Radiation response and other characteristics of the 9l rat glioma grown as an epigastric tissue...

Radiation Oncology Investigations 2:13+143 (1 994)

Radiation ResDonse and Other Characteristics of the 9L RaiGlioma Grown as an Epigastric

Tissue Isolate

Sydney M. Evans, v.M.D., and Cameron J. Koch, Ph.L).

Schools of Veterinary Medicine (S.M. E . ) and Medicine (C.J. K . ), University of Pennsylvania, Philadelphia, Pennsylvania

SUMMARY We studied the 9L tumor as a tissue isolated epigastric implant. Data supporting its applicability to this site, including tumor blood flow, glucose metabolism, plating efficiency, radiation response, and histopathologic appearance, are described. Im- plants were performed on 175-250 g male Fischer rats by tying 2-3 mm3 pieces of tumor onto the proximal segment of the epigastric branch of the femoral artery. Characteristics of the model include recovered cell yields of 6.1 2 4.2 x lo7 cells/g; 19-23% plating efficiency; 5% relative surviving fraction at 19.3 and 27.6 Gy in air breathing and euthanized rats, respectively; mean glucose utilization rate of 229 * 182 nmol/min/g; and tumor blood flow of 1.9-21.3 m l / m i n / l O O gin 0.4-3.3 g tumors. These characteristics, for the first time, provide a common framework for simultaneous evaluation of tumor metabolism and radiation response. Radiat Oncol Invest 1994;2:134-14.7. 0 1994 Wiley-Liss, Inc.

Key words: 9L, radiation, epigastric, rat

INTRODUCTION In the mid-l960s, a glioma tumor was induced in a male CD Fischer rat following weekly injections of N-nitrosomethylurea. After successive in vivo and in vitro transfers, a cell line was established (9L) which produced a gliosarcoma when implanted intracerebrally. Since that time, the 9L has been used extensively as a subcutaneous (SubQ) and intracerebral tumor model, especially for studies of radiosensitivity [ 1,2]. Typically, intracerebral and small SubQ 9L tumors are described as having minimal necrosis and no severe hypoxia [ 1,3]. How- ever, the 9L glioma has also been reported to con- tain uniform, moderate hypoxia [4-71.

The tissue isolated tumor with a well-defined vascular pedicle was developed primarily for access to and control over tumor blood flow (TBF). This model allowed drug administration systemically or via the arterial segment for measurement in the venous outflow [8]. The model was first described in 1961 [9]; tumor tissue was implanted in the kid- ney or ovary, and the neoplasm overgrew the normal organ resulting in a tumor attached to a vascular pedicle. In 1982, the Walker 256 carcinoma and several Morris hepatomas were grown on the epigastric branches of the femoral vessels in the rat’s inguinal region. Studies on amino acid, glucose, and lactic acid utilization were described [lo]. Glu-

Original received March 3 I , 1994; revised July 20, 1994; accepted August 1, 1994. Address correspondencelreprint requests to: Sydney M. Evans, V.M.D., Department of Radiation Oncology, Division of

Oncology Research, University of Pennsylvania, 195 John Morgan Building, 37th and Hamilton Walk, Philadelphia, PA 1 9 1 04-607 2.

Abbreviations: EMEM, Eagle’s minimal essential medium; IP, intraperitoneal; IV, intravenous; PE, plating efficiency; SIH, stress-induced hyperglycemia; TBF, tumor blood flow; SubQ = subcutaneous. 0 1994 Wiley-Liss, Inc.

Evans and Koch: Radiation of 9L Tissue Isolates 135

planted tumors acted as the initial source for tissue isolated implants on the epigastric vessels. Further implants during the following 3 months were from tumors implanted on the epigastric vessels.

Anesthetic and Temperature Considerations Rats were anesthetized with xylazine (1.3 mg/kg IP) (Haver Corp., Shawnee, KS) and ketamine (140 mg/kg IP) (Fort Dodge, Fort Doge, IA). Anesthe- tized animals were maintained on a water circulat- ing heating pad under a heating lamp. Body temperature was constantly monitored via an intrarectal probe and temperatures were maintained in this way between 37.5 and 36.5”C. During catheterization studies where the skin overlying the tumor was removed, the tumor surface was bathed regularly with warmed 0.9% saline and maintained under a warm, wet gauze.

Tissue Isolated Implantation Implants were performed on 175-250 g male Fis- cher rats. The recipient and donor rats were anesthetized for surgery as described above.

tamine was the most extensively utilized whole blood amino acid; glucose utilization was directly proportional to the rate of supply, and lactic acid was either produced or utilized, depending on supply concentration.

Tissue isolated tumors have a pattern of angio- genic architecture which is different from other commonly used models. The tissue isolated tumor implant begins on preexisting host vessels (in the epigastric tumor, it is the epigastric branch of the femoral vessels) and the new tumor microvascula- ture grows outward along with the tumor mass (the suture material used for implanting the epigastric tumor is inevitably found in the tumor’s center). These preexisting host vessels support the microcirculatory functions until the new tumor vessels are formed [ 1 1 ,121. In contrast, the blood supply of the SubQ tumor model must originate entirely from neovascularization. Thus, the tissue isolated epigastric implant has unique vascular architecture, providing a model for comparison to tumors grown in the SubQ location. These differences may be important in the response of tumor vasculature to modification and subsequent effects on nutrient delivery, including oxygen.

This summary emphasizes an interesting anomaly: virtually all of the tumor irradiation studies have been performed with less technically de- manding tumor implantation methods (such as subcutaneous), but most of the detailed tumor metabolism studies have been performed in the tumor isolated models. Practically no crossover data exist. We present data supporting 9L’s applicability to this site such that the interaction of the metabolism and radiobiologic response can be directly as- sessed in a single tumor model. TBF, glucose metabolism, plating efficiency (PE), radioresponse, and histopathologic appearance of the 9L tissue isolate in the epigastric location are described herein.

MATERIALS AND METHODS

Cell Lines The 9L cell line was obtained from Dr. K.T. Wheeler (Bowman Gray School of Medicine, Win- ston Salem, NC). Cells were thawed from frozen stock 4X yearly to start tumors anew. Cells were cultured (37°C 95% air and 5% carbon dioxide, 100% relative humidity) in the exponential phase of growth using Eagle’s minimal essential medium (EMEM) containing 12.5% v/v bovine calf serum and 1% antibiotics (GIBCO, Grand Island, NY), “complete medium.” At the time of inoculation, lo6 cells were injected subcutaneously over the flank of 175-250 g male Fischer rats. SubQ im-

Donors The tumor region was clipped and cleaned. A portion of viable tumor was excised and placed in iced EMEM. Fat, necrotic tissue and skin were dissected away and remaining viable tissues were transferred to clean iced EMEM containing gentamicin (Ly- phomed N0469-1000-60) and penicillin G (Pfizer Animal Health, New York, NY). Tumor specimens were then diced into 2-3 mm pieces and maintained on ice until implantation. The donor was euthanized with 120 mg of IP pentobarbital (Euthanasia 6 solu- tion, Veterinary Laboratories, Inc., Lenexa, KS).

Recipients The left inguinal region was clipped and cleaned. A small (1 cm) skin incision was made in the inguinal crease. The SubQ tissues were bluntly dissected away and the epigastric vessels running along the underside of the inguinal fat pad were located and isolated. Two 6/0 silk sutures were placed approximately 2-3 mm apart and tied. A piece of tumor was tied onto the proximal segment of the epigastric vessels and the vessels were cut between the sutures. A drop of gentamicin and penicillin each were placed in the wound. The inguinal fat pad was pulled over the surgery site and skin closed with hemoclips. The recipient rats were allowed to re- cover in a bed of clean shavings. Tumors were palpable 7-10 days following implantation. In this site, the tumors were typically close to spherical

136 Evans and Koch: Radiation of 9L Tissue LFolates

Suture w.

Fig. 1. The tumor grows from a stalk formed by the epigastric branches of the femoral vessels in the rat’s inguinal region. For measurement of TBF, the tumor outflow vein can be cannulated with PE50 tubing on the side proximal to the epigastric stalk. The cannula is secured with a ligature. The femoral vein distal to the epigastric stalk is closed off by

ligatures so that all venous blood drains from the tumor. Tumor venous outflow can be collected for measurement of TBF and/or metabolites/drugs. This catheter can be attached to a catheter draining the opposite femoral vein so that between collection times, the blood can be. recirculated into the systemic circulation. a , artery; v, vein.

(determined at autopsy), but it was difficult to accu- rately measure multiple orthogonal diameters due to overlying inguinal fat and skin. A diagram of the appearance and vascular anatomy of a mature epigastric implant is shown in Figure 1 .

Systemic Blood Sampling For comparison of glucose levels in systemic blood samples to tumor outflow glucose blood levels, a catheter was placed into the right femoral vein. These catheters were made of PE50 tubing and tied into place with 6/0 silk. Catheters were maintained by intermittent injection of 0.2 ml (10 IU/ml) heparin in saline. Blood samples were collected in pre-

weighed centrifuge tubes. These samples were maintained on ice until processed.

In Vivo Tumor Catheterization and Perfusion To perform an in vivo catheterization, the animal was anesthetized and maintained as described above. A skin incision was made approximately 0.5 cm distal to the tumor and the skin bluntly dissected off the tumor mass; bleeding was extremely rare, indicating absence of superficial vessel feeding the tumor mass. The tumor was gently isolated from surrounding SubQ and fatty tissue using forceps. The femoral vein proximal to the tumor was iso-


lated, and two 6/0 sutures were preplaced (Fig. 1). The femoral vessels distal to the epigastric tumor were ligated so that all blood collected from the femoral (tumor efferent) vein was draining the tumor. Immediately preceding catheterization of the vein draining the tumor, 0.2 ml heparin (1,000 IU/ ml) was given IV through the previously accessed right femoral vein. A catheter made of PE50 tubing with tapered tip and containing heparinized saline was then placed and tied in the femoral vein draining the tumor. In studies where TBF was to be measured over 30 min or longer, a reinfusion circuit was set up so that blood flowing out of the tumor was directly reinfused into the previously catheter- ized opposite femoral vein. At 10-30 min intervals, this circuit was opened and tumor outflow blood was allowed to flow freely for 1 min for collection in preweighed centrifuge tubes. The closed circuit was then reestablished. Blood samples were maintained on ice until processed. These samples were weighed to calculate TBF. The maximum amount of blood lost plus that collected during the entire experiment was no more than 7% of the blood volume. Withdrawal of this amount ensured that the rat’s blood volume was greater than the level where systemic hypovolemia could occur [ 131.

Plating Efficiency (PE) The preparation of tumor cells for PE proceeded based on a modification of the methods described by Howell and Koch [14]. Briefly, tumor tissue obtained by excisional biopsy was weighed and a 0.3-0.5 g aliquot was minced in a 1 ml cocktail containing 0.075% protease (Sigma P4806), 0.03% collagenase (type XI, Sigma C7657), and 0.03% DNase (Sigma D4527) dissolved in HEPES buff- ered saline. This tumor tissue was added to a trypsinizing flask containing an additional 9 ml cocktail. The tissue and cocktail were stirred at 200 rpm, 37°C for 30 min. At that time, an equal volume of iced complete medium was added and the flask shaken to release cells from any remaining tumor bits. Any undissociated tumor tissue was removed by filtering the suspension through a 70 pm nylon screen. The supernatant was spun for 10 min at 1,200 rpm and the resultant cell pellet resus- pended in iced medium and maintained on ice. For clonogenic assay, suitable numbers of cells were plated into 100 mm plastic Petri dishes containing 9 ml of complete medium. The number of cells plated was varied over a range in order to yield 100-200 colonies per plate. In this range, the number of colonies and the number of 9L cells seeded were linear. Multiple replicates were plated at each dilu-

0.4 1.3 1 .4 1 .4 1 .8 1.7 1.9 2.4 3.3

TUMOR WEIGHT (grams)

Fig. 2. TBF measured in nine 9L epigastric implants. For each tumor, 2-5 measurements were made over 1040 min. Each bar represents a separate tumor and the error bar represents the variation of TBF over the collection period. TBF ranged from 1.9 to 21.3 ml/min/100 g of tumor.

tion [3-51. The plates were incubated for 10-12 days, then fixed, stained, and counted.

Radiation Radiation was performed on an orthovoltage X-ray unit operated at 225 kV and 13 mA, 0.2 mm copper filter. The dose rate was 5.4 Gy/min. The tissue isolated 9L tumors in anesthetized (as previously described) air breathing or hypoxic (euthanized) rats were irradiated with doses of 0-30 Gy. Hy- poxia was induced in tumors by irradiation 10 min following euthanasia. Doses were estimated based on actual surface dosimetry for each tumor using thermoluminescent devices. Depth-dose determina- tions were made in a wax phantom.

Glucose Analysis Glucose analyses were performed on trichloroacetic acid (TCA) precipitated whole blood using Sigma diagnostic kit no. 510, based on glucose oxidase followed by peroxide measurement.

Histopathologic Analysis Tumor tissue was routinely fixed in 10% neutral formalin and prepared with hematoxylin and eosin. Fixed tumor tissue was examined by light micros- copy and subjectively evaluated for percentage and distribution of necrosis, number of capillaries, and presence of host cells (macrophages, neutrophils, eosinophils, etc.).

RESULTS AND DISCUSSION

Tumor Growth Sixty-eight tumors were studied for growth characteristics. Of the 68 tumors, 15 (22%) did not grow

138

Table 1. Measuring Techniques in Rat Tumors*

Implant type Cell type (mVmin/100 g) size Technique Reference

TI inguinal Walker 256 2.13-3 .0 4 g Direct 1161 SubQ foot DS sarcoma 97 -t 6 0.75 -t 0.3 g Krypton45 [331 IM flank R3230 adenocarcinoma 15.4 2 1.6 5-7 mm Microspheres WI Renal 40 -t 6.8 Small intestine 20.3 * 6.1 Mesentary 21.7 -t 4.9

Evans and Kocb: Rudiation of 9L Tissue Isolates

Literature Review of Varying Types, Sizes and Implant Sites, TBF Measurements, and

TBF Tumor

TI Walker 256 3.2-27.9 0.5-1.5 g Direct 1251 TI ovary R3230 adenocarcinoma 11-96 0 .61 .2 g 3H NMR-D2O 1351 TI renal DS sarcoma 25.0 2.5 g Direct I361

SubQ AH109A 18.6 -t 15.5 3 cm diameter Hz gas clearance [371

TI epigastric 9L 1.9-21.3 0.4-3.3 g Direct Evans

4.0 8.2 g

LYXO 22.0 L 15.8

*TI = tissue isolated: IM = intramuscular; NMR = nuclear magnetic resonance

within 30 days or had a morphology (abnormal vessels, hematoma, necrosis) that was unacceptable for the study planned. For the remaining 53 9L epigastric tumors, growth appeared to be exponential. However, we note the difficulty in measuring the volume of these tumors because of the presence of overlying inguinal fat and skin. Average tumor weight at 15 days was approximately 1 g corre- sponding to an average tumor volume of approximately 1,OOO mm’. Some tumors were allowed to grow to 5% of host body weight without evidence of overlying skin necrosis, metastasis, or host death.

Tumor Blood Flow Of 15 tumor implantations successfully prepared for epigastric tumors, 9 were successfully catheter- ized for measurement of TBF, for a success rate of 60%. TBF was measured by direct collection of tumor outflow; individual samples were 0.2-0.3 ml. Care was taken to keep total collection volume plus surgical loss to less than 7% of total blood volume, in order to avoid physiological effects of volume loss [ 131. Shown in Figure 2 are TBF data from nine 9L epigastric tumors. Each bar represents a separate tumor and the error bar represents the variation of TBF over the collection period (2-5 separate collections over 1040 min). Average TBF ranged from 1.9 to 21.3 ml/min/100 g in 0.4-3.3 g tumors. In all tumors studied, TBF remained relatively constant over the study period. We were ini- tially concerned about the effects of anesthesia and blood removal on TBF. Based upon our data, and supported by work in the literature [2,15], the xylazine/ketamine anesthetic combination appears to preserve TBF over the time period studied (less

than 1 hr). Similarly, if blood removal (loss plus sampling) is incremental over time and does not, in total, exceed 7% of the animal’s body weight, effects on TBF are minimal using the techniques described herein [ 131. These results are comparable to previous studies of TBF using a variety of techniques (Table 1). For example, in 4 g Walker 256 carcinoma epigastric implants, the mean TBF was 2.5 ml/min/100 g [ 161.

Glucose Metabolism One of the major advantages of the tissue isolated epigastric model is that it allows collection of tumor outflow blood for comparison to systemic blood. An example of the usefulness of such access is the evaluation of systemic glucose levels compared with tumor outflow glucose concentration. In all rats studied herein, the systemic blood glucose concentration increased during the study period (Fig. 3). At the time of the first systemic sample, the average glucose was 134 mg/dl. Over 60 min, the glucose rose to approximately 236 mg/dl (Fig. 3). This phenomenon is probably due to stress-induced hyperglycemia (SIH) and/or the effects of xylazine anesthesia [17]. SIH has been mentioned in previous studies using tumor isolates [9]; 3 decades ago, Gullino and Grantham [9] noted that the rats studied “showed an increase of the blood glucose levels.” SIH is also seen in other species such as cats (Evans, personal experience) and rabbits [ 181. This response is mediated via the sympathetic nervous system. The two most important mechanisms for initiation of hyperglycemia (and hyperlipidemia) are the release of adrenomedullary epinephrine to initiate hepatic gluconeogenesis and the inhibition of insulin release. Xylazine has been shown to in-

Evans and Koch: Radiation of 9L Tbme IsoIutes 139

500 I

250 -

200 -

150 -

y-1Y.1.7. n.2-o.m

1 0 0 , . , . I . I . I . I .

Fig. 3. Systemic glucose levels in male Fischer rats during tumor catheterization. Time 0 is approximately 15 min following administration of anesthesia. At time 0, the femoral catheter for systemic sampling has been placed.

El Y = 4 6 4 + 021. R12- 0890

crease blood glucose levels through its effects on the a,-adrenergic receptors of the pancreatic beta cells [ 191. Information on the systemic glucose levels is important for studies of tumor energy sub- strates, especially when metabolism-modifying techniques are used [20]. Since these data are ac- crued over time, investigators must constantly re- evaluate the “steady state” because critical parameters in the host are changing. The advantage of studying tumor metabolism compared to the animal’s own physiologic glucose response is the absence of any changes in systemic hypertonicity, fluid exchanges, etc. Investigators have infused glucose IV [21], IP [22], SubQ [23], and intratu- morally [24]. Interpretation of the results of these studies is complicated by the local and systemic physiological and hemodynamic consequences (vasoconstriction, decreased cardiac output, and changes in red blood cell rigidity) of such infusions [22,25,26].

Blood collected from the tumor vein was as- sayed for glucose concentration and compared to the systemic level. Mean glucose utilization rate was 229 & 182 nmol/min/g (n = 9). As shown in Figure 4, the tumor glucose utilization rate was directly proportional to the rate of supply to the tumor. Depending upon the glucose supply rate, a tumor may utilize 15-57% of the glucose supplied in vivo. Similar findings have been reported for in vivo glucose utilization and supply in rat tumors by other investigators [10,20]. In our studies, these parameters appear to be independent of tumor size. One interpretation of these data is that the variabil-

GLUCOSE SUPPLY (nrnol/mln/g)

I

Fig. 4. Glucose supply vs. utilization in nine 9L epigastric implants. The tumor glucose utilization rate is directly proportional to the rate of supply to the tumor.

c > 0 2 w 0 Y Y w

,001 0 5 1 0 15 20 25 30 35

RADIATION DOSE (Gy)

Fig. 5. PE of epigastric and subcutaneous 9L implants following 0-30 Gy radiation in air breathing compared to euthanized rats. (A) Epigastric, euthanized; (0) epigastric, air breathing; (m) subcutaneous, air breathing.

ity of glucose metabolism results from vascular heterogeneity, including arteriovenous shunting within these neoplasms. It has been previously shown that arteriovenous shunt flow in malignant tumor isolates can account for at least 30% of the arterial blood. This shunted blood may not take part in microcirculatory exchange processes [27-291.

The importance of glucose to the energy metabolism of solid malignancies has been known for many years [30]. By manipulating glucose metabolism, investigators have attempted to modify the cellular microenvironment, improve the tumor bioenergetic status [24,3 11, and create metabolic

140

Table 2. in Varying Locations*

Evans and Kocb: Radiation of 9L Tissue Isolates

Comparison of PE, Relative Survival, Cell Yield, and Tumor Size of 9L Tumors Grown

Epigastric Epigastric SubQ SubQ SubQ N, (Evans) air (Evans) air (Evans) air (Wallen) N, (Wallen)

Unirradiated PE (%) 22.5 19. I 20.5 15.6 15.6 Dose for 5% (relative) survival (Gy) 27.6 19.3 18.6 13.6 25.3

Tumor weight (g) 0.7-3.1 0.9-2.5 0.3-1.4 0 . s1 .4 0.9-1.4

*Data for SubQ tumors are taken from the literature 121.

n = 9 n = 18 n = 7

states more favorable to tumor cell killing. In gen- eral, improved delivery of glucose to the tumor has resulted in improved tumor bioenergetic status. The glucose extraction data presented herein agree with published studies showing an increased utilization of glucose when the tumor is presented with excess substrate in the blood [ 10,231. It is interesting that regardless of how the glucose is provided to the tumor, it will increase utilization proportional to supply. In the work of Gullino et al. [23], excess glucose was provided by SubQ dextrose injections; in the work presented herein, the elevated glucose levels resulted from SIH and/or secondary effects of anesthesia. The body’s response to pharmacologic or suprapharmacologic doses of glucose would include increased secretion of insulin. In contrast, the hallmark of SIH is decreased insulin secretion. Fur- ther studies on these phenomena are pending.

Cell Yield, PE, and Radiation Response The average cell yield of 15 9L epigastric tumors was 6.33 X lo7 (24 .3 X lo7). These very high cell yields are similar to those reported in the literature [ l ,2] and were obtained despite using a large range of tumor sizes (0.4-4.9 8). The PE of tumor cells from untreated 9L epigastric tumors was 19.1 * 4.67% (n = 5) in air and 22.5 2 7.85% (n = 4) in hypoxic (euthanized) rats. Since our unirradiated PE (19-21%) is in the same range as previously reported ( 15.6-27.9%) [ 1,3], the use of feeder cells was deemed unnecessary. Figure 5 and Table 2 demonstrate the radiation response of epigastric and SubQ 9L implants following 0-30 Gy radiation in air breathing compared to euthanized rats. These values are compared to literature values [3]. The radioresponse of our epigastric tumors in euthanized rats was similar to that reported by Wallen et al. [3] when corrected for our slightly higher PE: 22% vs. 15%. However, the epigastric 9L tumors in air breathing rats were slightly more radioresistant than those SubQ tumors reported by Wallen et a1 . [ 31 (Table 2).

The cell yields, PE, and radiation response of 9L tumor cells implanted intracerebrally and SubQ have been extensively studied [1,2]. There were differences in PEs and radiation response in these two sites; the authors concluded that these differences in radiation response were a result of the cellular microenvironment as determined by the tumor location. Our results with 9L tumors grown as epigastric and SubQ implants give cell yields and PEs (non-irradiated) which are similar to each other and to previous reports. The radiation response (Figure 5, Table 2) indicates somewhat greater resistance than was previously seen but the cells seem to be uniformly radioresistant. There is no evidence for a mixed population of aerobic and hypoxic cells giving the survival curve a shape characteristic of a sensitive and then resistant portion. Shonat and col- leagues [32] have measured the oxygen distribution within the tumor vasculature of small 9L tumors and found relatively low and uniform oxygen partial pressures. This has been the long-standing anomaly with the 9L tumor model; one would ex- pect large gradients of oxygen and nutrients from blood vessels. A partial explanation for these results may have been provided in a recent paper by Franko et al. [4]. They showed that binding of misonidazole, a metabolic indicator of relative oxygenation, was uniform over large regions of 9L tumors. Two explanations were provided by these authors. The first was that the oxygen consumption rate of the tumor cells was much less for tumor cells in situ vs. in vitro. Indeed, the reduced rate would have to be some 25-fold less than for virtually any other mammalian cell line studied. Another explanation was the possibility of a dramatic cycling of blood flow. Although our results do not shed any direct light on the discrimination of these possibili- ties, they do indicate that the degree of radiation resistance is quite variable within this tumor model.

Histopathologic Appearance Eleven 9L epigastric tumors following perfusion [4] or PE [7] studies were submitted for light mi-

Evans and Koch: Radiation of 9L Tism Isolates 141

Fig. 6. Histopathologic appearance of a 1.5 g 9L tumor grown as a tissue isolate on the epigastric vessels. The neoplasm was composed of similar spindle cells arranged in interwoven fascicles. The cells had round to oval to fusiform nuclei and variable amounts of eosinophilic cytoplasm with

croscopy. All neoplasms were composed of similar spindle cells arranged in interwoven fascicles. The cells had round to oval to fusiform nuclei and variable amounts of eosinophilic cytoplasm with indistinct borders (Fig. 6). Mitotic figures were common. Lymphocytes and plasma cells were prominent at the edges of the neoplasms. In gen- eral, as tumor size increased, the proportion of necrosis in the tumor also increased (Table 3). Subjec- tive evaluation of the number of capillaries did not appear to correlate with tumor size or presence of necrosis. In tumors with necrosis, these areas tended to be linear and occur in vessel-poor areas.

CONCLUSION The data presented demonstrate the simultaneous evaluation of radiation response and tumor energy metabolism in a single tumor model. The power of this model lies in the ability to simultaneously measure cell survival both in vivo and in vitro along with measurements of TBF and substrate consumption rates. Because these physiologic measurements can be manipulated and monitored directly in this model system, it may be possible to measure surviving fraction in individual tumors. Such studies

indistinct borders. On hematoxylin and eosin staining, less than 5% of the tumor was necrotic. Tumor tissue composed of fascicles of glioma cells and necrosis adjacent to a blood vessel are shown. x250.

Table 3. Tumor Histopathologic Analysis Vs. Weight*

Tumor No. (!z) Necrosis (%) CaDillaries Weight

92090 1 930603A20 9308 19A30 920804 9308 1 1 A20 920903 9308 13 930819N30 921 117 930805N20 921 124

0.41 1.06 1.10 I .26 1.30 1.35 1.52 1.56 2.50 2.70 4.00

<5 < 10 < 10 <I0

<5 10-25

<5 <I0

25-50 50

>50

Many Few Few Few Few Many Few Few Few Few Moderate

*Comparison of tumor weight and subjective evaluation of tumor necrosis and capillary density. There is a trend toward increased necrosis in larger tumors although even the smallest tumors have microscopic areas of necrosis.

would emphasize intertumor heterogeneity in a unique way.

ACKNOWLEDGMENTS We thank Yimei Cao and Kristine Laughlin for technical assistance and Thomas van Winkle for histopathologic analyses. This work was supported by grant CA-56679-01A1 from the National Insti-

142

tutes of Health, a Research Foundation Award at the University of Pennsylvania, and the Department of Radiation Oncology, Hospital of the University of Pennsylvania.

Evans and Koch: Radiation of 9L Tissue Isolates

REFERENCES 1 . k i t h JT, Schilling WA, Wheeler KT: Cellular radi-

osensitivity of a rat brain tumor. Cancer 35:1545-1550, 1975.

2. Wallen CA, Michaelson SM, Wheeler KT: Tempera- ture and cell survival variability across 9L subcutaneous tumors heated with microwaves. Radiat Res 85:

3. Wallen CA, Michaelson SM, Wheeler KT: Evidence for an unconventional radiosensitivity of rat 9L subcutaneous tumors. Radiat Res 85529-541. 1980.

4. Franko AJ, Koch CJ, Boisvert DPJ: Distribution of misonidazole adducts in 91 gliosarcoma tumors and spheroids: Implications for oxygen distribution. Cancer Res 52:3831-3837, 1992.

5. Wong KH, Wallen CA, Wheeler KT: Chemosensitiza- tion of the nitrosoureas by 2-nitroimidazoles in the subcutaneous 9L tumor model: Pharmacokinetic and struc- ture activity considerations. Int J Radiat Oncol Biol

6. Moulder JE, Rockwell SC: Hypoxic fractions of solid tumors: Experimental techniques, methods of analysis and a survey of existing data. Int J Radiat Oncol Biol

7. Rumsey WL, Schlosser C, Nuutinen EM, Robiolio M, Wilson DF: Cellular energetics and the oxygen depen- dence of respiration in cardiac myocytes isolated from adult rat. J Biol Chem 265: 15392-15399, 1990.

8. Jain RK, Wei J, Gullino PM: Pharmacokinetics of methotrexate in solid tumors. Pharmacokinet Biopharm

9. Gullino PM, Grantham FH: Studies on the exchange of fluids between host and tumor. I. A method for grow- ing tissue isolated tumors in laboratory animals. J Natl Cancer Inst 27:679493, 1961.

10. Sauer LA, Stayman JW, Dauchy RT: Amino acid, glucose, and lactic acid utilization in vivo by rat tumors. Cancer Res 42:4090-4097, 1982.

1 1. Kallinowski F, Tyler G, Mueller-Klieser W, Vaupel P: Growth-related changes in oxygen consumption rates of tumor cells grown in vitro and in vivo. J Cell Physiol

12. Peterson HI: Tumor Blood Circulation: Angiogenesis, Vascular Morphology and Blood FLOW of Experimen- tal and Human Tumors. Boca Raton, FL: CRC Press,

13. Waynforth HB, Flecknell PA: Experimental and Surgi- cal Technique in the Rat. San Diego: Academic Press, pp 6 8 4 9 , 1992.

14. Howell RL, Koch CJ: The disaggregation, separation and identification of cells from irradiated and unirradiated EMT6 mouse tumors. Int J Radiat Oncol Biol Phys

281-291, 1981.

Phys 18:1043-1050, 1990.

Phys 10~695-712, 1984.

7:181-194, 1979.

138: 183-1 9 1, 1989.

pp 45-56, 1979.

6:311-318, 1980.

15. Wixson SK, White WJ, Hughes HC, Lang CM, Mar- shall WK: A comparison of pentobarbitol, fentanyl- droperidol, ketamin-xylazine and ketamine-diazepam anaesthesia in adult male rats. Lab Anim Sci 37:72& 730, 1987.

16. Dauchy RT, Sauer LA: Preparation of tissue isolated rat tumors for perfusion: A new surgical technique that preserves continuous blood flow. Lab Anim Sci 36: 6 7 8 4 8 I , 1986.

17. Amouzadeh HR, Sangiah S: Effects of streptozotocin- induced diabetes on xylazine-ketamine anesthesia. Vet Hum Toxicol 32: 19-22, 1990.

18. Mossberg KA, Taegtmeyer H: Dihydroergotamine as a pharmacologic euglycemic clamp in the surgically trau- matized rabbit. Metabolism 40:594-599, 1991.

19. Hsu WH, Hummel SK: Xylazine induced hyperglycemia in cattle: A possible involvement of a-adrenergic receptors regulating insulin release. Endocrinology

20. Gullino PM, Grantham FH, Courtney AH: Utilization of oxygen by transplanted tumors in vivo. Cancer Res 27:102CL1030, 1967.

21. Volk T , Roszinski S, Jahde E, Glusenkamp K, Rajew- sky M: Effect of glucose-mediated pH reduction and cyclophosphamide on oxygenation of transplanted rat tumors. Int J Radiat Oncol Biol Phys 25:465471, 1993.

22. Kallinowski F, Vaupel P, Rungel S, Berg G, Fortmeyer HP, Baessler KH, Wahner K, Mueller-Klieser W, Walenta S: Glucose uptake, lactate release, ketone body turnover, metabolic micromilieu and pH distribu- tions in human breast cancer xenografts in nude rats. Cancer Res 48:7264-7272, 1988.

23. Gullino PM, Grantham FH, Courtney AH: Glucose consumption by transplanted tumors in vivo. Cancer Res 27:1031-1040, 1967.

24. Bhujwalla ZM, Constantinidis I, Chatham JC, Wehrle JP, Glickson JD: Energy metabolism, pH changes, and lactate production in RIF-1 tumors following intratu- moral injections of glucose. Int J Radiat Oncol Biol

25. Sevick EM, Jain RK: Blood flow and efferent blood pH of tissue isolated Walker 256 carcinoma during hyperglycemia. Cancer Res 48:1201-1207, 1988.

26. Ward-Hartley KA, Jain RK: Effect of glucose and ga- lactose on microcirculatory flow in normal and neoplas- tic tissues in rabbits. Cancer Res 47:371-377, 1987.

27. Endrich B, Hammersen F, Goetz A, Messmer K: Mi- crocirculatory blood flow, capillary morphology and local oxygen pressure of the hamster amelanotic melanoma A-MEL-3. J Natl Cancer Inst 68:475-485, 1982.

28. Vaupel P, Grunewald WA, Manz R, Swa W: Intracap- Nary HbO, saturation in tumor tissue of DS carcinosarcoma during normoxia. Adv Exp Med Biol94:367- 375, 1978.

29. Weiss L, Hulvom R, Tveit E: Blood flow characteristics in induced rat mammary neoplasia. Microvasc Res 17:119, 1979.

109:825-829, 1981.

Phys 22~95-101, 1992.

30. Warburg 0, Wind F, Negelein E: The Metabolism of Tumors. London: Constable & Co., pp 256, 1930.

31. Okunieff P, Vaupel P, Sedlacek R, Neuringer LJ: Eval- uation of tumor energy metabolism and microvascular blood flow after glucose or mannitol administration using 3'P NMR spectroscopy and laser Doppler flowme- try. Int J Radiat Oncol Biol Phys 16:1493-1500, 1989.

32. Shonat RD, Wilson DF, Riva CE, Pawlowski M: Oxy- gen distribution in the retinal and choroidal vessels of the cat as measured by a new phosphorescence imaging method. Appl Opt 33:3711-3718, 1992.

33. Kluge M, Elger B, Engel T, Schaefer C, Seega J , Vaupel P: Acute effects of tumor necrosis factor alpha or lymphotoxin on global blood flow, laser Doppler flux and bioenergetic status of subcutaneous rodent tumors. Cancer Res 52:2167-2173, 1992.


34. Hasegawa T, Song CW: Effects of hydralazine on the blood flow in tumors and normal tissues in rats. Int J Radiat Oncol Biol Phys 2O:lOOl-1007, 1991.

35. Eskey CJ, Koretsky AP, Domach MM, Jain RD: 3H Nuclear magnetic resonance imaging of tumor blood flow: Spacial and temporal heterogeneity in a tissue isolated mammary adenocarcinoma. Cancer Res 52: 6010-6019, 1992.

36. Vaupel P: Interrelationships between mean arterial blood pressure, blood flow and vascular resistance in solid tumor tissue of DS-carcinosarcoma. Experientia

37. Hori K, Suzaki M, Tanada S, Saito S, Shinozaki M, Zhang Q: Circadian variation of tumor blood flow in rat subcutaneous tumors and its alteration by angiotensin II induced hypertension. Cancer Res 52:9 12-9 16, 1992.

3 1 :587-588, 1975.

Radiation response and other characteristics of the 9l rat glioma grown as an epigastric tissue...

Documents

Transcript of Radiation response and other characteristics of the 9l rat glioma grown as an epigastric tissue...