4 B16 Murine Melanoma

Historical Perspective on the Development

ofa Solid Tumor Model

Enrique Alvarez, DVM, MA

CONTENTS
INTRODUCTION
HISTORICAL CONTEXT
B16 MELANOMA
CONCLUSIONS
REFERENCES

The determination of weight of a factor in producing metastases can not be judged

from single experiences on man, as it is impossible to eliminate conflicting conditions.
Only by the use of a homogeneous material which the size of the cells, their histologi-
cal and biological qualities, and the vascularity of the surrounding tissue, etc., are
practically constant can valid conclusions be drawn, and this elimination of variables
is possible to obtain only by the use of animal tumors of a long transplanted strain, so
that the morphological and biological characters are well known.
Dr. Leila C. Knox, 1922 (1)

1. INTRODUCTION
The development of reliable models of disease mechanisms largely depends on our
understanding of the characteristic processes of the disease being modeled. Syngeneic
tumors in mice offer an important model for oncology research. Although the use of
murine models of neoplastic disease has been raised to the level of a fundamental para-
digm in oncology research, it is to be considered with all the care and diligence
afforded to us by any biological model. Syngeneic murine tumors offer potential
advantages as well as limitations, which should always be present in the mind of the
investigator. Every year, countless studies are published describing new models and/or
new cell lines available to scientists. Many of these are highly specialized, and have a
narrow application to the broad community. Through time, several models are devel-
oped, which present us with a newer tool to truly advance our understanding. In many
instances, these models fill a specific unmet need. If fortunate enough, the model is

From: Tumor Models in Cancer Research

Edited by: B. A. Teicher© Humana Press Inc., Totowa, NJ
73
74 Alvarez

also relatively easy to reproduce, thus providing for rapid dissemination among the
community. The overall relative importance of an experimental model depends largely
on two important aspects. First, how does the model recapitulate the process it attempts
to emulate? Second, how does the model itself offer the flexibility to expand our
knowledge relating to the pathologic process being evaluated? This chapter pinpoints
how the B 16 murine melanoma line has proven itself a valuable model on both points.
This tumor line provides researchers with the ability to model the process of solid-
tumor formation and the following metastatic process seen in animals and man. Upon
establishment of this model of metastasis, a more detailed understanding of the steps
involved in tumor dissemination was gained. Astute manipulation of the cell line and a
rational use of experimental animal models were essential for this process. Overall, this
effort has helped the modem description of fundamental processes in metastasis, inva-
sion, and anti-tumor drug development. The body of knowledge derived from the B 16
murine melanoma line is ample, and even at present continues to grow. The emphasis
of this overview is limited to its origin and to the seminal work attributable to the early
work done with this tumor line and some of the tumor models that have arisen from it.
In the span of biological research, this model is relatively new. But within one lifetime,
it has helped shape our understanding of oncology. In science, many posed questions
are old and numerous, but the tools we need to thoroughly explore them and ask new
ones are in continuous evolution. These new tools encourage even more questions. The
B 16 melanoma is one of those tools.

2. HISTORICAL CONTEXT
The process of scientific discovery never occurs outside the context of contemporary
knowledge. Contemporary knowledge is relative to the time of the work itself. In the
early 1970s, at the time of the establishment of the B16 as a model for metastasis
research, the state of metastasis research from a technical standpoint was immature
when compared to today's standards. Although many recent developments in the area
of metastasis have depended on the use of relatively recent technological advances
(e.g., molecular biology, protein chemistry, and bioinformatics), many questions
regarding the nature of metastasis are very old. Up to the early 1970s, there had been
numerous qualitative (i.e., autopsies) and some quantitative studies (i.e., rodent mod-
els) relating to the natural history of the metastatic process, but a readily accessible
murine model was lacking-specifically, a model that would offer a predictable
metastatic pattern.
A periodic survey of the historical record serves to focus our attention to the correct
context of the research. Within this context, the significance of the questions asked
becomes clearer, as well as the importance of the techniques being used. The develop-
ment and current use of the B 16 melanoma line should be incorporated into a larger
scope of oncology research by looking at a previous generation of researchers and their
professional contributions to the field.
An often-quoted work by Dr. Stephen Paget was seminal in establishing our appreci-
ation for the complexity of the processes involved in metastasis (2). The concept of
Seed and Soil, as it applies to the tumor embolus and its potential site of invasion, is
firmly entrenched in the minds of researchers in the field of metastasis. Simple, allegor-
ical, yet effective at setting the concepts of a complicated pathological process, the idea
Chapter 4 I B16 Murine Melanoma 75

of seed and soil has been fundamental for over 110 years. The careful evaluation of
human autopsies derived from breast-cancer patients an obvious nonrandom pattern of
metastasis, noted by Paget, demonstrated that breast-cancer patients had a notable pre-
disposition for bony metastasis. This bony metastasis was nonrandom in its distribu-
tion, since, as Dr. Paget commented: "Who has ever seen the bones of the hands or the
feet attacked by secondary cancer?" This observation is critical in two important
aspects. First, the bony tissue clearly presents the tumor with a preferred invasion site
(soil to the seed). Second, bones are not just bones; there appears to be further discrim-
ination by the tumor embolus as to which bone offers an optimal site for colonization.
Effective tools, such as animal models that could be used to experimentally understand
the human condition, were not yet available to Paget. It would still be many years until
such specific disease models were widely available for use. Our contemporary descrip-
tion of the specific relationship between the cell and its host tissue uses the term
"tumor microenvironment" to essentially name the same phenomena described in
1889. Successful tumor metastasis is a complex, nonrandom, multifactorial event that
requires contributing components from both the cancer cell and its host.
As with many areas in science, in oncology we also find a periodic re-evaluation of
fundamental themes. The "Seed and Soil" concept has not been immune to this effect.
In 1982, Hart specifically reflected on the mechanisms of metastasis as they apply to
murine models (3). This work reiterated the clear importance of circulation and physi-
cal-cell distribution on the outcome of a metastatic event. Hart concluded:
Patterns of metastasis primarily appear to be a direct consequence of the delivery of
an optimal dose of tumor cells to the first organ encountered along the lymphatic or
venous pathway. Nonetheless, the very same tumors that use this mechanism as a pre-
dominant mode of spread will, on other occasions, exhibit true organ tropism.
This conclusion did not exclude the already noted metastatic preference of tumors to
certain organs, but gave a stronger importance to circulatory parameters in the final dis-
position of tumor metastasis. This attempt at revisionism occurred almost 100 years
after the publication by Paget. What had transpired in that interval to call for the re-
evaluation of the concept?
In the first half of the 1900s, there was a general effort to describe the nature of cir-
culating tumor cells in man and rodent models. In 1913, while studying a carcinoma
model of the Japanese waltzing mouse, Tyzzer found a correlation between the tumor
size, duration of tumor presence, peculiar conditions furnished by the host, and the
number of metastases (4 ). These important clinically applicable correlations were
being laid down by scientists using syngeneic models. In 1915, Iwasaki presented a
paper, which when carefully read can serve to introduce today's researcher to many of
the important areas of current interest (5). By using microscopy to describe the tumor
embolus-host interaction in necropsy cases, Iwasaki finely described the disposition of
several tumor types in the vessels of patients. Most of the work presents the reader with
a description of the relationship between the tumor embolus, thrombus formation/orga-
nization, and the formation of tumor metastasis. A particular description of tumor-ves-
sel interaction by Iwasaki is of particular interest today:
The tumour cell mass is here covered by a thin layer of endothelium continuous with
the intima. Though a small number of leucocytes may be found between the tumour
cells, the latter appeared to be normal: no sign of degradation can be seen. Some con-
76 Alvarez

nective-tissues fibres from the wall, as well as cells from the covering endothelium,
are thrust into the mass: these serve as stroma. I consider them as the first stage of
metastases formation; at the same time, I consider that the penetration of the wall by
the tumor cells from inside can sometimes occur without such an endothelial cover.
Eighty-five years ago we find an accurate description of the process of extravasation
by a metastatic cell, while at the same time describing a relationship between tumor
cells and endothelium. In the same paper Iwasaki also studied the fate of intravenous
(iv)-injected tumor cells in rats and mice.
In the cases of mouse carcinoma I again observed the appearances of which I have
described in cases of human sarcoma, that is, the tumour cell group on a vessel wall,
having a single endothelial layer directly covering it. The carcinoma cells of such
fixed emboli appear to be in healthy condition in every respect, and in the cases where
connective tissue bundles thrust into and divide them up, they have the common
appearance of alveolar carcinoma and are not to be considered as degenerating or
organizing at all.
This statement serves to offer initial validation of the use of murine models from a
comparative and histological perspective. When comparing his own tumor implanta-
tion results with that of fellow researchers (who obtained a lower tumor take-rate than
himself) Iwasaki concluded:
From my results I hold that it is not difficult to cause tumours by inoculating foci
directly into vessels. Nevertheless, all tumor cells introduced into blood vessels do not
necessarily disintegrate, as several authors believe, but if the technique is suitable, the
lung will be attacked in a very considerable number of cases.
Iwasaki's work was important in describing the lung as the preferred site of tumor
takes after iv injection. Much work followed to determine the reasons for this apparent
affinity.
Warren and Gates clearly demonstrated that the successful establishment of tumors
in rodent lungs after iv injection is strongly correlated with the cell viability of the sam-
ple being used (6). At that time, the authors were attempting to standardize the proce-
dure of iv tumor-cell implantation. This was demonstrated using the Walker 256
carcinoma line in rats. Cloudman in 1947 studied the-as they were formerly called-
organophilic tendencies of murine hepatic tumors (7). At that time, the author specifi-
cally looked at the dissemination patterns of two tumor lines (C954 liver carcinoma
and C198 reticuloendothelioma). The work employed the use of subcutaneous (SC)
implantations using a trochar as well as the use of parabiosis in mice. Interestingly,
Cloudman's method of tumor passage may have contributed to the organophilic ten-
dencies noted. The mode of tumor maintenance for the C198 line required that tumor-
affected pieces of liver from mice be SC-implanted in naive hosts. This SC tumor
would lead to disseminated disease in the mice. When the initial line was being estab-
lished, simple SC implantation led to poor tumor-take. But the hepatic metastatic
spread of the tumor offered an optimal tissue for reproducibly metastatic tumor when-
ever this tissue was SC-implanted. In the case of the C954 tumor line, it was main-
tained via simple SC passage. The C954 had no metastatic properties. Retrospectively,
it appears that Cloudman unintentionally selected for a tumor-line variant by only
using the tumor stock from metastatic sites. The research compared the metastatic
Chapter 4 I Bl6 Murine Melanoma 77

potential of the C 198 line vs the nonmetastatic C954 line. In addition to a direct com-
parison between both tumor lines, by using various mouse strains, there was a demon-
stration of the different metastatic patterns (organophilic tendencies) attributable to the
mouse strain being used. This led to the conclusion:
The appearance of tumor metastasis within a specific internal organ is probably
dependent upon a host-tumor interrelationship rather than upon either the tumor type
or the host type alone.
This is an important holistic approach to in vivo modeling. But strictly speaking, it
was probably not a comparison of equal lines. The successful establishment of the
tumor is attributed to both the tumor cell and the selected host.
In 1949, Coman et al. presented work, which in the context of Paget's hypothesis,
discussed the apparent inability of the V2 rabbit carcinoma to metastasize to muscle
(8). By injecting tumor cells into the arterial circulation and demonstrating tumor
growth in the muscle mass, the authors confirmed the ability of the tumor to recognize
the tissue as favorable. In their conclusion, the reason for the apparent lack of tumors in
certain organs was the result of a filtering effect by the lungs.
In 1950 and 1952, Zeidman studied important parameters for the use of a murine
tumor line in vivo at the University of Pennsylvania (9,10). Zeidman specifically stud-
ied the relationship between the number of viable tumor emboli iv-implanted and the
total number of resulting metastatic nodules. In 1950, using the mouse Sarcoma 241
line, Zeidman concluded:
That the number of metastases is directly proportional to the number of viable tumor
cell emboli released into the circulation. The longer a primary tumor existed the
greater the number of emboli released, as judged by the number of metastases
appearing in the lungs (9 ).
In his studies, the author also compared two rabbit tumor lines (V2 squamous-cell
carcinoma (SCC), Brown-Pearce carcinoma) and one rat tumor line (Walker 256 carci-
noma). One matter that still had to be resolved regarding iv injections of tumor cells
related to the pulmonary disposition of tumor cells. Were the lungs acting as a mere
sieve? To test this hypothesis, rabbits received an iv injection of tumor cells, while aor-
tic outflow was captured. This collected blood was then iv-injected into a naive host;
this second animal was then followed for tumor formation. Tumor formation in the sec-
ond hosts demonstrated that tumor cells successfully passed through the pulmonary
circulatory system. All three tumors were able to pass through the pulmonary vascula-
ture and form tumors in the second host, thus negating the pulmonary sieve hypothesis.
In 1961, Zeidman used microcinematic techniques to further evaluate the flow pat-
terns of tumor cells in circulation ( 11 ). In this work, he demonstrated the higher degree
of membrane flexibility/deformability of the Brown-Pearce tumor vs the V2 tumors.
This work was done by studying the vascular patterns in the mesenteric vessels of rab-
bits. It illustrated how the Brown-Pearce cells, upon reaching an arteriolo-capillary
junction, could deform and pass more freely than the V2 cells. This added to the body
of evidence for the tumor's enhanced ability for transpulmonary passage, thus high-
lighting an intrinsic cellular difference between both lines.
Descriptive pathology and in vivo work, as many of the works presented here have
been, continue to be essential for the development of our understanding of metastasis.
78 Alvarez

Even at the present time, in the era of genomics an attempt to elucidate the underlying
biological forces involved in the dissemination of prostatic carcinoma required the
careful evaluation of autopsies over 19,000 patients (12). Approximately 1,500 cases of
prostatic carcinoma were identified in this cohort. Of those cases, 35% showed evi-
dence of metastatic spread. Interestingly, over 100 years after the seminal work of Dr.
Paget, this paper offers an attempt to describe in detail the common metastatic patterns
of dissemination found in prostatic carcinoma patients using autopsy records.

3. B16 MELANOMA
In the context of the Seed-Soil hypothesis, the use of the B 16 murine melanoma line
as a model for both solid-tumor formation and metastasis was an important develop-
ment in oncology research. Although the introduction of this model for metastasis
research can be traced to 1970, the cell line itself had been identified and characterized
as a tumorigenic line years before. The B 16 murine melanoma cell line originated in
1954. The tumor spontaneously arose in a C57BL/6J mouse at the Jackson Laborato-
ries in Maine. There is no record of the sex of the originating mouse. The initial neo-
plastic lesion arose in the skin at the base of the ear. The following is a histological
description of the tumor from the Handbook on Genetically Standardized lax Mice by
Dr. Earl Green:
Gross: soft gray tissue, frequently hemorrhagic. Microscopic: tumor cells polyhedral
or spindle-shaped, arranged in perivascular mantles and diffuse masses; some cells
contain fine pigmented granules, a few are obscured by large, very dark globules of
pigment; stoma delicate and vascular. Pigment greatly decreased in comparison with
early-transplant generation (13).
This offers the first histological description of the tumor line in its host. The tumor
line was maintained at the Jackson Laboratories by continuous passage in vivo. At the
time of the report, the tumor is described as metastatic to lung, liver, and spleen. This
metastatic pattern was present after sc implantation.
Using the B16 melanoma line from the Jackson Laboratories, Dr. Isaiah Fidler care-
fully documented the final disposition of the melanoma cells after iv injection in mice in
1970 (14). A number of protocols used before this time to aid in the understanding of the
metastatic process proved to be either cumbersome, unreliable, or both. There was an
unmet need for a mouse line, and this line offered an important opportunity for the field.
In the 1970 study, B16 murine melanoma cells were initially cultured in vitro. The cells
used for iv injection were labeled with 125I-5-iodo-2-deoxyuridine. This radioactive label
provided a clear and specific way to monitor the organs in which tumor cells arrested.
This ability to monitor cells was specifically caused by the affinity of the radioactive label
for viable cells. Cell death would lead to the excretion of the label from the animal, thus
precluding the possibility of labeling the host's tissues. The iv injection of killed labeled
B 16 cells served to demonstrate the inability of the host to reutilize the radioactive label,
thus preventing a false-positive reading. In vitro, 200,000 labeled cells would produce an
average of 40,000 counts per min. After inoculating the cells into naive hosts, the number
of viable tumor cells in each organ was determined from radioactivity count, by the use of
the ratio of cpms to cells in the original inoculum. Simple and efficient, the labeling of
B 16 murine melanoma cells could now open the door for the description of a complex
tumor-host interaction. The B16 melanoma cells could now be iv-implanted and followed
Chapter 4/ Bl6 Murine Melanoma 79

Table 1
Fate of 12 5IUDR-Labeled Tumor Emboli Organ Distribution o£200,000 125IUDR-Labeled
Melanoma Cells Injected Intravenously into C57BU6J Mice
Number of cells*
Time of death Lung Liver Spleen Kidney Blood+ Urine++
1 min 136,750 2,230 200 300 3,750
2min 128,500 5,500 230 270 1,590
5min 106,700 7,600 260 270 2,200
7 min 103,500 7,570 280 290 2,300
10min 130,500 9,350 270 310 2,600 100
15 min 122,800 8,390 310 310 3,340 350
30min 117,900 4,260 250 370 3,500 4,300
45 min 105,000 4,140 390 460 3,900 4,100
1h 100,550 3,570 330 370 3,800 10,000
2h 89,590 5,830 680 530 4,300 7,000
4h 46,700 5,300 870 770 4,660 33,900
8h 18,000 1,340 320 300 4,500 23,500
12 h 5,500 700 580 230 1,050 10,500
1d 1,700 600 140 130 580 1,700
2d 610 260 160 130 140 1,400
3d 450 200 90 20 40 20
7d 450 230 40 0 0 0
14 d 400 0 0 0 0 0
* Mean number of cells (20 mice per time interval)
+ 1.0 cc of blood
++ Urinary bladder and contained urine

in the host animal, providing a faster, less cumbersome method than the previously used
histopathological approaches.
Tables 1 and 2 from the 1970 paper show the final organ of cellular arrest and tem-
poral distribution of the viable/dead and labeled B 16 murine melanoma cells after a
single iv injection (14). From the table, it can be easily established that in the earliest
postinjection time-points, the majority of the cells find themselves in the pulmonary
tissue, but some are also localized in other organs. After 14 d, only the lungs contain
labeled cells, now seen as tumor nodules. Liver, spleen, kidneys, and blood all showed
the early presence of the labeled cells, but none of these tissues show the establishment
of tumors at 14 d postinjection. Intravenously-injected B16 melanoma cells in mice
could lead to rapid accumulation of cells in the pulmonary tissue, lead to early high
levels of tumor cells in circulation caused by transpulmonary passage, or ultimately
produce a very low rate of tumor formation in the animals. In the case of the B16line,
this tumor formation was limited to the pulmonary tissue. As a control, the author used
killed B16 cells, which had had also been labeled with 1251-5-iodo-2-deoxyuridine.
Some of the obvious questions presented by this work include: Why are so few cells if
the injected cells able to form tumors? Why such apparent inefficiency? How do the
cells select the target organ for colonization? Again, in the case of the B16 tumor, the
lungs were not acting as a passive sieve. This work demonstrated that the mere pres-
80 Alvarez

Table 2
Fate of 125JUDR-Labeled Tumor Emboli Organ Distribution of200,000 l25JUDR-Labeled
Melanoma Cells Injected Intravenously into C57BLI6J Mice

Number of cells*
Time of death Lung Liver Spleen Kidney Blood+ Urine++
1 min 96,700 4,860 40 120 2,314
2min 97,250 5,420 100 570 845
5min 99,600 4,810 90 120 1,190
10min 91,700 5,700 300 270 2,070 345
15 min 64,400 4,000 220 200 2,600 380
30min 41,500 9,300 420 590 5,600 3,530
45 min 19,500 12,430 900 930 6,790 20,060
1h 2,100 8,800 940 740 9,340 25,000
2h 550 1,700 450 435 13,200 24,500
4h 200 2,310 640 360 4,010 22,290
8h 0 750 330 0 1,950 7,260
24 h 0 40 120 0 100 190
2d 0 30 40 0 0 120
7d 0 0 0 0 0 60
14 d 0 0 0 0 0 0
* Mean number of cells (10 mice per time interval)
+ 1 cc of blood
++ Urinary bladder and contained urine

ence of neoplastic cells in the circulation of the mice is not a guarantee of successful
tissue colonization by the tumor, confirming earlier work done with mouse Sarcoma
241 tumors in 1950 by Zeidman. (9). The work with the B16line is a good example of
how research is done in the context of an already established scientific framework.
But strictly speaking, the process recapitulated in the B16 model is only representa-
tive of what happens to neoplastic cells, which have escaped a primary tumor into cir-
culation. At this point in time, the iv model was not truly representative of the entire set
of steps now established as requirements for a cell to leave the primary tumor and suc-
cessfully colonize a distal site. In 1973, Dr. Fidler published another paper, which
described a model for metastatic neoplasia using B16 melanoma cells iv-injected in
syngeneic mice (15). Until this time, metastatic tumor models usually relied on the
desegregation of cells from solid tumors (a heterogeneous mixture of neoplastic and
normal cells). This study focused on the relationship between the number of implanted
cells, tumor emboli size, and the resulting pulmonary metastatic nodules. Cell selection
was aided by the use of in vitro culturing of the cells in order to select the most optimal
cell population for implantation. Building upon prior work by Zeidman, the author
studied the effects of cell viability and cell clumping in the formation of pulmonary
nodules (Tables 3 and 4). The value of this effort was as important as it was simple.
Using the B16 line, the results demonstrate a proportional increase in lung metastasis
formation in animals injected with higher numbers of cells. Although this is a rather
intuitive point (and one previously tested) it served to solidify a base of knowledge
around the B 16 as a murine model. In the context of the contemporary studies, this
Chapter 4 I Bl6 Murine Melanoma 81

Table 3
Relationship of the Number ofViable B16 Melanoma Cells Injected I.V.
in C 57 Mice to the Number of Resultant Pulmonary Metastases
Number of viable Average number of
B16 cells iv-injected pulmonary metastases*± S.D.
100 0.3 (0-1)
1,000 12.1 ± 3.4 (9-18)
10,000 71.6 ± 16.2 (52-96)
50,000 205.5 ± 34.3 (166-260)
100,000 394.8 ± 51.6 (338-502)
*Nine mice per each group. Pulmonary metastases were counted on d 14 post
iv injection with the aid of a dissecting microscope

Table 4
The Effect of B16 Melanoma Embolic Sze
on Resultant Pulmonary Metastases in C 57 Mice
Total number of embolic Number of cells per Average number of resultant
B16 cells iv-injected embolic clump pulmonary metastases*± S.D.
50,000 1 11.5 ± 3.4 (5-14)
10,000-12,000 4-5 33.3 ± 8 (21-41)
*Eight mice per each group. Pulmonary metastases were counted on d 14 post iv injection
with the aid of a dissecting microscope

effort established a standard to be followed by contemporary scientists. The careful

selection of viable single cells in this model was again shown to be essential for the
establishment of a reproducible model of in vivo metastasis. These 1970 and 1973
papers presented what should be considered, an introduction of the B 16 tumor line to
the scientific community (14, 15). Importantly, the works themselves clarified an opti-
mal protocol for the establishment of metastatic lesion in the lungs of C57-black mice,
using a relatively simple technical procedure.
Also published in 1973, the process that gave rise to the Bl6 (F1, FlO) sublines with
different metastatic potentials. This was a short two-page paper with important ramifi-
cations (16). The work describes the in vivo-in vitro selection process used to identify
cell variants with a high degree of preference for metastatic growth in the lungs. The
experimental protocol started by sc implantation of the tumor cells into mice. The cells
would grow into a tumor and spontaneously metastasize. The author then selected a
resulting metastatic nodule from the lung. This tumor was dissociated and cultured as a
monolayer in vitro. Upon expansion of the selected cells, these were then iv-implanted
into a new host. Again, the resulting pulmonary nodules were harvested and cultured in
vitro. After five cycles through the selection process, a line was derived. At this point
the B16-F10 melanoma line had been specifically selected to metastasize to the lungs
after iv injection. Table 5 shows the illustration used to explain the selection process. In
1973 Fidler concluded:
82 Alvarez

line
26

lung

27

lung

28

lung

29

30

Table 5
Schematic representation of the tissue culture and animal transplantation
system. The SC B16 melanoma tumor in the syngeneic mouse, C57BI/6J,
was adapted to grow in tissue culture as described previously (2,3 ). Conflu-
ent monolayers of tumour cells were collected by 2-min treatment of 0.25%
trypsin and vigorous shaking. Cell suspensions were diluted to give an
inoculum dose of 50,000 viable cells (trypan blue excluding cells) in 0.25
mi. Hank's basic salt solution (HBSS). Mice were injected iv and were killed
with ether 3 wk later, and submerged in consecutive washes of 7% iodine,
70% ethanol and sterile saline, and then placed in a laminar air flow hood.
Their lungs, which contained melanoma nodules, were removed aseptically.
Several pulmonary metastases were dissected free of the lungs, gently
pressed through a number 70 stainless-steel mesh sieve and filtered through
gauze. After centrifugation, the cells were resuspended in supplemented
media2, plated in several Petri dishes, and incubated at 37° C, 5% C0 2. Three
to four days later, small colonies could be observed with the aid of an
inverted microscope; one to five colonies with melanin granules were
selected in each dish and their position marked. All other cells or colonies
were removed by scraping and washed off with media. The selected attached
colonies were incubated for an additional 3-5 d, then trypsinized, combined,
and replated into 75C2m Falcon flasks (Falcon Plastics). When the cultures
became confluent, cells were collected, diluted to 25,000 cells 0.25 mi.- 1,
and injected iv into new C57 mice. Three weeks later, these mice were killed,
and their pulmonary metastases adapted to grow in culture as described
above. This procedure was repeated five times. The original line was desig-
nated as line No. 26 (our melanoma clone 26), and its daughter lines and
their progenies designated as lines 27, 28, 29 and 30. T.c., Grown in tissue
culture; injected intravenously.
Chapter 4/ Bl6 Murine Melanoma 83

As the tumour cell viability, size and homogeneity, and the syngeneic recipient did not
change from one preparation to another, the differences in metastatic incidence could
only be attributed to properties intrinsic to the various tumour eel/lines. The clonal
selection of tumours from successive metastases apparently results in cells better
capable of survival and formation of secondary growths. This indicates that survival
of circulating tumour emboli is not a random phenomenon ( 16).
Fifty years had passed since Dr. Leila Knox had proposed the criteria for an experi-
mental murine model of metastasis.
The B 16 model system was incorporated into the American T)rpe Culture Collection
in June 1978 (Mr. Andrew Redman personal communication). In the 1983 edition of
the Catalog of Transplantable Animal and Human Tumors published by the Division of
Cancer Treatment, National Cancer Institute (NCI) (Maryland, USA) a letter by Dr.
Fidler is included (17). This letter approves the distribution of the deposited B 16
melanoma lines by the NCI. The catalog shows on the summary sheets for each tumor
line that on October 11, 1979 the B16-F1, -FlO, -Fl()LR-6 and -BL-6 were incorporated
into the tumor repository. At this point, this important model was available to the entire
scientific community.
After the demonstration of the lung-colonizing ability of the B 16-F1 0 lines, many in
vivo lines were derived, using a similar selection approach. This approach of intra-
venous tumor cell administration would be followed for target organ seeding. Those
tumors arising at the target organ would be selected and again reimplanted into fresh
hosts, this would force a selection process. Examples of this methodology can be found
in Brunson et al. 1978, Nicolson et al. 1978, and Nicolson 1978 (18-20). These partic-
ular examples show how the selected lines would preferentially metastasize to the
brains of C57 mice. The brain-colonizing lines were shown to invade the meninges or
the forebrain (lines were designated B16-B10b or B10n). Line B16-Bl0n showed pref-
erence for cerebral vasculature. By this route, the cells would gain access to the cere-
bral cortex. In 1979, Brunson and Nicolson selected another variant of the B16
melanoma line. This time the selected lines would invade the ovarian tissue when
implanted in mice (21 ). An observation was made that the selection process produced a
less melanotic cell line than the originating cells. This ovary-colonizing variant was
designated the B17-010 line. In 1980, Raz and Hart used the cytochalasing B and
colchicines as selection agents in the B16 melanoma to promote the cytoskeletal
changes, which the authors suspected believed to be important in the development of
differential patterns of metastases in this particular line (22 ). The selected lines (desig-
nated B16-F10-B1, B2, and B3) the selected lines in addition to showing a higher
capacity for brain colonization. The cells also showed an increased mean number of
chromosomes when compared to the originating parental line. In addition to this selec-
tion process, which led to a line with higher propensity for brain colonization, the line
also demonstrated a much higher rate of pulmonary growth when iv-injected. In 1988,
another model of brain metastasis was presented using the B 16 melanoma line (23 ).
This time, the B 16 clones used produced metastatic spread to the brain parenchyma
from the vessels found in the leptomeninges.
In 1988, Arguello et al., using the B16 melanoma line, were able to modify its injec-
tion into mice to produce a model of bone-marrow metastasis (24 ). Injection of a rela-
tively small number of cells into the left ventricle produced a metastatic pattern
strongly directed to the axial skeleton and bone marrow. In the description of the
84 Alvarez

metastatic pattern of the line, the authors commented: Metastases were also commonly
found in the proximal large bones of the extremities. No metastases were ever seen in
the most distal small bones such as carpals and tarsals.
This pattern of dissemination is reminiscent of the pattern described by Paget (2).
Now a model closely resembling the seminal work of Pagewas available with the B 16
melanoma line.
In a recent publication by Dithmar et al., a novel selection of the B16 model was
again demonstrated (25). In this study, the B16-LS9line selected by Rusciano et al. is
used to establish a model of uveal melanoma in mice (26). The transcorneal implanta-
tion method used produces extra-ocular metastasis to lung and liver tissue. This pattern
is similar to the human uveal melanoma condition. Again, the use of the B 16 line
helped in the development of a model for human pathology.
Another area of cancer research that has directly benefited from the development of
the B16 tumor line as a model for metastatic disease is cancer immunology. In some of
the earliest work with the B 16 melanoma line, Fidler and Ziedman studied the effect of
host irradiation on the line's metastatic rate (27). At that time, the increased metastatic
rate of the cells in mice that had received whole-body irradiation was not attributed to
immunodepression, but to endothelial-cell damage. This increased colonizing ability
was referred to as "enhanced trapping effect." Interestingly, in their own discussion, the
authors cite how previous work had demonstrated that the use of cortisone could also
increase the metastatic ability of cells. It is likely that both the use of whole-body radi-
ation and cortisone treatment of the animals also led to a degree of immunosuppres-
sion, which enhanced lung colonization by tumor cells. In some of the earliest studies
of B16 melanoma cells and immune cells, the relationship between lymphocytes,
macrophages, and Bl6 melanoma cells (28-31). The direct repercussion of this work
can be seen in the development of various methods used to activate the patient's own
immune system against the circulating cancer cells. Specifically, it is seen in protocols
that use muramyl dipeptide or muramyl tripeptide in a liposome-based vehicle for iv
administration (32-34). Through activation of tissue macrophages, an immune-medi-
ated antitumor effect was elicited in vivo. This has led to a better understanding of the
complex host-immune response to tumor cells.
The B16 melanoma line was also instrumental in expanding our knowledge of the
ability of metastases to spread. With the use of parabiosis systems in mice, it was demon-
strated that metastatic spread can originate from a metastatic nodule. In a clever experi-
ment, a mouse was sc-implanted with B16 melanoma, and if this first animal underwent
surgical removal of the primary tumor site (leg amputation) and was then surgically
joined to a naive host, the naive host developed lung tumors. These lung tumors origi-
nated from metastatic nodules present in the first animal. This finding offers proof that
once a solid tumor has successfully spread to other tissues, the metastatic cascade per-
petuates itself in the host. This problem can serve to emphasize the basic problem with
solid tumors: metastases present the biggest therapeutic challenge.

CONCLUSIONS
A recurring theme of this chapter is that the work that we now see as a final product
is the summation of many people working at various points in time. This is evident
from Paget's hypothesis in the late 1800s to the current development of newer models
Chapter 4/ Bl6 Murine Melanoma 85

Fig. l. An endothelialized thrombus attaching to the vessel wall (arrow heads), 24 h after inoculation
of tumor cells. Part of a tumor cell (*)and [missing text] E, endothelium.

Fig. 2. A group of tumor cells completely surrounded by an endothelial covering (E) in the lumen of
an arteriole, 2 wk after inoculation of tumor cells. Scale bar: lOJ..Lm, x 1060.

of metastasis (2). As a final example, I would like to offer an image giving a visual
description of the now commonly accepted tumor-cell endothelialization extravasation
steps for the B 16-F1 0 melanoma line (Figs. 1 and 2). Lapis et al. published this sophis-
ticated microscopy work in 1988 (35). This is a clear example of how a new technology
86 Alvarez

Fig. 3. Experiment 37/189 E: iv inocoulation, March 3, 1915; killed 3d later. Commencing growth
of a young sarcoma embolus. Vacuolation of endothelial cells is seen at one point, and early disten-
sion of the artery is indicated by flattening of the folds of the elastic laminoe. Cells resuming spindle
shape. (x525.)

Fig. 4. Experiment 37!189B: iv inoculation, March 3, 1915; killed 5 d later. A later stage than Fig. 3.
Increasing distension of vessel and entrance of capillaries into ombolus on left upper surface. (x525).

was able to shed light on ongoing scientific research. But microscopy work- specifi-
cally, studying the relationship between the endothelium and circulating tumor cells in
a rodent model--<:an be traced to work presented in 1915 by Iwasaki (Fig. 3 and 4) (5).
While Iwasaki mostly studied human samples, a series of images were drawn of
murine tumors cells interacting with the vasculature. Back in 1915, Iwasaki recognized
that the importance of the tumor-endothelial interactions as essential for the establish-
Chapter 4 I B16 Murine Melanoma 87

ment of the tumor embolus. In Lapi's paper, newer technologies are applied to answer
old questions. Over 70 yrs had passed since the Iwasaki's description of extravasating
tumor cells. At that time, two different reactions of the endothelium were noted, and
contact of the tumor cell with the vessel wall would lead to either a separation of
endothelial cell or an engulfment by the endothelial lining. The images here present us
with a visual representation of works separated by many years, but of equal impact to
our knowledge.
In conclusion, the success of the B 16 melanoma tumor model is clearly evident
today. As an in vivo model for solid tumor formation and metastasis, the B16 has ful-
filled two important criteria presented earlier. The model has been instrumental in the
dissection of the many steps now associated with tumor establishment and the metasta-
tic cascade. A wide range of disciplines have originated from the use of this tumor line.
The eventual relevance of the many sub lines that have been selected so far still remains
unclear. The development of the solid-tumor model is a dynamic process, and one that
attempts to produce a very close representation of the disease process itself. Currently,
orthotopic tumor implantation appears to be gaining favor in the scientific community.
Knowledge regarding embolization, tumor-cell viability, cell number, organ of arrest,
and host strain are all important basic concepts required for the refinement of solid-
tumor models. Another important topic in the ultimate development of metastatic mod-
els is how the site of implantation of tumor cells in the host animal will influence the
metastatic pattern in murine models. Whereas many of the murine tumors that are sc-
implanted spread to limited organs (mostly lungs), tumors implanted orthotopically
generally behave in a manner more consistent with the natural metastatic progression
of the human counterpart for in-depth coverage on this topic. An argument has recently
been made regarding the need to use the advanced orthotopic tumor models for the
screening and development of chemotherapeutic agents (36,37). While the orthotopic
models might provide the researcher with a model that more accurately depicts the nat-
ural history of the tumor, the model itself is more technically demanding and costly
than syngeneic metastatic systems.
The knowledge gained from these efforts has directly led to the use of the B16 model
to ask more questions regarding the nature of metastasis, with the added benefit of time
to provide newer tools to answer such questions. It is also important to keep in mind that
the overall selection and the acceptance of the model in the scientific community has
been influenced by the already existing scientific framework of the time. The B 16
melanoma tumor line has most definitely made a profound impact in the field of oncol-
ogy research. It has served as a model for basic biological research, and has helped in the
identification of distinct pathways now available as potential therapeutic targets.

DEDICATION
In memory of E. Gregory MacEwen, a dedicated teacher and friend. Your enthusi-
asm will live in us.

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