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Acta Biochim Biophys Sin 2005,37:601-606 |
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doi:10.1111/j.1745-7270.2005.00083.x |
Hyaluronan Promotes Tumor
Lymphangiogenesis and Intralymphantic Tumor Growth in Xenografts
Li-Xia GUO, Ke ZOU, Ji-Hang
JU, and Hong XIE*
Laboratory of
Biotherapy, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Graduate School of the
Chinese Academy of Sciences, Shanghai 200031, China
Received:
March 8, 2005
Accepted:
June 3, 2005
*Corresponding author: Tel, 86-21-54921434; Fax,
86-21-54921436; E-mail, [email protected]
Abstract Hyaluronan (HA), a high
molecular weight glycosaminoglycan in the extracellular matrix, has been
implicated in the promotion
of malignant phenotypes, including tumor angiogenesis. However, little is known
about the effect of HA on tumor-associated lymphangiogenesis. In this study,
mouse hepatocellular carcinoma Hca-F cells combined with or without HA were
injected subcutaneously into C3H/Hej mice, then angiogenesis and
lymphangiogenesis of implanted tumors were examined by immunostaining for
platelet-endothelial cell adhesion molecule-1 and lymphatic vascular
endothelial hyaluronan receptor-1 respectively. Interestingly, we found HA
promotes tumor lymphangiogenesis and the occurrence of intratumoral lymphatic
vessels, but has little effect on tumor angiogenesis. Moreover, HA also promotes
intralymphatic tumor growth, although it is not sufficient to potentiate
lymphatic metastasis. These results suggest that HA, which is elevated in most
malignant tumor stroma, may also play a role in tumor progression by promoting
lymphangiogenesis.
Key words hyaluronan; tumor
lymphangiogenesis; intralymphatic tumor growth
Lymphangiogenesis, the formation of new lymphatics, is a fundamental
physiological process required for the development of the embryonic lymph
system and regeneration of lymphatic vessels in adults, for example, in wound
healing [1]. More recently, lymphangiogenesis has also been implicated in tumor
progression [2,3]. However, the regulation of tumor-associated
lymphangiogenesis is not well understood. Animal models demonstrated that
tumor-associated lymphangiogenesis can be induced by overexpression of
lymphangiogenic vascular endothelial growth factor (VEGF)-C or VEGF-D, and that
lymphangiogenesis may be involved in subsequent tumor lymphatic metastasis
[46]. Moreover, targeting of VEGF-C/VEGF-D and their receptor VEGFR-3 signaling
resulted in decreased tumor lymphangiogenesis and reduction of lymph node
metastases [69]. These data suggested the important roles of VEGF-C, VEGF-D and
their receptor VEGFR
extracellular matrix. HA has been shown to play important roles in tumor
progression. Most malignant solid tumors contain elevated levels of HA, and in
some cases HA levels were prognostic for malignant progression [10]; on the
other hand, HA has been implicated in regulating tumor malignant behaviors,
for example, anchorage-independent growth [10], tumor cell motility [11,12],
secretion of matrix metalloproteinases [13], as well as tumor angiogenesis
[14,15]. Despite the wealth of data, however, it has not yet been addressed
whether HA has any effect on tumor-associated lymphangiogenesis.
Experimental manipulations of HA concentration in tumor stroma by
overexpression of HA synthase or administration of exogenous HA have been used
to investigate the role of HA in tumor progression [14,15].
In this study, we examined the effect of exogenous HA on tumor
lymphangiogenesis, angiogenesis and lymphatic metastasis in a lymph node
metastatic model. The data showed that HA promotes the formation of
intratumoral lymphatic vessels and intralymphatic tumor growth. Our results
suggest that HA may be a potential new regulator in tumor lymphangiogenesis.
Materials and Methods
Cells
Mouse hepatocellular carcinoma Hca-F cells were injected i.p. into 8-week-old
C3H/Hej mice at 1 ml cell solution per mouse (107 cells/ml PBS) to
generate ascites. Ascites were then harvested and injected into new mice.
Tumorigenesis and tumor growth
assay
Hca-F ascites were harvested and washed twice with PBS, then resuspended
in PBS or 1 mg/ml HA (hyaluronic acid sodium salt from human umbilical cord;
Fluka,
Immunostaining for
lymphangiogenesis and angiogenesis
The tumors were excised and frozen in liquid nitrogen. Sections (5 mm) were immunostained with rat monoclonal antibody against
platelet-endothelial cell adhesion molecule-1 (PECAM-1) (PharMingen, Franklin
Lakes, USA) [16] or rabbit polyclonal antibody against mouse lymphatic vascular
endothelial hyaluronan receptor-1 (LYVE-1) (a kind gift from Dr. David G.
JACKSON, Oxford University, UK) [17]. The staining was detected with an ABC kit
(Vector Laboratories, Burlingame, USA) according to the manufacturer’s
instructions. The slides were stained with diaminobenzidine (Huamei,
Tumor metastasis assay
Superficial axillary, deep axillary and inguinal lymph nodes (on
right side) were removed, fixed in 10% neutral-buffered formalin and embedded
in paraffin. Sections (5 mm) were cut, stained with
hematoxylin and eosin, and examined microscopically. The rate of metastasis was
calculated as metastatic mice per total tested mice, and the rate of lymph node
metastasis was calculated as the number of positive lymph nodes per total
number of lymph nodes examined.
Statistical analysis
Data are presented as mean+/–SD. For statistical analysis, Student’s t-test
was performed. P<0.05 was considered to denote statistical
significance.
Results
HA induced tumor
lymphangiogenesis in xenografts
LYVE-1 is a highly specific molecular marker of lymphatic
endothelia and has been widely used in the detection of lymphangiogenesis
[17]. Here, we examined the effect of exogenous HA on lymphangiogenesis by
immunostaining for LYVE-1 to show the lymphatic vessels in implanted tumors.
Tumors were sectioned and subjected to immunostaining at day 21 after
implantation. In control tumors, peritumoral lymphatic vessels are present
along the boundary of the control tumor [Fig. 1(A)], and few
intratumoral lymphatics can be detected [Fig. 1(B)]. Furthermore, inside
the tumor body, there are only large tumor cells, and no LYVE-1 positive cells
are found [Fig. 1(C)]. In contrast, intratumoral lymphatic vessels are
present in the HA-treated tumors [Fig. 1(D–F)]. Those intratumoral lymphatic vessels appear heterogeneous,
because most of them congregate together in clusters [Fig. 1(D,E)].
Some of them are close to the tumor periphery [Fig. 1(D)], and some are
deep inside the area [Fig. 1(E)]. The intratumoral lymphatic vessels
should be hyperplastic, because LYVE-1+ endothelial cells are
frequently detected at the higher magnification of 400. These cells are flat in
shape and morphologically different from surrounding tumor cells [Fig. 1(F)].
The LYVE-1+ cells congregate together, and the
lymphatic vessel is taking shape [Fig. 1(F)]. These results demonstrate
that HA promotes the formation of lymphatic vessels in xenografts.
HA induced intralymphatic
tumor growth
Besides its effect on lymphangiogenesis, we found it interesting
that exogenous HA also promotes intralymphatic growth of tumor cells. In
control tumors, few tumor cells were found in either peritumoral or
subcutaneous lymphatic vessels [Fig. 2(A)]. However, in HA-treated
tumors, it can be clearly observed that intratumoral lymphatic vessels are
commonly infiltrated with tumor cells [Fig. 2(B)]. Furthermore, the
tumor cells also grow in neighboring subcutaneous lymphatic vessels [Fig.
2(C)]. Both the intratumoral and the subcutaneous lymphatic vessels filled
with tumor cells are strikingly enlarged and display irregular morphology [Fig.
2(B,C)]. In general, HA promotes intralymphatic tumor growth.
Effect of exogenous HA on
tumor angiogenesis and tumor growth
It has been reported that HA may be involved in the promotion of
tumor angiogenesis [18]. We next examined whether HA has any effect on
angiogenesis in our model. The blood vessels were detected by immunostaining
the frozen sections of implanted tumors with PECAM-1 (CD31), a specific
endothelial marker expressed mainly on blood vessels [16]. As shown in Fig.
3(A), the morphology of tumor blood vessels is apparently different from
lymphatic vessels, because the former is significantly smaller than lymphatic
vessels. The tumor angiogenesis was quantitatively assessed by counting the
numbers of PECAM-1+ spots in three areas
from five tumors of each group. It was shown that exogenous HA has little
effect on tumor angiogenesis, as quantified by the density of the tumor blood
vessels (2209 vessels/microscopic field for HA-treated tumors and 21611
vessels/microscopic field for control tumors) [Fig. 3(B)].
Previously, it was demonstrated that endogenous HA promotes tumor
growth by increasing tumor angiogenesis [14]. Therefore, we also examined the
effect of exogenous HA on tumor growth. As shown in Fig. 4, no
significant difference between the control tumors and HA-treated tumors was
found. This result is consistent with the fact that exogenous HA has no
significant effect on tumor angiogenesis.
Effect of exogenous HA on
lymph node metastasis of Hca–F
cells
It was suggested that tumor lymphangiogenesis might contribute to
tumor lymphatic metastasis [2,3], so we further examined whether exogenous HA
has any effect on lymphatic metastasis concomitantly with promoting
lymphangiogenesis. Previously, Hca-F cells have been demonstrated to develop
lymphatic metastasis selectively [19]. In this study, superficial axillary,
deep axillary and inguinal lymph nodes (on right side) were examined microscopically.
The percentage of lymph node metastasis, calculated as the number of positive
lymph nodes per total number of lymph nodes examined, was investigated. As
shown in Table 1, the percentage of lymph node metastasis for control
tumors is 53.3%, and 46.7% for HA-treated tumors. It was therefore concluded
that exogenous HA does not significantly affect lymph node metastasis of Hca-F
cells.
Discussion
Recent experimental studies have demonstrated the importance of lymphangiogenic
growth factors VEGF-C and VEGF-D in tumor lymphangiogenesis and lymph node
metastasis. In this study, we found that exogenous HA can induce tumor
lymphangiogenesis and promote intralymphatic tumor growth. These results
indicate that HA, in addition to VEGF-C and VEGF-D, may also play an important
role in regulating tumor lymphangiogenesis.
Our results show that exogenous HA induces tumor lymphangiogenesis
within xenografts. The HA-induced lymphatic vessels are hyperplastic, because
the number of lymphatic vessels is remarkably elevated and the frequent
presence of LYVE-1+ cells indicates that new lymphatic vessels are continuously coming
into being. Furthermore, our results demonstrate that HA selectively induces
tumor lymphangiogenesis without affecting tumor angiogenesis. The differential
effect of HA on tumor angiogenesis and lymphangiogenesis may be partly attributable
to the different receptors for HA on blood vascular endothelium (BEC) and
lymphatic vascular endothelium (LEC). CD44 and RHAMM are the receptors for HA
on BEC, and both of them can mediate the signaling of proliferation and
migration for BEC [20]. However, on LEC, the receptor for HA is LYVE-1, which
is homologous to CD44 but largely restricted to LEC [21]. Although the
function of LYVE-1 attracts much attention, it is not known whether LYVE-1 also
participates in lymphangiogenesis. The identification of the true function of
LYVE-1 will likely come from a whole animal study with an LYVE-1 knockout
mouse, which is currently underway [22]. Additional mechanisms may also be
involved in HA-induced lymphangiogenesis, owing to the unique properties of HA.
For instance, the increase of HA concentration in local tissue might change the
assembly of tumor stroma, which in turn facilitates tumor lymphangiogenesis.
Consistent with this hypothesis, Skobe et al. found considerable
heterogeneity of lymphatic vessel density within VEGF-C-overexpressed tumors,
the authors speculate that this may reflect variations in tumor microenvironment:
a promising microenvironment is likely to be critical for intratumoral
lymphangiogenesis [6].
VEGF family members have been implicated in angiogenic and
lymphangiogenic signaling. VEGF-A and VEGF-B are primarily involved in
angiogenesis, whereas VEGF-C and VEGF-D play predominant roles in the
regulation of lymphangiogenesis [23]. In additional experiments, we also
explored the potential role of HA in regulating the expression of
lymphangiogenic factors. The results showed that HA is able to increase the
expression of VEGF-D at both mRNA and protein levels in Hca-F cells; on the
other hand, HA has no significant effect on the expression of VEGF-A and VEGF-B
(data not shown). These results suggested a potential role of VEGF-D in
mediating HA-induced lymphangiogenesis, and also explain, at least partly, why
HA can promote lymphangiogenesis without affecting angiogenesis. Other
mechanisms may also contribute to this effect. It has been demonstrated that
the promotion of angiogenesis by HA is dependent on its molecular size [12]. It
was shown that HA oligosaccharides, but not the high molecular size of HA,
induces endothelium proliferation and migration in vitro and
angiogenesis in vivo. A related finding is that tumor cells often
exhibit elevated levels of not only HA itself but also hyaluronidase, which
renders tumor cells the promoted ability to internalize and degrade HA [24].
Because little effect of HA on tumor angiogenesis was detected in this study,
we postulated that HA might act in the form of high molecular size, instead of
the form of HA oligosaccharides.
The data showed that HA-induced tumor lymphangiogenesis is
accompanied with vast intralymphatic tumor growth. This phenomenon is very
similar to the observation in another experimental model with overexpressing
lymphangiogenic growth factor VEGF-C [9]. Tumor lymphangiogenesis has been
taken as the immediate cause for the intralymphatic tumor growth, because
lymphangiogenesis facilitates the interaction of tumor cells and lymphatic
vessels [9]. Thus, it is likely that HA promotes tumor cells to infiltrate into
lymphatic vessels indirectly through tumor lymphangiogenesis. Of course, we
currently cannot exclude the possibility that HA may alternatively promote the
intravasation of tumor cells directly by facilitating the interaction between
tumor cells and LEC: HA sequestered by LYVE-1 at the lumina surface and
intercellular junctions might provide en route a pathway for intravasation of
CD44+ tumor cells into the lymphatic vessels.
Consistent with this, a recent clinical report indicates that LYVE-1/HA may
play a role in tumor lymphatic invasion [25].
HA-promoted lymphangiogenesis was shown to have no association with
increased metastasis. Although lymphangiogenesis has been proposed to contribute
to metastasis, their relationship remains confused. Lymphangiogenesis can be
dissected from subsequent metastasis [25]. It was also shown that, in the
absence of HA and lymphangiogenesis, tumor cells can still display lymph node
metastasis, suggesting lymphangiogenesis is not necessary for metastasis.
Considering the fact that HA fails to increase the metastasis rate, we
postulated that lymphangiogenesis might play a minor role in the commitment of
metastasis. Notably, we do detect the intralymphatic growth of tumor cells in
the presence of HA, thus it can not be precluded that lymphangiogenesis may
contribute to metastasis extent instead of metastasis rate.
It is believed that the interaction between tumor cells and the
surrounding microenvironment plays a key role in tumor progression. Being the
basic constituents of this microenvironment, tumor stroma should be taken as a
functional pool for bioactive molecules rather than only a structural unit
[26]. In the present study, we provide first-hand evidence that HA, which is
usually elevated in malignant tumor stroma and multifunctional for malignancy,
may also play an important role in tumor lymphangiogenesis and promote
intralymphatic tumor growth. To our knowledge, our data provide the first insight
into the correlation between HA and tumor lymphangiogenesis. However, it is
necessary to further explore the underlying mechanisms, especially to determine
whether the interaction between HA and LYVE-1 is directly involved in tumor
lymphangiogenesis and the intravasation of tumor cells into lymphatic vessels.
Acknowledgements
We thank Dr. David G. JACKSON of
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