HETEROTRANSPLANTATION OF HUMAN CELL

LINES FROM BURKITT'S T U M O R S AND A C U T E

LEUKEMIA I N T O NEWBORN RATS
M. SOUTHAM,JOSEPH H. BURCHENAL,
CHESTER BAYARDCLARKSON,
ADRIENNETANZI,
ROSEMARY VIVIANMCCOMB
MACKEY,

The heterotransplantability of 22 human cell lines was studied by intravenous

inoculation into newborn rats. The cell lines included nine derived from
Burkitt (African) lymphoma, one from reticulum cell sarcoma, ten from
leukemia, one from infectious mononucleosis and one from a normal lymph
node. All were cultivated in vitro as suspension cultures. The usual inoculum
for each rat was 50 or 10 or 2 million cells. At the two higher doses four of
the Burkitt tumor lines (EB-2, EB-3, SLl, B35M), three of the leukemia lines
(SK-LZ, SK-L5, SK-LS), the infectious mononucleosis line C566, and the
cell line from a normal lymph node (SK-LNl) propagated and caused lethal
lesions in approximately half of the recipients. Inocula containing 2 million
cells of lines EB-2 or SL1 grew in a few rats but none of the other cell lines
grew at this dose. Brain, eye and kidney were the most common sites of growth.
Lung, adrenal, liver, and interscapular fat pad were less frequently involved,
and other tissues rarely. The results are discussed in relation to the biologic
significance of heterotransplantability and differences in the tissue distribution
of implants of these and previously studied human cell lines.

S EVERAL HUMAN CELL LINES FROM 8 U R K l T T

lymphoma5, 7 , 8. 11-13, 21. 31 a n d leuke-
inial-", 6. 7, 15, 23 have been established in con-
tinuous suspension culture in the past few
years. These cell lines are of particular inter-
est because they represent human neoplasms
which are responsive to chemotherapeutic
agents, which are suspected to be of viral
etiology, and which may be influenced by im-
munologic defense reactions.4 Established
lines of tumor cells permit repeated and
replicate studies which are not possible in the
human patient, but the extrapolation of data
from laboratory studies to the cancer patient
rests on the presumption that the cultivated
cells are truly the neoplastic cells.
As almost every specimen of tumor tissue
contains stroma, blood vessels and wandering
cells, there is a possibility that these non-
neoplastic cells propagate preferentially,
yielding a culture of mixed neoplastic and
From the Sloan-Kettering Institute for Cancer Re-
search, New York, N.Y. 10021.
This work was supported in part by a grant from
t h e National Cancer Institute. U.S. Public Health
Service (CA 08748).
Received for publication August 29, 1968.
28 1
non-neoplastic cells, or of non-neoplastic cells
only. Criteria to establish the identity of cul-
tured cells are necessary. When cytologic
criteria alone are inadequate for id'entifica-
tion, transplantation may be useful because
transplantation across genetic barriers gener-
ally occurs more readily with neoplastic cells
than with normal cells, and because growth
as a transplant provides a three-dimensional
tissue organization which can be evaluated
by the criteria of histopathology.
Use of the newborn rat for heterotrans-
plantation of human cancer has been de-
scribed in several reports from this labora-
tory. Several well-established cell lines which
grow as monolayers in culture grew progres-
sively in lungs and other organs following
intravenous inoculation into rats when they
were less than 24 hours of age.1.0. 20,, 30. 31 Sim-
ilar, but less frequent, growth occurred on
direct transplantation of cancer from patients
to newborn rats.33 Failure of the rats to reject
the heterologous tumor transplants is presum-
ably because of inadequate development of
immunologic rejection mechanisms at the age
when the cells are inoculated, and the conse-
quent specific immunologic t~lerance.~g This
282 Februayy 1969
CANCER
paper reports the use of this transplantation
technique for several lines of human cells de-
rived from leukemic blood, from Burkitt’s
tumor, or from other tissues of patients with
neoplastic and non-neoplastic diseases.
METHODS
,411 cell lines were grown at 37°C in sus-
pension witliout agitation in 4 liter Erlen-
meyer flasks with loosely fitting stainless stcel
tlowres in an atmosphere of 5% C 0 2 and
95‘5 air, or in 500 nil Erlenmeyer flasks or
16 oz medicine bottles with tightly fitting
screw caps. T h e culture medium for RCSI,
SK-LN1 and all 10 SK-L lines was McCoy’s
modified mctliuni supplemented with l-gluta-
mine (Grand Island Biological Co., Grand
Island, N.Y.) containing l0yo fetal calf serum,
with penicillin (200 units/ml) and strepto-
mycin, (100yg/ml).
T o harvest these cells from the culture
ineclium, they were first allowed to settle
oveinight in the culture bottles or in I-liter
centrifuge tubes. Then the cell-free superna-
tant medium was siphoned off, leaving a
concentrated cell suspension which was cen-
trifuged at 800 g for 10 minutes to p’oduce
a loosely packecl pellet of cells. For all other
cell lines the culture medium was Eagle’s min-
imal essential medium with glutamine (Mi-
crobiological Associates, Bethesda, Md.) with
lo?{, fetal calf serum, and penicillin and strep-
tomycin as above (no neomycin). Loosely
packcd cell pellets were prepared by centri-
fuging the cultures directly at 800 g for 10
to 20 minutes. After centrifrigation tlie cells
were 1 csuspended in either the original
medium or Puck’s saline A20 to give the
desired concentrations for injection into the
rats-usidly 50 million, 10 or 20 million,
and 2 million cells per 0.25 ml. Some lines
were not testcd at 50 million because trans-
plantation was successful with lower doses or
because insufficient numbei s of cclls were
available.
Rats were of the non-inbred Wistar strain,
bred in this laboratory for several genera-
rions. Within 24 hours after birth 0.25 mm of
cell suspension was injected by way of a lat-
eral tail vein, using 0.25 ml tuberculin type
syringe and 30-gauge needle. One or two
animals of each litter were kept as unin-
jected controls, and these were identified by
cutting off the tip of the tail. Rats given
different doses of cells were identified by toe
VOl. 29
clips. Litters were checked every 2 or 3 days
for evidence of illness or stunting and were
weighed weekly after the third week. Animals
that died within 3 days of inoculation were
excluded from the data. Animals that became
severely stunted or moribund were killed for
gross and microscopic examination. Of those
that grew normally, a few (two to five for each
cell line) were saved for long-term observa-
tion and the others were autopsied at 6 to
8 weeks of age. Histologic sections were made
of almost all abnormal appearing tissues and
from a sufficient selection of normal appear-
ing tissues to provide thorough representation
of major organs for each cell line.
RESULTS
Twenty-two cell lines were studied. Ten
were derived from lymphomas-nine from
Rurkitt’s tumors and one from a reticulum
cell sarcoma. T e n were derived from leu-
kemic tissues-nine from peripheral blood
leukocytes of patients with various types of
acute leukemia, and one from spleen of a
patient with chronic myelocytic leukemia. Two
cell lines were of non-neoplastic origin-one
from a patient with infectious mononucleosis
(line C566), and the other from a lymph node
of a patient with hemophilia who had n o
neoplastic disease (SK-LNl). T h e designa-
tion of each line, its origin, and key references
are listed in Table 1 or 2 together with data
on the extensiveness and results of the stud-
ies. T h e number of litters represented at each
dose, the number of experiments, i.e., studies
with a single harvest of cells, as well as the
number of individual rats, are recorded to
indicate the adequacy of the investigation
with each cell line. Biologic variation in the
recipients and in the cell suspensions does
influence results, so when only one experi-
ment w a ~performed the results may not
truly represent the potential of tlie cell line,
even if many animals were injected.
Nearly all of the rats survived the immedi-
ate effects of inocula containing up to 20
million cells. Inoculation of 50 million cells
caused severe cyanosis and dyspnea in most
recipients, but with most of the cell lines
which were tested at this dose some of the
recipients recovered after 20-30 minutes.
Histologic findings are illustrated i n Fig.
1-24. These are grouped by type of tissue or
organ, rather than by cell line, in order to
show a representative range of lesions in the
No. 2 HETEROTRANSPLANTATION
OF HUMAN
CELLLINES* S o u t h a m et al. 283
’Tint ri 1 Heterotransplantation of Human Cell Cultures t o Nemborn Rats by Intravenous Injection-
Cell Lines Derived from Lymphomatous Tumors or Lymph Nodes
___ _ _
No. of rats*
No. of
Key cells per Xo. of No. of Confirmed
Cell line Origin references rat X 106 expts. littcrs Total Lost tumor
EB-1 Burkitt tumor 11, 13 12 1 1 11 1 0
2 1 1 10 0 0
EB-2 Burkitt tumor 12, 13 10-20 5 7 23 1 9
2-4 3 4 23 2 1
EB-3 Burkitt tumor 13 10-20 5 7 42 6 11
2 1 1 7 0 0
Kudi Burkitt tumor t 2-4 1 2 17 0 0
Jijoye Burkitt tumor 25, 27 10-20 3 4 23 1 0
2 1 1 8 3 0:
Ogun Burkitt tumor t 10-20 3 4 28 1 0
2 1 1 10 1 0
Raji Burkitt tumor 25, 27, 28 10 1 1 8 4 0
2 1 1 9 0 0
SL-1 Burkitt tumor 31 10 1 1 2 0 2
2 1 1 9 0 3
B35M Burkitt tumor 21 50 1 1 3 0 1
10 1 1 11 1 0
SK-RCS1 Reticulum 6, 8 10-25 2 5 23 0 0
cell sa. 2 1 1 9 0 0
SK-LN 1 Normal node 6 50 1 1 4 1 I
hemophilia 10-25 2 4 22 2 3
-~ ~~~ ~ ~ ~-
* Rats that died before day 3 are omitted from all tabulations. Most such deaths occurred within a few hours
after inoculation of more than 20 million cells per rat. “Lost” rats includes those which died later than d a y 3 but
were too badly cannibalized or decomposed t o permit diagnosis of tumor growth, and some which disappeared.
For most cell lines two t o five rats which appeared well were retained for long-term follow-up.
+ Burkitt cell lines Kudi and Ogun were established by Pulvertaft and/or Osunkoya % * but are not specifically
described in a n y publication known to us.
1 One rat did have two small nodules in lung when autopsied on day 33. I t is not recorded as a successful trans-
plant because of histologic evidence of rejection and because of the lack of a n y other evidence of Jijoye cell
propagation in this or other rats.

various sites T h e lesions produced b) the
various cell lines were not individuall) dis-
tinctive in cytologic characteristics, but there
were differences in tissue distribution as tle-
scribed in Results and Discussion.
Lymphoma cell lznes: Of the nine lines
derived from Burkitt tumors, four (EB-2,
EB-3, SL-1, B35M) produced extensive tu-
morous growth in some rats, as described in
the following paragraphs and summarized in
Table 1. T h e other five Burkitt lines (EB-I,
Kudi, Jijoye, Raji, Ogun) and the reticulum
cell sarcoma line (RCS-1) were not trans-
plantable as judged by these studies. T h e
Rurkitt lymphoma line Kudi was tested only
at 2 4 million cells per rat, but EB-1, Jijoye,
Ogun, Raji and RCS-1 were tested at 10
or 20 million.
EB-2 grew in half of the rats which were
inoculated with 10 to 20 million cells. Some
rats died or were moribund after 2 to 3 weeks.
Some had intraocular hemorrhage or tumor,
or proptosis. Some had neurologic signs of
cerebellar damage. At autopsy, petechiae in
the brain and slightly enlarged pale kidneys
were the usual gross findings, and a few rats
liad scattered small nodules in the lungs.
Eleven rats had gross lesions but only nine
of these were studied microscopically. T h e
most frequent lesions were masses of tumor
cells within the brain parenchyma and in the
meninges and chorioid plexus (Fig. 1). T h e
eye lesions were due to growth of the tumor,
often accompanied by hemorrhage, in peri-
orbital tissues, between the sclera and retina,
in the iris or ciliary body, within the posterior
chamber (Fig. 7) and rarely within the an-
terior chamber. I n the kidneys masses of
tumor cells grew among the secretory tu-
bules but were not found within glomeruli. A
few rats had small nodules of tumor in the
lungs and adrenals (Fig. 17). One had gross
involvement of salivary glands and a mass
of EB-2 cells in the omentum. Histologic sec-
tions were prepared of liver and spleen of
nine rats that had gross evidence of growth
of the EB-2 cells in other tissues. All livers
were histologically normal. No EB-2 cells
were identified in spleens, but it was not
possible to be sure that there were none in
the splenic pulp. No gross or microscopic
evidence of EB-2 cells was found in pan-
284 February 1969
CANCER VOl. 23

TABLE 2. Heterotransplantability of Human Cell Cultures t o Newborn Rats by Intravenous Injection-

Cell Lines Derived from Leukemia or Infectious Mononucleosis
~~~~ ~

No. of rats*
No. of
Cell Diagnosis of Key cells per No. of No. of Confirmed
line donor references rat X 106 expts. litters Total Lost tumor
SK-L1 Acute myeloniono 7 50 2 0
leukemia 25 7 0
2 22 0
SK-L2 Acute lympho. 7 50-100 19 6
leukemia 10 4 50 5
2 14 0 0
SK-L3 Acute myelomono 7 50 7 0 Of
leukemia 10 14 0 0
2 9 0 0
SK-L4 Acute myelo. 50 1 2 0 0
leukemia 10-20 2 17 1 0
SK-L5 Acute myelo. 50 1 3 0 3
leukemia 10 2 14 0 0
SK-L6 Acute lympho. 50 1 2 2 0
leukemia 10-20 2 10 4 0
2 1 4 1 0
SK-L7 Acute myelo. 7 SO 3 5 0 Ot
leukemia 10-20 2 1 23 5 0
2 2 2 20 0 0
SK-L8 Acute myelomono 50 1 2 0 0
leukemia 20 2 15 0 0
SK-L9 Chronic myelo- 50 1 5 2 1
leuk. 10-25 24 2 1
SK-LlO Acute myelomono 10 1 11 0 0
leukemia
C-566 Infectious 10-20 1 36 1 7
mononucleosis 2 1 16 1 0
* Rats that died before day 3 are omitted from all tabulations. Most of such deaths occurred within a few hours
after inoculation of more tha 20 million cells per rat. "Lost" rats includes those which died later than day 3 b u t
were too badly cannibalized or decomposed t o permit diagnosis of tumor growth, and some which disappeared.
For most cell lines two t o five rats which appeared well were retained for long-term follow-up.
f Cell lines SK-L3 and SK-L7 did cause lesions in a few rats, as described in the text, but there was not any
evidence that they were continuing to propagate.

creas, intestinal tract, heart, testis, ovary, thy-
mus, lymph nodes, or interscapular brown
fat pads. Microscopic study of these organs
was limited to a few rats, but all those which
were examined had extensive involvement
of brain or kidney.
One of the 23 rats which was injected with
2 million EB-2 cells was killed because it
showed signs of cerebellar damage. I t had
no gross lesions but on microscopic exam-
ination its brain contained tumor cells. T h e
other 22 developed normally and had no
gross pathology when autopsied at about 6
weeks, so histologic studies were usually not
clone.
EB-3 caused lesions that were similar in
frequency, distribution, and in gross and mi-
croscopic appearance to those caused by EB-
2. Macroscopic lesions were present in 15 of
the 42 rats which were inoculated with 10
to 20 million EB-3 cells, and the 11 that
were studied histologically all had typical
transplant growth (Fig. 2, 14). These tu-
mor-bearing rats were stunted and died or
were moribund during the fourth or fifth
week after injection. Another six rats were
missing after the second week, and probably
died of tumor growth. T h e seven rats which
were inoculated with 2 million EB-3 cells
developed normally. They had no gross ab-
normalities at autopsy and were not exam-
ined microscopically.
T h e SL-1 line was studied only once. It
grew in both of the rats which received 10
million cells, and in three of the nine rats
which received only 2 million cells. T h e dis-
tribution and appearance of tumor growth
was essentially the same as for EB-2 a n d
EB-3 (Fig. 8, 13).
Line B35M was studied only once. One of
the three rats that received 50 million cells
did not grow as well as his littermates and
was killed on day 21. No gross lesions were
seen, but microscopic study showed extensive
growth of tumor cells in brain (Fig. 3) and
a small focus of tumor cells in the retina of
Yo. 2 HETEROTUANSPLANTATION CELLLINES* Southam et al.
OF HUMAN 285

Frc;. 1 (top). Low magnifica-

tion view of Burkitt cell line
EB-2 in medulla and fourth
ventricle. There is particularly
heavy growth of the EB-2 cells
between the ventricular epithe-
lium and the cerebellum (on
the right) and between the
ventricular epithelium and me-
dulla (to left and bottom of the
ventricle in this photograph).
'Ihere are also massive accumu-
lations of the EB-2 cells within
the capillaries of the chorioid
plexus, along the blood vessels
o f thc! medulla, and within thc
sul)rtrrnre of t h c medulla.

FIG. 2 (bottom). Low inagnili-

cation view of EB-2 cells in
brain. T h e large dark-staining
niasscs extending from right
margin into center of field and
a t lower right are collections of
EB-3 cells in the chorioid ves-
sels within the dilated lateral
vcntricle. At extreme upper left
a layer of EB-3 cells overlies
lhc cerehrurn in the subarach-
iioid space.

one eye. 'I here wa$ no tumor in lung, liler,
spleen, kidney or adrenal. T h e other two rats
which got 50 million cells and the ten that
got 10 million grew noimally and had no
gross pathology when sacrificed a t 8 weeks.
No histologic studies were made.
T h e Jijoye cell line is of particular in-
terest because its Herpes-like virus partitlea
have been extensilel) studied by electron
microscopy and serologic tec11niques.l~It was
studied three times in a total OC 31 rats in
four litters. T w o rats which were inoculated
with 50 million cells died immediately in
apnea and are not included in the tabulation.
286 February 1969
CANCER
Four of the 23 rats that received 20 million
cells died and were cannibalized during the
third week, but six others that appeared to
be developing less well than their normal
littermates were autopsied at intervals from
the second to fifth weeks and representa~ive
iissues were sectioned. T h e only gross or mi-
croscopic lesions that were found were two
nodules in the lungs of one rat which was
VOl. 23
FIG. 3 (top). Nunirrous foci of
Burkitt cell line B35M in brain
parenchyma. Many appear to
originate within or around
blood vessels.
FIG. 4 (bottom). Low magnifi-
cation vicw of leukemia cell
line SK-L5 showing heavy sub-
dural accumulation of the SB-
1.5 cells with perivascular ex-
tension deep into the cerebrum,
and areas of diffuse infiltration
within the cercbruni.
killed on day 33. These appeared to be the
sites of implantation of foreign cells which
had been rejected. Another 22 rats were
killed after 6 or 8 weeks. Gross findings were
normal and no histologic studies were made.
Leiiliernia lines: Of the ten lines derived
from leukemia (Table Z), only SK-L2 and
SK-L5 frequently produced gross and micro-
scopically confirmed growth. SK-L9 produced
No. 2 HETEROTRAhWLANTA I l O h OF H U M A N CELL L I N L b
uiiu5ual patterns of growth in a few rats.
T w o lines (SK-L9 and SK-L’i) did not prop-
agate but caused unusual lesions in a few
rats. These findings are described below. T h e
othcr five leukemia lines caused no gloss or
microscopic lesions in these experiments.
SK-L:! was the most aggressive of the leu-
kemia lines. Among the rats given 50 million
SK-12 tells, nearly 50% died duririg the sec-
* SOlltkllttl C t U l . 287
ond week, presumably due to growth of the
transplants. More than 25y0 were autopsied
because they were moribund at 2 to 4 weeks
and were found to have large pale kidneys
or petechial lesions in brain or both. Several
also had hemorrhages or opacities within the
eye and a few had gloss lesions of the liver.
However, the remaining 25% remaiiiecl
healthy and 1 1 x 1 no gros abnormalitie5 when

FIG.5 (top). Infectious niotio-

tiriclcosis cell line C566 growing
along and within blood ~essel h
and tliffrisely witliiii the rerc-
brit i n .

k i 6~ (botiorn). High power

view of a focal growth of SK-
LNI cells (normal human lymph
node origin) in the thalamic re-
gion.
288 February 1969
CANCER
autopsied at 4 to 6 weeks. Similar lesions were
found in about 10% of rats given 10 million
SK-L2 cells. Growth of the SK-L2 cells was
confirmed on microscopic examination for
all nine rats in which there was macroscopic
evidence of growth and in one other which
was cgrossly normal. There was tumor-like
growth of the transplanted cells in the brains
of all ten rats within the parenchyma, in the
subdural space and sometimes within the
chorionic villi, in kidneys of seven among the
secretory tubules (Fig. 15) (but only once
within a glomerulus), in adrenals of six (Fig.
18), in eyes of four (variously distributed in
retina, ciliary body, iris, anterior or posterior
chamber, and retro-orbitally), in liver of seven
(Fig. 21) and in lungs of six (Fig. 23).
SK-L5 grew in each of the three rats that
were given 50 million cells. Their growth
was retarded. One had signs of cerebellar
damage and the other two had tumor-like
growth in eyes. When killed at 3-5 weeks of
age the only macroscopic abnormalities were
the eye lesions. However, microscopic exam-
ination revealed extensive infiltrative growth
in the brains of all three (Fig. 4) in the
eyes of two (Fig. 9), and a small deposit in
the adrenal cortex of one. N o abnormalities
were found in lung, pancreas, spleen, or kid-
ney. One liver had areas of hepatocellular
degeneration near the central vein of some
lobules, but no growth of the transplanted
cells and no inflammation reaction. Rats
given 10 million SK-L5 cells developed nor-
mally and no pathologic changes were seen
grossly. T h e only one that was studied micro-
\topically appeared normal.
SK-L9, the line derived from chronic mye-
locytic leukemia, grew in only two rats, and
in unusual locations. I n one of the five rats
which received 50 million SK-L9 cells, slight
nodularity of the interscapular brown fat pad
was observed when autopsied at 20 days.
Microscopic study revealed focal deposits of
mitotically active tumor cells in the fat pad
(Fig. 11, 12) and a few small lesions in
brain. One of the 22 rats which received 20
million SK-L9 cells had a tumorous growth
approximately 5 x 5 x 10 mm at the base of
the tail where part of the intravenous in-
jection extravasated, and in two smaller ret-
roperitoneal masses, but not elsewhere. It
may be significant that two other rats that
received 50 million cells died during the
third week, but these could not be studied
11 i stologically.
Vol. 23
One of the rats which got SK-L7 cells
appeared sick and was killed on day 4. There
was a collection of cells, presumed to be SK-
L7 cells, among the renal tubules, but at this
age the kidney of the rats is in transition
from the fetal to the mature pattern of glo-
merular and tubular relationship, making the
recognition of scattered extrinsic cells un-
certain. Even if identification were positive,
however, the persistence of cells 4 days after
inoculation could not be taken as evidence
of growth. There was n o evidence of growth
of SK-L7 in other rats given 50 million or
fewer cells.
Four of the seven rats which were given
50 million SK-L3 cells appeared to have in-
traocular hemorrhages during the second and
third weeks, but when autopsied at 6 weeks
there was no abnormality in the eyes on gross
or microscopic study and the only other lesion
was a small cluster of unidentified cells, pos-
sibly SK-L3 cells, in one adrenal.
Cell lines of non-neoplastic origin: Sur-
prisingly, both of the cell lines that were
derived from patients with non-neoplastic
diseases produced tumor-like <growth in this
heterotransplant system.
C566 (Table 11) is a cell line derived from
peripheral blood of a patient with infectious
mononucleosis who recovered promptly and
is well at present, 2 years later (personal
communication from Benyesh-Melnick). It
caused the most striking gross pathology seen
in these studies. In addition to petechiae in
the brain and enlargement of the kidneys,
the livers of some of the rats were enlarged
and large pale areas were visible through
the capsule and on the cut surfaces. Histo-
logically there was massive focal infiltration
of the hepatic lobules by the transplanted
cells (Fig. 19, 20) and extensive hepatocell-
ular degeneration. There were also eye lesions
(Fig. lo), intertubular lesions in the kidneys,
focal lesions in the brain parenchyma and
subdural space and chorioid plexus (Fig. 5)
and a few deposits in lungs and adrenals.
These lesions occurred in six of the nine
rats in the two litters that were inoculated with
20 million cells in the first study that was
done with this cell line. I n two subsequent
studies, growth occurred in only one more
rat of a total of 27 that were injected at
this same dose.
SK-LN1 (Table 1) is the cell line deriletl
from a para-aortic lymph node obtained at
autopsy from a patient with hemophilia but
No. 2 HETEROTRANSPLANTATION C E L L LINES* SOwtha,?Z et
OF HUMAN Ul. 289

FIG. 7 (top). Low power pho-

tomicrograph of rat eye with
inass of EB-2 cells filling the
posterior chamber. T h e separa-
tion of layers of thc retina is
artifartitious.

FIG. 8 (bottom). High power

view of Burkitt cell line SLl
in posterior chamber of the eye
just behind the ciliary body.

n o neoplastic disease. Of the four iats inocu-
lated with 50 million cells of this cell line,
one died during the third week antl its tissues
could not be studied. Another developed
opacification of one eye antl on microscopic
study it had growth of the transplanted cells
in the eye, brain (Fig. 6) and kidneys (Fig.
IS), but the other two showed no evidence
of transplant growth. Three of the 22 rats
that received 10 to 25 million cells also had
growth in the brain. Two of these three rats
had n o other lesions, but one had generaliLed
and extreme enlargement of peripheral and
visceral lymph nodes due to active niultipli-
cation of large lymphoid cells. Tlie morpli-
ology of these cells was consistent with that
of SK-LN1 cells, but n o studies were done
to affirm that they were human rather than
290 February 1969
CANCER
rat cells. Lxcept foi this one rat, generaked
atleriopathy was xiever observed in this or
pietious studies in autopsies of several thou-
m i d rats which had been inoculated at birth
with various human cell lines.
DISUWON
Although the only apparent explanation
for the presence of the tumorou5 growths in
Vol. 23
FIG.9 (top). Leukemia cell line
SKL5 densely infiltrating the
choroid layer and separating the
retina from the sclera. T h e
sclera is at the top of the pho-
tograph. T h e dark staining band
running horixontally in the
middle of the field is the outer
nuclear layer of the retina.
FIG. 10 (bottom). Kctro-orhital
growth of the infectious mono-
nucleosis cell line. T h e C5G6
cells are a t the extreme right
in contact with the sclera. T h e
retina is intact and there are
no deposits of the hitman cells
within the orbit.
these rats is propagation of the injected cells,
strict proof that they were actually trans-
planted human cells require that they be
shown to contain either a human chromosomal
pattern or human tissue antigens. T h e latter
criterion was met for one rat which had
tumorous masses in the kidneys following
transplantation of EB-3 cells. A positive re-
action was demonstrated by the direct fluor-
No. 2 HETEROTRANSPLANTATION
OF HUMAN
CELLLINES* Southam et al.
escein-labelled antibody technique with a
pooled serum from rats which had been im-
munized as adults by repeated injections of
HEp 2 and RPMI 41 cells. Four other lines
oE evidence, although less stringent, indicate
that the ‘‘tumors’’ were composed of human
cells.
T h e presence of widespread tumor-like
giowth 50 soon after transplantation nlniost
29 I
certainly precludes the possibility that they
were induced rather than transplanted. T h e
cytologic characteristics of the cells were sim-
ilar in imprints of the tumorous organs and
in smears prepared from the suspension cul-
tures. Such preparations were compared lor
EB-2, ER-3, SK-L2 and SK-L5, and both the
transplanted and tissue cultured cell p el)-
iiration5 showed blast-like cell5 with niiiiier-

FIG. 11 (top). Low power view

showing foci of leukemia cell
line SK-L9 in lobules of the fat
pad.

FIG. 12 (bottom). Higher mag-

nification of Fig. 11. Note the
multiloculated brown fat cells.
 CANCER
February 1969
ous c j top1,isniic \a( uoule, and large nucleii
with prominent nucleoli. T h e human iden-
tity of the cells in cultuie has been established
for most of these lines b y recent chromosomal
studies.; And a human chromosomal pattern
was demonstrated for the tissue-cultured C566
cells following these lieter otransplantation
studies (personal communication, Dr. June
Biedler). Human ser uni globulins have been
demonstrated in the serum 01 rats bearing
Vol. 23
FIG. 13 (top). Low power pho-
tomicrograph of kidney with
large mass of Burkitt cell line
ER-2. Note that the glomeruli
appear normal.
FIG. 14 (bottom). Burkitt cell
line EB-3 in kidney. The trans-
planted cells are growing among
the tubules. None were found
in glomeruli.
transplants of some of these cell lines (un-
published data, C . M. Southam and A. G.
Levin).
T h e ability of newborn rats to survive after
intravenous injection of as much as 50 million
cells was surprising since such inocula were
roughly 10% suspensions and approximately
2576 of the rats blood volume. With pre-
viously studied cell lines such as HEp 2, J-
111, and RPMI 41,31 the maximum inocu-
so. 2 OF HUMAN
HETEROTRANSPLANTATION CELLLINES* Southam et al.
lum that was tolerated without a prohibitive
frequency of immediate asphyxia was 1-2
inillion cells. However, intravenous injections
of 20 million o r more normal rat lymphocytes
seldom killed the animals (unpublished ob-
servations). T h e difference is presumably due
to the extent of vascular obstruction by the
vaiious lines of cells, but this cannot be re-
lated solely to differences in the si7e of in-
293
dividual cells since the Burkitt and leukemia
cells are nearly a5 large as H E p 2, J-Ill and
RPMI 41 cells as judged by dried and
stained smears of cell suspensions. Cell lines
which grow as monolayers (HEp 2, 5-111 and
RPMI 41, etc.) tend to iorm cell clumps in
suspensions, in contrast to the Burkitt and
leukemia lines which d o not adhere to glass
o r plastic surfaces but grow in suspension as

I I C . 15 (top). Leukemia cell

liuc S K - I 2 in kidney. T h e “tu-
nioi” is intcrtubular displacing
but not invading or destroying
the renal tubiilca Glomeruli are
normal.

1 I(,. 16 (bottom). Intertubular

kidricy lesion similar to t h o x
in Fig. 13-15, but caused by
CK-LNI, the cell line of noimal
l p p h node origin.
294 CANCERFebruary 1969
single cells and small clusters which break
u p into single cells when shaken vigorously.
If these same characteristics of adhesion and
cohesion also pertain in vivo, the difference
in immediate lethality of these two groups
of cell lines might be related to the size and
persistence of intravascular cell aggregates
rather than to the si7e of the individual
Vol. 23
FIG. 17 (top). Deposit of EB-2
cells in atlrcnal medulla. T h e
normal medullary tissue is seen
as a wedge betwceti the cortex
and the “ t u m o r ” mass.
FIG. 18 ( Im t t ot n ) . Deposit of
SK-L2 cells in adrenal, occupv-
ing much of ttic medulla anti
the inner part o f the cortex.
cells. I t m a y be significant that these char-
acteristics also correlate with growth of im-
plants in the lungs. T h e cell lines which
grew as suspension cultures and which were
tolerated i n closes of 10 million and more
a n intravenous inoculation seldom grew in
the lungs, whereas the previoiisly studied cell
lines which grew in culture as highly cohesive
uo 2 H E ~ ~ R O T R A N ~ P L A N T I ~ T OF
IONH U M AN
and adhesive moriolayers and produced death
by asphyxia when inocula exceeded 1 or 2
million cells F e u most frequently and most
inassivelj in lungs.
Three of the lines derived from Burkitt
tumor (ER-2, LB-3, SL-1) and three ol
those derived from acute leukemia (SK-L2,
SK-L5 and SK-L9) were unequivocally trans-
plantable in several of the iecipients. T h e
tumorous g o w t h of these six liner would ap-
-
C E I I LINES Southurn ct nl. 295
pear to be evidence that they ai e truly ma-
lignant neoplastic cells, that is, Burkitt tumor
cells and leukemia cells, respectively. T h e
presence of small numbers of SK-LS cells
i n a rat sacrificed at 6 days, with n o evidence
of takes in animals killed at later times, may
result from simple persistence of the injected
cells rather than progressive growth. How-
ever, if cells are able to persist for a few
clays, i t is not unlikely that with larger inoc-

FIG. 19 (topj. 1 . 0 ~ power

. pho-
tomicrograph showing focal de-
posiLs of cell line C566 (infecti-
ous rnononuclcosis origin) in
liver.

Urc.. 20. Higlier inagnilicatioii

of Icsion shown iri 1;ig. 19. T h e
CSGti cells arc' concentrated
at-ourid ;
I cr.ritr;tl icin.
296 CANCERFebrua7.y 1969
ula progressive growth of these cell lines
might occasionally be observed.
T h e other cell lines of neoplastic origin
gave little or n o evidence of transplantabil-
ity, but both of the lines that were of non-
neoplastic origin did produce tumor-like
growth. These paradoxical findings raise ques-
tions concerning the validity of heterotrans-
plantability as a criterion of malignancy.
Vol. 23
FIG. 21 (top). Focal deposit of
SK-L2 cells in liver,
FIG. 22 (bottom). Cell line
C5G6 diffusely infiltrating sali-
vary gland. This and one rat
which was given En-2 cells were
the only instances of salivari
gland involvement obwvcd i n
thr present studies.
Certainly i t is possible that cultures derived
from Burkitt tumors and leukemia may ac-
tually originate from normal elements of
neoplastic tissue rather than from malignant
cells. T h e fact that some myelogenous leu-
kemia lines produce immunoglobulins17~3 5 ~ 3 6
also suggests this possibility for it seems un-
likely that cells of the myelogenous series
produce immunoglobulins. However, the be-
No. 2 HETEROTRANSPLANTATION
OF HUMAN -
CELLLINES Sozcthnrtz et al. 297
havior of SK-L5, which was derived from fail to grow i n any transplantation system, so
myeloblastic leukemia, contradicts even those the failure of most of these cell lines to grow
concepts, for it behaved as a malignant cell in the present studies does not prove that
on transplantation but produces immunoglob- they are not neoplastir. Morphology was not
ulins in culture,3G as expected of a lymphoid helpful for classification of cell origins, for
cell. Furthermore, many malignant tumors the histologic characteristics of tran5plants

FIG. 23 (top). Small nodule of

SK-L2 cells in lung. Lung was
seldom a major site of growth
of the cell lines included in
this study (see discussion).

FIG. 24 (bottom). Mass in sub-

cutaneous tissues at base of tail
due to SK-LNI cells. This and
a similar lesion in one rat which
was given SK-L9 cells were the
only subcutaneous masses 011-
served in these studies. They
probably resulted from partial
extravasation of the inoculum
with consequent growth of the
subcutaneous deposits in rats
made immunologically tolerant
b y the intravenous injectiori.
298 February 1969
CANCER
were quite the same for all these cell lines,
as is also true of their cytologic characteristics
in culture. Transplantability was not related
to the presence of virus particles. Jijoye cells,
which did not transplant, produce large num-
bers of herpes-like virus particles, as do EB-2
and EB-3 cells,S~16 which grew vigorously on
transplantation.
T h e heterotransplantability of the two
lines of non-neoplastic origin (C566 and SK-
LNI) could be the result of malignant trans-
formation of normal cells during cultivation.
Such transformation in vitro has been ob-
served in human cell lines such as F L am-
nion14 and Chang’s conjunctiva and appendix
lines.5, 22 Consistent with this possibility is
the observation that the C566 cell line was
tetraploid in studies performed by Dr. June
Biecller during the same period as these trans-
plantation studies (personal comni.). In prior
studies Benyesh-Melnick and coworkers ob-
served growth of C566 in 8 of 82 newborn rats
inoculated iv or into the Alternate ex-
planations for the “malignant” behavior of
these cells are that even normal human cells
might be transplantable in newborn rats if
they have appropriate but as yet undefined
characteristics, or that cell lines SK-LNl and
C566 were actually derived from malignant
cells even though tlie patients had no recog-
nized neoplasm. Is it possible that infectious
niononucleosis is more closely akin to leuke-
mia than we now suspect? Might the hemo-
philia patient, from whom the SK-LNl line
was obtained, have had an incipient lymphatic
neoplasm? At present the only possible conclu-
sion is that the relationship of transplaritabil-
ity to malignancy is moot. Tumor-like growth
of cells on heterotransplantation is suggestive
evidence that they are malignant neoplastic
cells but does not prove it. Failure of cells to
grow in transplantation throws doubt on the
neoplastic nature of the cells but does not
prove that they are normal.
T h e distribution of the “tumors” produced
by the human cell lines differed significantly
from that observed with other human cell
lines. T h e rarity of implants in the lungs is
remarkable since lungs are the major site of
growth for J-111, H E p 2, and numerous other
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