. These results suggest that the WBH might be useful for a treatment of large immunogenic tumors. However, the WBH is not a choice of treatment for possible micro-metastases.

Enhancement of thermal response of normal and malignant tissues by Corynebacterium parvum.

Further studies were carried out on the combined effects of Corynebacterium parvum and hyperthermia on animal tissues and cultured Chinese hamster ovary cells. Experimental animals were C3Hf/Sed mice derived from our defined flora mouse colony. Tumors were eighth-generation isotransplants of a spontaneous fibrosarcoma, FSa-II. Hyperthermia was given by immersing the mouse foot or culture flasks in the constant temperature water bath. Present experiments include thermal enhancement of C. parvum at different temperatures, effect of the agent on the kinetics of thermal resistance, and the mechanism of the thermal enhancement. The thermal enhancement by C. parvum was independent of temperature in a range between 42.5 and 46.5 degrees, and it increased with decreasing temperature. The analysis of the Arrhenius plot suggested a comparable activation energy for combined treatments and for heat alone between 42.5 and 46.5 degrees. The thermal resistance developed very rapidly in both normal and tumor tissues. Systemic administration of C. parvum failed to modify the kinetics of thermal resistance. Several experiments were attempted in order to disclose the mechanism. A single injection of C. parvum-induced macrophages failed to enhance thermal response of the mouse foot, while 3 daily injections of the macrophages enhanced the response, indicating that the enhancement by C. parvum is at least partly attributed to the C. parvum-induced macrophages. Whole-body irradiation of 6 Gy and/or administration of anti-mouse T-cell serum and histamine failed to inhibit the C. parvum enhancement of thermal response. No thermal enhancement was observed for Chinese hamster ovary cells treated at 43.0 degrees in vitro with C. parvum or thiomersalate , a preservative supplemented in C. parvum, although cytotoxic effect was shown at a high concentration of thiomersalate .

Quantitative studies on the transplantability of murine and human tumors into the brain and subcutaneous tissues of NCr/Sed nude mice.

The transplantability of experimental tumors into the brain (i.c.) and s.c. tissues of C3Hf/Sed and athymic NCr/Sed nude mice was examined using quantitative cell transplantation assays. Studies using the immune-competent C3H animals showed that brain is a more favorable site for the transplantation of syngeneic tumor than s.c. tissue and that this is true for nonimmunogenic as well as immunogenic tumors. The capacity of the brain to act as an immunological sanctuary can be overwhelmed by a strong, systemic, secondary immune response such as that evoked by the methylcholanthrene-induced sarcoma FSal. In studies performed using NCr/Sed nude mice, the allogeneic tumor MCaIV was found not to be demonstrably immunogenic. The cell dose required to transplant the tumor into 50% of recipients (TD50) could neither be increased by immunization procedures nor decreased by six Gy whole-body irradiation (WBI) prior to transplantation. Delayed-type hypersensitivity to this tumor was not expressed by nude mice after rechallenge with tumor antigen. The TD50 was again lower for i.c. than s.c. transplantation and the ratio s.c./i.c. was comparable to that found in syngeneic C3Hf/Sed hosts. Three human tumors have been similarly tested. They were: FaDu, a pharyngeal squamous carcinoma; HFSal, a fibrosarcoma; and U87, a malignant glioma. s.c. TD50 values were in all cases significantly higher than those obtained i.c. The ratios TD50 s.c./i.c. ranged from 6.4 to greater than 50 in five studies, substantially higher than those found for transplantation of murine tumors into either the syngeneic or the allogeneic recipients. Six Gy WBI reduced the s.c. TD50 for these tumors, but in each case the value remained significantly higher than that obtained i.c. 19.4 Gy WBI given in 10 equal fractions and followed by i.v. bone marrow rescue reduced further the s.c. TD50 for FaDu. NCr/Sed nude mice demonstrated cross-reacting delayed-type hypersensitivity against FaDu and HFSal. A small proportion of FaDu tumors (less than 2%) displayed a spontaneous halt in growth or even regression. When the host cell infiltrate of these tumors was analyzed, an increase was seen in the proportion of Thy 1.2 and asialo-GM1-positive cells as compared with progressively growing tumors. These data strongly suggest that a residual low level of immune reactivity exists in nude mice against xenotransplanted human tumors. This resistance to s.c. transplantation may be diminished by WBI and is less for intracerebral implantation.

Vascular endothelial growth factor receptor-2-blocking antibody potentiates radiation-induced long-term control of human tumor xenografts.

Antiangiogenic therapy can enhance radiation-induced tumor growth inhibition. However, the effects of combined antiangiogenic and radiation therapy on long-term tumor control and normal tissue response have not been reported. We treated mice bearing two different human tumor xenografts with anti-vascular endothelial growth factor receptor-2 antibody (DC101) and five dose fractions of local radiation and followed them for at least 6 months. DC101 significantly decreased the dose of radiation necessary to control 50% of tumors locally. The decrease was 1.7- and 1.3-fold for the moderately radiosensitive small cell lung carcinoma 54A and the highly radioresistant glioblastoma multiforme U87, respectively. In contrast to tumors, no increase in skin radiation reaction by the antibody was detected. Surprisingly, 44% of mice bearing 54A tumor developed clear ascites after DC101 treatment at its highest dose; this was fatal to 20% of mice. This adverse effect was seen only in mice that received whole-body irradiation 1 day before tumor implantation. The encouraging results on two human tumor xenografts suggest that vascular endothelial growth factor receptor-2 blockade merits further investigation to assess its potential as an enhancer of radiation therapy in the clinic.

Tumors arising in SCID mice share enhanced radiation sensitivity of SCID normal tissues.

We addressed the question of whether cancers arising in an abnormally radiation sensitive normal tissue are also abnormally sensitive to ionizing irradiation. Germ line mutation-carrying mice with an enhanced radiation sensitivity of the normal tissue, the severe combined immunodeficient (SCID), and normally radiation sensitive mice (C3H) were used to study the sensitivity of normal and tumor tissues in vivo and in vitro. The lethal dose for 50% of the irradiated animals after single dose whole body irradiation was 2.6-fold higher in C3H compared to SCID mice. The dose for an isoeffective acute skin reaction after single dose irradiation was end point dependent 1.7 to 3.7 times higher in C3H than in SCID mice. Embryonic fibroblast and methylcholanthrene induced soft tissue sarcomas derived from C3H and SCID mice were established in vitro and colony-forming assays after single dose irradiation were carried out. Choosing mean inactivation dose as the end point, SCID fibroblast lines were 3.0-fold and SCID tumor cell lines 2.7-fold more radiation sensitive than C3H fibroblast lines and C3H tumor cell lines. Tumor control and growth delay assays for 110-mm3 tumors were used to compare the radiation sensitivity of SCID and C3H tumors in vivo. The doses for 50% local tumor control and a growth delay of 40 days were 2.6 times higher in C3H tumors compared to SCID tumors. Tumors arising in an abnormally radiation sensitive normal tissue are also sensitive to irradiation. The difference in radiation sensitivity of normal tissues predicted the difference in tumor tissues in these two murine systems.

Effect of Corynebacterium parvum on the response to irradiation of a C3H fibrosarcoma.

A methylcholanthrene-induced fibrosarcoma of C3H mice has been used as 5th- to 6th-generation syngeneic transplant in an investigation of the role of Corynebacterium parvum as an adjuvant to the therapeutic effect of local irradiation of established tumors. The most effective route for administration of C. parvum in this tumor system was i.v., and the greatest effect of the C. parvum-mediated tumor graft rejection was observed for tumor growing intracutaneously or s.c. An intermediate level of effectiveness was obtained for tumor growing i.m. The combined C. parvum and local irradiation studies were performed using tumors growing in the leg muscle and measuring 8 mm in diameter at the time of local irradiation. Several routes of administration of C. parvum, dose levels of C. parvum, and time relationships between administration of C. parvum and irradiation were investigated. The outstanding finding was that very low doses of radiation were quite effective when administered to tumors growing in animals pretreated with C. parvum. This was true for radiation administered as a single or fractionated dose (10 equal doses spread over 18 days). For single-dose irradiation the effect was relatively dose independent over the range of 200 to 3000 rads. Some indication was obtained that local irradiation may impair the tumor graft rejection reaction. No evidence was obtained of an enhanced growth of tumor. Regression of tumor following irradiation was not modified by pretreatment with C. parvum. The mice that were unsuccessfully treated by radiation and C. parvum had a lower incidence of metastatic disease in the lung than did the mice treated unsuccessfully with radiation alone.

Taxane-induced apoptosis decompresses blood vessels and lowers interstitial fluid pressure in solid tumors: clinical implications.

Elevated tumor interstitial fluid pressure (IFP) is partly responsible for the poor penetration and distribution of therapeutic agents in solid tumors. The etiology of tumor interstitial hypertension is poorly understood. We have postulated that the solid stress generated by tumor cells growing in a confined space compresses blood vessels and increases tumor microvascular pressure and IFP. To test the hypothesis that neoplastic cell loss would decompress blood vessels and lower IFP, we induced apoptosis in tumors with paclitaxel and docetaxel. Taxanes inhibited the growth of the murine mammary carcinoma (MCa-IV) and of the human soft tissue sarcoma (HSTS-26T). Taxanes induced apoptosis and reduced the density of intact neoplastic cells in both MCa-IV and HSTS-26T. To determine whether neoplastic cell loss decompressed blood vessels, we measured the diameter of tumor vessels in HSTS-26T tumors implanted in transparent dorsal skin fold chambers. At 48 and 96 h after paclitaxel, the diameter of tumor vessels was significantly increased. The increase in vascular diameters was associated with reductions in microvascular pressure and IFP. The changes in neoplastic cell density and IFP were also correlated. In the human glioblastoma U87, which is resistant to paclitaxel, the IFP and cellular density were not modified by paclitaxel treatment. Collectively, these results support the hypothesis that solid stress generated by neoplastic cell proliferation increases vascular resistance and IFP. The increase in vessel diameter induced by paclitaxel and docetaxel suggests that taxanes could improve tumor response by increasing the vascular surface area for delivery of therapeutic agents.

Quantitative comparison between the transplantability of human and murine tumors into the subcutaneous tissue of NCr/Sed-nu/nu nude and severe combined immunodeficient mice.

In previous reports, nude mice have demonstrated residual immunoreactivity against xenografts. Severe combined immunodeficient (SCID) mice lack functional T- and B-cells. These animals are expected to be better hosts in which to perform preclinical studies on human tumors. The purpose of this study is to quantitate the advantage of SCID mice over nude mice in terms of transplantability of human and murine tumors and the importance of residual immunity in SCID mice. The transplantation assays are described by an assay based on the number of tumor cells required to transplant tumor into 50% of recipients (TD50). Seven human tumors of different histology and four murine tumor cell lines were used. Serial 2-10-fold dilutions of cells were injected (0.1 ml) into the flanks of normal and whole-body irradiated WBI nude and SCID mice. The results showed that in 6 of 6 human tumor cell lines studied, TD50S for SCID mice were 2.4 to 200 times lower than that of nude mice (significant in 5 cell lines). In contrast, in 2 of 3 murine tumors, TD50S in WBI SCID mice were significantly higher than that found in nude mice. When SCID and nude mice received WBI, TD50S were lower than those of nonirradiated animals in 5 of 5 xenografts (significant in 2 cell lines for nude mice and in 5 cell lines for SCID mice). We concluded that WBI SCID mice are significantly better recipients of human tumor xenografts than nude mice. There is a factor of 10-1625 gain in TD50S in favor of the WBI SCID mice when compared to nonirradiated nude mice. WBI has, however, an important effect on SCID mice which may suggest a detectable residual immunoreactivity, perhaps due to natural killer cells. These data demonstrate that WBI SCID mice are better models for human tumor transplantation that nude mice and, although WBI at 6 Gy suppressed significantly the immune system of nude mice, a certain level of immunoreactivity against xenografts is still maintained.

Quantitative comparison between the transplantability of human and murine tumors into the brain of NCr/Sed-nu/nu nude and severe combined immunodeficient mice.

We have demonstrated (A. Taghian et al., Cancer Res., 53: 5012-5017, 1993) that the take rate of human xenografts in the s.c. tissue of severe combined immunodeficient (SCID) mice is significantly higher than that of nude mice. Earlier, this laboratory reported that the transplantability of tumor xenografts was significantly higher for intracranial (i.c.) injection than for s.c. injection in nude mice. The purpose of this study is to assess: (a) the relative i.c. transplantability of human and murine tumors in comparison with s.c. tissue in SCID mice; (b) the relative i.c. transplantability in SCID mice in comparison to nude mice; and (c) the influence of whole-body irradiation on i.c. transplantability of SCID and nude mice. The assay based on the number of cells required to transplant tumors into 50% of recipients (TD50) was used to describe the transplantability assays. Five human and four murine tumor cell lines were used. Concurrent TD50 assays were performed i.c. in whole-body irradiated and nonirradiated SCID and nude mice. Serial 2-10-fold dilutions of cells were injected in a 10-microliters volume into the right parietal lobe 3 mm below the skin. The results showed that in all tumors studied the i.c. TD50S were significantly lower than the s.c. TD50S by a factor of 1.7-1580. The average enhancement ratio (s.c. TD50/i.c. TD50) in nude mice was twice that in SCID mice. No significant difference was found between the i.c. TD50S in SCID and in nude mice, contrary to the significant difference in s.c. TD50S between both strains of mice (A. Taghian et al., Cancer Res., 53: 5012-5017, 1993). Whole-body irradiation did not significantly affect the i.c. TD50 in nude mice; however, it did affect two of three xenografts in SCID mice. In conclusion, despite the significantly lower s.c. TD50S of human xenografts in SCID mice, i.c. TD50S were almost similar to those of NCr/Sed-nu/nu nude mice. This suggests the presence of different immunoreactivities between nude and SCID mice in s.c. transplantability; however, for i.c. transplantability, nude mice behaved equally as well as SCID mice. The significant enhancement ratio in SCID mice is further evidence that this strain of mice displays a residual systemic immunoreactivity, although the immunoreactivity is significantly lower than that of nude mice.

Timing of chemotherapy and surgery in a murine osteosarcoma model.

The sequential use of chemotherapy and surgery in the treatment of osteosarcoma developed in an empirical fashion without the benefit of investigations in animal models. The MGH-OGS murine osteosarcoma is a transplantable tumor that resembles the human disease with respect to histology, local invasiveness, metastatic characteristics, tumor ploidy, and its response to chemotherapy. We have used this tumor model to investigate the efficacy of preoperative, perioperative, and postoperative chemotherapy on the development of pulmonary metastases in three different experimental protocols. In each experimental design, perioperative chemotherapy demonstrated a significant advantage in preventing systemic relapse.