Locally Increased Metastatic Efficiency as a Reason for Preferential Metastasis of Solid Tumors to Lymph Nodes
GILES F. WHALEN, M.D., and SETAREH F. SHARIF, PH.D.
Metastases from solid tumors to lymph nodes do not portend as poor a prognosis as metastases to other sites. The authors wished to determine whether specific subpopulations of cells metastasized to lymph nodes and whether they have different properties than cells metastatic to visceral sites. Repetitive selection for "spontaneous" metastases of a B16 melanoma to either lung or lymph node increased the incidence of lymph node metastases. Cells derived from pulmonary and lymph node metastases were assayed for their ability to adhere to cryostat sections of lung and lymph node and respond to target organ-conditioned media in serum-free conditions. Both cell types were four times more adherent to lymph node than lung, and consistently attached to the hilar and subcapsular sinuses. Attachment of cells derived from pulmonary metastases to either tissue was threefold greater than that of cells derived from nodal metastases. Lung-conditioned media stimulated proliferation of both cell types, and transiently induced differentiated morphology in cells derived from lymph node metastases, but not in cells from pulmonary metastases. Neither effect was found in lymph-node-conditioned medium. These results suggest that cells metastasize to lymph nodes preferentially not because of a specific predilection for lymph node, but because it is an easy site to colonize. Adhesive interactions in the lymph node rather than trophic ones appear to account for this effect. Cells metastatic to lymph node may be less "malignant" than cells metastatic to visceral sites because less has been required for them to succeed as a metastatic focus.
n M j r ETASTASIS, THE PROCESS by which cells from a primary tumor establish secondary colonies at distant sites, may be described as a series of steps. Tumor cells must separate from the primary tumor mass, invade the microvasculature oftheir tissue of origin, survive in the circulation, reattach to endothelium in another organ, invade into the surrounding stroma of that organ, and finally proliferate in the new site. ' This process is biologically inefficient,2 probably because a cell thwarted Address reprint requests to Giles F. Whalen, M.D., F1906, New York Hospital-Cornell Medical Center, 525 East 68th St., New York, NY
10021. Accepted for publication July 29, 1991.
166
From the Department of Surgery, Surgical Oncology Research Laboratory, Cornell University Medical College, New York, New York
at any point cannot establish a metastatic focus. The process does not appear to be random, however. Evidence that metastases do not occur randomly is derived from a variety of clinical experiences with typical but anatomically discontinuous patterns of spread of common tumors (e.g., melanoma metastatic to intestinal submucosa), and from experiments with transplantable tumor models. Such experiments suggest that although host response modulates the site of metastasis, subpopulations of cells within the primary tumor have predilections for specific organs.35 Local-regional lymph nodes are a common site of metastasis in many clinically encountered carcinomas. Metastatic disease in the local-regional lymph nodes is a poor prognostic sign, but it is generally not so ominous as metastases to visceral organs. This difference in prognosis is usually thought to be due to differences between lymphatic and a more widespread hematogenous dissemination, or to a stepwise failure of host anti-tumor response, with local nodal defenses failing first. An alternative explanation is that specific subpopulations of cells succeed as lymph node metastases and that these are less malignant than cells that succeed as metastases to visceral organs. To test this hypothesis in vivo, we used the B16 mouse melanoma, a transplantable tumor model commonly employed in studies of organ-specific metastasis. We also used in vitro assays of organ-specific attachment and proliferation to investigate whether cells derived from lymph node metastases differ from cells derived from metastases to a visceral organ (lung) in their relative abilities to accomplish two important steps in a site-specific metastatic process: adhesion to their target organ, and growth in that target organ.
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Materials and Methods Selection of Tumor Cells The hypothesis that lymph nodes are the metastatic site of specific subpopulations of tumor cells was tested by selecting for either lung or lymph node metastases in vivo. We reasoned that metastases to a desired site would be increased by repetitive harvest and reinoculation of tumor cells from that site, if the major determinant of metastatic site was the predilection ofcertain cells for that site. The metastatic B 16-F 10 melanoma variant (provided by Dr. Bruce Zetter, Harvard Medical School, Boston, MA) was used to begin these experiments. Repetitive selection was performed as follows: A cell suspension (106cells/0. 1 mL medium) was inoculated into the web space of the right hind paw of 8- to 10-week-old C57BL/ 6J mice (Charles River Laboratories, Wilmington, MA). When the primary tumor was obvious (3 to 5 mm3), the foot was amputated. Mice were killed 4 to 6 weeks later. Cells from pulmonary metastases of animals without popliteal or inguinal lymph node lesions were harvested, expanded in culture, and reinjected into another set of mice. Similarly, cells from regional lymph node metastases in animals without pulmonary lesions were harvested, expanded, and reinjected into a new set of mice. Cell lines established from the third generation of pulmonary metastases (S3E) and from the third generation of lymph node metastases (S3C) were used in in vitro assays. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee. Cell Culture
Melanoma cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated calf serum (CS) (Hyclone Laboratories, Logan, UT), penicillin 100 IU/mL, and streptomycin 100 ,ug/mL (Irvine Scientific, Santa Ana, CA) at 37 C in 5% CO2. 3T3 cells were grown and maintained under the same conditions except that medium was supplemented with heat-inactivated fetal bovine serum. Adhesion
Organ-specific adhesion was assayed on fresh cryostat sections (4 ,m thick) of mouse lung and lymph node in the following manner: Frozen sections of each tissue were individually mounted on glass slides, encircled with a wax pencil (2 cm diameter), and covered with a 0. l-mL drop of a cell suspension. The cell suspension was prepared by trypsinizing confluent cells (0.05% Trypsin, 0.53 mmol/ L ethylenediaminetetra-acetic acid [EDTA]-Gibco). Washed cells were finally suspended in DMEM with 1% bovine serum albumin (1% BSA) (Sigma Chemical Co.,
167
St Louis, Mo.) at 5 X 105 cells/0.1 mL. Tissue sections covered with the appropriate cell suspension were incubated for 40 minutes at 5 C while being gently agitated on a rotating platform (7 cycles/minute), then rinsed in cold phosphate-buffered saline (PBS) to remove nonadherent cells, fixed with 1% gluteraldehyde, and stained with hematoxylin and eosin. Slides were analyzed by counting cells adherent to recognizable tissue in six random high-power fields (400X) per slide, and averaging this value for an individual slide. At least three slides for each cell/tissue combination were prepared in every experiment.
Proliferation The influence of local organ milieu on cells derived from different metastatic sites was assayed in a system designed to detect either stimulation or inhibition of proliferation on a matrix that had been deposited by the parental melanoma line. Matrix-coated plates were prepared as follows: 2 X 104 cells suspended in DMEM and 10% CS were plated into each well of a 96-well plate, allowed to grow for 36 hours, and removed with trypsin. The plates were washed with DMEM-10% CS and then twice with PBS. Test cells were plated in serum-free conditions on these plates (5 X 103 cells per well). Because of the presence of extracellular matrix, cells attached and spread within 2 hours, allowing assays of proliferation rather than indirect assays of attachment. After 4 hours, organ-conditioned medium was added to the wells and the plates were incubated at 37 C for 48 hours. Calf serum (10% final concentration) was used as a positive control. Cell number was determined by a described colorimetric assay that depends on the reduction of tetrazolium salt by living cells.6 Optical density correlated with cell number (r = 0.85) enough to use this colorimetric assay as an initial screen. Positive findings were confirmed by counting cells in triplicate wells on a Coulter counter (Coulter Electronics, Hialeah, FL) in repeated experiments. Differentiation was assessed in the same cell culture system; tests cells were plated in serum-free media on matrix-coated wells and examined at different time points after the addition of conditioned media. More stellate (as opposed to spindle) cell shape and increased pigmentation were judged to represent a greater degree of differentiation of the melanoma cells. These morphologic criteria have been shown to correlate with the presence of cell surface antigens found on mature, as opposed to fetal, melanocytes.7 Preparation of Conditioned Media The lungs and peripheral lymph nodes were removed from mice and extraneous tissue was trimmed. Remaining lung and lymph node was minced to 1- to 3-mm3 pieces and washed several times with PBS to remove blood and
debris. The tissue homogenate was placed into culture cells with medium (100 mL DMEM/ 15 mg tissue), and incubated at 37°C for 24 hours. Aliquots were removed at varying time intervals, centrifuged at 14,000g for 10 minutes, and the supernatant filter sterilized (22-,um pore size). This material then was stored at -80 C until used in the assays. In early experiments, 1 -mL aliquots were collected over a 24-hour period. Because peak mitogenic activity occurred in the first 4 hours, media was collected after 6 hours of conditioning in subsequent experiments. In addition, this material was dialysed (2000 molecular weight cut off) against unconditioned media for 12 hours before storage to restore small molecules depleted in the conditioning process.
Results In Vivo Selection for Site-specific Metastasis Selection for either lung or lymph node metastasis increased the incidence of metastases in general. Lymph node metastases were increased preferentially and equally by selection for either site. (Table 1)
Adhesion The cells derived from spontaneous pulmonary metastases (N3E) and the cells derived from spontaneous localregional lymph node metastases (S3C) both adhered to target organ tissue sections more avidly than control cells (3T3), and adhered four times more avidly to sections of lymph node than to sections of lung (Table 2). S3E (lung metastases) cells were threefold more adherent to tissue sections of either lung or lymph node than were S3C (lymph node metastases) cells. Cells attached to lymph node sections were predominantly localized to hilar, subcapsular, and medullary sinuses around germinal centers. This anatomic localization on lymph node sections oc-
TABLE 1. In Vivo Selection for Site ofSpontaneous Metastasis in B16 Melanoma
Selection
Lung Metastases
Lymph Node Metastases
Passage
Lung (%)
Lymph Node (%)
Lung(%)
Lymph Node (%)
1
5/19 (26) 4/41 (10)
4/19 (21) 13/41 (32)
5/19 (26) 1/13 (8)
4/19 (21) 4/13 (31)
3/9 (33) 6/41 (15)
5/9 (55) 25/41 (61)
3/9 (33) 2/10 (20)
5/9 (55) 6/10 (60)
2 3 4
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WHALEN AND SHARIF
168
There is a progressive increase in the percentage of mice with lymph node metastases regardless of whether lung metastases or lymph node metastases are repetitively selected (as described in Methods). Numerator represents number of mice with metastases to that site. Denominator represents total number of mice in that group.
TABLE 2. Adhesion of BJ6 Melanoma Cells Derived From Pulmonary (S3E) and Lymph Node (S3C) Metastases to Cryostat Sections of Their Target Organs
Cell Type
Cells/Lung Sections
Cells/Lymph Node Sections
S3E S3C 3T3
13.14 ± 2.31 4.74 ± 1.13 0.89 ± 0.21
99.13 ± 29.58 22.40 ± 9.69 1.76 ± 1.23
3T3 cells were used as controls. Both metastatic cell lines were more adherent to lymph node sections than to lung (p = 0.005), and the S3E line was more adherent to both tissues than the S3C line. Cells were counted in six random high-power fields (X400 per slide to obtain an average number of cells per field on that slide, three slides per group in each experiment. Mean data from three experiments presented ± SEM.
curred with both cell types. No specific anatomic pattern of attachment was apparent on the lung sections (Fig. 1). Proliferation Maximal proliferation stimulated by lymph-nodeconditioned medium was no greater than that stimulated by 10% CS (positive control) for either cell type. Lungconditioned media was more mitogenic. Initial experiments indicated that the peak mitogenic activity of lungconditioned medium (181.5% ± 46.6%; result expressed as a percentage of 10% CS-stimulated proliferation, in three separate experiments) occurred within the first 6 hours of conditioning. Subsequent experiments, employing pooled media from the first 6 hours of conditioning, showed the mitogenic effect of lung-conditioned medium to be dose dependent (Fig. 2). A more differentiated appearance of the melanocytes was serendipitously noted during assays of proliferation with lung-conditioned medium. The phenomenon occurred 12 to 24 hours after the conditioned medium was added to the wells and was lost by 48 hours (when proliferation was assayed). This effect of lung-conditioned medium was prominent on the lymph node metastasisderived S3C cells, not the lung metastasis-derived S3E cells, and was dose dependent (Fig. 3). Lymph node-conditioned medium did not induce this differentiated appearance in either cell type. Discussion The transformed cells that make up a primary malignant tumor are a heterogeneous population,7-9 varying also in their metastatic potential and propensity to metastasize to specific sites.5"'0"' It has been suggested that tumors progress and spread because selective pressures favor cells with a greater capacity to metastasize.8"2 Lymphatic spread to local-regional lymph nodes is common. Experiments that demonstrate rapid appearance of tumor cells in the circulation after inoculation into afferent lymphatic channels, however, suggest that traditional dis-
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METASTASIS OF SOLID TUMORS TO LYMPH NODES
169 .::.. ....:..
FIG. 1. Adhesion of metastatic B16 melanoma cells to cryostat sections of (A) lung; X237, scale bar 20 gm, and (B) peripheral lymph node; X73, scale bar 70 Mm. (C) Magnified (X237) view of attachment to lymph node section, scale bar 20 Mm. Arrows identify representative tumor cells that are in a slightly different focal plane than the tissue sections. Cells preferentially attached to subcapsular, hilar, and medullary sinuses in the lymph nodes.
tinctions between hematogenous and lymphatic spread may be artificial." '3 We therefore wondered whether subpopulations ofcells specifically metastasized to the lymph nodes and whether they were different from cells that metastasized elsewhere. Selection for lymph-node-metastasizing cells in vivo increased the incidence of lymph node metastases, but this result did not indicate that these cell have a predilection for lymph node specifically. Rather, equivalent increases in lymph node metastases after selection for lungmetastasizing cells indicated that the lymph node is simply an easier place to colonize than the lung, regardless of what predilections for other sites a cell might have. Assays measuring the ability of cloned tumor cells to adhere to specific tissues in vitro have generally correlated with the pattern of metastasis when those cells were inoculated intravenously." ", 4 We found that attachment in vitro also correlated with spontaneous metastases in vivo. Cells derived from spontaneous lymph node metastases adhered avidly to lymph node sections in vitro. Again, it
1 000
x
Cell Number
20 18 16 14 12 1 0
8 6 4 2 0
0
0.06 0.66 6.6 13.3 16.6 33.3 66.6 % concentration of conditioned medium
FIG. 2. Proliferation of B16 melanoma cells derived from pulmonary (U - *), and lymph node (E - O) metastases in response to increasing concentrations of lung-conditioned medium.
170
WHALEN AND SHARIF
Ann. Surg. * February 1992
FIG. 3. Differentiation of B16 melanoma cells derived from
lymph node metastases by increasing concentrations of lung-conditioned medium.
Treated cells showed increased pigmentation and dendritic morphologic features. Phase contrast micrographs of cells treated with (A) 13% conditioned medium, (B) 30% conditioned medium, (C) 10% CS, and (D) unconditioned DMEM without CS.
is unlikely that such attachment represented a specific attribute of the cells because cells derived from pulmonary metastases adhered to lymph node sections even more avidly. An important finding of these studies was the consistent attachment of both cell types to hilar and subcapsular sinus tissues on the lymph node sections, which correlates with the known sites of initial tumor cell arrest in vivo"5 and suggests that there is a concentration of adhesion molecules in these locations. Increased amounts of such molecules within the lymph nodes might explain the preferential adhesion (fourfold) to lymph node over lung that we observed for both cell types. Lymphokines such as tumor necrosis factor-alpha, which might be expected to be concentrated in lymph nodes, induce the appearance of at least one adhesion molecule (INCAM- 110) that can mediate melanoma attachment to endothelial cells.'6 Receptors on specialized venules within the lymph nodes mediating lymphocyte homing also might be able to mediate tumor cell attachment.'7-'9 Adherent cells also must thrive in their new environment. Preferential stimulation of site-specific metastasizing cells by trophic factors derived from their target organs has been reported for both lung and liver metastasizing tumors.20'2' We assessed the relative abilities of cells
derived from different metastatic sites to thrive in the milieu of their target organ by measuring their proliferative response to media conditioned with homogenates of that organ. Our experiments suggested that diffusible trophic factors in the lymph nodes do not mediate site-specific lymph node metastases because neither cell line was particularly stimulated by lymph-node-conditioned media. In contrast, lung-conditioned media had a dose-dependent mitogenic effect on both cell types. The trophic factor in our lung-conditioned media appear to be a molecule larger than 2000 kd because the dose-dependent effect survived dialysis with this pore size tubing. Purification of a tumorstimulating, lung-derived growth factor has resulted in identification of an approximately 66-kd glycoprotein that is not inhibited by antibodies against platelet-derived growth factor, epidermal growth factor, insulin-like growth factor 1, or granulocyte macrophage colony-stimulating factor.20 The dose-dependent differentiating effect of lungconditioned media on cells derived from lymph node metastases also remained intact after dialysis against unconditioned medium. Thus, the differentiating effect does not appear to be the result of nutrient depletion of the conditioned media. Generally, differentiation results in a slower rate ofproliferation. In these experiments, the lack of difference between the two cell lines in the amount of
METASTASIS OF SOLID TUMORS TO LYMPH NODES
Vol. 2 15 * No. 2
proliferation stimulated by lung-conditioned media may be due to the transient nature of the differentiating effect. After 36 hours, it was no longer apparent. Taken together our results indicate that (1) preferential metastasis to lymph nodes occurs because the lymph node is an easier place than lung to establish a metastatic focus, not because there are subpopulations of cells within the primary tumor that metastasize to lymph node specifically, and (2) increased adhesive properties, rather than trophic factors, are what make the lymph node a congenial site for metastases. Nevertheless, tumor cells metastatic to lymph nodes could still have a less malignant phenotype than cells metastatic to visceral sites because some cells could establish lymph node colonies despite a limited ability to metastasize elsewhere. We did find that B16 melanoma cells derived from spontaneous lymph node metastases were less adherent to their target tissue and were transiently more susceptible to a differentiating effect of another organ's local milieu than were the cells derived from spontaneous pulmonary metastases. Such findings help to explain the clinical observation that the appearance of metastatic lesions in viscera signifies widespread disease whereas the appearance of metastatic deposits in the localregional lymph nodes may not. Acknowledgments
6.
7. 8.
9.
10. 11.
12. 13. 14. 15.
16.
The authors thank Dr. G. Tom Shires for his encouragement and support.
17.
References
18.
1. Poste G, Fidler IJ. The pathogenesis of cancer metastasis. Nature
1980; 283:139-146. 2. Weiss L. Metastatic inefficiency. In Liotta LA, Hart IR, eds. Tumor Invasion and Metastasis. The Hague: Martinus Nijhoff, 1982, pp 81-98. 3. Nicolson GL, Belloni PN, Tressler RJ, et al. Adhesive, invasive and growth properties of selected metastatic variants of a murine large cell lymphoma. Invasion Metastasis 1989; 9:102-116. 4. Netland P, Zetter BR. Organ specific adhesion of metastatic tumor cells in vitro. Science 1984; 224:113-114. 5. Hart IR, Fidler IJ. Role of organ selectivity in the determination of
19. 20.
21.
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metastatic patterns ofB 16 melanoma. Cancer Res 1980; 40:2281 2287. Denizot F, Rita L. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986; 89:271-277. Houghton AN, Real FX, Davis LI, et al. Phenotypic heterogeneity of melanoma: relation to the differentiation program of melanoma cells. J Exp Med 1987; 164:812-829. Nowell PC. The clonal evolution oftumor cell populations. Science 1976; 194:23-28. Gorelick E, Fogel M, De Baetselier P, et al. Immunobiological diversity of metastatic cells. In Liotta LA, Hart IR, eds. Tumor Invasion and Metastasis. The Hague: Martinus Nijhoff, 1982, pp 133-146. Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells within a malignant tumor. Science 1977; 197:893-895. Nicolson GL, Winkelhake JL. Organ specificity of blood-bourne tumour metastasis determined by cell adhesion? Nature 1975; 255:230-232. Fidler IJ, Hart IR. Biological diversity in metastatic neoplasms: origins and implications. Science 1982; 217:998-1003. Fisher B, Fisher ER. The interrelationship of of hematogenous and lymphatic tumor cell dissemination. Surg Gynecol Obstet 1966; 122:791-798. Netland P, Zetter BR. Metastatic potential of B16 melanoma cells after in vitro selection for organ-specific adherence. J Cell Biol 1985; 101:720-724. Kurokawa Y. Experiments on lymph node metastasis by intralymphatic inoculation of rat ascites tumor cells, with special reference to lodgement, passage, and growth of tumor cells in lymph nodes. Gann. 1970; 61:461-471. Rice GE, Bevilacqua M. An inducible endothelial cell surface glyocoprotein mediates melanoma adhesion. Science 1989; 246: 1303-1306. Gallatin WM, Weissman IL, Butcher EC. A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature 1983; 304:30-34. Holzmann B, Weissman IL. Integrin molecules involved in lymphocyte homing to Peyer's patches. Immunol Rev 1989; 108: 45-61. Siegelman MH, Van De Rijn M, Weissman IL. Mouse lymph node homing receptor cDNA clone encodes a glycoprotein revealing tandem interaction domains. Science 1989; 243:1165-1172. Cavanaugh PG, Nicolson GL. Purification and some properties of a lung-derived growth factor that differentially stimulates the growth of tumor cells metastatic to lung. Cancer Res 1989; 49(14): 3928-3933. Sargent NS, Oestreicher M, Haidvogl H, et al. Growth regulation of cancer metastases by their host organ. Proc Natl Acad Sci USA 1988; 85:7251-7255.
GILES F. WHALEN, M.D., and SETAREH F. SHARIF, PH.D.
Metastases from solid tumors to lymph nodes do not portend as poor a prognosis as metastases to other sites. The authors wished to determine whether specific subpopulations of cells metastasized to lymph nodes and whether they have different properties than cells metastatic to visceral sites. Repetitive selection for "spontaneous" metastases of a B16 melanoma to either lung or lymph node increased the incidence of lymph node metastases. Cells derived from pulmonary and lymph node metastases were assayed for their ability to adhere to cryostat sections of lung and lymph node and respond to target organ-conditioned media in serum-free conditions. Both cell types were four times more adherent to lymph node than lung, and consistently attached to the hilar and subcapsular sinuses. Attachment of cells derived from pulmonary metastases to either tissue was threefold greater than that of cells derived from nodal metastases. Lung-conditioned media stimulated proliferation of both cell types, and transiently induced differentiated morphology in cells derived from lymph node metastases, but not in cells from pulmonary metastases. Neither effect was found in lymph-node-conditioned medium. These results suggest that cells metastasize to lymph nodes preferentially not because of a specific predilection for lymph node, but because it is an easy site to colonize. Adhesive interactions in the lymph node rather than trophic ones appear to account for this effect. Cells metastatic to lymph node may be less "malignant" than cells metastatic to visceral sites because less has been required for them to succeed as a metastatic focus.
n M j r ETASTASIS, THE PROCESS by which cells from a primary tumor establish secondary colonies at distant sites, may be described as a series of steps. Tumor cells must separate from the primary tumor mass, invade the microvasculature oftheir tissue of origin, survive in the circulation, reattach to endothelium in another organ, invade into the surrounding stroma of that organ, and finally proliferate in the new site. ' This process is biologically inefficient,2 probably because a cell thwarted Address reprint requests to Giles F. Whalen, M.D., F1906, New York Hospital-Cornell Medical Center, 525 East 68th St., New York, NY
10021. Accepted for publication July 29, 1991.
166
From the Department of Surgery, Surgical Oncology Research Laboratory, Cornell University Medical College, New York, New York
at any point cannot establish a metastatic focus. The process does not appear to be random, however. Evidence that metastases do not occur randomly is derived from a variety of clinical experiences with typical but anatomically discontinuous patterns of spread of common tumors (e.g., melanoma metastatic to intestinal submucosa), and from experiments with transplantable tumor models. Such experiments suggest that although host response modulates the site of metastasis, subpopulations of cells within the primary tumor have predilections for specific organs.35 Local-regional lymph nodes are a common site of metastasis in many clinically encountered carcinomas. Metastatic disease in the local-regional lymph nodes is a poor prognostic sign, but it is generally not so ominous as metastases to visceral organs. This difference in prognosis is usually thought to be due to differences between lymphatic and a more widespread hematogenous dissemination, or to a stepwise failure of host anti-tumor response, with local nodal defenses failing first. An alternative explanation is that specific subpopulations of cells succeed as lymph node metastases and that these are less malignant than cells that succeed as metastases to visceral organs. To test this hypothesis in vivo, we used the B16 mouse melanoma, a transplantable tumor model commonly employed in studies of organ-specific metastasis. We also used in vitro assays of organ-specific attachment and proliferation to investigate whether cells derived from lymph node metastases differ from cells derived from metastases to a visceral organ (lung) in their relative abilities to accomplish two important steps in a site-specific metastatic process: adhesion to their target organ, and growth in that target organ.
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METASTASIS OF SOLID TUMORS TO LYMPH NODES
Materials and Methods Selection of Tumor Cells The hypothesis that lymph nodes are the metastatic site of specific subpopulations of tumor cells was tested by selecting for either lung or lymph node metastases in vivo. We reasoned that metastases to a desired site would be increased by repetitive harvest and reinoculation of tumor cells from that site, if the major determinant of metastatic site was the predilection ofcertain cells for that site. The metastatic B 16-F 10 melanoma variant (provided by Dr. Bruce Zetter, Harvard Medical School, Boston, MA) was used to begin these experiments. Repetitive selection was performed as follows: A cell suspension (106cells/0. 1 mL medium) was inoculated into the web space of the right hind paw of 8- to 10-week-old C57BL/ 6J mice (Charles River Laboratories, Wilmington, MA). When the primary tumor was obvious (3 to 5 mm3), the foot was amputated. Mice were killed 4 to 6 weeks later. Cells from pulmonary metastases of animals without popliteal or inguinal lymph node lesions were harvested, expanded in culture, and reinjected into another set of mice. Similarly, cells from regional lymph node metastases in animals without pulmonary lesions were harvested, expanded, and reinjected into a new set of mice. Cell lines established from the third generation of pulmonary metastases (S3E) and from the third generation of lymph node metastases (S3C) were used in in vitro assays. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee. Cell Culture
Melanoma cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated calf serum (CS) (Hyclone Laboratories, Logan, UT), penicillin 100 IU/mL, and streptomycin 100 ,ug/mL (Irvine Scientific, Santa Ana, CA) at 37 C in 5% CO2. 3T3 cells were grown and maintained under the same conditions except that medium was supplemented with heat-inactivated fetal bovine serum. Adhesion
Organ-specific adhesion was assayed on fresh cryostat sections (4 ,m thick) of mouse lung and lymph node in the following manner: Frozen sections of each tissue were individually mounted on glass slides, encircled with a wax pencil (2 cm diameter), and covered with a 0. l-mL drop of a cell suspension. The cell suspension was prepared by trypsinizing confluent cells (0.05% Trypsin, 0.53 mmol/ L ethylenediaminetetra-acetic acid [EDTA]-Gibco). Washed cells were finally suspended in DMEM with 1% bovine serum albumin (1% BSA) (Sigma Chemical Co.,
167
St Louis, Mo.) at 5 X 105 cells/0.1 mL. Tissue sections covered with the appropriate cell suspension were incubated for 40 minutes at 5 C while being gently agitated on a rotating platform (7 cycles/minute), then rinsed in cold phosphate-buffered saline (PBS) to remove nonadherent cells, fixed with 1% gluteraldehyde, and stained with hematoxylin and eosin. Slides were analyzed by counting cells adherent to recognizable tissue in six random high-power fields (400X) per slide, and averaging this value for an individual slide. At least three slides for each cell/tissue combination were prepared in every experiment.
Proliferation The influence of local organ milieu on cells derived from different metastatic sites was assayed in a system designed to detect either stimulation or inhibition of proliferation on a matrix that had been deposited by the parental melanoma line. Matrix-coated plates were prepared as follows: 2 X 104 cells suspended in DMEM and 10% CS were plated into each well of a 96-well plate, allowed to grow for 36 hours, and removed with trypsin. The plates were washed with DMEM-10% CS and then twice with PBS. Test cells were plated in serum-free conditions on these plates (5 X 103 cells per well). Because of the presence of extracellular matrix, cells attached and spread within 2 hours, allowing assays of proliferation rather than indirect assays of attachment. After 4 hours, organ-conditioned medium was added to the wells and the plates were incubated at 37 C for 48 hours. Calf serum (10% final concentration) was used as a positive control. Cell number was determined by a described colorimetric assay that depends on the reduction of tetrazolium salt by living cells.6 Optical density correlated with cell number (r = 0.85) enough to use this colorimetric assay as an initial screen. Positive findings were confirmed by counting cells in triplicate wells on a Coulter counter (Coulter Electronics, Hialeah, FL) in repeated experiments. Differentiation was assessed in the same cell culture system; tests cells were plated in serum-free media on matrix-coated wells and examined at different time points after the addition of conditioned media. More stellate (as opposed to spindle) cell shape and increased pigmentation were judged to represent a greater degree of differentiation of the melanoma cells. These morphologic criteria have been shown to correlate with the presence of cell surface antigens found on mature, as opposed to fetal, melanocytes.7 Preparation of Conditioned Media The lungs and peripheral lymph nodes were removed from mice and extraneous tissue was trimmed. Remaining lung and lymph node was minced to 1- to 3-mm3 pieces and washed several times with PBS to remove blood and
debris. The tissue homogenate was placed into culture cells with medium (100 mL DMEM/ 15 mg tissue), and incubated at 37°C for 24 hours. Aliquots were removed at varying time intervals, centrifuged at 14,000g for 10 minutes, and the supernatant filter sterilized (22-,um pore size). This material then was stored at -80 C until used in the assays. In early experiments, 1 -mL aliquots were collected over a 24-hour period. Because peak mitogenic activity occurred in the first 4 hours, media was collected after 6 hours of conditioning in subsequent experiments. In addition, this material was dialysed (2000 molecular weight cut off) against unconditioned media for 12 hours before storage to restore small molecules depleted in the conditioning process.
Results In Vivo Selection for Site-specific Metastasis Selection for either lung or lymph node metastasis increased the incidence of metastases in general. Lymph node metastases were increased preferentially and equally by selection for either site. (Table 1)
Adhesion The cells derived from spontaneous pulmonary metastases (N3E) and the cells derived from spontaneous localregional lymph node metastases (S3C) both adhered to target organ tissue sections more avidly than control cells (3T3), and adhered four times more avidly to sections of lymph node than to sections of lung (Table 2). S3E (lung metastases) cells were threefold more adherent to tissue sections of either lung or lymph node than were S3C (lymph node metastases) cells. Cells attached to lymph node sections were predominantly localized to hilar, subcapsular, and medullary sinuses around germinal centers. This anatomic localization on lymph node sections oc-
TABLE 1. In Vivo Selection for Site ofSpontaneous Metastasis in B16 Melanoma
Selection
Lung Metastases
Lymph Node Metastases
Passage
Lung (%)
Lymph Node (%)
Lung(%)
Lymph Node (%)
1
5/19 (26) 4/41 (10)
4/19 (21) 13/41 (32)
5/19 (26) 1/13 (8)
4/19 (21) 4/13 (31)
3/9 (33) 6/41 (15)
5/9 (55) 25/41 (61)
3/9 (33) 2/10 (20)
5/9 (55) 6/10 (60)
2 3 4
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There is a progressive increase in the percentage of mice with lymph node metastases regardless of whether lung metastases or lymph node metastases are repetitively selected (as described in Methods). Numerator represents number of mice with metastases to that site. Denominator represents total number of mice in that group.
TABLE 2. Adhesion of BJ6 Melanoma Cells Derived From Pulmonary (S3E) and Lymph Node (S3C) Metastases to Cryostat Sections of Their Target Organs
Cell Type
Cells/Lung Sections
Cells/Lymph Node Sections
S3E S3C 3T3
13.14 ± 2.31 4.74 ± 1.13 0.89 ± 0.21
99.13 ± 29.58 22.40 ± 9.69 1.76 ± 1.23
3T3 cells were used as controls. Both metastatic cell lines were more adherent to lymph node sections than to lung (p = 0.005), and the S3E line was more adherent to both tissues than the S3C line. Cells were counted in six random high-power fields (X400 per slide to obtain an average number of cells per field on that slide, three slides per group in each experiment. Mean data from three experiments presented ± SEM.
curred with both cell types. No specific anatomic pattern of attachment was apparent on the lung sections (Fig. 1). Proliferation Maximal proliferation stimulated by lymph-nodeconditioned medium was no greater than that stimulated by 10% CS (positive control) for either cell type. Lungconditioned media was more mitogenic. Initial experiments indicated that the peak mitogenic activity of lungconditioned medium (181.5% ± 46.6%; result expressed as a percentage of 10% CS-stimulated proliferation, in three separate experiments) occurred within the first 6 hours of conditioning. Subsequent experiments, employing pooled media from the first 6 hours of conditioning, showed the mitogenic effect of lung-conditioned medium to be dose dependent (Fig. 2). A more differentiated appearance of the melanocytes was serendipitously noted during assays of proliferation with lung-conditioned medium. The phenomenon occurred 12 to 24 hours after the conditioned medium was added to the wells and was lost by 48 hours (when proliferation was assayed). This effect of lung-conditioned medium was prominent on the lymph node metastasisderived S3C cells, not the lung metastasis-derived S3E cells, and was dose dependent (Fig. 3). Lymph node-conditioned medium did not induce this differentiated appearance in either cell type. Discussion The transformed cells that make up a primary malignant tumor are a heterogeneous population,7-9 varying also in their metastatic potential and propensity to metastasize to specific sites.5"'0"' It has been suggested that tumors progress and spread because selective pressures favor cells with a greater capacity to metastasize.8"2 Lymphatic spread to local-regional lymph nodes is common. Experiments that demonstrate rapid appearance of tumor cells in the circulation after inoculation into afferent lymphatic channels, however, suggest that traditional dis-
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FIG. 1. Adhesion of metastatic B16 melanoma cells to cryostat sections of (A) lung; X237, scale bar 20 gm, and (B) peripheral lymph node; X73, scale bar 70 Mm. (C) Magnified (X237) view of attachment to lymph node section, scale bar 20 Mm. Arrows identify representative tumor cells that are in a slightly different focal plane than the tissue sections. Cells preferentially attached to subcapsular, hilar, and medullary sinuses in the lymph nodes.
tinctions between hematogenous and lymphatic spread may be artificial." '3 We therefore wondered whether subpopulations ofcells specifically metastasized to the lymph nodes and whether they were different from cells that metastasized elsewhere. Selection for lymph-node-metastasizing cells in vivo increased the incidence of lymph node metastases, but this result did not indicate that these cell have a predilection for lymph node specifically. Rather, equivalent increases in lymph node metastases after selection for lungmetastasizing cells indicated that the lymph node is simply an easier place to colonize than the lung, regardless of what predilections for other sites a cell might have. Assays measuring the ability of cloned tumor cells to adhere to specific tissues in vitro have generally correlated with the pattern of metastasis when those cells were inoculated intravenously." ", 4 We found that attachment in vitro also correlated with spontaneous metastases in vivo. Cells derived from spontaneous lymph node metastases adhered avidly to lymph node sections in vitro. Again, it
1 000
x
Cell Number
20 18 16 14 12 1 0
8 6 4 2 0
0
0.06 0.66 6.6 13.3 16.6 33.3 66.6 % concentration of conditioned medium
FIG. 2. Proliferation of B16 melanoma cells derived from pulmonary (U - *), and lymph node (E - O) metastases in response to increasing concentrations of lung-conditioned medium.
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WHALEN AND SHARIF
Ann. Surg. * February 1992
FIG. 3. Differentiation of B16 melanoma cells derived from
lymph node metastases by increasing concentrations of lung-conditioned medium.
Treated cells showed increased pigmentation and dendritic morphologic features. Phase contrast micrographs of cells treated with (A) 13% conditioned medium, (B) 30% conditioned medium, (C) 10% CS, and (D) unconditioned DMEM without CS.
is unlikely that such attachment represented a specific attribute of the cells because cells derived from pulmonary metastases adhered to lymph node sections even more avidly. An important finding of these studies was the consistent attachment of both cell types to hilar and subcapsular sinus tissues on the lymph node sections, which correlates with the known sites of initial tumor cell arrest in vivo"5 and suggests that there is a concentration of adhesion molecules in these locations. Increased amounts of such molecules within the lymph nodes might explain the preferential adhesion (fourfold) to lymph node over lung that we observed for both cell types. Lymphokines such as tumor necrosis factor-alpha, which might be expected to be concentrated in lymph nodes, induce the appearance of at least one adhesion molecule (INCAM- 110) that can mediate melanoma attachment to endothelial cells.'6 Receptors on specialized venules within the lymph nodes mediating lymphocyte homing also might be able to mediate tumor cell attachment.'7-'9 Adherent cells also must thrive in their new environment. Preferential stimulation of site-specific metastasizing cells by trophic factors derived from their target organs has been reported for both lung and liver metastasizing tumors.20'2' We assessed the relative abilities of cells
derived from different metastatic sites to thrive in the milieu of their target organ by measuring their proliferative response to media conditioned with homogenates of that organ. Our experiments suggested that diffusible trophic factors in the lymph nodes do not mediate site-specific lymph node metastases because neither cell line was particularly stimulated by lymph-node-conditioned media. In contrast, lung-conditioned media had a dose-dependent mitogenic effect on both cell types. The trophic factor in our lung-conditioned media appear to be a molecule larger than 2000 kd because the dose-dependent effect survived dialysis with this pore size tubing. Purification of a tumorstimulating, lung-derived growth factor has resulted in identification of an approximately 66-kd glycoprotein that is not inhibited by antibodies against platelet-derived growth factor, epidermal growth factor, insulin-like growth factor 1, or granulocyte macrophage colony-stimulating factor.20 The dose-dependent differentiating effect of lungconditioned media on cells derived from lymph node metastases also remained intact after dialysis against unconditioned medium. Thus, the differentiating effect does not appear to be the result of nutrient depletion of the conditioned media. Generally, differentiation results in a slower rate ofproliferation. In these experiments, the lack of difference between the two cell lines in the amount of
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proliferation stimulated by lung-conditioned media may be due to the transient nature of the differentiating effect. After 36 hours, it was no longer apparent. Taken together our results indicate that (1) preferential metastasis to lymph nodes occurs because the lymph node is an easier place than lung to establish a metastatic focus, not because there are subpopulations of cells within the primary tumor that metastasize to lymph node specifically, and (2) increased adhesive properties, rather than trophic factors, are what make the lymph node a congenial site for metastases. Nevertheless, tumor cells metastatic to lymph nodes could still have a less malignant phenotype than cells metastatic to visceral sites because some cells could establish lymph node colonies despite a limited ability to metastasize elsewhere. We did find that B16 melanoma cells derived from spontaneous lymph node metastases were less adherent to their target tissue and were transiently more susceptible to a differentiating effect of another organ's local milieu than were the cells derived from spontaneous pulmonary metastases. Such findings help to explain the clinical observation that the appearance of metastatic lesions in viscera signifies widespread disease whereas the appearance of metastatic deposits in the localregional lymph nodes may not. Acknowledgments
6.
7. 8.
9.
10. 11.
12. 13. 14. 15.
16.
The authors thank Dr. G. Tom Shires for his encouragement and support.
17.
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