Tumors induce coordinate growth of artery, vein, and lymphatic vessel triads
© Ruddell et al.; licensee BioMed Central Ltd. 2014
Received: 3 December 2013
Accepted: 16 May 2014
Published: 21 May 2014
Tumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other.
The murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels.
Each peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors.
Implanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.
KeywordsMelanoma Carcinoma Lymphangiogenesis Lymphatic vessel Angiogenesis Blood vessel
Blood and lymphatic vessels arise during embryogenesis and mature by an orderly program of gene expression, signaling, and morphogenesis that establishes a triad arrangement of lymphatic, vein, and artery throughout the body [1, 2]. The formation of each vessel type is regulated by different combinations of transcription factors (e.g. Notch, COUP-TFII, and Prox1), While some factors specifically regulate lymphatic (Prox1), vein (COUP-TFII), or artery formation (Hey1/2), other vascular signaling pathways show overlapping activities (e.g. Sox transcription factors, and vascular endothelial growth factors (VEGFs) [3–5]. Intercellular signaling of proteins including semaphorins, ephrins, and chemokines helps to pattern formation of the network of lymphatic and blood vessels, while VEGF family members are important to support growth of the lymphatic and blood vasculature. Mature triads of lymphatics, arteries, and veins are maintained in the adult unless activated by events requiring vascularization such as tumorigenesis .
Tumors induce extensive growth of blood vessels to obtain oxygen and nutrients essential for their continued expansion and progression [7, 8]. The resulting blood vessels are variable and abnormal, ranging in size and morphology from capillaries and small blood vessels to larger feeder arteries and draining veins surrounding the tumor vascular bed . The lymphatic vasculature can also expand [10–12], to increase lymph drainage from tumors featuring high interstitial fluid pressure [13, 14], and also to promote metastasis of tumors to draining lymph nodes (LNs) [15, 16]. Tumors can produce growth factors promoting angiogenesis (eg. VEGF-A, placental growth factor), as well as lymphangiogenesis (VEGF-C, -D), although the resulting vessels are highly abnormal relative to those of embryos [17, 18], presumably due to aberrant signaling [9, 19]. These vessels can actively increase blood circulation and lymph drainage, or they can become so abnormal that they are unable to transport blood  or lymph [14, 21].
Tumor-induced angiogenesis and lymphangiogenesis are generally studied separately, raising the question of whether the normal triad pattern of lymphatic, vein, and artery is used to support tumor vessel growth, or if blood and lymphatic vessels are independently generated and patterned during tumor growth. In this study we utilized two murine models to compare the growth of large peritumoral lymphatic and blood vessels. The syngeneic B16-F10 melanoma model  permits ready visualization of peritumoral vasculature on the flank skin of C57Bl/6 mice, and these tumors metastasize via the lymphatics to draining lymph nodes (LNs) [23–25]. The second model used was an autochthonous squamous cell carcinoma induced by DMBA mutagenesis, followed by repeated TPA promoter treatments of outbred mice . These naturally arising tumors first present as exophytic benign papillomas, which can convert to endophytic invasive carcinomas capable of metastasizing to draining LNs or to the lung. Both of these tumor models induced growth of large red-colored blood vessels in the skin extending several cm between the primary tumor and regional draining LNs. Immunohistochemical and functional analysis determined that these enlarged vessels included large arteries, veins, and one or more lymphatics, indicating that tumor-induced vessel growth utilizes the same patterning of vessel triads established during development.
Mouse tumor models
Mice were maintained in micro-isolator rooms under specific pathogen-free conditions. Experiments involving animals were approved by the FHCRC Animal Care and Use Committee. For the B16-F10 tumor model, four week-old C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). B16-F10 murine melanoma cells  from the American Type Culture Collection (Manassas, VA) were tested to confirm that the cells were free of mycoplasma contamination (Cambrex, Rockland, MD). Mice were anesthetized with isoflurane and subcutaneously injected in the left flank with 15,000 B16-F10 cells in 50 μl of Hanks’ buffered saline solution, while the control right flanks were injected with 50 μl of Hanks’ buffered saline solution [24, 25]. Mice were euthanized after 14–23 days, when tumors were 1–2 cm in diameter. Vessel diameter was measured using a ruler. Using a scalpel, single large red-colored blood vessels in the flank skin were dissected in 2 mm × 5 sections. Tissue was fixed in 4% paraformaldehyde at room temp for 4 h, incubated in 30% sucrose/PBS overnight at 4°C, and mounted in Tissue-Tek O.C.T. compound (Sakura; Torrance, CA) before cryosectioning at -25°C.
For the DMBA/TPA squamous cell carcinoma model, wild-type NIH/Ola or p19Arf+/- mice were treated on the shaved dorsal skin with DMBA, and then twice weekly with TPA for 15 weeks to induce papillomas and squamous cell carcinomas, as previously described [28, 29]. Mice were euthanized 28 to 32 weeks after DMBA treatment, or if moribund. More than a dozen papillomas or carcinomas arose on the back skin of each mouse, with diameters ranging from 2 to 15 mm. Large vessels draining papillomas or carcinomas were dissected from the interior surface of the skin in 2 mm × 5 mm sections with a scalpel, and mounted in O.C.T. for cryosectioning.
Functional labeling of lymphatic and blood vessels
Functional lymphatic vessels were identified by lymphography, injecting isoflurane-anesthetized mice subcutaneously in the base of the tail with 0.25% lysine-fixable Texas Red Dextran (Invitrogen; Gran Island, NY) and 2.5% Evans Blue (Sigma; St. Louis, MO) in 100 μl Hanks’ buffered saline solution, followed 20 min later by CO2 overdose and necropsy. The blood circulation was labeled by injecting mice intravenously in the tail vein with 0.05% Fluorescein isothiocyanate (FITC) – Lycopersicon esculentum lectin (Vector Laboratories; Burlingame, CA) in 100 μl Hank’s buffered saline solution in the tail vein, immediately followed by CO2 overdose, followed by dissection of vessels. Vessels were mounted in OCT for cryosectioning.
Immunostaining analysis and vessel quantitation
Twelve μm cryosections were fixed in 4% paraformaldehyde for 10 min, followed by incubation in 0.3% hydrogen peroxide in methanol for 30 min. Sections were then immunostained with CD31 (BD Pharmingen), MECA -32 (BD Pharmingen), LYVE-1 (Ebioscience), or 10.1.1 , as previously described . Immunohistochemical staining was detected using horseradish peroxidase-labeled secondary antibodies and Vector VIP detection, followed by brief methyl green counterstaining at room temperature (Vector Laboratories). In some experiments, sections were immunostained with 10.1.1 and Prox1 (Millipore; Billerica, MD), and detected with Alexa 568-anti-hamster and Alexa 488-conjugated anti-rabbit antibodies (Invitrogen; Sparks, MD), respectively, followed by mounting in Prolong Gold (Invitrogen).
For measurements of 10.1.1- or CD31-positive lymphatic, vein, and artery size, three sections from each vessel were viewed at 100x magnification, and vessels were outlined to measure the area using the NIS Elements BR 3.0 program (Nikon, Inc.; Melville, NY). Area measurements were analyzed for significance by Mann–Whitney U two-tailed test.
Peritumoral blood and lymphatic vessels are enlarged
We tested whether this angiogenesis also involves lymphatic vessels, as the SE blood vessel between the ING and AX LNs tracks with a major lymphatic vessel draining from the ING LN . A functional assay was used to identify lymphatic vessels associated with the tumor blood vessels, by injecting Evans Blue dye subcutaneously into the base of the tail to label the ING and AX LN lymphatic drainage on both flanks . This injection identified the blue collecting lymphatic vessel adjacent to the SE blood vessel (Figure 1b). The labeled SE lymphatic vessel was much larger on the tumor-draining flank (Figure 1d) relative to the contralateral control flank (Figure 1c). These tumors therefore induced enlargement of the SE lymphatic vessel adjacent to the SE blood vessels. The ING and AX LNs on each side also labeled with Evans Blue (Figure 1b), indicating that this lymphatic drainage is not blocked by tumor growth. The second-tier AX LN preferentially filled with Evans Blue dye on the tumor-draining side relative to the control side (Figure 1b), consistent with our previous reports that B16-F10 tumors accelerate lymph drainage [24, 33].
Identification of functional lymphatic and blood vessels
Dye injection assays were used to functionally distinguish these vessels. Intravenous injection of FITC lysopersicon esculentum lectin  labeled the vascular endothelium and erythrocytes of the SE artery and vein, but not the lymphatic vessel of normal control mice (Figure 2c). Subcutaneous injection of lysine-fixable Texas Red Dextran (15 kd MW) into the lateral tail base  preferentially labeled the draining lymphatic vessel (Figure 2d). These functional immunostaining assays demonstrate that intravenously injected FITC lectin labels CD31-positive blood vessels, while subcutaneously-delivered Texas Red Dextran identifies 10.1.1-positive lymphatic vessels.
Peritumoral growth of lymphatic, artery, and vein triads
Dye was injected subcutaneously into the tail to test whether the enlarged tumor-draining lymphatic vessels are able to transport fluid, as tumor-draining lymphatics can be compressed and nonfunctional . Injected Texas Red Dextran labeled the 10.1.1 Ab-positive lymphatic vessel, indicating that it is functional in draining lymph from the tail past the tumor and through the ING LN to travel through the efferent SE lymphatic vessels (Figure 3c). In addition, the artery and vein were labeled with Texas Red Dextran (Figure 3c), indicating that the dye traveled from the lymphatic drainage to the heart and into the blood circulation 20 minutes after injection. . This is consistent with our previous reports that dye reaches the vascular circulation within minutes in mice with cancer, due to increased lymph flow [33, 36]. The circulation of subcutaneously injected lectin in lymphatic as well as blood vessels reveals that both types of vessels are functional.
Prox1 immunostaining was used to confirm the lymphatic identity of the abnormally enlarged 10.1.1 Ab-positive SE lymphatic vessel, as Prox1 is a transcription factor expressed in lymphatic but not vascular endothelium . The endothelial cells of the SE lymphatic vessel of Figure 3c and adjacent lymphatic vessels co-expressed surface 10.1.1 (Figure 3d) and nuclear Prox1 (Figure 3e, f), confirming that these abnormal vessels are lymphatics. These findings identify the 10.1.1 antibody as a marker of collecting lymphatic vessels, in addition to its previously described utility for identification of initial lymphatic vessels .
Coordinate growth of peritumoral lymphatic and blood vessels
The IF vessel dissected from the tumor-bearing flank also included a lymphatic, vein, and artery (Figure 5d, e), that were all enlarged relative to the contralateral control vessels (Figure 5a, b). Subcutaneously delivered Texas Red Dextran identified the vein and artery but not the lymphatic vessel (Figure 5f). Tumor growth in this particular mouse therefore blocked IF lymphatic drainage, while other tail-draining lymphatic vessels instead delivered Texas Red Dextran to the blood circulation.
The ODD vessels also consisted of triads of enlarged lymphatics, veins, and arteries by immunostaining for CD31 (Figure 5g) and 10.1.1 (Figure 5h). In the example shown, the lymphatic and blood vessels were all functional, as subcutaneously injected Texas Red Dextran labeled lymphatic as well as blood vessels (Figure 5i). There are no vessels visible in comparable regions of the contralateral control flank (Figure 1c) so that it remains to be determined whether these ODD vessels are derived by expansion of pre-existing very small vessel triads, or by some other mechanism.
Pre-metastatic tumors sometimes block lymph drainage
Coordinate growth of lymphatic, vein, and artery triads in an autochthonous tumor model
Vessels draining benign papillomas from 3 mice were dissected, sectioned and immunostained, to identify triads including thick-walled arteries and veins (Figure 7g), as detected by immunostaining for vascular endothelial marker MECA-32 . Multiple lymphatics were detected by 10.1.1 immunostaining (Figure 7h). These slowly growing benign tumors thus also induce coordinate growth of large vessel triads, with the arteries particularly enlarged relative to the veins and lymphatics. There are no major vessels in the dorsal skin of mice (Figure 7d), so that the origin of these peritumoral vessels is not obvious.
Immunostaining analysis of the red-colored vessels surrounding DMBA/TPA-induced carcinomas from 6 mice also identified triads of vein, artery, and lymphatics by CD31 (Figure 7i) and 10.1.1 (Figure 7j) immunostaining. These vessels were greatly enlarged and abnormal, resembling the vessels draining B16-F10 melanoma tumors (Figure 3a). The lymphatic vessel shown in Figure 7j included 10.1.1 Ab-positive flaps that likely are valves typical of collecting lymphatic vessels [41, 42], which were a common feature of these peritumoral lymphatics (data not shown). Other carcinoma-associated vessels showed more extreme enlargement and disorganization, although they maintained clustering of multiple blood vessels (Figure 7k) and lymphatics (Figure 7l). These findings identify coordinate growth of peritumoral lymphatic and blood vessels in autochthonous papillomas and carcinomas.
Discussion and conclusions
The enlarged red-colored vessels arising over some distance from spontaneous or implanted tumors include clustered lymphatics, veins, and arteries, demonstrating that the triad arrangement typical of large vessels is utilized during tumor-induced angiogenesis and lymphangiogenesis. The peritumoral SE and IF vessels likely grow by enlargement of the pre-existing normal vessel triads. Blood and lymphatic vessels can both enlarge by endothelial proliferation, incorporation of bone marrow-derived cells [43, 44], and/or by thinning and dilation . The “odd” vessels that appear surrounding melanomas, papillomas, and carcinomas could similarly be generated by expansion from very small vessel triads, or they could potentially arise by recapitulation of their initial formation in embryos [3, 46, 47]. The latter possibility could involve assembly of endothelial progenitors, as occurs in development of the dorsal artery and cardinal vein [48, 49], or sprouting of lymphatic vessels from small veins [50, 51]. Further studies of the generation of these “odd” vessel triads growing around tumors will be required to identify the mechanisms guiding this coordinate blood and lymphatic vessel growth.
Many of the peritumoral vessels grow toward the regional LNs in the melanoma and squamous cell carcinoma models, raising the question of how this expansion is related to lymph node metastasis. Only the lymphatic vessel would directly connect the tumor to the draining LN to deliver tumor cells, so that the enlargement of functional lymphatic vessels could facilitate LN metastasis. In fact, prostaglandin and VEGF-D-induced enlargement of collecting lymphatic vessels has recently been demonstrated to promote metastasis to draining LNs . Other studies have found that VEGF-C and VEGF-D- expressing tumors induce lymphangiogenesis and increase metastasis [10–12]. The coordinate growth of large blood and lymphatic vessel triads thus not only supports tumor growth and fluid drainage, but also likely promotes lymphatic metastasis to draining lymph nodes.
The large blood vessels in the vicinity of B16-F10 melanomas were always functional in circulating blood, however lymphatic vessels were sometimes blocked. Blocked lymph drainage was readily identified upon necropsy after Evans Blue subcutaneous injection into the tail base, with dye usually reaching the ING LN but not the second tier draining AX LN, and this blockade could be confirmed by subcutaneous injection of Texas Red Dextran followed by immunostaining analysis. Our identification of altered lymphatic drainage is relevant for lymphography mapping of the sentinel tumor-draining LN in human cancer patients, for pathology analysis to diagnose whether the tumor has metastasized [52, 53]. The accuracy of sentinel LN identification has been improved [54, 55], however the difficulties in correctly identifying the tumor-draining LN could be caused by blocked lymphatic drainage as experimentally demonstrated here.
Recent studies have identified effects of large LN metastases to block tumor lymphatic drainage in mouse tumor models [56, 57]. In our study, mice showing blocked lymph drainage did not yet contain metastases in the ING or AX LNs, even though melanotic metastases consisting of less than 1,000 cells can readily be visualized on the LN cortical surface . Those tumors demonstrating blocked lymph drainage were not larger or located closer to the SE or IF vessel triads relative to tumors with functional lymphatics, so that blocked drainage is not readily explained by tumor overgrowth. However, these tumors could potentially compress the lymphatic vessels to impair lymph drainage. Alternatively, in-transit metastases in tumor-draining lymphatic vessels have been identified in murine and human melanomas [58, 59], which could potentially block lymph drainage before lymph node metastases are detectible.
Both of the murine tumor models investigated here exhibited coordinate growth of tumor-draining lymphatic and blood vessel triads, suggesting that tumors produce factors promoting growth of both types of vessels. Members of the VEGF family of endothelial growth factors (VEGF-A, -C, -D) show overlapping specificity for VEGF receptors expressed on lymphatic and blood vessels [16, 60], which could potentially drive the concerted growth of both types of vessels [19, 61, 62]. Our RT-PCR analysis of B16-F10 melanoma cells identified abundant VEGF-A, VEGF-C, and VEGF-D expression at the mRNA level (data not shown), and B16-F10 melanomas  and DMBA-TPA-induced papillomas  produce high levels of VEGF-A protein. It remains to be determined whether these VEGFs or other endothelial growth factors coordinate growth of the blood and lymphatic vessel triads. Investigation of the mechanisms coordinating growth of these peritumoral vessel triads could help to define therapeutic strategies to effectively block both tumor angiogenesis and lymphangiogenesis.
AR, AC, KK, and MF conceived and designed the experiments. AR, AC, KK, and MF performed experiments. AR and CK wrote the manuscript. All authors read and approved the final manuscript.
Reverse transcription-polymerase chain reaction
Vascular endothelial growth factor.
We thank Maribel Harrell, Sheila Ganti, Kay Gurley, and Kendra Byrd for their advice and assistance. This work was supported by National Institutes of Health Grant NCI R01-CA68328 (AR), and NCI Mouse Models of Human Cancer Consortium U01 CA141550 and NIEHS grant RO1 ES020116 (CJK).
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