Lymphatic metastasis

Cancer Metastasis Reviews 2:307-317 (1983) © 1983, Martinus Nijhoff Publishers. Printed in the Netherlands

Lymphatic metastasis

Ian Carr St. Boniface General Hospital," Department of Pathology, University of Manitoba, Winnipeg, Manitoba

Keywords: lymphatic metastasis, footpad-lymph-node mode of lymphatic metastasis, lymphography, tumor cell heterogeneity

Summary

Lymphatic metastasis is an important mechanism in the spread of human cancer. During its course, tumor cells first penetrate the basement of membrane of the epithelium, in which they arise, and then the underlying connective tissue, carried partly by h),drostatic pressure. They enter the lymphatic partly by active movement, pass up the lymphatic trunk; they then settle and proliferate in the subcapsular sinus, penetrate its endothelium and proliferate and destroy the node. There are .v, aried forms . of immune response in the node and in human nodes often a complex fibrous and vascular response. The degree of lymphocytic response may be important for prognosis. The nodal reaction may be stimulated by release of antigens from the tumor.

One of the most studied animal models of lymphatic metastasis is that which occurs in the politeal node after injection of tumor into the footpad. This model has been used to show that tumor cells enter lymphatics through gaps in endothelium, probably between endothelial cells, and that lymph nodes can destroy small numbers of tumor cells. Local immunol(herapy and chemotherapy can sterilize a lymph node of tumor cells; the modes of treatment used have included intralymphatic injection and encapsulation of chemotherapeutic agents in liposomes. Prior radiotherapy may accelerate metastasis possibly by making tumor cells shed into lymphatic vessels. Lymph nodes are rather poor barriers to tumor cells. The prognostic significance of lymph node metastasis varies within tumor type; if hematogenous metastasis is early, then the presence of lymph node metastasis is of lesser prognostic significance. Lymph nodes can probably destroy only small numbers of tumor cells.

Tumor cell heterogeneity is of importance in many aspects of metastasis; while clonal variation may be of importance in determining lymph node metastasis, it is not yet clear how important this is, nor whether specific clones metastasize specifically to lymph nodes.

Lymphography is well established in diagnosis of lymphatic metastasis. A recent interesting development has been to inject antibodies labeled with a radioactive label, and image the label in lymph nodes with a gamma-camera. If anti-tumor antibodies are used in this way it may be possible to detect lymph node metastasis.

Within the expanding field of tumor metastasis, lymphatic metastasis needs much more attention, particularly in relation to the diagnosis and treatment of the lymphatic spread of human cancer.

Address for reprints: Dr. I. Carr, Department of Laboratory Medicine, St. Boniface General Hospital, 409 Tache Avenue, Winnipeg, Manitoba R2H 2A6

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Lymphatic metastasis

The common human cancers spread and kill first by lymphatic metastasis; most work on animal tumor metastasis, on the other hand, has been done with models of hematogenous metastasis. After the pioneering study of lymphatic metastasis carried out by Zeidman and Buss (1), there was a lull for about 16 years. Since then, much more work has appeared (2-4 for reviews). The present essay aims not to repeat previous reviews in detail, but rather to summarize some important facts re- lating to the lymphatic metastasis of human cancer, to recapitulate some of the conclusions arising out of animal work and point out the differences between natural and experimental lymphatic metastasis, to review recent work on the selective delivery of therapeutic agents in lymphatic metastasis and lastly to indicate the gaps in present knowledge.

Metastasis along lymphatic pathways occurs in stages; these may usefully be described as:(A) premetastatic invasion, (B) approach, (C) penetration, (D) translocation, (E) intranodal settling, (F) growth and destruction of the lymph node and (G) metastasis to further nodes. (A) Pre-metastatic invasion. This may involve penetration of the basal lamina of an epithelium and penetration of connective tissue spaces (5--6). There may be pathways of preferred orientation down which tumor cells might more readily travel (7). The have been described as pre-lymphatic pathways. The interstitial pressure of tumors is high (8) and this may lead to a tide of oedema fluid. Hydrostatic pressure of edema fluid may be important in carrying the cells toward the lymphatic capillary. (B) Approach. The tumor cells come into close contact with the lymphatic endothelium. (C) Penetration. The tumor cells, one way or other cross the endothelial barrier. (D) Transloca- tion. The tumor cells pass up the lymphatic trunk. (E) Intranodal settling. The cells settle in the sinusoids of the node and start to proliferate. (F) Growth and destruction of the lymph node. The tumor cells penetrate and/or destroy the endothelium of sinusoids, and then proliferate within the parenchyma of the node. (G) Further seeding to

secondary nodes. This occurs at a relatively early stage - often before destruction of the node. Tumor cells may penetrate the blood stream either in the primary, by lymphatic or venous connections in a lymph node or by passage up the lymphatic chain to the thoracic duct.

It is interesting how little is known of several of these stages, and how very little of what is known derives from the study of human tumor metastasis. It is notable that at each stage the conditions are different, the vessel wall is different, the medium and flow characteristics are different, the secondary site is different, and the availability of reacting cells is different.

Aspects of human lymphatic metastasis have been reviewed elsewhere (9). The early phases of lymphatic metastasis are difficult to identify in human material. It is very rare to see tumor cells actually in the act of penetrating lymphatic vessels at the edge of a neoplasm. They may sometimes, though uncommonly, be seen in lymphatic vessels; this, in the absence of metastasis, has been corre- lated with a poor prognosis (10). If tumor cells have reached the node it is not significant whether they are present in the pre-nodal lymphatic or not.

Lymphatic trunks are rarely examined, but occasionally tumor cells can be seen in the extracapsular lymphatics of the node. These cells may block the vessel and form an extracapsular mass. Within the node, tumor cells can sometimes be found only in the subcapsular sinus, either free or adherent to the sinus lining cells. Thereafter they move down the sinusoids, distending them as they go; ultimately they invade and destroy the adjacent lymph node. The node may be completely replaced by tumor or may show focal capsular invasion. Tumor cells may sometimes be seen in the efferent sinusoids. The tumor stimulates the appearance of stroma similar to that seen in the primary; thus, there may be dense fibrous tissue or a prominent blood capillary plexus. The tumor may show extensive degenerative changes (such as mucinous changes in adenocarcinoma) or frank necrosis, presumably ischemic. In differentiated adenocarcinomas tumor may seed down the sinusoids as single cells, maturing into acinar elements later and including a characteristic stroma. The

reaction in a node may include polymorphs, macrophages and giant cells and may rarely simulate sarcoidosis. A node containing a small metastasis may show marked reactive changes - and occasionally nodal enlargement may be due to reactive change and not to the presence of tumor cells per se. Thus, a lymph node metastasis of a human cancer is often a complex structure, not merely an aggregate of tumor cells in a scarcely modified lymph node.

Lymph nodes react to antigenic or inflammatory stimuli in their drainage area by a B cell response, enlargement of lymphoid follicles and formation of germinal centres, by a T cell response, increase in paracortical areas due to entry and proliferation of lymphocytes and by increased prominence of sinus macrophages - sinus histiocytosis (11-15).

Probably the patterns of lymphocytic reaction in the node are the most important in relation to prognosis, because of the immunologic response implied. Four histologic patterns have been described - lymphocytic predominance, where the extrafollicular parts of the node are stuffed with lymphocytes, follicular reaction, an intermediate average state and lymphocyte depletion, often with some hyaline fibrosis. In carcinomas in many sites, e.g., uterus, cervix, breast and upper re- spiratory carcinoma, lymphocyte predominance is associated with good prognosis and lymphocyte depletion with bad prognosis. This is not true however of other sites - e.g., colonic carcinoma, where blood spread to the liver is likely to obscure the effects of lymphatic spread. Congruent with these observations is the demonstration that lymph nodes with metastatic deposits contain few- er T-lymphocytes (11-15).

The histiocytic reaction in draining nodes is in- dependent of the lymphocytic response and has also been related to tumor prognosis. The precise nature of sinus histiocytosis is not clear; it may represent a combination of proliferation and maturation of resident macrophages with increased monocyte traffic through the node. Ultrastructural studies have suggested that it may occur in phases - initially the macrophages are small, later they enlarge and finally there is a

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degenerative phase (16). These may not be func- tionally identical. Most of the work in this area has been done on breast cancer and arose from observations (17) that sinus histiocytosis was associated with a better prognosis. In the years since, there has been considerable dispute on this subject. On balance it is doubtful whether sinus histiocytosis is a good prognostic index (18). Sar- coid reaction, i.e., proliferations of large epithe- lioid macrophages, rarely occur in nodes draining cancers. Their significance is not clear. These reactions may be responses to areas of necrosis and inflammation in a tumor, or they may be reactions to shed tumor antigens.

The binding of tumor antibodies has been studied (19) scintigraphically and autoradiographical- ly by making purified antibodies to CEA and labeling them with 1311. The antibody was then injected subcutaneously in an appropriate site - the web of the fingers, the perineum, or the peri- prostatic tissues, depending on the lymph nodes to be examined. Sequestration of the labeled antibody was demonstrated in lymph nodes draining tumors both in the presence and in the absence of metastases. Nodes which sequestered labeled antibody showed lymphoid hyperplasia. It seems likely from this study that release of antigens by tumors may stimulate important nodal reactions.

The size of a nodal metastasis may affect its prognostic significance. Small occult metastases can be found by step sectioning of lymph nodes but are of little prognostic importance (20). In- itially metastasis follows normal patterns of lymph flow, but destruction of a lymph node by tumor may lead to blockage of normal patterns of lymphatic flow and drainage by abnormal lymphatic channels.

Traditionally, carcinoma tends to metastasize by lymphatics and sarcoma by the blood stream. Like carcinoma, both melanoma and synovial sarcoma metastasize by the lymphatics. Such tumors as liposarcoma, fibrosarcoma, osteogenic sarcoma, leiomyosarcoma and rhabdomyosarcoma were traditionally held rarely, if ever, to metastasize by lymphatics. Embryonic rhabdomyosarcoma metastasizing from the palm of the hand to the axillary nodes is an exception. Recent studies on

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metastasis in patients with sarcoma treated by chemotherapy show that lymphatic metastasis does, however, occur in most types of advanced neopla- sia, if the patient survives long enough (21).

A wide variety of experimental animal models now exists; for a fuller bibliography, reference may be made to earlier reviews (2-5). These animal models differ significantly from the human phenomenon. An ideal model would involve repro- ducible metastasis of a primary tumor to a defined lymph node that drained only that tumor, repro- ducible at a constant rate, metastasizing further to kill the animal by hematogenous spread. Such an ideal tumor would further be transplantable in a truly syngeneic strain, and its venous and lymphatic effluents could be cannulated readily. Such a model does not presently exist. The earliest stage, the penetration of lymphatics by a primary tumour, is unstudied. The models that do exist involve either injection of cell suspensions or in- troduction of tumor fragments or mushes subcutaneously usually into a footpad, thigh muscle or flank, with study of the draining popliteal ing- uinal or axillary lymph nodes. It is essential to exclude the possibility of direct intralymphatic injection of tumor cells; this is best done by careful examination of histologic sections of nodes a few hours after injection. A brief account will first be given of basic anatomic pathologic patterns, ex- emplified by the work of myself and several col- laborators; this will be followed by comments on several models which have yielded valuable information (as opposed to the mere demonstration that lymphatic metastasis happens).

The model which I have studied has been the footpad-lymph-node model in which a suitable number (usually 5 million) viable tumor cells are injected into the foodpad and metastasis is studied in the draining ipsilateral popliteal lymph node, the para-aortic lymph nodes and the lungs. This has been done with several tumors, notably the Rd/3 tumor induced by dimethylbenzanthracene in inbred rats, the Walker rat carcinoma and the 13762 rat mammary adenocarcinoma in syngeneic F344 rats (22-26). Metastasis is consistent - more so in the syngeneic tumors, where it is over 95% successful, less consistent, 90% or more in the

allogeneic Walker rat carcinoma. Tumor cells can be seen in lymphatic vessels in the simulated primary tumor, can be obtained by cannulation of the lymphatic trunk and can be identified in the extracapsular lymphatic vessels of the node. Thereafter they can be identified in the radial and medullary sinusoids; they leave the sinusoids in a manner as yet incompletely determined and destroy the lymph node (27). The rate of metastasis varies from tumor to tumor; in general no cells are seen at 6 hr, a few at 24 hr or 48 hr, and by 9 days the node shows at least partial destruction. Tumors which are composed of single cells tend to metastasize more rapidly than tumors like adenocarcinomas composed of clusters of cells. When the Rd/3, a single cell tumor, was studied in detail, it was found that after injecting 5 x 106 cells into the footpad, there were about 1.5 x 102 cells in the popliteal node in one day, 2.3 x 104 in two days, 4.5 x 104 in three days and 5 x 105 in four days (28). Progressive metastasis occurred if the footpad was removed any later than 24 hr after injection of tumor. This implies that the lesion at 24 hr is a micrometastasis which can be treated or may even regress. When similar small numbers of tumor cells were injected directly into the node, they did not produce progressive metastasis (24).

Examination of the simulated primary (the footpad) showed tumor cells protruding processes between endothelial cells, and migrating between endothelial cells, clustered in the case of an adenocarcinoma. The tumor cells were accompanied by lymphoreticular cells. Only in the case of the allogeneic Walker rat carcinoma was there extensive necrosis of lymphatic endothelium. The major problem about these experiments is that find- ing where tumor cells are penetrating lymphatics means cutting very large numbers of blocks. Gaps between endothelial cells were not evident in the absence of migrating cells, and the reasonable in- terpretation is that the tumor cells can induce opening of lymphatic inter-endothelial junctions; it is possible, however, that the tumor cells found small numbers of open junctions. The rarity of the appearance implies that tumor cells migrate either seldomly or rapidly. The endothelial necrosis may be an allogeneic artifact.

The lymph draining such tumors, obtained by cannulation, contains both lymphoreticular cells and tumor cells. In the allogeneic Walker rat carcinoma, the lymphatic endothelium becomes necrotic and tumor cells flood in; in this case, where the immune response is presumably more evident, lymphoreticular cells are present in large numbers. (25) Tumor cells from a differentiated adenocarcinoma are found in the lymph both singly and in clumps (26). The lymph nodes although eventually destroyed do show some sign of a mixed lymphoreticular reaction - a variable early proliferation of sinus macrophages, followed by paracortical lymphocyte immigration, and enlargement and proliferation of germinal centers. These reactions have been analyzed morphometri- cally in a model of mouse mammary carcinoma, metastasizing from the footpad (29). There was a successive increase in paracortical area and number of germinal centers. The reaction in distant nodes was similar. From morphologic and func- tional studies it seemd clear that the first draining node triggers the immune response but that later the whole lymphoreticular system reacts. The nodes do not, however, show the prominent fib- rogenic response seen in some human nodal metastases.

Setting up models of lymphatic metastasis is time consuming. Models include hamster lympho- ma, guinea-pig hepatoma, rat hepatoma, mouse and rat mammary adenocarcinoma, oesophageal carcinoma, prostatic carcinoma, mouse fibrosarcoma and squamous carcinoma (see references in 2 and 4). Relatively few of these models have been studied over a long enough period to produce significant biological information, as opposed to the mere demonstration that lymphatic metastasis can happen. The most interesting way in which these models have been used is the study of local administration of chemotherapy, or immunbther- apy along lymphatic patways. Notable among these are the guinea-pig hepatoma model and the 13762 rat mammary adenocarcinoma model.

In general such models are not good replicas of human metastasis in the relative simplicity of the metastasis induced. Tumor cells proliferate, burst out of sinusoids and destroy the node. There is a

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varying but usually trivial degree of lymphoreticular response. Experimental lymphatic metastasis of VX2 carcinoma in rabbits (30) shows that there is an increased vascularity in the node for about four weeks. The stromal fibrotic response which is an important reaction in many human metastatic cancers is not seen, and indeed the nodal metastasis is usually merely a proliferation of tumor cells in a non-reactive node.

The guinea-pig hepatoma model (31-33) involved the use of hepatoma induced in a strain of guinea pigs by injection of dimethylnitrosamine. The hepatoma was then converted into an ascites tumor. Progressive tumor growth was induced by injection of 105 tumor cells intradermally. The animals died of metastasis to vital organs after inoculation of 106 tumor cells. Tumor cells were seen in the subcapsular marginal sinus of the su- perficial distal axillary node at seven days though not at four days. There was therefore a situation analogous to the human situation where micrometastasis was present in the draining node and where the simulated primary could be removed by excision, leaving behind a micrometastasis. This tumor responds to immunotherapy with myco- bacterial vaccines and tumor cell vaccines; eli- mination of micrometastasis is best achieved by injection of the vaccine near to each metastatic site; that is, in addition to the generation of systemic immunity, for the best result a local reaction is needed.

The 13762 rat mammary adenocarcinoma model (34-37) was originally induced with 7.12 dimethybenzanthracene in F344 rats and adapted for ascites passage. After initial injection of 1 million tumor cells on the dorsolateral thorax, axillary nodes were infiltrated by tumor by the 7 th day and animals died about the 40th day with extensive pulmonary infiltrates, pleural effusions and metastasis in mediastinal and axillary nodes. Surgical excision after the 7th day was rarely cura- tive because of the presence of small numbers of tumor ceils in the node. Again, therefore, there is a possibility of studying nodal micrometastasis. Nodal micrometastasis in this model was curable by injection of BCG into the tumor followed by excision of tumor on day 20; similar effects were

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obtained with injections of Corynebacterium parvum. The results were dose dependent and accompanied by the development of systemic immunity - but protection was attained in only 30-40% of the animals. Tumor recurred in about a quarter of these, killing the animals. There is an important resemblance here to human cancer in that treatment is partially successful.

Systemic chemotherapy may have a potent and even perhaps preferential effect on lymphatic metastasis (38).

Attempts have been made to increase the effect by using local lymphatic pathways, usually with a retardant to slow perfusion of the chemotherapeutic agent. Such a retardant may be a simple lipid emulsion (39) which blocks lymphatics and has been used in both animal models and humans. More recently, chemotherapeutic agents entrap- ped in liposomes have been effective in the therapy of lymphatic metastasis of mouse and rat mammary tumors (40,41). Direct intralymphatic injection of 5-fluorouracil has been shown to be effective in sterilizing the regional lymph node of metastasis, although not curing the animal (42). The technique of intralymphatic injection in small animals is tricky, but intralymphatic injection of liposomes containing chemotherapeutic agents seems likely to be a potent way to treat lymph node metastasis.

The intralymphatic route may also be used for immunotherapy, thus concentrating the effect in the lymph nodes, where it is required. In human malignant melanoma there is evidence that the chance of local recurrence in draining lymph nodes was reduced by giving endolymphatic BCG two or four weeks after excision; the patients five- year survival however, was, not affected (43). The same workers studied the effects of intralymphatic BCG on nodal metastasis in the rabbit VX2 carcinoma; this was effective but intracutaneous or intravenous BCG was ineffective (43). Intralym- phatic immunotherapy has been tried; irradiated autologous tumor cells were injected into lymphatics in dogs with spontaneous tumors. Reduction or stabilization of tumor mass was claimed, but the methods of assessment of these were not clearly defined (44). Recently, non specific intraly-

mphatic immunotherapy has been studied in humans by treating randomly assigned patients with pre-operative intralymphatic methanol extract- able BCG. In this study 1 patient of 13 so treated had recurrence and died (45). It is clear that intralymphatic immunotherapy is by no means established, but in appropriate circumstances is a suitable subject for controlled trials.

The lesson to be derived from these studies is that it may be possible to obtain local cure of a local lesion by local lymphatic therapy and that intralymphatic infusion or liposome concentration may concentrate therapy locally. This must always be regarded as only adjuvant to obtaining systemic cure of systemically disseminated neoplasm. Optimal results could be expected from appropriate combinations of local and systemic immunotherapy, chemotherapy and radiotherapy, after appropriate surgical reduction in tumor bulk.

The effects of radiotherapy on lymphatic metastasis are of some significance in view of the clinical use of adjuvant radiotherapy. Radiotherapy is effective in treating lymphatic metastasis of radiosensitive tumors; radiosensitivity decreases with nodal size because of the presence of a higher hypoxic fraction (46). The effects of prior radiotherapy on the metastasis of the KHT sarcoma, a spontaneously arising tumor in C3H mice, have been investigated in some detail (47,48). Metastasis was present in the draining node at day 3. Metastasis was identified either histopathologi- cally or by transplantation into other animals. When local radiation in doses of 2400-3600 rad was given to the footpad one day before inoculation, there was a very significant increase in nodal metastasis, as determined either by lymph nodal weight, and in lung metastasis. Secondary metastasis occurred only after the popliteal lymph node was considerably enlarged implying that there was marked trapping of tumor cells in the popliteal lymph node. The effect was probably not related to the immune response since these tumors were not significantly immunogenic. Some of the effects may be related to early inflammatory changes in the irradiated skin - vascular leakage, increased interstitial tissue pressure and edema, increased patency of lymphatic endothelium and increased

lymphatic flow with increased shedding of tumor cells into lymphatic vessels. Very similar findings were obtained in other models (49).

Spontaneous regression of lymph node metastasis is rare. There is however one remarkable series of experiments (50) in which regression occured after infection of the tumor cells with virus. When 5 million uninfected tumor cells were injected into the footpad, there was consistent metastasis to popliteal lymph nodes, thence further up the lymphatic chain, killing the rats in about 18 days. In the node, tumor cells spread in the usual way from the subcapsular sinus, deeper into the node, thence destroying it. When the tumor was infected with Friend virus, however, degenerative changes in the tumor cells accompanied by lymphocytic and histiocytic infiltration were identified at day 6. Thereafter tumor cells were seen to degenerate till at ten days no tumor cells were identified. There is considerable dispute in the literature as to whether lymph nodes can act as an effective 'barrier' to the spread of tumor. After infusion into lymphatic trunks VX2 carcinoma cells were held up for about three weeks (1). Most workers (22,51-53), however, agree that tumor cells pass through quite quickly, in some circumstances within a few hours. An extreme view is that there is continuous lymphatic dissemination but that only relatively few tumor cells stay in the lymph node (52,53).

The speed with which tumor cells pass through the node has relevance for the significance of lymphatic metastasis. If tumor cells pass quickly through, the actual metastasis in the node might be incidental; the important prognostic factor would be whether or not early blood spread had occurred. This must vary from situation to situation. In general, the presence of neoplasm in a lymph node is rather a marker of how far the neoplastic cells have gone, than a cause of serious physiologic upset. The latter can happen, however, if for instance grossly enlarged lymph nodes in bronchial carcinoma block the superior vena cava. Nodal metastasis means hematogenous metastasis either early by penetration of lymphatico-venous connections in or around the node, or later by passage of tumor cells from the thoracic duct into

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the blood circulation. The significance of lymph node metastasis in

human breast cancer has been studied in some detail (54). Clinical staging of nodal metastasis is insufficiently accurate. The pathologic examination of ten or less nodes seems to give adequate information and to predict local axillary recurrence. Failure of treatment at five years does not seem to be related to the number of axillary nodes removed and examined. This has led to the hypothesis that 'regional lymph nodes are primar- ly indicators and not instigators of distant disease' (54). This is keeping with the evidence that hematogenous metastasis occurs very early in human breast cancer; clearly lymph nodes are not 'way stations' in such dissemination. There is , however, evidence (summarized in 55) that the prognosis in carcinoma of colon and melanoma deteriorates with increased numbers of lymph nodes involved. The view has been expressed that 'Negative lymph nodes reflect conditions that, in addition to pre- venting nodal growth of tumors, also inhibit metastases from occuring elsewhere. Positive nodes reflect an inter-relation that permits the development of metastases' (54). This hypothesis clearly deserves further exploration. The retention of tumor cells in lymph nodes in experimental animals can be reduced and tumor dissemination activated by cortisone therapy, or exercise of the tumor-bearing limb (56,57).

It is uncertain whether tumor cells are destroyed in a lymph node. In one model in which there was concomitant viral infection, this certain- ly occurred (50); and there is inferential evidence that it can occur during chemotherapy. It may be possible even in the absence of therapy (58,59). In our own experiments (24), there was good inferential evidence that about 250 tumor cells might be destroyed. In human lymph nodes evidence of degeneration of tumor cells is very rare. The node carrying a large burden acts as a new primary, shedding tumor cells into the blood (60).

There is now good evidence that the ceils in tumors are heterogenous in relation to many properties (61), and it is widely held that there are consistent differences between primary tumors and their metastases. There is much evidence that

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successful metastasis Is related to selection for properties which are advantageous in this process (61), but this is not universally accepted (62). The evidence that lymphatic metastasis is due to such selection is inadequate as yet, although clonal variations in ability to metastasize successfully to nodes have been demonstrated; thus, when a mouse fibrosarcoma was injected into the ear, the parent tumor metastasized successfully, while of two clones studied one reached the nodes but did not persist there, and another was inefficient at reaching nodes (63).

It has recently been shown that tumor cell lines derived from the rat mammary adenocarcinoma 13762 may be more metastatic than the parent tumor; lines derived from lung and from lymph node were more metastatic to both lung and lymph node. Similarly cloned cells varies in their ability to metastasize. One cloned line derived from a lung metastasis was particularly successful at metastasizing to lymph nodes. Much more evidence, however, is needed as to whether cells in a lymph node metastasis have a particular propensi- ty for lymph node metastasis (64).

An imaginative picture may be drawn of the situation around a lymphatic in a metastasizing neoplasm. Tumor cells may be single, or adherent in groups. Those which lie singly may be borne towards the lymphatic along pre lymphatic pathways of orientation in connective tissue. Their transport may involve a tide of fluid. Tissue matrix and basement membrane matrix may be broken down by enzymes released by the tumor cells or lymphoreticular cells. The invading cells may move actively. And rapidly dividing cells may bud off into adjacent tissue - so-called mitotic pressure. Since lymphatics lie only at the edge of tumors, these factors can operate only in a geog- raphically pre-selected part of the tumor - the edge. The tumor cells adjacent to the lymphatic move along the external surface of the endothelium led by fine cytoplasmic processes. The processes either find the small number of open inter- endothelial gaps, or induce opening of closed gaps: by the release of soluble mediators. The tumor cells then move actively into the lymphatic vessel between endothelial cells. The frequency of this

phenomenon is low. In some tumors lymphatic endothelial cells may degenerate and die, though it is not certain whether this is an allogeneic artifact. If lymphatic endothelial gaps are wide open, relatively large numbers of tumor cells may gain access. Where tumor cells are adherent in clusters the same factors may operate in a manner modified by adhesion. Thus, division of a tumor.cell behind the head of the column will push the whole column forward. Presumably the leading cell in the column will operate as a single cell, but will guide or pull the rest of the column. Whole clusters of cells penetrate between endothelial cells and must then break off. The single or clustered tumor cells move passively up the lymphatic trunk to the draining node. There clumps lodge more readily than single cells. The tumor cells proliferate (perhaps facilitated by the environment of the node), pass down the radial sinusoids and burst out of the sinusoids by mechanisms which must be similar to those involved in their penetration of the lymphatic. Within a few days tumor cells are found in the efferent sinusoids. Tumor-killing mechanisms operative in the node are effective only when a few cells are present. There has been very little work done in vitro to investigate these speculations due to the lack of in vitro models which relate to lymphatic metastasis.

Other recent experimental work has focused on the diagnosis of lymphatic metastasis. An established clinical method of evaluating whether lymph nodes contain neoplasm is lymphography: a radiopaque substance is injected into a drainage area, and the area and its nodes X-rayed (reviewed in 19). Another variant is to use radionuc- lides and scan the area with a y camera. When 99-technetium was injected into the feet of mice bearing a syngeneic mammary carcinoma in the footpad, and of rabbits similarly bearing VX2 carcinoma in the footpad, the draining lymph nodes took up less technetium than normal. This was not apparently associated with lymphatic obstruction and was attributed to depression of medullary macrophage function. Similar changes occured in human patients; the precise relationship to histo- logically demonstrable lymph node metastasis was not clear (65,66). In an attempt to obtain tumor

specific homing of a demonstrable substance, an antibody to CEA was labeled with131I and injected into an appropriate site (19), e.g., the hand in the case of carcinoma of breast. Nodal metastasis was then detected by gamma imaging. In addition the labeled antibody may bind the nodes c.on- taining free CEA, usually in hypertrophic germinal centers. The most interesting recent study has been to inject into the feet of mice a monoclonal antibody to a major histocompatibility antigen (K ~) which binds specifically to one class of sites on K k + spleen cells. A third of the dose injected into a foot concentrated in the draining node in a specific manner as imaged with a y camera. This technique was used with an anti-tumor antibody to localize metastases of an adenocarcinoma in a lymph node (67). It seems likely that immuno- scinti-graphic techniques, as applied to local lymphatic drainage, will become more important in the study of lymphatic metastasis.

The things that we do not know about neoplastic invasion and metastasis are legion (68). Less is known about lymphatic metastasis than about hematogenous metastasis. For instance, we do not know how tumor cells reach lymphatic vessels; we know too little about how they penetrate lymphatics - whether, for instance, chemical mediators are released. We know little about how nodes destroy tumor cells, or how tumor cells leave lymph node sinuses. We know too little about cellular selection for lymphatic metastasis, if it happens, where it happens. Many of these defects relate to lack of basic cellular, as opposed to whole animal models to study the subject. Most of what we do know about lymphatic metastasis relates to animal models. We do not know how much of this information relates to human lymphatic metastasis. And we are only at the begin- ning of knowing how to treat lymphatic metastasis in human cancer. The author's work is supported by the National Cancer Institute of Canada and the St. Boniface Research Foundation.

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