role of parathymic lymph nodes in metastatic tumor development

NON-THEMATIC REVIEW

Role of parathymic lymph nodes in metastatictumor development

Gaspar Banfalvi

Published online: 2 December 2011# Springer Science+Business Media, LLC 2011

Abstract Parathymic lymph nodes as potential sites oftumor progression have been neglected in humans. We haveestablished a rat renal capsule–parathymic lymph nodemodel to study in vivo metastasis. Epithelial liver carcino-ma (HeDe) and mesenchymal mesoblastic nephroma(NeDe) cell lines have been established after inducingchemical carcinogenesis in newborn Fisher 344 inbred ratsby N-nitrosodimethylamine. Implanting the exact numberof tumor cells (HeDe, NeDe) under the renal capsuleallowed the standardization and timing of metastaticdevelopment. Tumor cells released from the primary tumorin the peritoneal cavity were drained to the parathymiclymph nodes (PTNs) as sentinel lymph nodes. Similarly,tumor cells injected i.p. were engulfed by macrophages,drained through the transdiaphragmatic channels, andtransported to the thoracal lymphatics, primarily to PTNs.Tumor cells after transdiaphragmic drainage can enter bothanterior mammary and parathymic sentinel lymph nodes.The potential common origin can shed new light on themetastatic cell progression of PTNs and mammary tumors.

Keywords Carcinogenesis . Tumor cell lines . Lymphnodes . Rat tumor model .Metastasis

1 Introduction

Tumors in the anterior portion of the mediastinum are morelikely to be malignant than those in other compartments [1,

2] and are probably in the first line of metastatic tumordevelopment. The role of regional lymph nodes in cancermetastasis at the early phase of tumor growth is indicatedby the induction of T cells in these nodes. When a smallnumber of tumor cells enter a regional lymph node, thisnode operates as a temporary barrier against tumor growth.The suppressor activity induced by the tumor cells in theregional lymph node will facilitate tumor development andlead to lymphatic metastasis.

Needle biopsy specimens taken from the anteriormediastinum has predominantly centered on the thymusencompassing a whole range of tumorous diseases includ-ing hyperplastic conditions, benign and malignant neo-plasms. In addition, other neoplastic diseases typical forthis location may be encountered, such as extragonadalgerm cell tumors; lymphomas; soft tissue tumors; andconditions that simulate tumor development, cyst forma-tion, and inflammatory conditions [3]. Lymphoma repre-sents a common malignancy to selectively target this nodalchain, which usually results in the contiguous spread fromthe anterior mediastinal or paratracheal area to othermediastinal lymph node groups [4]. But is it really thealtered thymus development that is responsible for all ofthese neoplastic changes?

The cytotoxic T lymphocyte antigen 4 (CTLA-4) alsoknown as CD152 protein interacts with B7 ligands thattransduce inhibitory signals to T7 lymphocytes and playsan important regulatory role in the primary immuneresponse. Mice homozygous for null mutation in CTLA-4not able to express this protein suffered from lymphopro-liferative disorder under normal thymocyte development,leading to the conclusion that CTLA-4 is primarily involvedin the regulation of peripheral T cell activation. However, itturned out that the apparent alteration in thymocytes and theabnormal Tcell expansion in CTLA-4-deficient mice were not

G. Banfalvi (*)Department of Microbial Biotechnology and Cell Biology,University of Debrecen,Egyetem Square,Debrecen 4010, Hungarye-mail: [email protected]

Cancer Metastasis Rev (2012) 31:89–97DOI 10.1007/s10555-011-9331-y

due to altered thymocyte development rather than to theenlargement of parathymic lymph nodes (PTNs) [5]. Theoverestimated role of thymus in tumor development isrelated to the difficulty to separate PTNs from the thymus.The best way to distinguish the thymus from PTNs inrodents was to inject India ink intraperitoneally. Thymusexcluded whereas parathymic lymph nodes took up thecolloidal ink [5, 6].

2 Lymphatic drainage of colloidal particles

2.1 Mediastinal lymph nodes

Mediastinal lymph nodes are located along the trachea,esophagus, and between the lung and the diaphragm. Theselymph nodes are in tight connection with abdominal lymphnodes draining lymph to the left subclavian vein. The lymphnodes of the thoracic cavity are the parathymic and posteriormediastinal lymph nodes. PTNs lie in the proximity of thethymus. The common arrangement of rodent parathymiclymph nodes was reported to be located dorsolaterally to eachlobe of the thymus. In mice, the parathymic nodes weighingone tenth to one twentieth of the thymus are found in thecapsule, whereas in rats the nodes lie on the capsule [7]. Thenumber of parathymic lymph nodes varied between individ-ual rats, but was regularly between three to four on each side[8]. The mapping of the lymphatic system of rats revealedthat the lymph to the parathymic lymph nodes comes fromthe peritoneal cavity, liver, pericardium, and from the thymusand pours its content into the mediastinal lymph trunk thatenters the subclavian vein [9]. The three to four small murinenodes lying immediately behind the thymus can be removedonly with the thymus [10]. Due to the poor visibility ofmurine parathymic lymph nodes, reports of phenotypicalterations in the thymus [11, 12] could be explained bythe inclusion of parathymic lymph nodes obscuring thepicture. To avoid such ambiguities, it is advised to use rats tostudy parathymic lymph nodes that are positioned outside thethymus [7]. The presence of thymic lymphatics and thymus-specific lymph nodes have been reported in other animals,namely, in sheep [13] and in guinea pigs [14]. Humanparathymic lymph nodes have been reported only in twostudies; fetal thymic lymphatics have been published first acentury ago [15]. These small lymphatic organs are locatedon the thymus, but covered with the thymic capsule [16].

2.2 Translocation of colloidal particles from abdominalto thoracal cavity

To investigate the lymphatic drainage pattern from theperitoneal cavity, colloidal ink particles are preferentiallyused. Before further discussion, the colloidal particles have

to be distinguished from nanoparticles, although they areoften used as synonyms. Contrary to their overlappingrange, the size of nanoparticles (1–100 nm) and colloidalparticles (1 nm–10 μm) is different. Colloidal andnanoparticles including viruses, bacteria, yeast cells,cancer cells, cell remnants, carbon particles (e.g., Indiaink), thin fibrous crystals of asbestos, etc., are recog-nized by organisms as foreign.

When colloidal particles (e.g., Rotring ART 5912 dye)were injected intraperitoneally to rats, after 1 week, thecolloidal carbon deposited mainly in the parathymic lymphnodes and a smaller amount was collected in the leftposterior mediastinal lymph node [8]. Tilney [9] reportedthat colloidal carbon injected intraperitoneally drained tothe thorax and was extracted exclusively by the parathymiclymph nodes. That parathymic lymph nodes represent thedraining nodes for the peritoneal cavity [17] was confirmedby injecting Pelikan ink i.p. into rats [18]. It was found thatperitoneal macrophages laden with carbon particles passedacross the diaphragm and deposited the colloidal carbonparticles in the parathymic lymph nodes. The lung was notthe predominant route of clearance of the peritoneal lymph[18]. When India ink was not injected intraperitoneally butintravenously to mice, the blackened PTNs stood outprominently in contrast to the thymus [7].

The lymphatic drainage in the peritoneal cavity after i.p.inoculation of bacteria (Listeria monocytogenes), similarly tothe drainage of India ink, revealed that both agents weretransported by intraperitoneal macrophages toward thethoracic lymphocentrum (Lymphocentrum thoracicum ven-trale) and to the lymph nodes of the mediastinal lymphocen-trum (Lymphocentrum mediastinale) [19]. Peritoneal cellsmigrated through the diaphragmatic “stomata” to the para-thymic lymph glands via mediastinal lymphatics [20].Intraperitoneal injection of viral particles, such as porcinecircovirus type 2, were phagocytosed by intraperitonealmacrophages and transported toward the sternal lymph nodesof the ventral thoracic lymphocentrum [21]. Sternal glandsare located at the anterior ends of intercostal spaces by theside of the internal mammary artery. Sternal glands deriveafferents from the mamma, from the anterior abdominal wall,from the upper surface of the liver through a group of glandsbehind the xiphoid process, and from the anterior part of thethoracic wall. The parasternal lymph node chain alone wasrarely involved in mammary cancer, but frequently affectedalong with the axillary lymph nodes. When the parasternallymph nodes were involved in cancer, the survival rate wasextremely poor, even after their surgical removal [22].

2.3 Phagocytosis of foreign colloidal particles

The professional phagocytes include cells known asneutrophils, monocytes (macrophages, dendritic cells), and

90 Cancer Metastasis Rev (2012) 31:89–97

mast cells. Professional phagocytes in the blood areneutrophils and macrophages; in the thymus, macrophagesand monocytes, which can differentiate either to macro-phages or to dendritic cells. Different types of macrophagesdetermined by their location have specific names (Table 1).

Professional phagocytes will attack foreign colloidalparticles. When such small particles are administeredintraperitoneally, they will be attacked by white blood cells(granulocytes, monocytes, lymphocytes) and will be carriedto the thoracic lymph nodes, primarily to parathymic lymphnodes. The immune response in the parathymic lymphnodes of rats was studied after the administration ofantigens into the peritoneal cavity. Upon i.p. injection of aparathyphoid vaccine (killed forms of Salmonella para-typhus B), the primary immune response was their drainageto parathymic lymph nodes [23], confirming the notion thatimmune cells recognize bacteria as antigens and translocatethem from the peritoneal cavity to the parthymic lymphnodes. The translocation of colloidal particles (ink, bacteria,virions) from the peritoneal cavity to the parathymic lymphnodes raises the question whether or not the lymphaticdrainage of tumor cells as antigens to thoracic lymph nodes,particularly to parathymic lymph nodes, would also occur.We have answered this question by the establishment of ametastatic tumor model in rats and have followed the fate ofcolloidal particles after different types of administration.

2.4 Effect of particle size and mode of administrationon the transport of colloids

2.4.1 Dissolved particles

Dissolved particles (<1 nm) smaller than colloids circulatewith the blood after i.v. administration. The dissolvedmaterial may or may not enter cells upon s.c. injectiondepending on the permeability of the dissolved substance.As we deal here with undissolved colloidal particles, thefate of dissolved particles will not be discussed.

2.4.2 Particles larger than colloids

Particles larger than colloids (>10 μm) injected i.v. maycause microembolization in microvessels connecting arte-rioles and venules [24]. Microembolization causes focalmicroinfarcts with leukocyte (10–15 μm) infiltration. Wehave studied earlier factors influencing 113mIn–Fe(OH)3macroaggregate formation and have generated micro-embolization in the lung of rats [25]. The heterogeneityof the 113mIn–Fe(OH)3 macroaggregate containing largerthan 10 μm particles made these preparations unsuitablefor lung scintigraphy when injected i.v. to rats, causingtemporary occlusions in vessels of vital organs such as theheart and lung. Coronary microembolization provoked by 25-μm microspheres not only induced patchy microinfarcts inpigs with leukocyte infiltration but also elevated the expres-sion of tumor necrosis factor-α in monocytes/macrophages[26]. To avoid microembolization, intravenously adminis-tered particles should not exceed the colloidal size range.Large particles causing microembolization are out of thescope of this review.

2.4.3 Intravenously injected colloidal particles

We have measured earlier the distribution of 113mIncolloidal particles in rats after i.v. administration and havefound that most of the colloid was extracted by the Kupffercells of the reticuloendothelial system (RES) of the liver.RES is better known today as the mononuclearphagocytic and lymphoreticular system. The distributionof 3.7 MBq 113mIn colloid 10 min after injection into thefemoral vein was measured in Fisher 344 rats [27]. Thedistribution of colloidal particles in the liver (Kupffer cells98%), spleen (1.5%), and lung (0.2%) indicated that theprimary sites of extraction of colloidal particles from thecirculation were not the primary lymphoid organs (bonemarrow, thymus), but the secondary lymphoid organs (liver,spleen, lymph nodes, tonsils, mucosa associated lymphoidtissue (MALT), microglia of central nervous system). Theextraction of colloidal particles after i.v. administration wasindependent of their size within the colloidal range [27].

2.4.4 Subcutaneously injected colloidal particles

Subcutaneous administration is one of the most appropriateroutes for delivering nanoparticles (1–1,000 nm) and antigensthat can drain directly from the injection site to the nearbysecondary lymphoid organs, primarily to the nearest lymphnode. The fate of particles following s.c. administration isknown to be size-dependent. Moreover, the transport ofsubmicron particles into draining lymphatic capillaries occursthrough different mechanisms, again depending on particlesize. Similarly to s.c., peritumoral and intratumoral injection

Table 1 Strategic location of macrophages in the body

Location Phenotypes of macrophages

Blood Neutrophils, macrophages

Connective tissue Histiocytes

Neural tissue Microglia

Lymphoid tissue Macrophages (free and fixed), dendritic cells

Alveoli (lungs) Dust cells

Thymus Macrophages (free and fixed), monocytes

Liver Kupffer cells, monocytes

Spleen Sinusoid lining cells, dendritic cells

Bone Osteoclasts

Cancer Metastasis Rev (2012) 31:89–97 91

of nanocolloids help identify the nearest lymph nodes, alsoreferred to as the sentinel lymph nodes.

2.4.5 Intraperitoneally injected particles

The transport of intraperitoneally delivered peritonealparticles seems to be independent of size; consequently,the fate of cells larger than colloids can also be followed.Further discussion will focus primarily on colloidal par-ticles and tumor cells after their homotopic or heterotopictransplantation into rodents.

3 Metastatic tumor model

For the transplantation of tumor cells, rodent models arepreferred. Tumor cells can be transplanted into rodentseither as tumor cells or as tumor slices. The site of thetransplanted cells depends on the mode of the administra-tion by injecting tumor cells into (1) the blood vessels i.v.,i.a., or i.c. and (2) the tissues by s.c., i.d., i.m., or injectingdirectly into the specific tissue.

Homotopic or orthotopic transplantation is the transplan-tation of tumor tissue or cells from a donor into its normalanatomical position in the recipient. Heterotopic transplan-tation is the transplantation of the tumorous tissue or cellstypical of one area to a different recipient site. Thecharacterization of the metastatic rat tumor model is asfollows:

1. Establishment of liver and kidney tumor cell linesfrom primary tumors. Chemical carcinogenesis wasinduced in newborn Fisher 344 inbred rats by N-nitrosodimethylamine [28]. Tumors were originallymaintained by the orthotopic or heterotopic transplanta-tion of tumor slices into rats. Hepatocarcinoma (HeDe)and mesenchymal nephroblastoma (NeDe) tumor celllines have been developed from primary tumors generatedby chemical carcinogenesis in Long–Evans rats [29, 30].The establishment of tumor cell lines from primarytumors is viewed schematically in Fig. 1.

2. The implantation of an exact number (103–106) oftumor cells under the left renal capsule (Fig. 2a)resulted in primary tumor (Fig. 2b) which containeddisruptions filled with blood and tumor cells (Fig. 2c).Tumor cells were released through these disruptionsinto the peritoneal cavity and were translocated to thethoracal parathymic lymph nodes.

3. Timing of tumor formation. Due to the exact number oftumor cells implanted, tumor development in rats wasstandardized (Table 2).

4. Necrosis in the hypoxic central part of the growingtumor. Positron emission tomography (PET) with 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) has beenparticularly useful to study lymph node metastases incancer patients. We have carried out PET in rats [31].The radiotracer glucose analog 18F-FDG uptake in theorgans and tissues of tumor-bearing rats was expressedas the differential absorption ratio (DAR):

DAR ¼ accumulated radioactivity=g issueð Þtotal injected radioactivity=body weightð Þ

The biodistribution of the radiotracer glucose analog18F-FDG in HeDe whole tumor was 18 times higherthan in the resting muscle, taken as a unit. The pixeldensity in the living part of the HeDe tumor was 23times and in the necrotic part four times higher than inthe muscle. Glucose metabolism was almost six timeshigher in the outer living portion than in the innernecrotic region of the tumor. Similar results wereobtained with NeDe tumor, where the tumor accumu-lated 14 times more 18F-FDG than the muscle. Theradiotracer uptake in the living part of the tumor was 17times and in the necrotic part 2.8 times higher than inthe muscle. The ratios between the living and necroticparts were similar in the HeDe and NeDe tumors [31].

5. Lack of angiogenesis inside the primary tumor. In theprimary tumor, the lack of angiogenesis was manifestedas a pink stream of interstitial fluid carrying red bloodcells and tumor cells to disruptions inside and outside

Fig. 1 Establishment of tumor cell lines from HeDe and NeDetumors. a Hepatocarcinoma tumor slices were placed heterotopicallyand nephroblastoma tumor slices orthotopically under the renalcapsule of rats. b Primary tumors in rats were maintained earlierexclusively by the implantation of tumor slices in rats. c Tumor slicesminced into small pieces were digested in collagenase medium and theprimary tumor cells were grown, isolated, and kept as primary cellcultures. d After 20 subcultures, tumor cell lines (HeDe, NeDe) wereestablished and the exact number of tumor cells were implanted,resulting in a controlled tumor development

Fig. 2 Subrenal capsule assay. a GelasponR sponge containing 106

NeDe cells were implanted under the capsule of the left kidney ofLong–Evans rats. b Tumor development. c Tissue section of tumorafter hematoxylin–eosin staining showing disruptions filled withblood. Bars, 1 cm (a, b); 100 μm (c)


the tumor [32]. Disruptions were full of red blood cellsand tumor cells (Fig. 3). The presence of tumor cells inPTNs was verified by transplanting slices of enlargedPTNs under the capsule of the kidney of rats, leading totumor formation. After the transplantation of enlargedPTN slices into rats, the appearance of tumor cells inparathymic lymph nodes took 6 days [30].

6. Tissue disruptions and metastasis in PTNs. Tumor cells(106, NeDe) implanted under the renal capsule of ratsgenerated primary tumor on the kidney and metastasisin PTNs (Fig. 4). Parathymic lymph nodes wereretrieved by performing thoracotomies on anaesthetizedrats and dissecting them from the adjacent thymic tissue[32]. Relative to the control (Fig. 4a), at day 12 aftertumor cell (106) implantation, the PTNs were enor-mously (70–80 times) enlarged (Fig. 4b). In the tissueof the control parathymic lymph node (Fig. 4c), therewere no red blood cells. Hematoxylin/eosin-stainedtissue disruptions were seen in tumorous parathymiclymph nodes. Enhancement of the eosinophil stainingrevealed the presence of aggregations of red blood cellsin PTNs (Fig. 4d).

7. Location of the first site of metastasis. Localization of thesentinel lymph node was done by whole body autoradi-ography using the radiotracer glucose analog (18F-FDG).The metabolic activity was measured by 18F-FDGuptake in the primary tumor, in different organs, tissues(heart, liver, kidney, spleen, lung, thymus, blood), and inparathymic lymph nodes [31]. After the implantation ofIndia ink, similarly to tumor cells (HeDe, NeDe), dyeparticles appeared in PTNs [32].

4 Relationship between mammary and parathymiclymphatics

The two main nodal chains of the breast are parasternal(internal mammary) and axillary lymph nodes. One wouldexpect that internal tumors drain toward the internalmammary lymph nodes and lateral tumors toward theaxilla. However, this is not always the case, contrary to thefact that the location of the tumor may influence thefrequency of the metastasis of internal mammary nodes. Asfar as the thymic tumors are known, their thymic metastasesare quite rare, but parathymic lymph nodes may haveconnections with upstream nodal chains. The downstreamelements of parathymic lymph nodes have been indicatedby the finding that after acute gastroenteritis of rats, thecells of the exudative ascites appeared in the parathymicglands [33]. Earlier experiments related to staining and X-ray contrast analysis of materials clarified that thelymphatic vessels of the peritoneal cavity perforate thediaphragm. After penetration on the aspect of thediaphragm, the retrosternal vessel is running in closeproximity to the internal mammary artery (arteriamammaria interna) and reaches the upper mediastinallymph nodes [34–36]. The fact that colloidal particles canmove from the peritoneal to the thoracal cavity and fromthoracal lymph nodes to internal mammary and para-thymic lymph nodes raises the important question: whatkind of connection may exist between parathymic lymphnodes and mammary lymph nodes. Lymphatic vessels ofthe thymus in man are located by the side of the internalmammary artery and drain among others to the parasternal(internal mammary) lymph nodes, deriving afferents fromthe mamma, from portions of the abdominal and anteriorthoracic walls, and from the surface of the liver through asmall number of glands behind the xiphoid process [37,38]. The diaphragmatic lymphatic plexus draining into thelarge internal thoracic lymphatics consists of a pair oflongitudinally linked chains of nodes on either side of thesternum, usually four to five nodes in total on each side.Internal thoracic lymphatics enter the parathymic lymph

Fig. 3 Renal tumor formation and appearance of tumor cells inparathymic lymph nodes. a Lagging angiogenesis outside and necrosisinside the enlarged tumor (schematic view). b. Disruptions in theprimary tumor (small black arrows). c Small boxed area in (b) isenlarged. Tumor and blood cells breaking away through the disruptedtumor (indicated by the black arrow in c). d Detached cells of theprimary tumor enter the interstitial fluid, seen as channels indicated by

the black arrow. The lack of angiogenesis and the presence of cancercell are shown by the stained interstitial fluid and by the accumulationof red blood cells (upper right corner) around tissue disruptions. e Theappearance of red blood cells (black arrow) in the parathymic lymphnodes indicates the formation of secondary tumors (metastases).Modified with permission from [32]

ImplantedHeDe cells

Metastaticdeath (weeks)

106 cells 3

105 cells 4

104 cells 5

103 cells ∼6

Table 2 Timing of metastasisformation in rats


nodes. Mediastinal ducts upstream to the parathymic andposterior mediastinal nodes enter the subclavian veins.

Tumor cells after transdiaphragmic drainage can enterboth anterior mammary and parathymic sentinel lymphnodes. The common origin of their drainage suggests acloser relationship between the parathymic and mammarylymph nodes. Further discussion will focus on this potentialrelationship.

1. Similarly to tumor cells, Pelikan ink given to rats i.p.passed across the diaphragm and colloidal carbonparticles deposited not only in the parathymic lym-phatics but also in mediastinal internal mammarylymph nodes [19]. This experiment led to the conclu-sion that transdiaphragmatic lymphatic channels draininto the intrathoracic lymphatics, which in turn entereither anterior mammary or parathymic lymph nodes.Unfortunately, no efforts have been made to verify theinformation obtained by the histological examination ofmammary node metastases improved by the examina-tion of retrosternal nodes, especially those of para-thymic lymph nodes. One could argue that we do nothave evidence from the real-life situation of humanpatients regarding the connection between parathymicand mammary glands. Another argument could be thatlymphoscintigraphy of sentinel lymph nodes followinginjection of radioisotope particles could not distinguishbetween the parathymic lymph nodes and the thymictissue. Since parathymic lymph nodes of man have notbeen studied, this does not come as a surprise.

2. Lymph nodes act as filters for possible noxious agents[39, 40]. White blood cells attack any bacteria, viruses,cancer cells they find in the lymph as it flows throughthe lymph nodes. The parathymic lymph nodes prob-ably serve the same filtrating purpose when there is aperitoneal inflammation. The filtration capacity of theanterior mediastinal lymph nodes has been demonstrat-ed not only in animals but also in man, observingbacteria in these lymph nodes after peritoneal infection[41]. The murine peritoneal inflammatory cell responsepasses the parathymic lymph nodes, which removethese inflammatory cells from the afferent lymph [17].

It was also found in rats that the phagocytosis ofinflammatory peritoneal cells in the parathymic lymphnodes is undertaken by the macrophages of the subcap-sular and medullary sinuses. Polymorphonuclear leuko-cytes and eosinophils were phagocytosed by themacrophages of the medullary sinus and the medullarycord macrophages phagocytosed plasma cells [33].

Cancer cells broken away from the tumor normallybecome stuck in the nearest lymph nodes. The firstlymph node (or group of nodes) reached by metastasiz-ing cancer cells is called the sentinel lymph node (SLN).Doctors normally check the lymph nodes first to find outhow far a cancer has grown or spread. In breast cancer,the lymph node status remains the major conventionalprognostic factor, especially since some investigatorshave reported that lymph node metastases have notnecessarily been associated with hematogenous dissem-ination [42–44]. Besides the lymph node status which isa generally accepted prognostic indicator, the combina-tion of average microvessel count and blood vesselinvasion also provides a prognostic index for hematog-enous dissemination of tumor cells [45]. Related totumor development, there are several other biologicmarkers, such as angiogenesis, p53, proliferating cellnuclear antigen and c-erbB-2 oncoprotein, apoptosis,mitosis, and necrosis [45], the controversies of which ontumor hematogenous metastasis have not been clarifiedand prevented their use as prognostic markers for breastcancer patients [46–51].

3. Returning to the controversy of lymphoscintigraphy,during the identification of sentinel lymph nodesfollowing radioisotope injection in human patients, itis not known whether tumor cells in mammary lymphnodes would follow a metastatic route similar to thespread of primary tumors from the peritoneal cavity tothe parathymic lymph nodes. One reason of the limitedknowledge may be related to the pitfalls of thedetection of sentinel lymph nodes demonstrated bythe peritumoral injection of colloidal particles inmammary lymphoscintigraphy. The detection dependson the particle size, similar to the s.c. injection of

Fig. 4 Tumor formation in parathymic lymph node. a Controlparathymic lymph node. b Parathymic lymph node 12 days after theimplantation of 106 NeDe cells under the capsule of the left kidney ofthe rat. c Hematoxylin–eosin staining of the tissue section of control

PTN. d Hematoxylin staining of a tissue section of PTN 12 daysNeDe cell implantation. Bars, 0.5 cm (a, b); 100 μm (c, d). Modifiedwith permission from [30]


colloids with different particle sizes. The particle sizeof radiopharmaceuticals used for lymphoscintigraphy isthe subject of much discussion. As a consequence ofthe smaller particle size, the SLNs could not bevisualized when the average particle size of theperitumorally injected 99mTc-ReS was ∼100 nm, whilelymphoscintigraphy using larger 99mTc-ReS colloidparticles (∼500 nm) enhanced the visualization ofsentinel lymph nodes in breast cancer [52]. It wasthought that a larger particle size (often taken up byonly one node) was more suitable for SLN identifica-tion [53, 54]. The preferential use of 99mTc-ReSradiopharmaceutical is related to its particle size (5–80 nm), enabling adequate migration from the injectionsite and lymph node uptake [55]. To the contrary, inonly 39% of the patients with lymph node visualizationdid a single lymph node appear during 99mTc-ReSlymphoscintigraphy, while in 61% of the patients, twoto eight lymph nodes were seen [56]. These authorscame to the conclusion that the insufficient penetrationof larger particles in lymphatic capillaries minimizedthe number of traceable radioactive lymph nodes,leading to the underestimation of the number ofsentinel lymph nodes [56]. The same authors alsopointed out the importance of the injection site. Whileintratumoral and peritumoral administration of theradiotracer enabled mapping the axillary and non-axillary drainage, subcutaneous and intracutaneousinjections were other limiting factors draining predom-inantly to the axilla. Restricted detection can maskfurther lymph nodes, although tracing them by lym-phoscintigraphy could be important [56]. The detectionof these lymph nodes could light up a whole chain ofvessels and reveal connections showing the contiguousspread of tumor cells to other mediastinal lymph nodegroups.

Depending on the stages of the mammary cancer,tumor cells can spread to axillary lymph nodes orenlarge internal mammary lymph nodes. The idea ofturning on lymph nodes of a chain was supported bythe lymph drainage in cats injecting India ink inside themammary parenchyma. After opening the thoraciccavity, the coloration of lymph nodes and vessels was92%, indicating the connection between mammary andthoracal lymph nodes [57]. In man, the diaphragmaticnodes are located on the thoracic surface of thediaphragm. The anterior nodes can drain to the internalmammary nodes alongside the xiphoid and provide aroute for the retrograde spread of breast cancer to theliver, when the upper internal thoracic trunks areblocked [58]. From the disrupted liver, cancer cellscan flow back to internal mammary and PTNs throughthe diaphragmatic nodes.

5 Conclusion

The kidney capsule–parathymic lymph node complex thatwe originally proposed as a suitable metastatic model forthe isolated in vivo examination of tumor developmentprovides a reasonable explanation for the formation ofmetastases not only in parathymic but potentially also inother thoracic lymph nodes. Chemical carcinogens mayexert their carcinogenic activities in different tissues andorgans, with the liver as the major organ of detoxificationand one of the most frequent sites of tumor formation. Liveror kidney tumor cells released from disrupted tumors enterthe lymphatics of the diaphragm and form a specializedsystem which is draining tumor cells through the stomata,the drainage channels for absorption from the peritonealcavity. The clinical importance of these channels is thatthey provide escape for tumor cells, pathogens, and toxinsalike from the peritoneal cavity. Colloidal particles includ-ing tumor cells (HeDe, NeDe) engulfed by macrophagesand drained through the transdiaphragmatic channels aretransported to the thoracal lymphatics and accumulateprimarily in PTNs. Although parathymic lymph nodes areregarded as the sentinel lymph nodes that have beenreached by cancer cells from the primary tumors throughthe peritoneal cavity, one cannot escape the thought thattumors cells may also be targeted to the mammary lymphnodes. This notion is supported by the i.p. administration ofcolloidal carbon particles to rats that deposited not only inthe parathymic lymphatics but also in mediastinal internalmammary lymph nodes [19].

Finally, that the connection between thymic and mam-mary glands is probably not simply an anatomical relation-ship deserves mention, although anastomotic connectioncannot be ruled out. Functional changes in the stromal cellsinvolved in the abnormal thymic development occur duringthe mammary tumor development of mice [59]. In rats,splenectomy reduced the tumorigenic response in chemi-cally induced mammary tumors, while it increased thefrequency of tumor formation in parathymic lymph nodes,in conformity with the notion that PTNs actively participatein this compensatory reaction of the immune system ofanimals [60] and, by analogy, probably also in man.

Acknowledgment This work was supported by the HungarianScientific Research Fund (OTKA grant) T 42762 grant to G.B.

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role of parathymic lymph nodes in metastatic tumor development

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