B Lymphocytes Promote
Lymphogenous Metastasis of
Lymphoma and Melanoma1
Alanna Ruddell*,†, Maria I. Harrell*,
Momoko Furuya*, Sara B. Kirschbaum*
and Brian M. Iritani*,†
*Fred Hutchinson Cancer Research Center, University
of Washington, Seattle, WA, USA;†Department of
Comparative Medicine, University of Washington,
Seattle, WA, USA
The prognosis of patients with many types of cancers correlates with the degree of metastasis to regional lymph
nodes (LNs) and vital organs. However, the mechanisms and route of cancer cell metastasis are still unclear. Pre-
vious studies determined that B-cell accumulation in tumor-draining LNs (TDLNs) induces lymphatic sinus growth
(lymphangiogenesis) and increases lymph flow, which could actively promote tumor dissemination through the
lymphatic system. Using young Eμ-c-Myc mice that feature LN B-cell expansion as hosts for tumor transplants,
we show that subcutaneously implanted lymphomas or melanomas preferentially spread to TDLNs over non-TDLNs,
thus demonstrating that these tumors initially metastasize through lymphatic rather than through hematogenous
routes. In addition, the rate and amount of tumor dissemination is greater in Eμ-c-Myc mice versus wild-type hosts,
which correlates with LN B-cell accumulation and lymphangiogenesis in Eμ-c-Myc hosts. The increased lymphatic
dissemination in Eμ-c-Myc hosts is further associated with rapid hematogenous tumor spread of subcutaneously
implanted lymphomas, suggesting that TDLN metastasis secondarily drives lymphoma spread to distant organs.
In contrast, after intravenous tumor cell injection, spleen metastasis of lymphoma cells or lung metastasis of mela-
noma cells is similar in Eμ-c-Myc and wild-type hosts. These studies demonstrate that the effect of Eμ-c-Myc hosts to
promote metastasis is limited to the lymphatic route of dissemination. TDLN B-cell accumulation, in association with
lymphangiogenesis and increased lymph flow, thus significantly contributes to dissemination of lymphomas and
solid tumors, providing new targets for therapeutic intervention to block metastasis.
Neoplasia (2011) 13, 748–757
Metastasis is the major cause of death from cancer; hence, an under-
standing of the mechanism of cancer cell trafficking to secondary or-
gans could inform the development of therapeutic interventions to
improve patient survival. Surprisingly, the actual route of tumor dis-
semination is still controversial in both mice and humans. The lym-
phatic system was initially proposed to be the main conduit for tumor
spread because the tumor-draining lymph nodes (TDLNs) are com-
monly the first site where metastasis is detected (reviewed by Kawada
and Taketo  and Nathanson ). In support of this hypothesis, the
identification of tumor cells in sentinel LNs (SLNs) has become the
most accurate predictor of metastasis to distant organs [2–4]. SLN dis-
section improves survival of patients in some studies , presumably
by limiting distant metastasis . However, other studies have shown
that extensive LN dissection does not reduce metastasis to distant or-
gans [6,7], leading to the proposal that SLN metastases are merely in-
dicators, rather than governors, of metastasis (reviewed by Sleeman
and Thiele ). In the latter interpretation, the detection of tumor
cells successfully seeding LNs would indicate that cancers have ac-
quired the ability to metastasize through the bloodstream.
In animal models, evidence supporting a lymphatic route of metas-
tasis has been obtained by manipulation of the expression of lymphatic
Abbreviations: TDLN, tumor-draining lymph node; SLN, sentinel lymph node; TRP,
tryosinase-related protein 1
Address all correspondence to: Alanna Ruddell, PhD, Fred Hutchinson Cancer Research
Center,MSC2-023,1100 FairviewAve N,Seattle,WA98109.E-mail:firstname.lastname@example.org
1This work was supported by the National Institutes of Health (National Cancer In-
stitute RO1 grant CA68328 to A. Ruddell and National Center for Research Re-
sources grant 5K26RR-024462 to B. Iritani).
Received 25 May 2011; Revised 13 June 2011; Accepted 15 June 2011
Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1522-8002/11/$25.00
Volume 13 Number 8August 2011pp. 748–757
endothelial growth factors VEGF-C or -D, to demonstrate that over-
expressed factors promote tumor lymphatic vessel growth (lymphangio-
genesis) and metastasis to draining LNs [9–11], whereas inhibition of
these factors blocks tumor lymphangiogenesis and dissemination [12–
14]. In addition, SLN removal in mice developing melanoma reduces
hematogenous tumor spread [15,16]. However, tumor lymphangio-
genesis is not a consistent feature in murine tumor models [17,18],
and some mouse tumors can spread through the hematogenous route
[19,20]. Analysis of human cancers has also yielded mixed results, with
tumor expression of VEGF-C or -D or tumor-associated lymphangio-
genesis sometimes but not always predicting poor prognosis [21–23].
Hence, an understanding of both lymphogenous and hematogenous
routes may be required to fully describe the mechanisms of primary
tumor spread to distant organs in animals and in humans.
Our previous studies of mouse models of metastatic cancers iden-
tified highly specific alterations in LNs that could be important for
tumor dissemination. First, our studies of Eμ-c-Myc transgenic mice
developing B-cell lymphomas identified LN B-cell accumulation,
lymphatic sinus growth, and increased lymph flow that preceded the
appearance of disseminated lymphomas . We then used a mela-
noma model to test whether TDLNs of solid tumors develop similar
changes. Wild-type mice bearing B16 melanomas in the rear footpad
develop metastasis to the draining popliteal LN and then to the lungs
after several months [20,25]. The TDLNs of these mice also showed
enlargement, B-cell accumulation, extensive lymphangiogenesis, and
increased lymph flow, before metastases were detected in the LN
[26,27]. TDLN alterations and LN metastasis did not develop in B-
cell–deficient mice, indicating that B cells are required for these effects.
Using murine models of squamous cell carcinoma, we found that wild-
type mice developing tumors similarly showed TDLN B-cell accumu-
lation and lymphangiogenesis and that the extent of these alterations
predicted progression from benign papillomas to metastatic carcino-
mas . Taken together, these findings led us to hypothesize that
B-cell accumulation in the TDLN induces TDLN lymphangiogenesis
and increased lymph flow, which actively promotes metastasis through
The availability of immune-competent mouse cancer models al-
lows us to examine the involvement of B-cell–induced LN alterations
in tumor metastasis through the lymphatic or blood circulation, in a
manner that is not possible in human cancer patients. The LNs of
young preneoplastic Eμ-c-Myc mice feature B-cell accumulation, lym-
phangiogenesis, and increased lymph flow , providing a model sys-
tem to test the involvement of these LN alterations in tumor spread.
Importantly, the lymphatic vasculature of young Eμ-c-Myc mice seems
normal, except for the lymphatic sinus growth within LNs . In this
study, the metastasis of tumors implanted into Eμ-c-Myc and wild-type
hosts was measured to identify any effects of the Eμ-c-Myc LN altera-
tions on lymphogenous or hematogenous tumor dissemination. We
report that the B-cell–induced LN alterations of young Eμ-c-Myc mice
drive metastasis of lymphomas as well as solid tumors.
Materials and Methods
Eμ-c-Myc B6 (Cg-Tg(IghMyc)22Bri/J)  and GFP (C57BL/
6-Tg(UBC-GFP)30Scha/J)  transgenic mice on C57BL/6 back-
ground (Jackson Laboratory, Bar Harbor, ME) were bred and main-
tained in microisolator rooms at the Fred Hutchinson Cancer Research
Center animal facility and were handled in accordance with the pro-
tocols approved by the Fred Hutchinson Cancer Research Center
Animal Care and Use Committee. GFP-expressing lymphoma cells
were obtained from the LNs of double-transgenic Eμ-c-Myc/GFP mice
bearing lymphomas, minced, filtered, and stored at −70°C in B-cell
media . For subcutaneous tumor implantation, 250,000 lym-
phoma cells were thawed and injected subcutaneously into the flank
of 4-week-old Eμ-c-Myc or wild-type littermates in Hank’s balanced
salt solution (HBSS) and allowed to grow for 11 days. HBSS was in-
jected into the control contralateral flank. For systemic tumor injection,
mice were intravenously injected in the tail vein with 250,000 cells in
HBSS, and 10 days later, organs were collected after CO2euthanasia.
LNs and spleens were minced, dispersed, and filtered, and spleens were
additionally treated to remove red blood cells , followed by flow
cytometry analysis using a Calibur flow cytometer (Becton Dickinson,
Franklin Lakes, NJ). Gated lymphocytes were analyzed using FlowJo
software (Tree Star, Inc, Ashland, OR).
For the analysis of Eμ-c-Myc lymphoma cell division, cells were
labeled with 5-(and -6)-carboxyfluorescein diacetate succinimidyl
ester (CFSE; Invitrogen, Carlsbad, CA) and subcutaneously injected
into the flank of Eμ-c-Myc or wild-type recipients . Four days later,
lymphomas were analyzed by flow cytometry to detect CFSE-positive
lymphoma cell division. Propidium iodide staining was used to mea-
sure DNA content for cell cycle analysis of lymphoma cells, using a
Vantage II cytometer (Becton Dickinson) .
B16-F10 murine melanoma cells  from the American Type
Culture Collection (Manassas, VA) were grown in Dulbecco modified
Eagle medium with 10% fetal calf serum and were tested for virus
(Research Animal Diagnostic Laboratory, University of Missouri,
Columbia, MO) and Mycoplasma contamination (MycoAlert; Cambrex,
Rockland, ME) before injection into littermates. Melanoma cells
the other footpad was injected with HBSS alone, and mice were then
incubated for 20 days before euthanasia and autopsy . For systemic
tumor induction, 20,000 B16-F10 cells were injected into the tail vein.
After 14 days, mice were autopsied to identify any visible black metas-
tases. Lungs were fixed in Fekete solution (100 ml of 70% ethanol,
10 ml of neutral buffered formalin, and 5 ml of glacial acetic acid), and
colonies were counted .
Tissues were cryosectioned and immunostained, followed by de-
tection using Vector VIP with Methyl Green counterstaining (Vector
Laboratories, Burlingame, CA), as described [24,26]. Antibodies used
were c-Myc (Oncogene Research Products, La Jolla, CA), LYVE-1
(eBioscience, San Diego, CA), and HRP-labeled goat secondary anti-
bodies (Southern Biotechnology, Birmingham, AL).
Real-time Reverse Transcription–Polymerase Chain Reaction
RNA was purified and reverse-transcribed using random primers
. Real-time polymerase chain reaction (PCR) was performed using
FastStart Universal SYBR Green Master Mix (Roche, Indianapolis,
IN) and primers for the tryosinase-related protein 1 (TRP) gene
. β-Actin was amplified from each sample to check that comple-
mentary DNA synthesis efficiency was the same for each sample. To
measure the percent of B16-F10 cells, the Ctvalues of experimental
samples were compared with a standard curve, prepared from B16-F10
cells mixed with NIH 3T3 cells in varying proportions (1 B16-F10
Neoplasia Vol. 13, No. 8, 2011B Lymphocytes Promote Lymphogenous MetastasisRuddell et al.
cell in 1 × 105NIH 3T3 cells up to 1 × 105B16-F10 in 0 NIH 3T3
cells) to maintain the same total cell number. The number of cells/LN
was calculated by dividing the total amount of RNA/LN by 0.06 ng
of RNA/cell, and then the absolute number of B16-F10 cells/LN
was calculated by multiplying the percent B16-F10 cells by the total
Eμ-c-Myc Hosts Promote Lymphoma Metastasis
We proposed that TDLN alterations (B-cell accumulation, lym-
phangiogenesis, increased lymph flow) associated with the formation
of metastatic cancers actively promote tumor spread through the
lymphatic system . To test this hypothesis, young 4-week-old
preneoplastic Eμ-c-Myc mice, which already feature these LN altera-
tions, were used as hosts for murine lymphoma cells to determine
whether they show accelerated tumor metastasis relative to wild-type
hosts. First, the dissemination of GFP-tagged Eμ-c-Myc B-cell lym-
phoma cells was analyzed after subcutaneous implantation into the
flank of young Eμ-c-Myc or normal littermate hosts. Organs were
harvested 11 days later to assess dissemination by flow cytometry
analysis of GFP-lymphoma cells. The lymphoma cells formed dis-
crete flank tumors of similar size in Eμ-c-Myc and wild-type hosts
(Figure 1A), indicating that primary lymphoma growth is similar
in Eμ-c-Myc or normal littermates. Lymphocytes and GFP-positive
lymphoma cells from the primary tumor and from spleens of im-
planted Eμ-c-Myc and normal littermate hosts were further analyzed
by flow cytometry (Figure 1B). GFP-positive lymphoma cells were
abundant in the primary tumor and in spleens of Eμ-c-Myc hosts;
however, they were rare in spleens of wild-type hosts (Figure 1C).
Quantitation of the abundance of GFP-positive lymphoma cells from
a number of mice revealed a 10-fold increase in lymphoma cells in
spleens of Eμ-c-Myc relative to wild-type mice (Figure 1D), demon-
strating that lymphoma dissemination to the spleen is accelerated in
To distinguish whether lymphoma dissemination to the spleen is
promoted by lymphatic or hematogenous pathways, we compared
the ipsilateral TDLNs versus contralateral nondraining LNs for signs
of metastasis. We reasoned that, if metastasis predominantly oc-
curred through a hematogenous route, then both LNs should con-
tain equal numbers of lymphoma cells, whereas if the metastasis
occurred through a lymphatic route, then the TDLNs should pref-
erentially accumulate metastatic cells (Figure 2A). Flow cytometry
of gated lymphocyte populations (Figure 2B) was used to measure
the percentage of GFP-positive lymphoma cells in LNs (Figure 2C).
In wild-type hosts, flank lymphoma cells preferentially trafficked
to the draining left inguinal and axillary LNs, whereas few cells
were detected in the nondraining right LNs (Figure 2D), indicating
that lymphoma cells primarily spread through the lymphatics. This
Figure 1. Metastasis of subcutaneously implanted lymphoma in Eμ-
c-Myc hosts. (A) The growth of Eμ-c-Myc lymphoma tumors (black
dashed line) subcutaneously implanted into the left flank is similar
in Eμ-c-Myc and wild-type littermate hosts. (B) Flow cytometry gat-
ing of lymphocytes demonstrates enlarged Eμ-c-Myc lymphoma
cells with increased forward light-scatter obtained from primary
flank Eμ-c-Myc lymphoma and spleen of an Eμ-c-Myc host but
not from wild-type host spleen. (C) Flow cytometry measurement
of GFP-positive lymphoma cells in primary lymphoma or in spleens
of Eμ-c-Myc or wild-type hosts. (D) GFP-Eμ-c-Myc lymphoma me-
tastasis to spleen is increased 10-fold in Eμ-c-Myc relative to wild-
type littermates bearing flank tumors, comparing eight Eμ-c-Myc
and eight wild-type mice. Standard errors are shown. This differ-
ence is statistically significant by 2-tailed t test (***P < .0001).
B Lymphocytes Promote Lymphogenous MetastasisRuddell et al. Neoplasia Vol. 13, No. 8, 2011
ipsilateral spread was accentuated in Eμ-c-Myc hosts, where lym-
phoma cells also preferentially accumulated in the draining left LNs,
as shown by four-fold increases in lymphoma cell abundance relative
to wild-type littermates. This is likely an underestimation of lym-
phoma cell accumulation because Eμ-c-Myc LNs are enlarged and con-
tain twice as many lymphocytes as wild-type mice , so that the
absolute number of lymphoma cells likely increased eight-fold in
LNs of Eμ-c-Myc hosts.
Hematogenous lymphoma dissemination to nondraining right
LNs (Figure 2D) was also increased in Eμ-c-Myc hosts relative to
wild-type mice, so that lymphoma cells were six times more abun-
dant in the right inguinal and axillary LNs in Eμ-c-Myc versus nor-
mal littermate hosts. Again, the absolute increase in lymphoma cell
number may approach 12-fold given the 2-fold larger size of LNs
in Eμ-c-Myc mice. This systemic spread may be caused by lympho-
genous delivery of lymphoma cells into the thoracic duct and blood-
stream or by hematogenous spread from the draining LN or primary
tumor. These findings indicate that, in wild-type hosts, initial lym-
phoma dissemination is mainly restricted to the lymphatic circula-
tion, whereas in Eμ-c-Myc hosts, lymphatic metastasis to draining
LNs is enhanced to a greater degree, which is additionally associated
with systemic spread to distant organs.
Eμ-c-Myc and Wild-type Hosts Support Similar
Lymphoma Cell Growth, Lymphoma Cell Survival,
and Hematogenous Metastasis
The observed enhancement of LN metastasis in Eμ-c-Myc mice
could potentially be due to a host’s effect on the tumor environment
to promote tumor cell proliferation rather than to a specific effect on
lymphoma spread through the lymphatic system. This possibility was
tested by subcutaneously implanting CFSE-labeled lymphoma cells
into the flank of Eμ-c-Myc or wild-type hosts, followed by compar-
ison of the in vivo proliferation of lymphoma cells in Eμ-c-Myc and
wild-type hosts. Four days after injection, lymphoma cells were
subjected to flow cytometry to assess cell division of the transferred
cells. Injected lymphoma cells proliferated to the same extent in wild-
type and Eμ-c-Myc mice (Figure 3A), as indicated by similar dilution
of CFSE at each generation. Flow cytometric analysis of propidium
iodide–stained cells also indicated that lymphoma cells were cycling
equivalently in Eμ-c-Myc and wild-type hosts (Figure 3B). These
Figure 2. Lymphogenous metastasis of subcutaneously implanted
Eμ-c-Myc lymphoma in Eμ-c-Myc hosts. (A) A lymphogenous route
of metastasis is indicated by selective spread from the left flank
tumor to draining left inguinal and axillary LNs, before delivery into
the pulmonary circulation and heart. Hematogenous metastasis is
indicated by tumor spread from flank implant to both draining and
nondraining LNs and through the bloodstream to the spleen. (B)
Flow cytometry gating of lymphocytes demonstrates enlarged
Eμ-c-Myc lymphoma cells with increased forward light-scatter in
inguinal TDLN of an Eμ-c-Myc host but not in wild-type host in-
guinal TDLN. (C) Flow cytometry measurement of GFP-positive
Eμ-c-Myc lymphoma cells in inguinal TDLN of Eμ-c-Myc and
wild-type hosts. (D) GFP-labeled Eμ-c-Myc lymphoma cells pref-
erentially accumulate in draining left inguinal (L ING) and axillary
(L AX) LNs but not in nondraining right LNs (R ING and R AX) of
wild-type or Eμ-c-Myc hosts. Eleven Eμ-c-Myc hosts were com-
pared with 11 wild-type hosts. Statistical significance by 2-tailed
t test is indicated (*P < .03x, **P < .003x).
Neoplasia Vol. 13, No. 8, 2011B Lymphocytes Promote Lymphogenous MetastasisRuddell et al.
findings demonstrate that the enhanced metastasis in Eμ-c-Myc mice
is not explained by increased lymphoma cell growth in these hosts.
The Eμ-c-Myc host could potentially enhance hematogenous lym-
phoma metastasis by a mechanism distinct from its effects on LN
lymphatic sinus growth. This possibility was directly tested by intra-
venously injecting GFP-labeled lymphoma cells into Eμ-c-Myc and
wild-type hosts. Ten days later, flow cytometric analysis revealed a
modest 1.5-fold increase in lymphoma cell abundance in the spleens
of Eμ-c-Myc versus wild-type littermates (Figure 3C). Eμ-c-Myc hosts,
therefore, exert minimal effects on hematogenous lymphoma metas-
tasis, relative to the large effects on lymphatic metastasis to draining
LNs (Figure 2D). These findings suggest that the increased hematog-
enous spread of lymphoma cells to the nondraining LNs and spleen
in Eμ-c-Myc hosts likely results from accelerated LN metastasis, lead-
ing to increased systemic delivery.
Eμ-c-Myc Hosts Show Enhanced Lymphogenous Metastasis
Our finding that lymphoma metastasis to draining LNs is en-
hanced in Eμ-c-Myc mice provides support for our hypothesis that
B-cell accumulation within LNs promotes tumor spread through
the lymphogenous route. This effect could be specific to lymphomas,
or these LN alterations could also promote solid tumor metastasis. To
examine this question, we used a model whereby syngeneic B16-F10
melanoma cells were injected into the footpads of Eμ-c-Myc or normal
littermates. In the B16-F10 model, melanoma cells implanted in the
rear footpad metastasize initially to the draining popliteal LN, followed
several months later by the appearance of lung metastases [25,39], as
depicted in Figure 4A. We found that melanomas implanted in the
footpad were similar in appearance and growth in Eμ-c-Myc and
normal littermate hosts (Figure 4B). Measurement of the area of the
pigmented melanomas indicated that tumors were 1.5-fold larger in
Eμ-c-Myc mice (Figure 4C), indicating a modest effect of the Eμ-c-
Myc host on footpad tumor growth. However, the major difference
in Eμ-c-Myc versus wild-type littermates was the increased appearance
of metastases in the draining popliteal LN of Eμ-c-Myc hosts. Small
melanotic metastases were visualized as black dots on the LN capsule
from a number of Eμ-c-Myc hosts (Figure 4D, left panel, arrow),
whereas in some Eμ-c-Myc hosts, the melanoma cells overgrew the en-
tire LN (Figure 4D, middle panel). In contrast, most LNs from wild-
type hosts were free of black melanotic cells (Figure 4D, right panel).
Most Eμ-c-Myc hosts developed visible TDLN metastases (11/16),
whereas few wild-type hosts developed visible metastases (3/14), dem-
onstrating a statistically significant increase in the incidence of TDLN
metastasis in Eμ-c-Myc hosts (Figure 5A). These results indicate that
melanoma cells metastasize much more readily in Eμ-c-Myc hosts rel-
ative to wild-type hosts.
To measure the degree of melanoma metastasis to LNs in each host,
we developed a quantitative real-time reverse transcription (RT)–PCR
assay, which measures levels of the melanocyte-specific TRP messenger
RNA expressed in B16-F10 melanomas, as a surrogate method of cell
counting . A standard curve of RNA, prepared from known
numbers of TRP-expressing B16 cells spiked into TRP-negative
NIH 3T3 cells (0.01%-100%), was measured by real-time RT-PCR
analysis. This assay provided a sensitivity of detection of approximately
0.01% or 10 melanoma cells/LN (Figure 5B). Real-time RT-PCR
comparison of TDLNs from Eμ-c-Myc and wild-type hosts revealed
striking differences in melanoma metastasis to the draining popliteal
LN. The abundance of melanoma cells was, on average, 158 times
greater in TDLNs from Eμ-c-Myc relative to wild-type hosts (Fig-
ure 5C). Calculation of the number of melanoma cells per TDLN also
revealed many more tumor cells/LN in Eμ-c-Myc hosts (Figure 5D).
In wild-type hosts, the number of tumor cells in TDLN metastases
ranged from 108 to 707 cells/LN, whereas in Eμ-c-Myc hosts, the num-
ber of tumor cells in TDLNs ranged from 61 to 37,000,000 cells/LN.
In mice with detectable metastasis, the mean was 308 cells/LN for
wild-type mice, and 3,500,000 cells/LN for Eμ-c-Myc mice, indicating
an 11,000-fold enhancement of melanoma cell accumulation in the
TDLN of Eμ-c-Myc mice. These findings are in good agreement with
our visual autopsy findings (Figure 4D). Eμ-c-Myc hosts thus not only
increase the incidence of lymphatic metastasis from the primary tumor
Figure 3. Lymphoma growth and hematogenous metastasis are
similar in Eμ-c-Myc and wild-type hosts. (A) Proliferation rates
are similar in CFSE-labeled lymphoma cells subcutaneously im-
planted into flanks of Eμ-c-Myc or wild-type hosts, and analyzed
by flow cytometry 4 days later, to detect CFSE diminution by cell
division in each generation. (B) Lymphoma cell cycle is similar in
Eμ-c-Myc or wild-type hosts, as measured by propidium iodide
DNA staining and flow cytometry. (C) Hematogenous metastasis
of intravenously injected lymphomas to spleen is modestly en-
hanced in eight Eμ-c-Myc versus 8 wild-type hosts. This difference
is statistically significant by 2-tailed t test (P < .004).
B Lymphocytes Promote Lymphogenous MetastasisRuddell et al.Neoplasia Vol. 13, No. 8, 2011
(Figure 5A) but also accelerate the accumulation of melanoma cells
within the TDLN (Figure 5D). The secondary draining iliac or in-
guinal LNs, nondraining LNs, lungs, or other distant organs did not
contain detectable melanoma cells, indicating that the metastasis-
promoting effect of Eμ-c-Myc hosts is restricted to the first-tier TDLN,
at least in this short-term study.
Eμ-c-Myc Mice Develop Peripheral LN Lymphangiogenesis
The ability of Eμ-c-Myc hosts to accelerate metastasis of mela-
noma cells to the popliteal TDLN could be due to B-cell–induced LN
Figure 5. The incidence and the rate of TDLN metastasis of mela-
nomas are enhanced in Eμ-c-Myc hosts. (A) The increase of visibly
detectable tumor-draining popliteal LN metastasis in Eμ-c-Myc ver-
suswild-typehostsat20 daysafter tumorimplantation isstatistically
significant(Fisherexact 2-tailed test, P < .025). (B) Real-time RT-PCR
measurement of TRP messenger RNA levels in B16 melanoma cells
mixedwithnon–TRP-expressingNIH 3T3cellsin various proportions
to develop a standard curve for quantitation. (C) The abundance of
B16 melanoma cells in the draining popliteal LN is greatly increased
in Eμ-c-Myc hosts,and thisincreaseisstatistically significant (Mann-
Whitney 2-tailed test, P < .004). (D) The estimated number of mela-
noma cells per draining LN is greatly increased in Eμ-c-Myc hosts,
and this increase is statistically significant (Mann-Whitney 2-tailed
test, P < .001). Medians are indicated.
Figure 4. The Eμ-c-Myc host promotes melanoma metastasis to
the draining popliteal LN. (A) B16-F10 melanoma implanted in
the rear footpad of wild-type mice begins to metastasize to the
draining popliteal LN after 20 days and, several months later, to
the lungs. (B) Melanoma tumor growth (blue dashed line) is similar
in the footpad of Eμ-c-Myc and wild-type hosts. (C) Footpad mela-
noma area is modestly enhanced in 17 Eμ-c-Myc versus 17 wild-type
hosts at 20 days after implantation and is statistically significant by
2-tailed t test (*P < .02). (D) Melanotic melanoma metastasis (arrow,
left panel) is detected on the surface of the draining popliteal LN
(blue dashed line), whereas in some cases, melanoma cells over-
grow the popliteal LN of an Eμ-c-Myc host (middle panel). The LN
is typically tumor free in a wild-type host (right panel).
Neoplasia Vol. 13, No. 8, 2011B Lymphocytes Promote Lymphogenous MetastasisRuddell et al.
lymphangiogenesis, which we first described in visceral mesenteric LNs
of Eμ-c-Myc mice . Immunostaining was used to test whether the
peripheral popliteal LNs of young Eμ-c-Myc mice also accumulate
c-Myc–expressing B cells and undergo lymphatic sinus growth. Popliteal
LNs of Eμ-c-Myc mice were filled with purple-staining c-Myc–expressing
B cells, whereas LNs from normal littermates contained lymphocytes
with normal low c-Myc expression (Figure 6A). Popliteal LNs from
Eμ-c-Myc mice reproducibly exhibited lymphangiogenesis throughout
the cortex and medulla, whereas in wild-type mice, the normal LN lym-
phatic sinuses were restricted to the cortex, detected by immunostaining
with the LYVE-1 lymphatic endothelial antibody (Figure 6B). The in-
guinal LNs of Eμ-c-Myc mice also accumulated c-Myc–expressing B cells
(Figure 6C) and showed extensive lymphatic sinus growth (Figure 6D).
These findings demonstrate that the peripheral as well as visceral LNs
of Eμ-c-Myc mice accumulate c-Myc–expressing B lymphocytes and
develop extensive lymphangiogenesis. Importantly, these LN alterations
are already developed in 4-week-old Eμ-c-Myc mice, at the age when
they were used as hosts for the tumor implantation experiments.
The enhanced lymphogenous metastasis of melanomas in Eμ-c-Myc
hosts could potentially be mediated by enhanced tumor-associated
lymphatic vessel growth within or around the primary tumor rather
than by LN lymphangiogenesis. However, we found that the number
and size of lymphatic vessels in the melanoma footpad tumors
(Figure 6E, arrows) or in the peritumoral dermis (Figure 6E, arrow-
heads) were similar in Eμ-c-Myc and wild-type hosts. These findings
indicate that lymphatic sinus growth within LNs is the major lymphatic
vessel alteration associated with promotion of lymphogenous metastasis
in Eμ-c-Myc hosts.
Eμ-c-Myc Hosts Do Not Promote Hematogenous Metastasis
The ability of Eμ-c-Myc hosts to accelerate melanoma metastasis
could be restricted to the lymphogenous route of metastasis, or the
Eμ-c-Myc hosts could promote both lymphogenous and hematoge-
nous spread. To examine the ability of Eμ-c-Myc hosts to promote
tumor metastasis through the bloodstream, melanoma cells were in-
jected into the tail vein, and the number of melanoma colonies was
assessed in the lung . Melanotic B16 cells form visible colonies
on the lung, thus allowing the size and number of metastatic tumors
to be measured. Host effects to promote growth of metastases can be
evaluated by assessing the diameter of the lung colony. Whereas the
size of lung colonies was somewhat variable, on average, the colony
diameters were indistinguishable between Eμ-c-Myc and wild-type
hosts at 14 days after injection (Figure 7A). This indicates that Eμ-
c-Myc hosts do not preferentially promote growth of melanoma cells.
The average number of lung colonies per mouse was also the same
in wild-type and Eμ-c-Myc hosts (Figure 7B), indicating that the abil-
ity to establish metastases is not altered in Eμ-c-Myc mice. In addi-
tion, 2 of 14 wild-type mice developed metastases in the peritoneal
cavity, whereas 1 of 16 Eμ-c-Myc mice developed a smaller peritoneal
metastasis (data not shown), indicating that hematogenous metastasis
to other organs was also similar in wild-type and Eμ-c-Myc hosts. B16
metastases were not identified in LNs from wild-type or Eμ-c-Myc
hosts, indicating that B16 melanoma cells delivered through the blood
circulation do not preferentially colonize LNs. Taken together, these
findings demonstrate that the Eμ-c-Myc host does not preferentially
stimulate melanoma cell growth, survival, or metastasis through the
hematogenous route, relative to wild-type littermates. Furthermore,
the strong effect of the Eμ-c-Myc hosts to promote metastasis is specific
Figure 6. Lymphangiogenesis in peripheral LNs of Eμ-c-Myc mice.
(A) The cortex (C) and medulla (M) of a representative popliteal LN
from a 4-week-old Eμ-c-Myc mouse is filled with purple-stained
c-Myc–expressing B cells, whereas c-Myc is expressed at normal
low levels in lymphocytes of popliteal LNs from wild-type mice, by
immunohistochemical staining with c-Myc antibody followed by
Methyl Green nuclear counterstaining. (B) An Eμ-c-Myc popliteal
(M), whereas a wild-type LN shows normal lymphatic sinuses con-
fined to the cortex, by immunostaining with LYVE-1 antibody. (C) The
cortex and medulla of an inguinal LN from a 4-week-old Eμ-c-Myc
in an inguinal LN from Eμ-c-Myc mouse. (E) LYVE-1 antibody-positive
dermal lymphatic vessels (arrowheads) and intratumoral lymphatic
vessels (arrows) are similar in footpad melanomas of Eμ-c-Myc and
wild-type hosts. Scale bars are indicated.
B Lymphocytes Promote Lymphogenous MetastasisRuddell et al.Neoplasia Vol. 13, No. 8, 2011
to the lymphogenous route of metastasis. LN B-cell accumulation and
associated lymphangiogenesis and increased lymph flow in Eμ-c-Myc
hosts are likely responsible for the active and immediate promotion
of tumor metastasis to the draining LN, which can then drive further
tumor spread to distant organs, as illustrated in Figure 7C. In wild-
type hosts bearing tumors, B-cell accumulation and TDLN alterations
can gradually arise for weeks to months after tumor formation, to
eventually promote metastasis to LNs.
Our studies on lymphoma and melanoma metastasis in mice provide
evidence that these tumors preferentially spread through the lym-
phatic route. We show that lymphoma cells, when delivered locally,
spread initially to the draining LNs in wild-type mice. In addition,
we found that Eμ-c-Myc hosts, which feature preexisting LN B-cell
accumulation and lymphangiogenesis, promote increased metastasis of
lymphomas to the draining inguinal and axillary LNs relative to wild-
type hosts. Using the B16-F10 melanoma model, we found strongly
increased lymphogenous metastasis to the draining popliteal LN in
Eμ-c-Myc versus wild-type mice, demonstrating that Eμ-c-Myc hosts
also promote LN metastasis of solid tumors. Primary lymphomas or
melanomas showed similar growth rates in Eμ-c-Myc hosts and wild-
type hosts, suggesting that enhanced tumor growth did not account for
the increased LN metastasis in Eμ-c-Myc mice. Melanoma cells also
did not spread to nondraining LNs, indicating that general LN tro-
pism does not explain preferential spread of melanoma cells to drain-
The peripheral LNs of young Eμ-c-Myc hosts feature abnormal ac-
cumulation of c-Myc–expressing B cells throughout the cortex and
medulla, which is accompanied by extensive lymphatic sinus growth
and increased lymph flow. These same changes also arise gradually
for weeks to months in the TDLNs of wild-type mice developing
metastatic melanoma  or squamous cell carcinoma . B-cell
accumulation is required for lymphatic sinus growth and increased
lymph flow because TDLNs from B-cell–deficient mice implanted
with melanomas do not exhibit lymphangiogenesis or increased lymph
flow, and melanoma cells do not metastasize to TDLNs in B-cell–
deficient mice . Thus, we proposed that B cells residing in TDLNs
stimulate lymphangiogenesis and increase lymph flow, which pro-
motes metastasis by physically increasing cell trafficking through the
lymphatic drainage. Our finding that metastasis through the lymphatic
route is strongly increased in Eμ-c-Myc hosts, which feature preexisting
LN B-cell accumulation, lymphatic sinus growth, and increased lymph
flow, provides strong support for this hypothesis.
It is not yet clear how LN lymphangiogenesis in Eμ-c-Myc mice in-
creases lymph flow through the popliteal LN more than 20-fold .
Our previous studies demonstrated that dermal lymphatic vessels in
Eμ-c-Myc mice appear normal, whereas mesenteric LN lymphatic si-
nuses show extensive growth . Here, we report that the lymphatic
vessels in and around the implanted melanoma footpad tumors are
similar in Eμ-c-Myc and wild-type hosts, whereas the draining popliteal
LNs of Eμ-c-Myc but not wild-type mice feature extensive lymph-
angiogenesis. The restriction of B-cell–induced lymphatic vessel growth
to the LNs in Eμ-c-Myc mice supports the notion that LNs somehow
regulate lymph flow, perhaps by influencing intrinsic contractile activ-
In contrast to the strong increase in lymphogenous metastasis to
LNs in Eμ-c-Myc mice, we observed minimal effects of Eμ-c-Myc
hosts on hematogenous metastasis after tail vein injection. Our anal-
ysis of intravenously injected B16 melanomas was particularly re-
vealing because both the size and the number of lung colonies were
comparable in Eμ-c-Myc and wild-type hosts. In addition, intrave-
nously injected lymphoma cells showed similar growth in the spleens
of Eμ-c-Myc and wild-type hosts. These results demonstrate that the
metastasis-promoting effects in Eμ-c-Myc hosts are essentially limited
to the lymphatic system and are not due to effects on primary tumor
formation or growth. Our finding that the growth of subcutaneous pri-
mary tumors is similar in Eμ-c-Myc and wild-type hosts also indicates
Figure 7. Hematogenous melanoma metastasis is similar in Eμ-c-
Myc and wild-type hosts. (A) Representative lungs from Eμ-c-Myc
and wild-type hosts injected intravenously with B16-F10 melanoma
cells. The average size of melanoma colonies is the same in Eμ-c-
Myc and wild-type lungs. (B) The average number of melanotic lung
colonies is similar in eight Eμ-c-Myc and eight wild-type hosts. (C)
Model of active promotion of initial lymphogenous route of metas-
tasis of tumors to draining LNs by B-cell–induced LN lymphangio-
genesis and lymph flow, such as that seen in young Eμ-c-Myc mice,
which can subsequently promote hematogenous dissemination of
theTDLN accumulates B cells toinducelymphatic sinus growth and
increase lymph flow.
Neoplasia Vol. 13, No. 8, 2011B Lymphocytes Promote Lymphogenous Metastasis Ruddell et al.
that defective immunosurveillance does not contribute to the pheno-
type of increased metastasis in Eμ-c-Myc mice.
The B-cell accumulation and associated alterations in Eμ-c-Myc
LNs could also potentially influence some aspect of the antitumor
immune response to enhance LN metastasis. For example, antitumor
immunoglobulins promote squamous cell carcinoma formation and
metastasis in a chronic inflammation model . However, B16 mel-
anomas induce minimal humoral immune responses in wild-type mice
[35,42], and our tumor induction experiments are too brief to involve
tumor-specific antibody production. Tumor-infiltrating B cells can
promote prostate cancer growth ; however, we have not observed
B cells infiltrating B16 melanomas . Immune function in Eμ-c-
Myc mice seems to be normal , although subtle alterations in
maintenance of B-cell tolerance have been described . LN lym-
phatic sinus endothelium has recently been shown to present self-
antigens to maintain peripheral immune tolerance . These results
suggest that LN lymphangiogenesis in Eμ-c-Myc mice could reinforce
TDLN immune tolerance to tumor growth within the LN. Analysis
of the immune microenvironment of Eμ-c-Myc LNs will be required
to determine whether any local alterations in antitumor immune re-
sponse contribute to increased TDLN metastasis in Eμ-c-Myc mice.
The high rate of metastasis of subcutaneously implanted Eμ-c-Myc
lymphomas to draining LNs was accompanied by increased hematog-
enous spread to the spleen. This enhanced systemic dissemination
could result from the secondary spread of lymphoma cells from the
TDLN through the lymphatic drainage into the thoracic duct, heart,
and then to the blood circulation. Alternatively, lymphomas could
spread more directly to distant organs by entering the venous blood
circulation from within the TDLN or from the primary tumor.
These potential mechanisms of secondary tumor dissemination from
TDLNs cannot be distinguished from our current studies. How-
ever, Eμ-c-Myc lymphoma cells predominantly fill lymphatic sinuses
within TDLNs , suggesting that the lymphatic route can support
further dissemination from the first-tier TDLN. Progression and
prognosis of non–Hodgkin or Hodgkin lymphomas is categorized
on the basis of tumor localization in regional LNs (i.e., stage I)
and further spread to nondraining LNs and distant organs (stage
IV [46,47]). The LNs of patients with malignant lymphoma exhibit
LN lymphangiogenesis , suggesting that inhibition of LN lym-
phangiogenesis could be an efficient strategy to limit systemic dis-
semination of human lymphomas. We were unable to determine
whether metastasis of melanomas to distant organs is also accelerated
in Eμ-c-Myc hosts after melanoma implantation in the footpad be-
cause lung metastases take several months to appear after initial LN
metastasis and footpad tumors outgrow acceptable limits before sec-
ondary metastasis . However, it is likely that the B-cell–induced
LN alterations of Eμ-c-Myc hosts also promote solid tumor spread
to distant organs because LN B-cell accumulation and lymphangiogen-
esis predicts the metastatic potential of squamous cell carcinomas to
spread to draining LNs and lungs over a longer period .
B-cell accumulation, lymphangiogenesis, and increased lymph
flow within regional draining LNs also arise in a B-cell–dependent
manner in mice responding to acute bacterial infection  and in
mice with rheumatoid arthritis [50,51]. Our studies of Eμ-c-Myc
mice support the idea that metastasis is promoted by B-cell accu-
mulation and LN lymphangiogenesis, thus providing a potential link
to explain the association of chronic inflammation and cancer pro-
gression . In support of this idea, a recent study demonstrated
increased mammary carcinoma metastasis in mice with autoimmune
arthritis . In humans, patients with arthritis show normal inci-
dence of many types of cancers, but they exhibit increased cancer
mortality , consistent with the proposal that TDLN alterations
promote metastasis. TDLN enlargement, lymphocyte accumulation,
and sinus expansion are often diagnosed as tumor-reactive lymph-
adenopathies in human cancer patients, although their connection
to metastasis is not clear . Our findings suggest that inhibition
of TDLN B-cell accumulation and associated lymphangiogenesis
merit investigation as a therapeutic approach to block metastasis of
lymphoma or solid tumors.
The authors thank Tina Albershardt, Tania Habib, Lee Hartwell,
Chris Kemp, and Paul Neiman for their advice; and Daniel Diolaiti,
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