distribution of fluorescent tracers in heterologously...

The diagnostic use of radioactive isotopes in the localization of the intracranial tumors has stimulated a search forradioactive tracers which will localize in such tumors (2,11, 12, 18, 20, 29). However, such studies are concernedmainly with the concentration of the tracer in the tumorrelative to that in the brain tissue, and not with themorphologic localization of the tracers in the tumor.

The earlier studies on the localization of tracer substances used azo dyes in a variety of spontaneous andtransplantable animal tumors (1 , 3, 7, 13, 32). Thesestudies revealed a gross accumulation of dye which wasactually limited to the stroma and necrotic cellular debris.Moore (19) first reported the use of sodium fluorescein inidentifying intracranial tumors at surgery, and its localization in an experimental tumor was described by Shapiroand Landing (28). Although sodium fluorescein can bedetected in a lower concentration than the azo dyes (21),it is highly diffusible and too soluble to be localized accurately at the microscopic level. Since the azo dyes bindto circulating proteins in varying degrees (23), tracerstudies with the different azo dyes may involve both aprotein-bound and a free dye component, depending onthe dye used (6, 30). This problem is minimized withsodium fluorescein because only 40 per cent of the dye isbound, and that weakly, to proteins (31). Fluoresceinlabeled albumin, which still retains the properties of nativealbumin (26), can be localized grossly and microscopicallywith good preservation of the morphologic structures.

In the present study, the localization of fluorescent

* This work was supported by USPHS Grant C5482 and by a

Special Fellowship (BT 793) from the National Institute of Neurological Diseases and Blindness, Public Health Service.

Received for publication June 4, 1964.

tracers, i.e., sodium fluorescein and rhodarnine- or fluores

cein-labeled albumin, in tumors was investigated. Byinjecting either sodium fluorescein or a fluorescent-labeledalbumin in animals bearing established transplantabletumor lines, it is possible to compare the distributions of asmall, negatively charged molecule and a protein moleculein different tumor lines.

MATERIALS AND METHODS

The four tumors employed in this investigation havebeen maintained in this laboratory for many generationsby the method of Greene (4) as heterologous intracerebraltransplants in guinea pigs. The first tumor, HC305, wasestablished by Dr. Edward Krenetz from a human glioblastoma transplanted directly into the guinea pig anteriorchamber and subsequently to the brain (5). Tumor 178is from a human glioblastoma and was first established intissue culture (17), then transferred subcutaneously toirradiated and steroid-conditioned hamsters and subsequently to the brains of untreated guinea pigs (16). Thethird tumor is an amelanotic melanoma derived from direct transplantation of a human tumor into unconditionedguinea pigs, and its biology will shortly be reported by oneof the authors.' The last tumor was induced in the cerebrum of C3H mice with methylcholanthrene (27), and wastransplanted once subcutaneously in C3H mice and thento the brains of unconditioned guinea pigs. This is anundifferentiated tumor with the characteristics of anependymoblastoma.

Guinea pigs bearing these heterologous intracerebraltransplants were given injections intraperitoneally of

1 E. E. Manuelidis, in preparation.

1749

Distribution of Fluorescent Tracers in HeterologouslyTransplanted Intracerebral Tumors*

MARTIN R. KRIGMAN AND ELIAS E. MANUELIDIS

(Department of Pathology, Yale University Medical School, New Haven, Connecticut)

SUMMARY

The macro- and microscopic distribution of sodium fluorescein and rhodamine- orfluorescein-labeled albumin was investigated in four different intracerebral tumorsheterologously transplanted to guinea pigs. Three of the four tumors were human tumors : two glioblastomas multiforme (Tumors 178 and HC305) and one amelanotic melanoma; the fourth tumor was an ependymoblastoma induced with methylcholanthrene in C3H mice. The results indicate that all of the fluorescent tracerswere localized in the small necrotic foci ; the sodium fluorescein was distributed intracellularly ; the rhodamine- or fluorescein-labeled albumin was localized in variousstromal elements ; and the labeled albumins were located intracellularly in the viabletumor cells of the HC305 tumor and ependyrnoblastoma, extracellularly in the viablecells of the melanoma and Tumor 178, and intracellularly in all the tumor cells showingregressive changes.

on April 9, 2019. © 1964 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

FLUORESCENT TRACERTRAcER DOSAGE(MG/100 c@)No.

ot ANIMALSIN ZAcETUMORGROUP

Mela- Tumor HC3OS Ependy.noma 178 tumor moss

Sodium fluoresceinFluorescein-labeled

albuminRhodamine-labeled

albuminFluorescein-labeled

albuminNoninjected controls10

50

50

50 X 36

10

2

2

26

10

2

2

26

8

2

2

26

6

2

2

2Total22222018

1750 Cancer Research Vol. 24, November 1964

TABLE 1SUMMARY OF EXPERIMENTAL GROUPS

RESULTSGROSS OBSERVATIONS

The four tumors were similar in gross appearance. Thetumors were limited to the side on which they were originally transplanted, the right side of the brain, in all cases;they extended from the frontal lobe to the basal gangliaand ranged from 0.5 to 1.5 cm. in diameter. They oftenextended up to the dorsal and/or the basal pial surfacesand to, and occasionally into, the right lateral ventricle.The melanoma, Tumor 178, and HC305 tumor were pinkgray and translucent, with multiple gray-white opaque fociof necrosis, whereas the ependymoblastoma was gelatinous, translucent, and friable. The tumors appearedto be sharply demarcated from the host brain tissue, andgrowth appeared to be by expansion, with distortion ofthe adjacent structures.

The tumors normally fluoresced blue to silver-blue underultraviolet light, while with the extravascular distributionof the tracers the tumors fluoresced yellow-green withsodium fluorescein and fluorescein-labeled albumin andorange with rhodamine-labeled albumin (Figs. 1â€”8). Themelanoma and ependymoblastoma fluoresced more intensely with sodium fluorescein (Figs. 1 and 7) than Tumor178 (Fig. 5), and the HC305 tumor showed the leastfluorescence (Fig. 3). With the labeled albumins, TumorsHC305 and 178 fluoresced slightly (Figs. 4 and 6), theependymoblastoma the most (Fig. 8), and the melanomashowed either no or barely perceptible fluorescence (Fig.2). The small necrotic foci, irrespective of the tumor orthe fluorescent tracer, fluoresced with an intensity equal to,or greater than, the apparent viable tumor. However,the occasional large necrotic focus, 4 mm. or more, showeda nonfluorescent center with an intensely fluorescent rim.

MIcRoscoPIc OBSERVATIONS

The green-yellow fluorescence of the fluorescein-labeledalbumin and the orange-red fluorescence of the rhodaminelabeled albumin contrasted sharply with the normal violetblue autofluorescence of the tumors and the silver-whiteautofluorescence of the basement membranes, reticulin,and collagen. There was no difference between the distribution of the fluorescein- or rhodamine-labeled albuminsin the tumors. In the animals that had been given injections for 3 days, the labeled albumin showed the samedistribution in the tumors as in the animals given only oneinjection.

Melanoma.â€”The melanoma consisted of a sheetlikegrowthof large epithelial-like mononuclear cells and multinucleated giant cells sharply demarcated from the adjacent brain tissue (Fig. 9). Hypertrophied astrocytesfrom the host brain were distributed throughout the tumorwith an increased number about the necrotic foci (Fig. 10).The necrotic foci consisted of separated pyknotic cellsand cellular debris and were located predominantly inthe central portions of the tumor. Many of the tumorcells surrounding these foci were swollen and vacuolated.The stroma was scanty and consisted predominantly ofregularly distributed vessels, which varied from capillarystructures to large vessels with thin collagenous walls.There was a network of reticulin fibers associated withthe vessels which extended into the tumor proper.

Fluorescent microscopic examination revealed in the well

either 10 mg. of sodium fluorescein or 50 mg. of fluoresceinor rhodamine-labeled albumin and sacrificed 1 or 24 hr.later. A small group of animals was given 50 mg. offluorescein-labeled albumin/100 gm/day for 3 days andsacrificed 24 hr. after the last injection. A total of 82guinea pigs was examined. The experimental groups aresummarized in Table 1.

The sodium fluorescein was prepared as a 1 per centsolution in normal saline. The labeled albumins wereprepared as 10 per cent protein solutions by conjugatingeither fluorescein isothiocyanate or rhodamine B isothiocyanate with crystalline bovine albumin, all three reagentsobtained from Nutritional Biochemicals Corp. The unreacted fluorescent material was removed by passing theconjugate through a Sephadex 25M column (24), and thesolutions were Sterilized through a Berkfield filter.

The animals were sacrificed with ether; the brains wereimmediately removed, and multiple coronal sections 2â€”3 mm. thick were cut. Alternate sections were placeddirectly in Sorensen buffered (jH 7.2) 10 per cent formalincooled to 4Â°C., and the opposing surfaces were observedand photographed under visible light and long-waveultraviolet light from a CH-4 mercury spot lamp with aWood filter (Eastern Equipment Co.) and then placed inbromformalin.

Sections for fluorescent microscopy were prepared onlyfrom the animals that had received injections of labeledalbumin; the sodium fluorescein is highly diffusible andtoo soluble for preparing satisfactory sections for fluorescent microscopy. The tissue was fixed for 24 hr. in coldbuffered formalin, rapidly dehydrated, and embedded ineither Tissuemat or Paraplast; sections were cut at 6 and10 Jh, mounted on precleaned, nonalbuminized slides,cleared in xylene, and mounted in Fluoromount. Alternate sections were cut and stained with hematoxylin andeosin and Laidlaw's connective tissue stain. Sections forCajal gold sublimate stain for astrocytes and triple silverimpregnation by the method of Scharenberg (25) wereprepared from the material fixed in bromformalin.

A Reichert Zetopan microscope equipped with an HBO200 ultraviolet light source, cardioid dark field condenser,Schott BG2 and Corning 5-mm.-thick no. 5970 transmitting ifiters, and Wratten 2A and Zeiss 0G4 barrierfilters was employed for the fluorescent microscopy.



KRIGMAN AND MANUELIrnsâ€”Fluorescence in Intracerebral Tumors 1751

preserved tumor, including the giant cells, a linear fluorescence which outlined the cells and appeared to be limitedto the cell membranes (Fig. 15), whereas the necrotictumor cells were intensely and diffusely fluorescent, andthe tumor cells at the margin of the necrotic regions,presumably undergoing regressive changes, showed focalcytoplasmic fluorescence. The stromal vessels showed auniform fluorescence in their basement membranes, collagenous walls, and the associated reticulin network.There was a uniform fluorescence located in and/or on theendothelial cells. This problem of localizing the fluorescent-labeled albumins in or on the endothelial cells wasalso encountered with the other tumors. The astrocytesshowed a cytoplasmic fluorescent beading which was moreintense and consisted of larger droplets near necrotic foci(Fig. 16).

Tumor 178.â€”Tumor 178 consisted of lobules formed bycords of polyhedral epithelial cells with some pleomorphism. The cells in the periphery of the lobules wereclosely packed, whereas the central cells showed a tendencyto separate, lose their angulated outline, and progress topyknotic or swollen vacuolated cells floating freely in aproteinaceous matrix in the center of the lobule (Fig. 11).These necrobiotic changes were found in all the lobulesand constituted one-half to two-thirds of a given lobule.The lobules were separated by a loose, highly vascularstroma which contained fibroblasts, collagen and reticulinfibers, histiocytes, and tortuous dilated vessels. The yessels extended into the lobules as capillary structures running between the cords of tumor.

Fluorescent microscopy revealed a linear fluorescenceoutlining the closely packed cells in the periphery of thelobule (Fig. 17). In the central regions, the partiallyseparated cells showed fluorescent foci in the cytoplasm;the completely separated cells showed extensive cytoplasmic fluorescence, and the pyknotic necrotic cells showed ahomogeneous fluorescence including the nucleus (Fig. 18).The proteinaceous matrix in the center of the lobules had adiffuse but less intense fluorescence. The interlobularfibrovascular stroma showed an intense fluorescence alongthe basement membranes and adventitia of the vessels,the cytoplasm of the fibroblasts, the reticulin and collagenfibers, and coarse fluorescent granules in the histiocytes(Fig. 19). In the vessels, there was a homogeneousfluorescence located in and/or on the endothelial cells.The capifiaries between the cords of tumor cells had finefluorescent droplets over and/or in the endothelial cellsand an intense fluorescence in the basement membranes.

Tumor HC305.â€”The HC305 tumor consisted of regular,fusiform, spongioblastlike cells growing in a loose whorlingpattern, sharply demarcated from the adjacent brain tissue(Fig. 12). The ends of the cells tapered off into single,delicate, threadlike processes which often extended toadjacent vessels. The cells were lined up about necroticfoci, simulating pseudopalisading, and showed coarsetortuous end processes. These necrotic foci were multiple,relatively small, and predominantly located in the centerof the tumor; they consisted mainly of pyknotic cells andcellular debris. The stroma consisted of fibrovascularbands with both simple vessels lined by plump endothelialcells and glomeruluslike vessels. Separate aggregates of

glomeruluslike vessels were also seen, predominantly inthe periphery of the tumor (Fig. 13).

Fluorescent microscopic examination of the tumor revealed fluorescent droplets in the cytoplasm of the tumorcells and a homogeneous fluorescence along the taperedcell end processes (Fig. 20). The necrotic cells showed anintense homogeneous fluorescence, and the cells borderingthe necrotic foci appeared to have more and coarser fluorescent cytoplasmic vacuoles. The glomeruluslike vascularstructures showed a moderate uniform fluorescence in theendothelial cells and an intense homogeneous fluorescencein the basement membranes (Fig. 21). The fibrousstromal components showed an intense uniform fluorescence in the cytoplasm of the fibroblasts and in the reticulin and collagen fibers and coarse fluorescent droplets inthe histiocytes.

Ependymoblastoma.â€”The ependymoblastoma consistedof sheets of small uniform cells with an indistinct margin,made up of tumor cells and astrocytes, between tumor andadjacent brain parenchyma. The tumor cells had an ovalhyperchromatic nucleus, indistinct cytoplasmic tags (Fig.14), and one prominent process visible in the silver impregnations. Throughout the tumor, there were manybroad regions of regressive change; the tumor cells wereswollen and showed a clear watery cytoplasm outlinedby a distinct cell membrane. In the center of these regions, the cells were pyknotic, poorly defined masses.Hypertrophied astrocytes were distributed throughout thetumor and were most prevalent in the periphery of thetumor. The vascular stroma was sparse and consistedof both dilated simple vascular channels with a scantyadventitial reticulin and clusters of vessels, with prominentendothelial hyperplasia, which were irregularly distributedin the periphery of the tumor.

Fluorescent microscopy revealed a beaded fluorescencein the cytoplasmic tags of the well preserved tumor,whereas the poorly preserved cells showed cytoplasmicfluorescence accentuated along the cell membranes (Fig.22). The necrotic cells showed a homogeneous fluorescence, including the nuclei as well as the cytoplasm. Theastrocytes contained dense fluorescent droplets in theirperikaryon and processes. The vessels showed fluorescence in and/or on proliferating endothelial cells and ahomogeneous fluorescence along the basement membranes.

DISCUSSION

Abnormal permeability or the breakdown of the bloodbrain barrier in the tumors is evident from the gross andmicroscopic extravascular distribution of the fluorescenttracers. Since the gross fluorescent appearance reflectsthe distribution of the tracers in the tumor cells, stroma,and necrotic foci, the role that each of these constituentsplays must be considered.

The ependymoblastoma and HC305 tumor show intracytoplasmic accumulations of fluorescein- and rhodaminelabeled albumins, in contrast to the extracellular localization of the fluorescent-labeled albumins on the cellmembranes of the melanoma and Tumor 178. However,the tumor cells showing regressive changes, irrespective ofthe type of tumor, contain coarse fluorescent cytoplasmicvacuoles. The presence or absence of labeled albumin inthe well preserved tumor cells indicates a difference in the



1752 Cancer Research Vol. 24, November 1964

ability of the different tumors to accumulate albumin,presumably by pinocytosis. However, this difference isno longer evident in the tumor cells undergoing regressivechanges. This may be compatible with the finding ofNairn et al. (22) that it is only the injured liver parenchymal cells which will accumulate albumin. In all thetumors, the necrotic cells and cellular debris show an intense homogeneous fluorescence in the cytoplasm andnucleus. This fluorescence of the necrotic cells and debrisindicates a complete cell membrane breakdown, whichhas been referred to by Holtzer and Holtzer (8) as theâ€œinjuredcell reaction.â€• This homogeneous fluorescenceof necrotic cells has been observed in tissue culture (8, 9)and in different experimental studies in vivo (10, 22) withfluorescent-labeled proteins.

The accumulation of labeled albumin in the stroma ofthe tumor, diffuse fluorescence along the collagen andreticulin fibers, and the cytoplasmic fluorescence of fibroblasts and histiocytes have essentially the same distribution as in connective tissue of normal animals thathave been given injections of fluorescein-labeled albumin(14, 15, 26). The homogeneous fluorescence outliningthe lumina of the stromal vessels could not be accuratelylocalized in reference to the endothelial cells. Only in theendothelial cells of the glomeruluslike vessels was theintraendothelial localization of the labeled albumin apparent. This problem of localizing the fluorescein- orrhodamine-labeled albumin in or on the endothelial cellwas also reported by Schiller et al. (26) in their studieswith normal vessels of the systemic circulation. Thepresence of labeled albumin was also observed along thebasement membranes of the vessels in the tumor. Otherobservers have noted similar distribution on the basementmembranes in vessels which are normally permeable to

albumin (14, 15, 26). In the regions of normal brain,where the vessels are not permeable to albumin, we foundno fluorescence along the basement membrane.

Astrocytes are found admixed with the tumor cells in themelanoma and ependymoblastoma. These are presumably derived from the host neuroglia. Why the astrocytesare found only in these two tumors cannot be readily cxplained. The accumulation of labeled albumin by theseastrocytes is similar to that observed in tissue culture (9)and in tracer studies in vivo (10) with fluorescein-labeledalbumin.

The differences among the tumors in their gross fluorescence with the labeled albumins can be attributed to thedegree of stromatization, extent of necrobiotic changes,and pinocytotic activity of the tumor cells. The melanoma has a sparse stroma and few foci of necrosis, andthe viable tumor cells lack pinocytotic activity ; withTumor 178 this lack of pinocytosis is compensated for bythe extensive regions of regressive change and abundantinterlobular stroma. Although the ependymoblastomahas a sparse stroma, the intracellular accumulation oflabeled albumin and the extensive regions of regressivechange make this the most fluorescent of the tumors. TheHC305 tumor, which shows intracytoplasmic accumulationof the labeled albumin and has an abundant stroma, isonly intermediate in its fluorescence because the necroticfoci are few and limited to the central regions of tumor.

The prominent gross fluorescence in the melanoma withsodium fluorescein and the minimal gross fluorescence withthe fluorescein- and rhodamine-labeled albumins suggest adifference in morphologic localization of the sodium fluorescein compared to the labeled albumins. Since the labeledalbumins are located predominantly extracellularly inthe melanoma, the sodium fluorescein may be distributed

Fios. 1â€”8.â€”Grossphotographs of coronal sections of brainsbearing heterologously transplanted tumors taken with longwave ultraviolet light. All of the specimens are enlarged by afactor of 2.5.

FIG. 1.â€”Melanoma. The tumor shows a yellow-green fluores

cence which is accentuated in central necrotic regions. Sodiumfluorescein.

FIG. 2.â€”Melanoma. The tumor shows a normal blue autofluorescence ; the white-yellow-green fluorescence in the centerrepresents labeled albumin in necrotic foci. Fluorescein-labeledalbumin.

FIG. 3.â€”HC305 tumor. The tumor shows only a slight applegreen fluorescence with accentuation in necrotic foci. Note theintense fluorescence in the white and gray structures adjacent tothe tumor. Sodium fluorescein.

FIG. 4.â€”HC305 tumor. There is a slight apple-green fluorescence in the tumor which is accentuated in central necrotic regions. The fluorescence is more intense in adjacent and distalsegments of white matter. Fluorescein-labeled albumin.

FIG. 5.â€”Tumor 178. The tumor shows a uniform blue-green

fluorescence. Sodium fluorescein.FIG. 6.â€”Tumor 178. There is a mottled apple-green fluores

cence throughout the tumor, representing the extensive necroticfoci. The brown autofluorescence of the neural tissue in thisfigure and Figure 8 is due to an additional barrier filter, WrattenG@ 15. Fluorescein-labeled albumin.

FIG. 7.â€”Ependymoblastoma. The tumor shows a yellowfluorescence which is accentuated in the multiple necrotic focithroughout the tumor. The multiple segments of yellow-whitefluorescence around the tumor are located predominantly in whitestructures. Sodium fluorescein.

FIG. 8.â€”Ependymoblastoma. The tumor shows a uniform

yellow-orange fluorescence. Rhodamine-labeled albumin.



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Fi;. 9.â€”Melanoma. The sheetlike pattern of mononuclearepithelial cells and multinucleated giant cells is demonstrated.Hematoxylin and eosin, X 110.

FIG. 10.â€”Melanoma. Astrocytes in the tumor which areaccentuated about a necrotic focus. Cajal gold sublimate stain,x 110.

FIG. 11.â€”Tumor 178. The lobular pattern is demonstrated.The well preserved cords of polyhedral cells are in the peripheryof the lobules, and the progressive regressive changes occur towardthe center. Hematoxylin and eosin, X 110.

FIG. 12.â€”HC305 tumor. The fusiform spongioblastlike morphology is demonstrated. Hematoxylin and eosin, X 150.

FIG. l3.â€”HC305 tumor. Glomeruluslike vascular structuresin the tumor are demonstrated. Hematoxylin and eosin, X 150.

FIG. 14.â€”Ependymoblastoma. The sheetlike growth patternof small uniform cells with indistinct cytoplasm is demonstrated.

Hematoxylin and eosin, X 150.

1754



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FIGS. 15â€”22.â€”Fluorescentphotomicrographs.FIG. 15.â€”Melanoma. The apple-green fluorescence of the

labeled albumin along the cell membranes of the well preservedtumor cells is demonstrated. Fluorescein-labeled albumin, X 800.

FIG. 16.â€”Melanoma. Astrocytes in the tumor showing applegreen fluorescence in their perikaryons and processes. Fluorescein-labeled albumin X 800.

FIG. 17.â€”Tumor 178. Cords of well preserved tumor in theperiphery of a lobule showing the apple-green fluorescence oflabeled albumin along their cell membranes. Fluorescei n-labeledalbumin, X 320.

@ 18.â€”Tumor 178. The center of a lobule showing focal

orange fluorescence in cells undergoing regressive changes anddiffuse fluorescence in necrotic cells. Rhodamine-labeled albumin, X 800.

FIG. 19.â€”Tumor 178. Interlobular stroma showing applegreen fluorescence on collagen and reticulin fibers and connectivetissue investments of a vessel. Fluorescein-labeled albumin,x 200.

F1;. 20.â€”HC305 tumor. Tumor cells showing focal cytoplasmieorange fluorescence and diffuse fluorescence along end processes.Rhodamine-laheled albumin, X 800.

FIG. 21.â€”HC305 tumor. Glomeruluslike vessels in the tumorshowing apple-green fluorescence along the basement membranesand in the endothelial cells. The yellow fluorescence is labeledalbumin in the lumina. Fluorescein-labeled albumin, X 300.

Fig. 22.â€”Ependymoblastoma. Tumor cells showing the cytoplasmic localization of the apple-green fluorescence. Fluoresceinlabeled albumin, X 800.

1756



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KRIGMAN AND M@uELIDIsâ€”Fluorescence in Intracerebral Tumors 1759

intracellularly. Moore (20) has observed an intracellulardistribution of sodium fluorescein in a few tumors examined by squash preparations. However, the solubilityof this tracer and its rapid diffusion make any exact Jocalization difficult. If this tracer is distributed intracellularly, the slight fluorescence seen with the HC305tumor as compared with the other tumors may reflect theindependence of the plasma and tumor concentration asdemonstrated by Matthews and Molinaro (18) for intracellularly distributed tracers. The intense fluorescenceobserved in the ependymoblastoma and the moderatefluorescence in Tumor 178 with the sodium fluoresceinreflect the combined effects of an intracellular localizationin cellular tumors and an accumulation in many extensiveregions of necrosis.

The majority of the visible necrotic foci are small, ranging from 0.5 to 1 mm., and fluoresce intensely, irrespectiveof the tracer used, while the large necrotic foci, 4 mm. ormore, have nonfluorescent centers. The latter phenomenon corresponds to the observation by Moore with sodiumfluorescein (19) and similar observations with azo dyetracers (3, 7, 32). In prolonged experiments with theazo dyes, the necrotic foci ultimately become stained (3,7, 32). Shapiro and Landing (28) interpreted the relativestaining of viable and nonviable regions as a doseâ€”durationphenomenon, as there is little, if any, active circulation inthese necrotic foci and the extent of distribution of thetracer is dependent upon diffusion.

Although the results of this study are only qualitativeand subject to the limitations of fluorescent tracer technics, they indicate that in considering the localization of atracer substance, whether a dye or a radioactive isotope,many factors must be taken into account. The localization of a tracer in the tumor is affected by whether thetracer is distributed intra- or extracellularly (18). However, this differentiation may not always hold for differenttumors and may not exist in regions where the tumors areundergoing regressive changes. Lastly, the extent ofnecrosis and type and degree of stroma will affect thedistribution of the tracers.

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2. DAvis, L.; MARTIN,J.; ASHKENAZY,M.; LEROY,G. V.; andFIELDS, T. Radioactive Diiodofluorescein in Diagnosis andLocalization of Central Nervous System Tumors. J. Am.Med. Assoc.,144:1424â€”32,1950.

3. DURAN-REYNALS,F. Studiesonthe Localizationof DyesandForeign Proteins in Normal and Malignant Tissues. Am. J.Cancer, 3598â€”107,1939.

4. GREENE, H. S. N. The Transplantation of Tumors to theBrains of Heterologous Species. Cancer Res., 11:529â€”34,1951.

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6. GREGERSEN,M. I., ANDRAwSON, R. A. The Disappearance ofT-1824 and Structurally Related Dyes from the Blood Stream.Am. J. Physiol., 138:698â€”707,1943.

7. HESS, M. The Localization of Acid-Azo Dyes in Tumors. J.Pathol. Bacteriol., 51:309â€”11,1940.

8. HOLTZER, H., ANDHOLTZER,S. The In Vitro Uptake of Fluorescein Labeled Plasma Proteins. I. Mature Cells. Compt.rend. tray. lab. Carlsberg, 31:373â€”408,1961.

9. KLATZO,I., ANDM.IQUEL,J. Observations on Pinocytosis inNervous Tissue. J. Neuropathol. Exp. Neurol., 19:475â€”87,1960.

10. KLATZO,I.; MIgUEL, J.; ANDOTENASEK,R. The Applicationof Fluorescein Labeled Serum Proteins (FLSP) to the Studyof Vascular Permeability in the Brain. Acta Neuropathol.,2:144â€”SO,1962.

11. LOCKSLEY,H. B. ; SWEET,W. H. ; POWSNER,H. J. ; ANDDow, E.Suitability of Tumor-bearing Mice for Predicting RelativeUsefulness of Isotopes in Brain Tumors. Comparative Clinicaland Laboratory Study in Localization and Treatment ofBrain Tumors with P-32, Na-24, K-42, and Sodium Borate.A.M.A. Arch. Neurol. Psychiat., 71:684â€”98,1954.

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1964;24:1749-1759. Cancer Res Martin R. Krigman and Elias E. Manuelidis Transplanted Intracerebral TumorsDistribution of Fluorescent Tracers in Heterologously

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