invasionofskeletalandsmoothmuscle byl1210leukemia1...[cancerresearch 27part1,2159-2178,november1967]...

[CANCER RESEARCH 27 Part 1, 2159-2178, November 1967]

Invasion of Skeletal and Smooth Muscleby L1210 Leukemia1

DAVID BRANDES,2 ELSA ANTON, AND BRIAN SCHOFIELD

Departments of Pathology, The Johns Hopkins University School of Medicine, and Baltimore City Hospitals, Baltimore, Maryland

SUMMARY

Invasion of skeletal and smooth muscle fibers by L1210 leu-kemie cells was studied by electron microscopy and histochemis-try. In the host-tumor interzone, degenerative changes were firstdetected in the tumor cells, and their cytoplasmic components,especially ribosomes, appeared free in the interstitial spaces.Many of the images strongly suggested that such tumor cellcomponents may have been taken up by the muscle cells, especially in areas of fusion between normal and malignant elements. In some areas, the presence of tumor cell material seemedto exert a stimulatory effect on the normal structures, reflectedby hyperplasia of cell organelles rather than cellular multiplication. Degenerative changes and lysis of muscle fibers occurredpredominantly in areas of tumor cell degradation, rather than inthe presence of healthy tumor cells, indicating that productsderived from altered tumor cells may play an important role inneoplastic invasiveness. Our results also indicate that in the caseof this particular tumor, lysosomes probably do not play a rolein the destruction of normal structures.

INTRODUCTION

The actual mechanisms of invasion and replacement of normalstructures by malignant cells has given rise to much speculationin the past, based greatly on inferences derived from histologieobservations (6). The invasive capacity of neoplastic cells hasbeen attributed to their power of progressive and rapid multiplication, to their motility and phagocytic capacity, to the elaboration of lytic or toxic products, or to the loss of growth restraintnormally exercised by cells on each other. These classic ideas havebeen reviewed by Willis (33) and later by Berenblum (6).

More recently, the mechanisms of tumor invasion have beenreexamined in animal experimental studies and with the aid ofelaborate biologic models. Many of the results have pointed tothe importance of proteolytic enzymes and toxic substancesfrom tumor cells as factors which may facilitate the destructionof normal elements and the progression of the neoplasia [seeVasiliev (31, 32) and Sylvan (27, 29) for reviews].

The investigations reported here, which form part of a compre-

1This investigation was supported by (jrants CA 08518 fromthe National Cancer Institute, and HD 00042from the NationalInstitute of Child Health and Human Development, NIH, USPIIS.

2Recipient of a Career Development Award, K3-CA-21,756-04.National Cancer Institute, USPHS.

Received April 11, 1967; accepted July 11, 1U67.

hensive study on the LI 210 leukemia in our laboratory, wereundertaken to determine whether electron microscopic and histo-chemical examination of invaded muscle would provide furtherinformation on the events at the host-tumor interzone that wouldshed some light on specific factors related to invasiveness.

MATERIALS AND METHODS

The solid and ascitcs forms of the LI 210 leukemia were carriedin DHA/2 mice weighing approximately 20 gm. In the case of thesolid form, 0.1 ml of a saline suspension (1:10) of the spleen froma leukemic donor was injected intramuscularly. For the ascitesform, a 1:10 saline suspension of ascites fluid from a leukemicdonor was prepared, and 0.1 ml was injected intraperitoneally.Twenty-four animals were injected in each group and were sacrificed on the fifth and sixth day after inoculation.

The local tumor developed in the skeletal muscle at the site ofinoculation of the solid form, and areas of infiltration of theintestinal wall and the diaphragm in the mice injected with theascites cells were rapidly removed. All tissues were fixed in 3%glutaraldehyde in cacodylate buffer (24) for 2 hours at approximately 4Â°Cand washed and stored overnight in the same buffer.

For electron microscopy, the tissues were dehydrated in alcohols,embedded in Epon 812, and ultrathin sections were cut with theLKB Ultrotome or with the Porter-Blum microtome. The procedures for the histochemical preparations, both at the light andelectron microscopy levels, have been described in detail in aprevious paper (9). The grids were counterstained with uranylacetate and were observed and photographed with the RCAEMU-3F electron microscope.

RESULTS

The Fine Structure and Histochemislry of LeukemicCells

The L1210 leukemic cells were characterized by the presence oflarge nuclei occupying a great portion of the cell (Fig. 1). Abundant free ribosomes were found in the cytoplasm, but very fewcisternae of the rough endoplasmic reticulum could be seen inthese cells. Prominent Golgi complexes and virus iÂ¡articleswereseen in most cells. The ]Â¡articleswere located exclusively in thecytoplasm, either in the matrix or within cisternae of the endoplasmic reticulum. On the basis of their morphologic appearanceand outer diameters, the viruses have been designated as Aparticles in accordance with the proposed classification of Dalton

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David Brandes, Elsa Anton, and Brian Schofield

(12).3 A more detailed description of the L1210 leukemie cells

and of the virus particles characteristic of this neoplasia are to befound in two recent publications (7, 8).

In light microscope preparations stained by the acid phos-phatase technic for the demonstration of lysosomes (Fig. 2), theleukemie cells appeared predominantly negative, but a strongpositive reaction was observed in macrophages. The electronhistochemical preparations (Pig. 3) confirmed the paucity oflysosomes in leukemie cells, which usually contained but a fewof those particles. In the preparations thus far examined, neitherlysosomes nor acid phosphatase activity could be seen in thehost-tumor interzone.

The Host-Tu mor Interzone

Light Microscopy. The degree of infiltration and destructionof skeletal muscle and smooth muscle on the fifth day after transplantation of the leukemia are illustrated in light micrographs(Figs. 4, 5). In both cases, the neoplastic cells have infiltratedand widened the spaces between the muscle fibers, many of whichappear as isolated fragments.

Electron Microscopy. Skektal Muscle Invasion. Before theonset of recognizable changes, the plasma membrane of theleukemie cells, as well as the sarcolemma of the muscle fibers,appeared as continuous dense structures (Fig. 6). The sarcolemmawas externally coated by a basement membrane, and a moderateamount of connective tissue fibers were seen between the leukemiecells and the muscle. Aggregates of ribosomes, apparently derived from leukemie cells, were frequently detected in the interstitial spaces t>etween leukemie cells and muscle, before anymorphologic alterations occurred in either cell type (Fig. 7).

Progressive alterations in leukemie cells appeared to precedethe changes in muscle fibers. Partial or almost complete disappearance of the plasma membrane occurred in many of the leukemie cells (Fig. 8), and their cytoplasmic content, especiallyribosomes, appeared free in the interstitial spaces in close apposition to the surface of the muscle fibers. Several degenerativechanges including pycnosis and frank necrobiosis (Fig. 9), wereobserved in leukemie cells in the vicinity of morphologically intact muscle fibers, but virus particles could still be detectedamong the debris of the neoplastic cells (Fig. 9, inset).

Several changes were observed at the surface of the musclefibers in areas where the plasma membrane of the leukemie cellsbecame discontinuous or disappeared, or in the presence of debrisfrom necrobiotic leukemie cells. Increased micropinocytoticactivity and the development of areas of hyperplastic marginalsarcoplasm were among the earlier detectable changes in musclefibers which otherwise appeared normal.

The micropinocytotic vesicles appeared arranged in rows alongthe muscle surface and contained structureless material of low-

electron density (Figs. 8, 10, 11). The contents of the areas ofhyperplastic marginal sarcoplasm varied in the different portions

3 It has been proposed, more recently, that the particles in thecytoplasmic matrix be designated as Â¡ntracytoplasmic A particles,ami those in the eiuloplasmic reticulum as intracisternal A particles. Suggestions for the Classification of Oncogenic RNA Viruses.Informal Meeting, New York, February 9, 19fi(i (J. Nati. CancerInst,, 37: 395-397, 1966).

of the invaded muscle. In some instances they contained myelinbodies which seemed to be derived from degenerating cell or-ganelles such as mitochondria and ribosomes (Figs. 10, 11). Thehyperplastic areas frequently appeared as protrusions whichcontained predominantly abundant ribosomes (Fig. 11) or hyper-trophic mitochondria and hyperplastic Golgi elements (Figs. 12,13). Degenerative changes were detected in many of the marginalhypertrophie mitochondria (Fig. 13). The membranes of thecristae lost their sharpness, and in many instances the mitochondria were occupied by interlacing bands of amorphous material. Structures which seemed to represent residues of degenerating mitochondria also appeared in these areas.

Progressive alterations in skeletal muscle during leukemie cellinvasion, which appeared directly related to their breakage andreplacement of muscle fibers by tumor cells, were observed.

The sarcolemma disappeared, and the muscle showed a frayedirregular surface (Fig. 14). In more advanced stages the surfaceshowed a frank moth-eaten appearance and the myofibrils hadbegun to disintegrate (Fig. 15). The loss of muscle substance gaverise to the formation of lacunar spaces, and as this process advanced deeper into the muscle, the fibers became thinner andirregular (Fig. 16) and showed areas of constriction that appearedto lead to actual fragmentation and disintegration (Figs. 17, 18).

Intestinal Smooth Muscle Invasion. As in the case of skeletalmuscle invasion, some of the smooth muscle fibers showed signsof stimulation reflected in enhanced pinocytosis, mitochondrialaggregates, and hyperplastic Golgi complexes (Fig. 19). Abundantclusters of free ribosomes in the interstitial spaces were observedin the host-tumor interzone, and very frequently the hyperplasiaof cell organelles occurred in the vicinity of the areas with extracellular ribosomes (Fig. 20).

Degenerative changes in the leukemie cells were also seen toprecede alteration in the smooth muscle fibers. In the neoplasticcells (Fig. 21), the perinuclear cisternae appeared very dilated,the mitochondria were swollen, and most of the cristae had disappeared. The plasma membrane was no longer visible, and thevarious cytoplasmic components of the leukemie cells, especiallythe ribosomes appeared free in the interstitial spaces. In someareas, the plasma membrane of the smooth muscle cells appearedinterrupted, and the fusion of host-tumor cell cytoplasm becameestablished (Fig. 21).

The areas of tumor cell-muscle fusion seemed to constitute a"no man's land" with cell organelles "intruding" into both sides

of the line, for mitochondria and myofibrils were seen to lodgesimultaneously in muscle and leukemie cells (Fig. 22). Pinocytoticvacuoles, externally coated or containing ribosomes apparentlyderived from those present in the interstitial spaces, were observed in muscle cells, and in areas of fusion, tumor cell ribosomes appeared to gain access into muscle fibers (Fig. 23). Initiallytic changes were observed in such areas (Fig. 23), and patternsof disintegration of the normal structures in contact with alteredleukemie cells were similar to those described in relation to theinvasion and replacement of the skeletal muscle by malignantcells.

The apparent stimulative effect of tumor cell material on normal structures appeared to be exerted also on small blood vesselspresent in the areas of tumor cell invasion. In areas containingtumor cell debris, the cytoplasm of vascular endothelial cellsappeared broadened and contained abundant cell organelles,

2160 CANCER RESEARCH VOL. 27



Invasion of Muscle by LI210 Leukemia

particularly ribosomes (Fig. 24). The luminal surface was throwninto complex projections and folding's, indicative of intense

pinocytosis, and hyperplasia of the Golgi apparatus and of therough endoplasmic reticulum was also prevalent (Fig. 25).

DISCUSSION

The present results have indicated that during invasion ofskeletal and smooth muscle, alteration of the leukemic cells,including necrobiosis, occurs before the onset of degenerativechanges in the normal tissues.

Partial or complete disappearance of the plasma membranesof the neoplastic cells exposed the surface of the muscle fibersdirectly to the cytoplasmic components of the leukemic elementand particularly to the ribosome aggregates.

In such areas, increased pinocytotic activity was detected atthe surface of the muscle fibers, which may indicate the uptakeof material derived from the altered leukemic cells. More directevidence pointing to interchange of material between normal andneoplastia elements was derived from the presence of numerousareas of fusion between both cell types, in which cell organellesappeared to protrude from one cell type into the other.

The presence, in the interstitial spaces, of material derivedfrom altered leukemic cells appeared at first to exert a stimulative effect on the muscle fibers and also on the small bloodvessels present in the areas of tumor cell invasion. In the skeletalmuscle fibers, the apparent stimulative effect was reflected in thedevelopment of areas of marginal sarcoplasmic hyperplasia,increase in the number and size of the mitochondria, and hypertrophy of the Golgi elements. Aggregates of mitochondria andhypertrophy of the Golgi components were also observed in thesmooth muscle fibers, and in some instances, both types of hyper-plastic organelles appeared in close association. The stimulativeeffect on blood vessels occurred in areas where the interstitialspaces were occupied by cytoplasmic products derived fromneoplastic cells. The cytoplasm of the capillary endothelial cellsappeared broadened and studded with ribosomes, the endoplas-mic reticulum and Golgi appeared unusually prominent, and theluminal surface was thrown into elaborate infoldings and finger-like projections which seemed to be actively engaged in theformation of pinocytotic vacuoles.

A stimulatory effect of tumor cells or their products on normaltissues, including blood vessels and stroma, has been reported innumerous instances, both in vivo (4, 14, 17, 23, 30, 32), and invitro (17-19, 23). In such instances, however, tumor cells or

their extracts stimulated the proliferation of normal cells, but inour case only hyperplasia of cell organelles was encountered,which may have been due to the nondividing nature of thetissues examined in this study.

What the biologic changes are that occur at the host-tumorinterzone that lead from a condition of stimulation to one ofdegradation and lysis of normal structures are not known withcertainty, but have attracted much interest and have been thesubject of extensive experimental studies (see Ref. 11 for reviews).

In our light microscopic preparations, the areas of muscle cellinvasion were packed with tumor cells, and it appeared thatcompression alone, as suggested in classic textbooks of pathology, may have been a cause in the atrophy and breaking downof the normal structures.

At the level of resolution of the electron microscope, however,the gap between tumor cells and skeletal muscle was considerablylarger, and the alterations of the normal structures appeared tooccur in areas where tumor cells were breaking down, ratherthan in relation with vigorously multiplying leukemic cells.

Noi (21) failed to detect morphologic or histochemical changesin kidney cells situated in front of or even in direct contact withgrowing tumor cells, and he questioned the idea that extracellularproteolysis or other destructive processes in normal tissues mightprecede tumor invasion.

Those studies, however, were based on light microscopy observations, and it is possible that the author failed to detect thepresence of tumor cell debris in areas of invasion and the initialsubcellular changes described in this study.

Alteration and lysis of skeletal and smooth muscle fibers werealso detected in areas not immediately in contact with growingtumor cells, suggesting that substances extruded from alteredneoplastic cells may have acted via the interstitial fluid. Lyticchanges have been observed at distances greater than 1(X)ÃŸfrom tumor cells, and on the basis of histochemical and biochemical evidence, this process has been referred to as "extracellularproteolysis" (25, 26), indicative of local abnormal characteristics

of the interstitial milieu prevailing in and around tumors (27).Increased proteina.se activity at the host-tumor interzone, and

possible activation of these enzymes through imbalance of regulatory mechanisms, have been related to the destructive capacityof neoplastic cells (27, 29). The possibility that cathepsin andother lysosomal enzymes may be involved in tissue breakdownin areas of tumor invasion has been discussed by Pearse andHess (22), but these authors failed to detect high acid phospha-tase activity in tumor cells, except in stromal macrophages.Similar findings are reported here, where our preparations at thelight and electron microscopic levels showed very few lysosomesin the leukemic cells and no acid phosphatase activity in areasof muscle tissue breakdown.

Structural defects in the organization of tumor-cell plasmamembrane and increased permeability of this structure (1-3, 5,27, 28), have been held res]x>nsible for the release of cytoplasmicmaterial including enzymes, but these studies tend to indicate amore severe process, that is, the actual disappearance of the cellmembrane in the more peripheral tumor cells (type A cells ofSylvÃ©nand Malmgren) (29).

It is possible that some of the cytoplasmic constituents of theleukemic cells, penetrating the normal structures in areas offusion or by pinocytosis may have been responsible for the ensuing degenerative changes and final lysis of the muscle fibers.The marginal sarcoplasm of striated fibers as well as in some ofthe smooth muscle fibers contained more ribosomes than areusually found in normal conditions. Many of our electron micrographs seemed to indicate that some of the cell ribosomes werepresent in the interstitial spaces, and that incorporation into themuscle fibers may have occurred by pinocytosis or at the areasof fusion of malignant and normal cells.

The material extruded from tumor cells which had lost theirplasma membrane may have served other purposes than to actas a toxic agent involved in the lysis of normal elements in thecourse of invasion. As suggested by other authors (16, 31), thematerial released from the tumor cells may have contained aconditioning or growth-stimulating factor required for the suc-

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cessful establishment of the isologous tumor graft, or may haveserved as a source of nutritional material for other tumor cells.It may therefore be postulated that cell products, including enzymes and cytoplasmic organelles, liberated by the tumor cellsmay have fulfilled several functions such as stimulation of normalelements, conditioning the establishment of the tumor graft andserved as a source of nutrition for other neoplastic cells. Such afeedback-like mechanism as stimulation of one Å“il type byproducts derived from the breaking up of other cell types hasbeen considered as an extremely economic intercellular controlmechanism (10). A lytic action on the invaded structures byactivated enzymes or through the toxic effect of tumor cellcomponents would then constitute one facet of the many properties attributed to the breakdown products derived from tumorcells.

Some of the alterations observed in skeletal and smooth musclefibers in the areas of tumor cell infiltration greatly resembledchanges described in various muscle diseases.

Mitochondrial aggregates, such as those seen in both types offibers, have been described in patients with myotonia congenita,paramyotonia congenita, hypokalemic periodic paralysis, andadynamia episodica hereditaria (13). Hypertrophy and increasein mitochondrial population in perinuclear and subsareolemmicregions such as those seen in this study have also been observedin rat skeletal muscle after thyroidectomy and thyroid hormonestimulation (15). Areas of marginal sarcoplasmic hyperplasiacontaining abundant ribosomes such as seen in this study havealso been described in striated muscle in the case of trichinosis(20).

In our studies, these changes appeared to be localized exclusively in the areas of tumor cell infiltration, thus indicating alocal reaction to the invading neoplastie celLs rather than asystemic disease as in the case of most of the above conditions.

ACKN OWLEDGMKNTS

We wish to thank M. D. Tuduhl, Sr., for assistance in thephotography.

REFERENCES

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10. Klein, G., and Klein, E. The Evolution of Independence FromSpecific Growth Stimulation and Inhibition in MammalianTumor Cell Populations. Symp. Soc. Exptl. Biol., //: 305-328,1957.

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22. Pearse, A. G. E., Hess, R., and Snyder, R. Stromal and TumorCathepsins and Their Relationships to Metabolic Activityand Invasiveness. In: M. J. Brennan and W. L. Simpson (eds.),Biological Interactions in Normal and Neoplastic Growth;Henry Ford Hospital International Symposium, pp. 057-670.Boston- Little Brown and Co., 1962.

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Invasion of Muscle by L1210 Leukemia

Vasiliev, J. M. The Role of Connective Tissue Proliferation inInvasive Growth of Normal and Malignant Tissues : A Review.Brit. J. Cancer, 18: 524-536, 1958.Vasiliev, J. M. The Local Stimulatory Effect of Normal TissuesUpon the Growth of Tumor Cells. In: M. J. Brennan and W. L.Simpson (eds.), Biological Interactions in Normal and Neoplastic Growth; Henry Ford Hospital International Symposium, pp. 299-309. Boston: Little Brown and Co., 19(i2.Vasiliev, J. M., and Guelstein, V. I. Local Cell Interactions inNeoplasms and in the Foci of Carcinogenesis. Progr. Exptl.Tumor Res., 8: 26-65, 1966.Willis, R. A. The Direct Spread of Tumors. In: Pathology ofTumors, Ed. 2, pp. 147-165. London: Butter-worth and Co.(Publishers Ltd.), 1953.

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David Brandes, Elsa Anton, and Brian Schoficld

FIG. 1. Normal appearance of L1210 leukemic cells. The nuclei (.V) occupy a large portion of the cell. The cytoplasm is studdedwith ribosomes and shows virus particles (V), some of which are of the intracisternal type (arrows). The Golgi complex ((?) is welldeveloped, hut the rough-surfaced endoplasmic reticulum (REIt) is scarce, a, X 10,500; 6, X 20,500; inset, X 30,000.

FIG. 2. Light micrograph. A positive reaction is seen in a few macrophages (arrows). The muscle fibers (asterisks) and the invadingleukemic cells show no apparent activity. Acid phosphatase stain, X 350.

FIG. 3. Electron micrograph. The leukemic cells (LK) contain few small lysosomes (Ly) which were not resolved in the light micrographs. Neither the muscle fiber (SK) nor the host-tumor (HT) interzone show any apparent activity. A', nuclei. Acid phosphatase

stain, X 11,500.FIGS. 4 AND5. One-micron sections from blocks embedded in Kpon from which ultrathin sections were then cut forelectron micros

copy. Toluidine blue stain.FIG. 4. Invasion of smooth muscle of intestinal wall. Fragmented muscle fibers (arrows) are surrounded by leukemic cells. X 350.FIG. 5. Invasion of skeletal muscle. Some fragmented fibers (arrows) are surrounded by leukemic cells, but others (asterisks) are seen

in areas showing no leukemic infiltration. X 350.FIG. 6. Portion of a leukemic cell (LA') infiltrating a space between skeletal muscle fibers (SK). The plasma membrane (Pni) is still

intact and so is the sarcolemma (SI). The latter is externally coated by a thin layer of amorphous material with the appearance of abasement membrane (Urn). The myofibrils (My) appear normal. N, nucleus; V, virus particles; Li, lipid droplets; Ct, connective tissuefibers. X 20,000.

FIG. 7. Host-tumor interzone showing clusters of ribosomes (arrows) in the interstitial space. The plasma membrane of the leukemiccell (LK) and the sarcolemma (SI) are still intact. JV, nucleus of leukemic cell; .V, nucleus of skeletal muscle fiber. X 10,500.

FIG. 8. Degenerating leukemic cell (/>K) in the midst of skeletal muscle fibers (SK). The nucleus (X) is undergoing pycnosis. Theplasma membrane is no longer visible, and the cytoplasm is in direct continuity with the interstitial space (arrows). The sarcolemma.(Si) is intact and shows numerous pinocytotic vesicles. Virus particles are still present (V) in the tumor cell cytoplasm. X 21,500.

FIG. 9. Two necrobiotic leukemic cells (LK\, AA'2). Cytoplasmic debris have spread through the interstitial space (asterisk). An

adjacent muscle fiber (SK) appears normal and the sarcolemma (SI) is intact. Virus particles (V) are still seen in the necrobiotic leukemic cell. They are shown at higher magnification in the iipper right inset. Part of a macrophage (MP) is also visible. X 9,500.Inset, X 18,500.

FIG. 10. Cytoplasmic materials from disintegrating leukemic cells are in continuity with the interstitial space. The nuclei (N) areundergoing pycnosis. The skeletal muscle fiber (SK) contains numerous myelin bodies (My). X 18,500.

FIG. 11. A skeletal muscle fiber (SK) in contact with leukemic cell debris (asterisk) shows an area of hyperplastic marginal sarco-plasm (star) containing abundant ribosomes and myelin bodies. X 20,000.

FIG. 12. Skeletal muscle fiber (SK) in an area of invasion. Hyperplastic marginal sarcoplasm containing an accumulation of hyper-trophic mitochondria (M), abundant ribosomes, and a prominent Golgi complex (G). X 23,500.

FIG. 13. Area similar to that shown in Fig. 12 shows degenerative changes in mitochondria (arrows). A prominent Golgi complex (G)is visible. SK, skeletal muscle fiber. X 21,500.

FIG. 14. Initial steps in the process of skeletal muscle disintegration. The sarcolemma is no longer visible and the surface of themuscle fibers show a frayed appearance (arrow). A degenerating leukemic cell (LK) devoid of plasma membranes is present in this area.X 11,500.

FIG. 15. The process of skeletal muscle degeneration is more advanced. The sarcolemma has disappeared, and the frayed appearanceof the surface is accentuated (arrows). The myofibrils start to disintegrate (asterisk). Abundant debris of tumor cell is seen in the interstitial space. X 23,500.

FIG. 16. Skeletal muscle fibers (SKi) in the process of fragmentation. The myofibrils are interrupted in an area of constriction (arrows). A second fiber (SKt) shows incipient fraying of the surface. No neoplastic cells are seen in this area, but the interstitial spacecontains cell debris. N, nucleus of muscle cell. X 14,500.

FIG. 17. Isolated skeletal muscle fragments (SK) in an area which contains no tumor cells. Cellular debris are present in the interstitial spaces. N, nucleus of muscle cell. X 10,000.

FIG. 18. Diaphragmatic muscle in an area of ascites tumor cell invasion. The larger fragment (.4) contains mitochondria] aggregatesand shows striai ed fibrils (SF). Pinocytotic and finger-like projections at the cell surface are abundant. Small portions of muscle remainattached to larger fragments by narrow Cytoplasmic bridges (arrows). No tumor cells are present in this area. X 10,000.

FIG. 19. Smooth muscle (SM) from intestinal wall, in the neighborhood of an area of leukemic infiltration. Aggregates of mitochondria(M), hypertrophie Golgi elements (G), and increased pinocytotic activity are observed in many muscle cells. X 20,000.

FIG. 20. Smooth muscle fibers (SM) surrounded by leukemic cells (LK). Abundant rosettes of ribosomes are free in the interstitialspaces (asterisk) between both cell types. Mitochondria, some with altered cristae, Golgi elements (G), myelin bodies (arrows), and ribosomes are concentrated near such areas. X 21,500.

FIG. 21. Altered leukemic cell (AA'i) with marked dilation of the perinuclear cisterna (Pc) and swollen mitochondria (M). The plasma

membrane has almost completely disappeared, and areas of fusion of tumor cell and muscle cells are frequent (asterisk). Mitochondrialaggregates are seen in such areas. The plasma membrane (Pm) of another leukemic cell (LA%) is intact. X 30,500.

FIG. 22. Extensive fusion of altered leukemic cell (LK) and smooth muscle (SM). Morphologic alterations in leukemic cell mitochondria (asterisk) are more obvious. Tumor cell ribosomes are in contact or within muscle cytoplasm which also shows vacuoles (Va) containing ribosomes. Initial lytic changes are seen in the muscle fiber at the area of the fusion with the leukemic cells (arrow). X 21,500.

FIG. 23. a, a mitochondrion (M) is partially within a leukemic cell (LK) and partially within a smooth muscle filier (SM). b, Arrowshows where sarcolemma (SI) becomes interrupted. Muscle fibrils (star) have also penetrated the leukemic cell cytoplasm, a, X 10,500;b, X 18,500.

FIG. 24. Capillary in area containing tumor cell debris. The endothelium is hypertrophie and shows enhanced pinocytotic activity(arrous). X 11,500.

FIG. 25. Hyperplasia of Golgi elements (G) and of rough endoplasmic reticulum in capillary endolhelium in the area of tumor cellinvasion. X 18,500.




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1967;27:2159-2178. Cancer Res David Brandes, Elsa Anton and Brian Schofield Invasion of Skeletal and Smooth Muscle by L1210 Leukemia

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