JOURNAL

OF SURGICAL

RESEARCH

18, 137-161 (1975)

Electron

Microscopic

on Formation WILLIAM

Observations

of Pulmonary

Metastases

F. SINDELAR, M.D., PH.D., TOMMIE SUE TRALKA, AND ALFRED S. KETCHAM, M.D. Surgery Branch and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Received November 8. 1974

The development of pulmonary metastases after local surgical control of malignant tumors has represented a major cause of treatment failure in a variety of neoplasms. The metastatic process has been a subject of considerable interest, investigation, and speculation for many years, but it remains as a poorly characterized and little understood phenomenon. The formation of blood-borne metastases is generally considered to involve several steps, including: (1) growth of a primary tumor with eventual shedding of viable malignant cells into the blood, either directly through vascular invasion or indirectly by lymphatic drainage; (2) transport of tumor cell emboli through the circulation to distant organs; (3) arrest of blood-borne malignant cells in distant vascular beds; (4) penetration of vascular walls by embolized tumor cells; and (5) growth of metastasized malignant cells in the parenchyma of target organs. Histologic investigations of the metastatic process were undertaken early [ 181.Various initial studies of experimental metastases in animal neoplasms indicated that once malignant cells shed from a primary tumor reach the venous circulation, they are carried to the capillary bed of target organs where the cells become arrested prior to invasion of the target organ parenchyma [ 11, 24,251. The initial attachment of circulating tumor cells to the capillary endothelium of distant organs has been regarded as a crucial

step in metastasis [6, 131.Numerous investigations have suggested that circulating tumor cells embolize to target organs, where they arrest in vascular channels and attach to the endothelium prior to invading the surrounding tissue parenchyma [4, 5, 26, 271. Various workers have proposed that the attachment of embolized malignant cells to the vascular walls is largely dependent upon entrapment of the tumor embolus within a thrombus or fibrin meshwork [2, 8, 17, 28, 291. Such a thrombus might serve to stabilize the tumor emboli within the vesselsuntil the cells are able to penetrate the endothehum and establish metastatic foci within the perivascular connective tissue. Despite active research efforts, there have been relatively small amounts of data concerning the mechanisms involved in the arrest of circulating tumor cells and their method of growth in metastatic sites [9, 16, 301. Recently, electron microscopic investigations have attempted to characterize events in metastasis formation through examination of embolic tumor cells arrested within vascular beds [12, 141.Jones and coworkers [12] investigated the sequence of events in experimental pulmonary metastases of the Walker 256 carcinosarcoma in rats, utilizing both electron microscopy and fibrin-specific immunofluorescence. These investigators demonstrated that after intravenous injection, tumor cells were arrested in pulmonary capillaries and were surrounded by a thrombus formed from ag-

137 Copyright SI 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

138

JOURNAL

OF SURGICAL

RESEARCH

gregated platelets and unpolymerized fibrin. The thrombus dispersed within 8 hr, and the arrested tumor cells were observed to attach to the capillary endothelium. By 48 hr after cell injection, all tumor cells were found in perivascular locations where they gave rise to metastatic foci by cell division. Tumor cells were thought to cross the vascular wall in the regions in which they were arrested, although the cellular events in breaching the endothelial membrane were not precisely characterized. The present study represents an attempt to describe by electron microscopy the cellular behavior during early pulmonary metastasis formation in a murine fibrosarcoma. Specific areas of descriptive interest concentrated upon the initial tumor cell attachment within the pulmonary capillary, the role of platelets and fibrin in tumor cell arrest, the method of vascular penetration by the arrested tumor cells, and the characteristics of tumor growth at metastatic sites. MATERIALS AND METHODS Tumor Preparation A methylcholanthrene-induced murine fibrosarcoma, coded M19, was used for all experiments. The tumor was induced in 1971 and was maintained in the laboratory by serial passage to the thigh musculature of mature C57BL/6J mice. Transplant generation 22 was used in the present study. Suspensions of individual tumor cells were prepared by a modification of the tissue dissociation method of Madden and Burk [15]. Tumors growing in the thigh muscles were excised from donor mice, removed of necrotic debris, and finely minced in Earle’s balanced salt solution. Tumor fragments were then treated for 15 min at 37°C with 10 vol of Earle’s salt solution containing 0.25% trypsin. The tissue was washed and resuspended in 10 vol of Earle’s solution containing 0.25% trypsin and 0.01% deoxyribonuclease. Addition of the DNase was necessary to prevent the precipitation of large flocculates of nucleic acid released by

VOL.

18, NO. 2, FEBRUARY

1975

cells damaged during the dissociation procedure. The tissue was agitated for 1 hi at 24°C on a gyratory shaker (1 cm diameter of rotation, 80 rpm) to break up the tissue fragments. The tissue was strained through a loo-mesh sieve to yield the dissociated cell suspension. Cells were washed three times with Hanks’ balanced salt solution and adjusted to a final concentration of 1.0 x 10’ cells/ml. Microscopic examination confirmed dissociation of the tissue into uniform single cell suspensions, with only rare cellular clumps or debris. Cell viability as determined by exclusion of 0.05R trypan blue was consistently in excess of 95%. Tumor Inoculation In all experiments, adult male C57BL/6J mice were used as tumor host recipients. l.C x lo6 viable dissociated Ml9 fibrosarcoma cells in 0.1 ml Hanks’ solution were injected intravenously through the lateral tail vein. Host animals were sacrificed for study at intervals varying from 15 min to 14 days after cell injection. Mice were anesthetized with ketamine (0.5 mg/g body wt), and the lungs were removcc while the pulmonary circulation was still intact. Preliminary experiments showed the development of significant alterations of ul trastructural detail in both arrested tumor emboli and in pulmonary vascular endothehum within a few minutes after any interrup tion of blood circulation to the lungs. In each experiment, groups of five mice were sacrificed at the following intervalr after tumor inoculation: 15 and 30 min; 1, 3. 6, 9, 12, and 18 hr; 1, 2, 3, 5, 7, 10, and 14 days. Each experiment was performed thra times. Lung tissue removed from each ex. perimental animal was prepared for botl light and electron microscopic examination In each experiment, 10 mice were inoculated with tumor cell suspension and served ac controls. These control animals were autop sied 3 wk after inoculation, and the numberr of pulmonary metastaseswere recorded.

SINDELAR,

TRALKA,

AND KETCHAM:

Light Microscopy

Portions of lung tissue from experimental animals were fixed for 24 hr in neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin for routine light microscopy. Sections were examined for tumor cell emboli. Electron Microscopy

Fragments of lung taken from mice inoculated with Ml9 fibrosarcoma were immediately fixed for 1 hr in 2% gluteraldehyde in Sorensen’s buffer (pH 7.4). Tissue was then postfixed for 1 hr in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon 812. All tissue blocks were step-sectioned at various levels. Thick sections (1 pm) were stained with 2% azure B and saturated basic fuchsin and were examined by light microscopy to localize areas with tumor cells. Tissue blocks containing tumor cells were

ULTRASTRUCTURE

OF METASTASES

139

prepared for electron microscopic examination. Ultrathin sections were cut on an LKB Ultrotome III microtome. Approximately 20 serial thin sections were prepared of each area suspected to contain tumor cell emboli. Sections were mounted on uncoated copper grids and were stained with 10% methanolic uranyl acetate [20] and 0.1% lead citrate [23]. Prepared sections were examined with a Siemens Elmiskop 1A electron microscope. RESULTS Ultrastructure

of the Ml9 Fibrosarcoma

Portions of M 19 fibrosarcoma growing in the thigh musculature were excised from tumor-bearing mice and prepared for microscopy. Routine light microscopic examination showed the tumor to be highly cellular, with considerable pleomorphism and nuclear hyperchromicity (Fig. 1). The

FIG. 1. Ml9 fibrosarcoma growing intramuscularly in C57BL/6J mouse. The stroma is highly cellular. Cells are pleomorphic and have hyperchromatic nuclei. Scale 100 pm. Magnification 250x.

140

JOURNAL

OF SURGICAL

RESEARCH

VOL.

18, NO. 2, FEBRUARY

1975

which is surFIG. 2. Ml9 librosarcoma cell in fragment of tumor tissue fixed in situ. Cell has a large nucleus(N) rounded by a prominent nuclear membrane (NM) and which contains a single nucleolus (Nu). Cytoplasm is relatively scant, and the cell is limited by a cell membrane (CM) devoid of specialized intercellular junctions. Mitochondria (M) are present in perinuclear locations, and membranous degeneration within mitochondrial cristae is frequently seen. Vesicles of smooth endoplasmic reticulum (SER) are present in small amounts, and scattered short strands of rough endoplasmic reticulum (RER) are seen. Numerous ribosomes (R) are found throughout the cytoplasm. Some bands of extracellular collagen (Co) are seen. Scale I pm. Magnification 15,000~.

tumor showed typical sarcomatous stroma. Tumors were not highly vascularized and contained only small amounts of collagen matrix. Electron microscopic examination of M 19 cells revealed distinguishing ultrastructural features (Figs. 2-4). Nuclei were large and often lobulated. The nuclear ground

substance was finely dispersed; however, after preparation of individual cell suspensions, dissociated tumor cells frequently showed chromatin aggregation at the nuclear periphery. Nucleoli displayed a targetlike appearance, with a prominent pars amorpha surrounded by a densely aggregated nucleolonema. Nuclear pores were

~---^_---

_--~ .--,.-... ,., *,-_-_--___ .-.-~~ -i___--

., ,.,

.,

,, ” ,, “,

,.

,, ,.”

,,. ., ,I,

,,,

,, “, “,”

FIG. 3. Ml9 fibrosarcorra cell. Nucleus(N) is multilobulated, and nucleoli (Nu) show the typical target-like appearance with.a prominent pars amorpha surrounded by a dense nucleolonema. The nuclear ground substance is finely dispersed, with no chromatin aggregation. The cytoplasm is largely devoid of specialized organelles. Mitochondria frequently show membranous degeneration forming myelin figures (MyF). Scale I pm. Magnification 15,000~.

,.

142

SINDELAR,

TRALKA,

AND KETCHAM:

prominent in many cells. Perinuclear cytoplasmic fibrils were present. Cytoplasm of Ml9 cells was relatively scant and devoid of complex organelles. Golgi complexes were present but inactive. The endoplasmic reticulum was not extensively developed, although cells showed scattered strands of rough reticulum and occasional smooth reticulum vesicles in perinuclear locations. Free ribosomes were found throughout the cytoplasm, and polysomes were seen with regularity. Mitochondria were present in moderate numbers, usually clustered on opposite sides of the nucleus in polarized fashion. Mitochondria showed a typical double-membrane configuration with large cristae. Membranous degeneration was frequently observed within mitochondria, and intramitochondrial myelin figures were commonly seen. Adjacent tumor cells were separated by

ULTRASTRUCTURE

OF METASTASES

143

wide (over 1000 A) intercellular spacesover much of their surface areas, although scattered points of cellular contacts with typical 300 A spacing of adjacent plasma membranes were present. The points of close contact were considered to represent areas of intercellular attachment. No specialized membrane structures of cell adhesion were seen;no desmosomesor tight junctions were present. After cell dissociation, the individual cells displayed extremely active ruffled surfaces, with extensive pseudopodial processes present over virtually the entire free surface membranes of the cells (Fig. 4). Control Experiments Thirty control mice were intravenously inoculated with 1.0 x lo6 viable trypsindissociated tumor cells and were observed for 3 wk. The animals were sacrificed, and pulmonary metastases were counted. All

FIG. 5. Multiple intravascular thrombi in mouse lung 1 hr after intravenous injection of Ml9 fibrosarcoma cells. Thrombi (arrows) are found in many pulmonary capillaries and arterioles within 15 min of tumor cell inoculation and persist as long as 18 hr. The thrombi were most frequent at 1-3 hr. Scale 100 pm. Magnification 100x.

144

JOURNAL OF SURGICAL

RESEARCH VOL. 18, NO. 2, FEBRUARY

1975

FIG. 6. Platelet thrombus in pulmonary arteriole 1 hr after injection of Ml9 fibrosarcoma cells. Vessel is completely occluded with a denseplatelet thrombus (arrows). Scale 50 pm. Magnification 500x

animals developed multiple metastases, with an average value of 17.4 f 1.47 metastases (mean f standard error) and a range 9-56. In order to compare the metastatic potential of trypsin-dissociated cells to nonenzymatically disrupted tumor tissue, 30 mice were inoculated intravenously with 1.0 x IO6 viable cells prepared by mechanical dissociation of Ml9 tumor fragments [19]. Pulmonary metastases were counted at autopsy after 3 wk: all animals developed metastases, with average value 19.0 f 1.68 and range 10-62. No statistical difference in the number of lung metastases was present between animals injected with trypsinized or with nontrypsinized tumor cells. This result was interpreted as indicating that the enzymatic method of preparation of dissociated cell suspensions did not significantly alter the metastatic potential of the M 19 fibrosarcoma.

Light Microscopic Observations Specimens of mouse lung were removed and processed for routine light microscopy at various intervals after intravenous injection of Ml9 tumor cells. A prominent feature which appeared within 15 min of cell injection was the presence of thrombi in several of the pulmonary capillaries and arterioles (Fig. 5). Thrombi were most numerous l-3 hr after cell injection and were thereafter seen with decreasing frequency, although some persisted as long as 18 hr. In paraffin sections the thrombi appeared to be formed of loosely packed erythrocytes in an eosinophilic granular matrix. Examination of Epon-embedded sections prepared for light microscopy gave greater cytologic detail and revealed that the thrombi were largely constituted by aggregated platelets. The thrombi frequently filled pulmonary vessels with compact platelet masses (Fig.

SINDELAR,

TRALKA,

AND KETCHAM:

6), and occasional entrapped intravascular tumor cells could be identified (Fig. 7). Examination of serial sections through thrombus-containing vessels often demonstrated arrested tumor cells in small pulmonary vessels with surrounding platelet aggregates that propagated proximally within the vessels.Thus, it appeared that the platelet thrombi were consistently associated with the initial arrest of tumor emboli. Examination of sections prepared 18-48 hr after tumor cell injection revealed the dissolution of the intravascular thrombi. Occasionally malignant cells could be detected attached to the walls of small pulmonary vessels. These cells were adherent to endothelial linings but were not surrounded by other cells or proteinaceous matrix. Nests of tumor cells were noted in perivascular positions, beginning by 48 hr after cell injection.

ULTRASTRUCTURE

OF METASTASES

145

Such nests were interpreted to arise from the penetration of vascular walls by arrested tumor cells and from the subsequent growth of tumor cells in the perivascular connective tissue. By 3-5 days after cell injection, microscopic metastatic tumor foci were detectable scattered through pulmonary parenchyma. Thereafter, the metastases were observed to enlarge, resulting in the formation of typical pulmonary metastatic nodules, composed of closely packed malignant cells of similar histologic appearance to the cells of the primary Ml9 fibrosarcoma (Fig. 8). Electron Microscopic

Observations

Arrest of circulating tumor cells. Circulating Ml9 fibrosarcoma cells were identified in pulmonary vesselswithin 15 min of cell injection. Approximately 50 pilot microscopic sections were scanned for each

FIG. 7. Platelet thrombus containing tumor cell emboli. Pulmonary arteriole is occluded by platelet thrombus which formed 1 hr after intravenous injection of fibrosarcoma cells. The thrombus contains three tumor cells (arrows) which have become entrapped in the mass of aggregated platelets. Scale 25 pm. Magnification 800x.

146

JOURNAL OF SURGICAL

RESEARCH VOL. 18, NO. 2, FEBRUARY

1975

FIG. 8. Ml9 fibrosarcoma metastasis in mouse lung 14 days after tumor cell inoculation. Section is taken through an early nodular fibrosarcoma metastasis at the periphery of the pulmonary parenchyma. The metastasis showsthe typical sarcomatous cellular pattern seenin the M 19primary tumor. Scale 100pm. Magnification 100x.

tumor cell encountered. The free-floating fibrosarcoma cells displayed extensive surface ruffles and pseudopodia. Free tumor cells were present within the pulmonary vasculature for as long as 3 hr after injection, but the majority of the circulating cells were seen during the first 30 min. Tumor cells appeared to attach to the vascular endothelium at the tips of the surface pseudopodia. The arrest of tumor cells in the pulmonary vessels was followed by massive platelet aggregation at the site of the tumor emboli. Such platelet aggregations formed dense masses which often completely filled pulmonary capillaries and which appeared to propagate proximally within the vessels from the initial point of tumor cell arrest (Fig. 9). In many instances, tumor cells could be identified adhering to the vascular endothelium by means of short surface

pseudopodia, with platelets aggregating around the adherent cells (Fig. 10). Fibrin could not be morphologically identified in association with the arrested tumor cells or with the platelet aggregates. Many circulating tumor cells appeared structurally damaged. Approximately 40% of the unarrested circulating fibrosarcoma cells showed ultrastructural signs of degeneration, including nuclear pyknosis and loss of peripheral cytoplasm (Fig. 11). Such damaged cells were observed during the first hour after cell injection and were seen only rarely thereafter. These degenerating cells did not arrest in the pulmonary capillaries. Such cells were thought to be nonviable. Certain tumor cells which did arrest in pulmonary vessels displayed signs of chromatin margination and possibly early pyknotic nuclear changes. However, no cytoplasmic damage was observed in such cells

FIG. 9. Platelet thrombus. Densely packed platelets (Pl) are present in pulmonary capillary 15 min after intravenous injection of tumor cells. Platelets are tightly aggregated but do not appear to be adherent to the capillary endothelium (En) and do not show any adhesive junctions between platelet outer membranes. Platelets exhibit no signs of activation or degeneration. The alveolar epithelium (Ep) and alveolar space (Alv) are seen. A fibrocyte (Fc) is present. Scale I pm. Magnification 15$00x.

_..

._ _ . -

.

II

-

FIG. 10. Tumor cell embolus in pulmonary capillary. Section shows tumor cells (TC) present in dense platelet (PI) thrombus 30 min after intravenous dissociated fibrosarcoma cell suspension. One of the tumor cells appears to be adherent to the capillary endothelium (En) along a free surface (arrows) slight ruffling of the cell membrane. Scale 1 pm. Magnification 18.000~.

injection at points

of of

FIG. 1I. Circulating tumor cell displaying nuclear degeneration and loss of cytoplasm. Many freely circulating tumor cells display signs of degeneration, including nuclear pyknosis and loss of peripheral cytoplasm, suggesting that only a portion of the circulating tumor cells remain viable and retain the potential for arrest and growth at metastatic sites. Cells with severe degenerative changes do not arrest in pulmonary vessels. Section shows a fibrosarcoma cell circulating free within the lumen of a pulmonary arteriole 30 min after tumor cell injection. Ceil shows moderate chromatin condensation and almost complete loss of cytoplasm. Scale 1 pm. Magnification 21,000~.

.

FIG. 12. Fibrosarcoma cell impacted in pulmonary capillary 1 hr after cell injection. Section shows malignant cell filling the lumen of a small capillary. The cell has a few fine surface membrane ruffles, and several points of adherence are present (arrows) between the tumor cell and the vessel wall. The cell shows chromatin condensation and nuclear features of early pyknosis. Tumor cells which’exhibit some signs of nuclear degeneration but’which do not show cytoplasmic loss occasionally imnart in nnlmnnarv vesek Thtihilitv andmrttastatir notential d ulr&rtiaUv deLtPeneratim.ceJAs ic nne&ianah)~ T& manillarv PmIntbpJi~lm CFn\ ad.ihr nlwnlnr

.

151

152

JOURNAL

OF SURGICAL

RESEARCH

and points of cytoplasmic adherence to the vascular endothelium were sometimes identified (Fig. 12). Such tumor cells with slight nuclear morphologic changes were considered to be questionably viable. Arrested tumor cells displaying no ultrastructural alterations were considered to have the greatest potential for initiating metastasis formation. The intravascular platelet thrombi underwent dissolution, beginning at about 3 hr and continuing until 18 hr after cell injection. The individual platelets did not degenerate, but the platelet clusters appeared to disaggregate. After dissolution of the platelet thrombus, tumor cells could be identified adhering to the endothelial linings of pulmonary capillaries and arterioles (Fig. 13). The adherent cells attached to the endothelium by means of short pseudopodia. These surface processes usually took the form of short (0.1-0.5 pm) rufflings of the plasma membrane, but the pseudopodia often extended into elongated filaments up to 5 pm in length (Fig. 14). Although mul-

VOL. 18, NO. 2, FEBRUARY

1975

tiple points of attachment occurred betwee the tumor cell and endothelial membrane along the surfaces of the longer pset dopodia, the shorter cell processestypical1 displayed only a single point of attacl ment at the tips of the pseudopodia. Adjz cent cell membranes at the points of al tachment were closely approximated, wit intermembranous distances typically les than 100A. Vascular penetration by arrested tumo cells. Between 1 and 3 days after injectior tumor cells which had become adherent tj pulmonary capillaries were observed pene trating the endothelium. Cells appeared tl flatten against the endothelial surfaces an initiate vascular penetration between neigh boring endothelial cells (Fig. 15). The tumo cells seemedto migrate through areas wher’ the attenuated processes of adjacent endo thelial cells joined. The characteristic junc tional complexes in these regions were fre quently separated or broken down. Althougl occasional intravascular platelets were associated with the migrating tumor cells

FIG. 14. Fibrosarcoma cell attaching to capillary wall 3 hr after injection. Tumor cell (TC) has adhered to the endothelium (En) of a small pulmonary capillary by means of pseudopodial (Ps) surface processes. The pseudopod appears to have multiple points of attachment (arrows). Most adhering tumor cells adhere to the capillary endothelium by short (0.5 pm) pseudopodia, but some attachments occur in elongated surface filaments which may exceed 5 pm in length. Scale 1 pm. Magnification 18,000 x.

FIG. 15. Tumor cell penetrating capillary wall. A large tumor endothelium (En) 24 hr after intravenous injection of dissociated ment to the endothelium (arrows). Penetration frequently occurs thrombus has disintegrated by the time breaching of the vascular period during which they remain adherent to and penetrate the great alveolar cell (GAC) with characteristic laminated osmiphilic

cell (TC) which was adhering to the wall of a pulmonary capillary (Cap) has begun penetration of the fibrosarcoma cell suspension. The tumor cell appears to be initiating penetration at points of attachat the junctional regions between adjacent endothelial cells. Although the characteristic platelet wall takes place, occasional platelets (PI) can be associated with arrested tumor cells throughout the endothelium. Section shows an intact alveolar epithelium (Ep). The alveolar lumen (Alv) is seen. A bodies is present. Scale 1 pm. Magnification 12,000~.

154

JOURNAL OF SURGICAL

RESEARCH VOL. 18, NO. 2, FEBRUARY

1975

FIG. 16. Fibrosarcoma cell free in alveolar space. Section taken 24 hr after cell injection shows tumor cell (TC) which apparently has penetrated through the wall of a neighboring vessel and which lies free in the alveolar space (Alv). The tumor cell has numerous surface processes and displays some chromatin condensation. The tumor cell contains na lysosomes, thereby distinguishing it from an alveolar macrophage. Another tumor cell (TC) is present and is adherent to the endothelium (En) of a pulmonary capillary at several points (arrows). The alveolar epithelium (Ep) is intact. Collagen (Co) deposits are present adjacent to the capillary. Scale 1 pm. Magnification 12,000x.

no platelet thrombi and no strands of polymerized fibrin were present at any stage. Penetration of the vascular wall apparently occurred rapidly, since relatively few tumor cells (approximately one cell in every 250 pilot sections examined) were observed fixed during the process of actually crossing the

endothelium, when compared to the greater numbers of cells seen either prior to penetration (adhering to the endothelium) or after breaching of the capillary (lying in subendothelial positions). Although occasional tumor cells completely crossed the vascular wall early and

SINDELAR,

TRALKA,

AND KETCHAM:

ULTRASTRUCTURE

OF METASTASFS

155

FIG. 17. Perivascular fibrosarcoma cells 3 days after tumor inoculation. Section shows a pair of tumor cells (TC) within the connective tissue adjacent to a pulmonary capillary (Cap). No intravascular tumor cells are present. A great alveolar cell (GAC) is seen in close proximity to the tumor cell nest. Scale 5rm. Magnification 5000x.

were found free within the alveolar space (Fig. 16), most of the migrating cells appeared to lodge at least temporarily immediately beneath the endothelium. By 3 days after cell injection, all tumor cells had penetrated the endothelial cellular junctions and their basement membranes. Cells at this stage were uniformly found in perivascular connective tissue, frequently in locations adjacent to collagenous stroma (Fig. 17). Growth of pulmonary metastases. After penetration of the walls of the vessels in which they had been arrested, tumor cells migrated into perivascular connective tissue. Fibrosarcoma cells were identified within the pulmonary parenchyma as early as 24 hr

after injection but were most numerous at 3 days. At that time both individual cells and small cell clusters were found perivascularly (Fig. 17). Tumor cells within the pulmonary connective tissue exhibited none of the morphologic signs of nuclear or cytoplasmic degeneration which were present in some of the cells of questionable viability that were arrested intravascularly during the first 3 hr after injection. Mitotic figures were observed among the tumor cells located in the perivascular parenchyma (Fig. 18), suggesting that after capillary penetration the tumor cells proliferated to form cell nests that ultimately developed into macroscopic pulmonary

156

JOURNAL OF SURGICAL

RESEARCH VOL. 18, NO. 2, FEBRUARY

1975

FIG. 18. Perivascular fibrosarcoma cell undergoing division. Tumor cell (TC) is seenin mitosis 3 days after cell injection. The fibrosarcoma cell is located in the pulmonary parenchyma between a capillary (Cap) and the alveolar space (Alv). Scale 5 pm. Magnification 5000x.

metastases. Metastatic nodules were grossly visible in all experimental animals at 7 days after tumor inoculation. Most of the nodules were located at or near the lung periphery. Sections through metastatic foci revealed fibrosarcoma cells similar in ultrastructural appearance to those in the primary tumor. Mitotic figures were relatively common in the metastases and were most frequent near the peripheries of the nodules. No capsules were identified. Often, some infiltration around the metastases by chronic inflammatory cells was identified. This infiltrate was not prominent with routine light microscopy, but in electron micrographs lymphocytes and plasma cells were frequently identified in the peripheral regions of pulmonary metastatic deposits (Fig. 19). DISCUSSION Tumor Cell Identification Electron microscopic study of the Ml9 fibrosarcoma was initially performed in

tissue fragments removed from intact, transplantable primary tumors. This was undertaken in order to establish ultrastructural criteria to enable the identification of tumor cells which were later found in lung tissue after intravenous injection. Certain features which typically characterized the Ml9 cells in electron micrographs included: large and frequently lobulated nuclei, “target-like” nucleoli, perinuclear fibrils, polarized mitochondria often containing myelin figures, and cytoplasm generally lacking in complex organelles. After the injection of cell suspensionsand the preparation of lung tissue for microscopy, fibrosarcoma cells were usually identified within the lung sections without difficulty. However, in approximately lO15% of the presumptively identified tumor cells, the plane of the tissue section failed to include all of the characteristic ultrastructural features of the Ml9 tumor.

SINDELAR,

TRALKA,

AND KETCHAM:

ULTRASTRUCTURE

OF METASTASES

157

FIG. 19. Section through the periphery of an established pulmonary metastasis 14 days after tumor inoculation. The section was taken through the peripheral region of a small pulmonary metastasis. Tumor cells (TC) are present. Several lymphocytes (L) and a plasma cell (PC) are seen.A mononuclear cell infiltrate often is associated with the periphery of metastases but usually does not extend deeply into the metastatic deposit. Scale 5 pm. Magnification 6000x.

The usual structure excluded in the section plane was the target nucleolus. These presumptive tumor cells resembled those of the lymphocytoid series in the host animals because of their large nuclei and their undifferentiated cytoplasm. Thus, it was not possible to absolutely distinguish tumor cells from large lymphocytes or from circulating macrophages in every case. Although possible ambiguity of cell type identification is a criticism of this investigation, the validity of the reported results is not affected, since the vast majority of observations at all stages of the experiment involved the behavior of cells that were unequivocally identified as tumor emboli. Such cells showed all of the subcellular fea-

tures seen in cells of the Ml9 primary tumor. These cell types were studied within the pulmonary vessels and perivascular connective tissue. No observation of tumor cell behavior was considered characteristic unless that observation was repeated in at least five separate experimental animals. No conclusions were based solely on the behavior of isolated cells whose precise identification could not be established. Validity of the Experimental Model All conclusions on the mechanisms of Ml9 tumor cell attachment to vessels, vascular penetration, and growth within the pulmonary parenchyma were based on the behavior of intravenously injected single cell

158

JOURNAL

OF SURGICAL

RESEARCH

suspensions.These conclusions are proposed to reflect the cellular events occurring during spontaneous hematogenous metastasesdeveloping from a primary tumor. There is no assurance that the characteristics of tumor cell lodgement and growth in target organs are similar for both injected cells and spontaneous metastases. However, it is likely that the basic mechanisms of cell adherence to vascular linings, endothelial penetration, and migration into connective tissue remain unchanged for circulating tumor cells, irrespective of whether they were injected intravenously or shed intravascularly from a primary site. This proposal is supported by the results of control experiments which showed the consistent production of pulmonary metastatic foci after intravenous inoculation of tumor cells. These tumor foci were identical to spontaneous Ml9 metastases arising in animals bearing primary intramuscular tumors. Ideally, studies on the behavior of embolized tumor cells should be performed on spontaneous metastases. However, such a study on Ml9 fibrosarcoma metastases is not technically feasible with the electron microscope. Spontaneously shed circulating tumor cells are likely to present in small numbers [9]. Since lung sections must be randomly selected for examination, it would be extremely fortuitous to find ultrathin sections containing spontaneously shed tumor cells. Even in the artificially produced Ml9 tumor emboli, where large numbers of tumor cells were intravenously injected at a known time, over 50 pilot tissue samples were sectioned and examined for each individual arrested tumor cell found. Most of the past work on the behavior of intravascular tumor emboli has been produced performed with artificially metastases by intravenous cell injection [4, 5, 24, 27, 291. Although much information can be gained about tumor cell arrest, vascular penetration, and growth, the validity of any general conclusions drawn from the behavior of experimental tumor emboli must await final correlation with the cellular

VOL.

18, NO. 2, FEBRUARY

1975

events of spontaneous tumor cell metastases. Tumor Cell Arrest in Pulmonary Vessels Role of pseudopodia. A consistent finding throughout the study was the extensive pseudopodial surface activity displayed by the Ml9 fibrosarcoma cells, both in dissociated single tumor cell suspensionsand in free circulation within the pulmonary vessels after intravenous inoculation. These pseudopodia were thought to play a crucial role in the adhesion of tumor cells to the endothelial linings of the pulmonary vessels. Tumor cells that became arrested intravascularly during the first hour after cell injection were observed to be attached to the endothelium by short pseudopods or surface membrane ruffles. Even in association with the platelet thrombi which developed soon after initial tumor cell arrest, the embolized fibrosarcoma cells appeared to continue to adhere to the vascular walls by means of short projections of their surface membranes. After dissolution of the platelet aggregates, the arrested tumor thrombi remained attached to the endothelium by pseudopodia. Thus, the attachment of tumor cells to the vascular endothelium appeared dependent on the adhesiveness of surface projections. Pseudopodia are known to be important in adhesive interactions between cells in many systems, particularly in tissue culture [ 1,211 and in embryonic morphogenetic movements [7, 10, 221. Surface processes and membrane ruffles have been proposed to be the general organelles of cell adhesiveness, based on the electrochemical attractive behavior of cell membranes at points with small radii of curvature [3]. In view of the general evidence that pseudopodia mediate cellular adhesiveness, it is reasonable to propose that attachment of circulating tumor cells to vascular endothelium may be dependent on surface processes spun out by the tumor cells. Consequently, avenues of possible experimental prevention of blood-borne metastases might

SINDELAR,

TRALKA,

AND KETCHAM:

ULTRASTRUCTURE

OF METASTASES

159

involve attempts to prevent pseudopod tion or prevention of spontaneous meformation (as perhaps by metabolic poisons) tastases. Role of fibrin. Several experimental or to modify the adhesive groups at the tips studies have been performed in attempts to of surface ruffles (such as by changing surstudy the arrest of circulating tumor cells in face charges on the cell membrane). Role of platelets. Platelets played a con- vascular beds [6,8,9, 11, 13, 18,25,26, 301. spicuous role in the events of early tumor Certain workers have suggestedthat fibrin is cell arrest. Dense platelet thrombi formed necessary for the attachment of tumor around embolized tumor cells almost im- emboli to the vascular walls [2, 28, 291. mediately after adhesion of the tumor cells Wood [27, 281, using microcinematography to the vascular wall. The platelet thrombi in the rabbit ear chamber, demonstrated the appeared to propagate proximally in the formation of fibrin thrombi around V2 pulmonary vessels to fill the capillaries and tumor cells which were arrested in arterioles with a dense aggregate which capillaries after intraarterial injection of a persisted for as long as 18 hr before tumor cell suspension, Antimetastatic dispersing. Platelet aggregation around effects of various anticoagulants which tumor emboli has been previously described prevent fibrin deposition have been demby electron microscopy [12]. However, there onstrated in some experimental animal tuhave been no reports of the formation of mors [4, 17, 291, thereby suggesting a role large platelet masses as those seen sur- for fibrin in the metastasis of tumor emboli. rounding M 19 cell emboli. However, no direct involvement has been demonstrated of a fibrin thrombus in the The platelets aggregated after the initial arrest of tumor cells in the vessels. Thus, actual adhesion of embolized tumor cells in platelet aggregation did not appear to be the capillary beds or in the vascular penetration method of initial entrapment of tumor of tumor emboli into the surrounding conemboli. However, the platelet thrombi might nective tissue. function in stabilizing the initial adhesions In an electron microscopic study of between embolized tumor cells and the endo- pulmonary tumor emboli using a rat thelium, perhaps by mechanically holding hepatoma, Ludatscher and her co-workers the arrested tumor cells against the endothe- [17] noted no signs of fibrin deposition at the lium until more stable attachments can be sites of tumor cells arrested within vessels. made. Some evidence for this proposal is Jones et al. [ 121,using Walker 256 carcinogiven by the observation that the initial at- sarcoma, showed positive staining for fibrin tachments of tumor cells were mediated by by immunofluorescence at the sites of arshort fragileappearing pseudopodia, while rested pulmonary tumor emboli. However, later attachments, after disaggregation of these workers were unable to demonstrate the platelet thrombi, were formed by longer fibrin deposits in the area of tumor emboli and more extensive cell surface processes. by electron microscopy. They concluded Experimental alteration of platelet that fibrin was probably present in function might affect the metastatic monomeric form but that no actual fibrin potential of tumor cell emboli, perhaps by thrombus was formed. acting to prevent or disrupt the adhesionThe observations reported here are consisstabilizing platelet thrombus. Experiments tent with the previous electron microscopic studying possible antimetastatic effects of studies of tumor emboli [12, 141. No fibrin platelet antagonists are presently underway. deposits were detectable at any inIf intact platelet function is necessary for the travascular sites of tumor cell arrest. Alinitial stages of tumor cell embolization and though large platelet aggregates were attachment, the use of platelet inhibitors formed around tumor emboli, no might have therapeutic benefit in the reduc- polymerized fibrin was formed. Thus, fibrin

160

JOURNAL

OF SURGICAL

RESEARCH

deposition did not appear to be necessary for metastasis formation in the Ml9 fibrosarcoma system. Vascular Invasion and Growth of Tumor Cells Tumor cells which had become arrested in the pulmonary vessels were observed to initiate penetration of the capillary walls by 24 hr after cell injection. The actual breaching of the vascular wall took place between the junctions of the adjacent endothelial cells, with the penetrating tumor cells appearing to cause some separation of the endothelial cytoplasmic processes. Previous electron microscopic studies of experimental pulmonary metastases [12, 141 have indicated that the crossing of the vascular wall by tumor cells probably took place between endothelial cells. However, documentation of this proposal has been lacking, due to the difficulty of finding tumor cells fixed in the process of breaching the vascular wall. Marchesi and Florey [16] have studied acute inflammatory reactions with the transmission electron microscope and have described the extravascular migration of leukocytes as occurring by penetration between endothelial cells. The observations reported here substantiate earlier proposals by certain investigators [12, 14, 27, 28, 291 that actual vascular penetration by embolized tumor cells occurs directly between adjacent endothelial cells. After vascular penetration, fibrosarcoma cells were observed to invade the perivascular pulmonary parenchyma. Once within the connective tissue, viable cells underwent mitosis to form cell nests that presumptively gave rise to metastatic tumor nodules. Growth of these tumor foci within the parenchyma represented the last stage in the formation of pulmonary metastases in the experimental Ml9 fibrosarcoma system. ACKNOWLEDGMENTS Sincere thanks are due to Dr. G. T. O’Conor for valuable advice and for making available electron microscopic facilities. The technical assistance of Mr. E. R. Henson is gratefully acknowledged.

VOL.

18, NO. 2, FEBRUARY

1975

REFERENCES 1. Abercrombie, M. The bases of locomotory behavior of fibroblasts. Exp. Cell Res. Suppl. 8:188198(1961). 2. Agostino, D., Grossi, C. E., and Cliffton, E. E. Effect of heparin on circulating Walker carcinosarcoma 256 cells. J. Nat. Cancer Inst. 27:17-24 (1961). 3. Bangham, A. D., and Pethica, B. A. The adhesiveness of cells and the nature of the chemical groups at their surfaces. Proc. Roy. Sot. Edin. (B) 28:43-52 (1960). 4. Baserga, R., Kisieleski, W. E., and Halvorsen, K. A study on the establishment and growth of tumor metastases with tritiated thymidine. Cancer Res. 20910-917 (1960). 5. Baserga, R., Putong, P. B., Tyler, S., and Wartman, W. B. The dose-responserelationship between the number of embolic tumor cells and the incidence of blood-borne metastases. &it. J. Cancer 14:173-185 (1960). 6. Baserga, R., and Saffiotti, U. Experimental studies on histogenesis of blood-borne metastases. Arch. Parhol. 59:26-34 (1955). 7. Bellairs, R. Differentiation of the yolk sac of the chick studied by electron microscopy. J. Embryol. Exp. Morphol. 11:201-225 (1963). 8. Fisher, B., and Fisher, E. R. Anticoagulants and tumor cell lodgment. Cancer Res. 27:421-425 (1967). 9. Griffiths, F. D., and Salsbury, A. J. Circulafing Cancer Cells. Thomas, Springfield, 1965. IO. Gustafson, T., and Wolpert, L. The cellular basis of morphogeneis and sea urchin development. Inr. Rev. Cytol. 15139-214 (1963). Il. Iwasaki, T. Histological and experimental observations on the destruction of tumour cells in the blood vessels. J. Pathol. Bacreriol. 20:85-105 (1915).

12. Jones, D. S., Wallace, A. C., and Fraser, E. E. Sequence of events in experimental metastases of Walker 256 tumor: Light, immunofluorescent, and electron microscopic observations. J. Nat. Cancer Inst. 46:493-m (1971). 13. Lee, Y. Experimental studies of metastases. A review. J. Missouri State Med. Assoc. 65:205-210 (1968). 14 Ludatscher, R. M., Luse, S. A., and Suntzeff, V. An electron microscopic study of pulmonary tumor emboli from transplantable Morris hepatoma 5123. Cancer Res. 27:1939-1952 (1967).

15. Madden, R. E., and Burk, D. Production of viable single cell suspensions from solid tumors. J. Nat. Cancer Inst. 27:841-861 (1961).

16. Marchesi, V. T., and Florey, H. W. Electron microscopic observations on the emigration of leucocytes. Quart. J. Exp. Physiol. 45:343-348 (1960).

SINDELAR,

TRALKA,

AND KETCHAM:

17. Ryan, J. J., Ketcham, A. S., and Wexler, H. Reduced incidence of spontaneous metastases with long-term Coumadin therapy. Ann. Surg. 168:163168(1968). der 18. Schmidt, M. B. Die Verbreitungswege Karzinome und die Beziehung Generalisierter Sarkome zu den Leukamischen Neubildungen. Fischer,

Jena, 1903. 19. Snell, G. D. A cytosieve permitting sterile preparation of suspensions of tumor cells for transplantation. J. Nur. Cancer Inst. 13:1511-1515 (1953). 20. Stempak, J. G., and Ward, R. T. An improved staining method for electron microscopy. J. Cell Biol. 22:697-701(1964).

21. Taylor, A. C., and Robbins, E. Observations on microextensions from the surface of isolated vertebrate cells. Develop. Biol. 7:660-673 (1963). 22. Trinkaus, J. P. Mechanisms of morphogenetic movements, in Orgunogenesis (DeHaan, R. L., and Ursprung, H., Eds.), pp. 55-104. Holt, Rinehart, and Winston, New York, 1965. 23. Venable, J. H., and Coggeshall, R. A simplified lead

ULTRASTRUCTURE

OF METASTASES

161

citrate stain for use.in electron microscopy. J. Cell Biol. 25:407-408 (1965).

24. Warren, S., and Gates, 0. The fate of intravenously injected tumor cells. Amer. J. Cancer 27:485-492 (1936).

25. Weil, R. The intravenous implantation of rat tumors.J. Med. Res. 2&497-508 (1913). 26. Willis, R. A. The Spread of Tumours in the Human Body. 3rd ed., Butterworth, London, 1973. 27. Wood,‘S. Pathogenesisof metastasis formation observed in vivo in the rabbit ear chamber. Arch. Puthol. 66:550-568 (1958). 28. Wood, S. Experimental studies of the intravascular dissemination of ascitic V2 carcinoma cells in the rabbit, with special reference to fibrinogen and fibrinolytic agents. Bull. Schweiz. Akad. Med. Wiss. 2092-121 (1964).

29. Wood, S., Holyoke, E. D., and Yardley, J. H. Mechanisms of metastasis production by bloodborne cancer cells. Canad. Cancer Con& 4:167-223 (1961).

30. Zeidman, I., and Buss, J. M. Transpulmonary passage of tumor cell emboli. Cancer Res. 12:731733 (1952).