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Keywords:

  • oral tongue cancer;
  • regional lymph node metastasis;
  • cancer-associated fibroblasts;
  • epithelial-mesenchymal transition;
  • tumor microenvironment

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We examined cancer-associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial-mesenchymal transition (EMT) in 19 pair-matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α-Smooth muscle actin (α-SMA) was studied to identify CAFs. EMT was studied with syndecan-1, Cadherin-11, fibroblast-specific protein (FSP)-1, secreted protein acidic and rich in cysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E-cadherin and Ki-67) and their relationship to the CAFs (α-SMA). We found that metastatic RLNs hosted CAFs similarly as in pair-matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki-67 positive) downregulate E-cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan-1, Cadherin-11, FSP-1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.

Metastatic squamous cell carcinoma (SCC) to regional lymph nodes (RLNs) plays a pivotal role in initial diagnosis, staging and management of oral SCC and is the single most important prognosticator for these patients.1–3 Approximately 30% of patients with intraoral SCC present with positive RLNs. Oral tongue SCC (OTSCC) is associated with the highest rate of metastasis as compared with other tumor sites in the oral cavity.4

Recently, it became apparent that carcinomas recruit benign microenvironment supporting cells to facilitate invasion and metastasis.5 Cancer-associated fibroblasts (CAFs) are tumor-associated fibroblasts with myofibroblast-like phenotype.6, 7 They are important facilitators of invasion, producing extracellular matrix proteases and angiogenic factors; they also modulate tumor drug-sensitivity.8 We have previously demonstrated the coevolution of CAFs in an in vivo carcinogenesis model9 and in patient specimens.10 Moreover, we found that CAFs were significantly correlated with local recurrence in tongue cancer; this has also been demonstrated in colorectal carcinoma.11, 12 To date, no study has examined CAFs in the context of metastatic disease in patients with OTSCC.

Carcinomas can invade as single cell migration13 or as multicellular aggregates in a process known as collective cell migration, in which the carcinoma cells may retain their epithelial characteristics.14 Epithelial-mesenchymal transition (EMT) refers to the loss of carcinoma epithelial phenotype and the acquisition of mesenchymal-associated features, which plays a relevant role in tumor invasion and metastasis.15 Transformation to tumor spindle cells can be associated with EMT but is not a requisite feature. EMT enables malignant epithelial cells to move, invade, metastasize and modify adjacent extracellular matrix.16, 17 Markers of EMT include Cadherin-11 and fibroblast-specific protein (FSP)-1, which are associated with increased motility,15, 18 and Twist that has a key role in repressing E-cadherin transcription.15, 19 Downregulation of syndecan-1 enhances the impact of Cadherin-11 and FSP-1, further increasing capability for motility and invasion.15 Finally, tumor upregulation of secreted protein acidic and rich in cysteine (SPARC) indirectly represses E-cadherin and also regulates matrix deposition and turnover in the tumor microenvironment, which paves the way to invasion and metastases.20, 21

The new microenvironment of distant metastatic sites might not support cancer growth; cancer survival will, therefore, depend upon intrinsic features such as the ability to engage in effective molecular cross-talk with the new microenvironment thereby modulating the supporting tissues.22 The cellular composition of benign supporting cells at metastatic sites has not been well studied.22, 23 We speculate that CAFs in RLNs not only facilitate development of metastasis, but support cancer survival. Our aim is to examine the frequency of CAFs in paired primary and metastatic OTSCC and to study the relationship between EMT transformed tumor cells and adjacent CAFs using EMT markers (syndecan-1, Cadherin-11, FSP-1, SPARC, Twist), and triple immunostaining with E-cadherin (epithelial phenotype), α-SMA (myofibroblastic-like phenotype) and Ki-67 (cell proliferation).12

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nineteen patients with metastatic RLNs were studied, 14 from the Chaim Sheba Medical Center in Israel, and 5 from Oulu University Hospital in Finland. The study was approved by the institutional review boards of both institutions.

Study protocol required histological confirmation of lymph node metastases. Clinically occult metastases were excluded. Matched sections from metastatic lymph nodes and primary tumors were examined immunohistochemically with antibodies of EMT, including syndecan-1 (clone B-B4, Serotec, Oxford, UK; 1:100) and Cadherin-11 (polyclonal, Zymed; Invitrogen, Carlsbad, CA; 1:100) as markers of cell surface proteins; FSP-1 (polyclonal, Abcam, Cambridge, UK, ready-to-use), as a marker of the cytoskeleton; SPARC (clone NCL-O-NECTIN, Novocastra, Newcastle upon Tyne, UK; 1:80), as an extracellular matrix regulator, and Twist (polyclonal, Abcam, Cambridge, UK; 1:500), as a transcription factor.15, 17, 21, 24 In addition, RLNs were submitted to a triple immunostaining procedure with E-cadherin (clone NCH-38, DakoCytomation, Glostrup, Denmark; 1:50), Ki-67 (polyclonal, Dako A/S, Glostrup, Denmark; 1:50) and α-SMA (clone 1A4, Dako A/S, Glostrup, Denmark; 1:100). The detailed procedure for this was described elsewhere.12 As a result, the E-cadherin staining was visualized as a bright blue color, cytoplasmic α-SMA as brown and nuclear staining with Ki-67 as a red fuchsin color. RLNs that were free of metastatic tumor taken from the same level as that of the metastasis-positive RLNs were used for comparative purposes in each case.

Semiquantitative analysis of immunoreactivity was examined in both the primary and metastatic cancers, as well as adjacent supporting microenvironment (fibroblasts, endothelium, inflammatory cells, and extracellular matrix).

CAFs

Identified by α-SMA immunoreactivity and appeared as plump spindled cells with brown cytoplasmic staining. The frequency of these cells was classified into CAF-rich or CAF-poor patterns. The CAF-rich pattern described numerous plump CAFs dispersed within the carcinoma and adjacent to it. CAFs usually overlapped with one another and were intermixed within islands of carcinoma, especially at the perimeter of tumor islands. The CAF-poor pattern referred to sparse CAFs that were less-plump and featured a spindle-shaped morphology that focally accompanied the periphery of the carcinoma component and were not abundant in either the primary tumor and/or metastatic RLN. The pattern was assigned according to the most prevalent pattern.

Syndecan-1

Only membranous staining in cancer cells was considered as positive, analogous to syndecan expression in normal oral epithelium.25 Cytoplasmic staining in benign microenvironment cells was interpreted as positive. Staining scores were calculated by multiplying staining intensity (1, weak; 2, moderate; 3, strong) by the percentage of stained cells (0, no stain; 1, 1-25%; 2, 26-75%; 3, >75%), with a range of possible scores between 0 and 9.26–28 Only scores of ≥3 were considered as positive.

Cadherin-11

The antibody used targets the intracellular domain of the protein,29 and as such both cytoplasmic and nuclear localization was expected for malignant and benign cells.30 The cut-off for cadherin-11 staining was set at >10% of positive cells.31

FSP-1

Both cytoplasmic and nuclear staining patterns were evaluated. Immunoreactivity in ≥20% in malignant and benign cells was considered positive.32

SPARC

Cytoplasmic staining pattern was evaluated in both malignant and benign cells and quantified similarly as for syndecan-1.26 Only scores of ≥3 were considered as positive.

Twist

Cases were interpreted as positive when they demonstrated strong nuclear express ion of ≥50% cells.33

Triple immunostaining with E-cadherin, Ki-67 and α-SMA was assessed only qualitatively. Membranous E-cadherin immunoreactivity within the metastatic tumor (blue color) was regarded as positive. Tumor cells devoid of E-cadherin could be identified by nuclear pleomorphism, usually highlighted by Ki-67 staining (red-purple). α-SMA immunoreactivity, reflected as brown cytoplasmic staining within spindle, plump and epithelioid cells within the microenvironment closely associated with the metastatic carcinoma, was regarded as indicative of CAFs.

McNemar statistical test was performed to find differences in the frequency of cases positively stained with the various markers in the microenvironment versus the carcinoma, and in the RLNs versus the corresponding primary tumors. Statistical significance was set at p < 0.05. Calculations were performed using the SPSS software version 15 (Chicago, IL).

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Nineteen lymph nodes were studied immunohistochemically for 6 markers. Only 4 markers were examined in one case, and only α-SMA was examined in 2 cases, due to limited tissue.

General

The mean age of patients with metastatic RLNs was 60.4 ± 18 years (range, 23-82 years). Metastatic RLNs were most frequently (13 cases, 62%) found at level II of the neck dissections.

CAF pattern

Eleven metastatic tumors were classified as CAF-rich and 8 metastatic tumors were CAF poor. (Figs. 1a and 2, respectively). Classification into these 2 categories was not associated with size of metastases. There was general agreement in CAF classification between paired primary and metastatic tumors (p > 0.05): 8 pairs were CAF rich (Fig. 1b) and 5 pairs were CAF poor. Discordant patterns were seen in 6 tumors. In the control tumor-free RLNs, plump myofibroblasts expressing α-SMA were observed only around parenchymal blood vessels and in the capsule and some septae of the RLNs. (Fig. 3). Plump myofibroblasts were not present within the parenchyma of any benign lymph nodes.

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Figure 1. (a) Metastatic carcinoma associated with the CAF-rich pattern in the RLN (α-SMA, scale bar 500 μm). (b) The matched pair primary tumor of the metastasis presented in (a) showing a CAF-rich pattern (α-SMA, scale bar 200 μm).

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Figure 2. Metastatic carcinoma associated with the CAF-poor pattern (arrows) in the RLN (α-SMA, scale bar 200 μm).

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Figure 3. A lymph node free of metastatic carcinoma showing α-SMA staining only in the capsule and not within the node parenchyma. Inset: α-SMA staining highlighting positive cells within the fibrous capsule and in blood vessel walls (α-SMA, scale bar 1 mm).

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Table 1 summarizes the distribution frequency for expression of syndecan-1, Cadherin-11, FSP-1, SPARC and Twist. Examples of IHC results are illustrated in Figure 4. Cadherin-11 and Twist were most often expressed in both primary and metastatic carcinomas. SPARC expression was significantly increased in the primary carcinoma as compared to the metastasis (p = 0.039). FSP-1 was also upregulated in primary carcinoma as compared to the metastasis, albeit not achieving significance.

Table 1. The frequency of cases positive for the selected panel of antibodies in the carcinoma versus microenvironment in the regional lymph nodes and primary tumors
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Figure 4. Syndecan-1 in the primary tumor (a) is focally preserved in a membranous pattern (arrow). In the RLN (b) it shows a considerable staining of the stromal cells (arrow heads) in addition to focal membranous staining of the carcinoma cells (arrow). Cadherin-11 in the primary tumor (c) and RLN (d) showing nuclear staining of the majority of the cells within the carcinoma and tumor microenvironment. FSP-1 in the primary tumor (e) and lymph node metastasis (f) showing both nuclear and cytoplasmic staining in the carcinoma cells (arrows) and adjacent microenvironment (arrow heads). SPARC staining pattern in the primary tumor (g) is noticeable for its location at the periphery of the tumor, which is in close contact with the microenvironment. In the lymph node (h), the staining pattern highlights the continuity between the carcinoma and its microenvironment. Twist is remarkable for its nuclear staining of the carcinoma cells in both the primary tumor (i) and lymph node metastasis (j). The cells within the microenvironment are rarely positive. (aj, scale bar 200 μm).

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In the supporting tissue microenvironment, expression of the “EMT” markers was commonly seen in benign fibroblasts, endothelial cells and inflammatory cells as well as extracellular matrix. In some cases, tumor and microenvironment expression blended into one another. Both Cadherin-11 and FSP-1 were significantly upregulated in the microenvironment at the primary site as compared with the metastatic site (p = 0.031 and p = 0.008, respectively).

The triple immunostaining showed considerable loss of membranous E-cadherin by the metastatic carcinoma (Fig. 5). There were areas in which positively stained E-cadherin tumor islands were often closely surrounded by layers of α-SMA-stained CAFs at their periphery. In other areas, E-cadherin expression was completely or almost completely lost and carcinoma cells could be usually identified by the pleomorphic, Ki-67 positively stained nuclei. In these areas in particular, CAFs were interspersed among the tumor cells, usually with indistinguishable borders between the CAFs and the carcinoma cells.

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Figure 5. Triple immunostaining with E-cadherin, Ki-67 and α-SMA in a RLN illustrates a tumor island with preserved membranous E-cadherin staining (blue color) in most of the cells (a). Many tumor cells, both positive and negative for E-cadherin, possess Ki-67 positive nuclei (red-purple color). The tumor island is partially surrounded at its periphery by a delicate layer of α-SMA-positive CAFs (brown color, arrow). A small lymphocytic follicle is seen adjacent to the tumor island. In another area of the metastatic deposit (b), most of the tumor cells are E-cadherin negative with only remnants of membranous staining (center). A rich network of CAFs is interspersed among the tumor cells and is intimately associated with them. At the right lower corner there are 2 small lymphocytic follicles. (a, b: scale bar 100 μm).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

EMT has been classified into 3 categories; type 1 EMT is associated with implantation, embryo formation, and organogenesis, processes requiring cellular differentiation into diverse phenotypes, and cell migration, within a limited time frame. Type 2 EMT is associated with wound healing and tissue regeneration, producing repair-associated mesenchymal cells/fibroblasts. Type 3 EMT refers to carcinomas, which have undergone genetic alterations and have acquired the capacity of movement and invasion into the adjacent tissues, resulting in the scattering of cancer cells and metastatic spread.15In vitro models suggest that the acquisition of these features alone may be insufficient to produce cancer invasion, as both mechanical force, and protease-mediated matrix remodeling activities are also required.14 CAFs are key players in leading collective invasion of the carcinoma cells as they are capable of both remodeling extracellular matrix and providing the mechanical propulsive force to facilitate invasion.

Here, we demonstrate that markers of EMT, associated with increased motility, are common in both primary and metastatic OTSCC. Cadherin-11 is a mesenchymal cadherin, (as opposed to E-cadherin, an epithelial cadherin) which is associated with invasion. Cadherin-11 regulates actin cytoskeletal scaffolding, important to actin-based signaling modules and the dynamic formation of F-actin (filamentous actin) protrusions. Cadherin-11 also regulates α-catenin turn-over at the adherens junctions, facilitating the dynamic remodeling of cell-cell contacts which is important to intercellular invasion.34, 35 FSP-1 facilitates motility by activating non-muscle myosin.36, 37 We found high expression of Cadherin-11 and FSP-1 in both primary and metastatic carcinomas. Syndecans are transmembrane domain proteins that carry 3 to 5 heparan sulfate and chondroitin sulfate chains which bind ligands such as fibroblast growth factor, vascular endothelial growth factor, transforming growth factor-beta, fibronectin, and antithrombin-1. Downregulation of syndecan-1 is associated with decreased intercellular adherence and acquisition of motility.38 Accordingly, its expression has been reported to be lost in about 60% of primary tumors25 as opposed to its ubiquitous expression in nonmalignant oral mucosa,25, 27 This is consistent with our finding of loss of syndecan-1 expression in approximately 59% of primary tumors, in contrast to ubiquitous synecan-1 expression in adjacent histologically benign squamous mucosa (unpresented data). Twist is a basic helix-loop-helix transcriptional factor which is an essential marker of EMT as it represses E-cadherin expression.15 In our sample, Twist was highly expressed in both the primary and metastatic carcinoma cells. SPARC is a matricellular protein involved in tissue repair at the level of procollagen processing, deposition of extra-cellular matrix, metalloproteinase expression.20, 21 The function of SPARC varies with the tissue of origin. SPARC facilitates EMT by downregulating E-cadherin and upregulating matrix metalloproteinases through the SNAIL pathway, which is a key-role transcription factor in the initiation of EMT.39 The expression of SPARC in the primary and also in the metastatic carcinomas could join the effect of Twist and further contribute to the loss of expression of E-cadherin.

Concomitant with EMT, CAFs were also commonly identified in the microenvironment. This is the first report of CAFs within the microenvironment of metastatic RLNs. The pattern of CAF distribution in the RLNs was usually similar to that in the primary tumors, which raises the issue of their origin. As benign RLNs are devoid of CAFs, we hypothesize that the OTSCC are responsible for bringing CAFs to the lymph nodes. Analogous to microenvironmental changes at the primary site,7, 8 metastatic tumor can exert a paracrine effect to recruit and activate local resting fibroblasts or induce transdifferentiation of a wide range of cells, including smooth muscle cells, pericytes, adipocytes and inflammatory cells.23, 40 Experimental evidence demonstrates that fibroblasts at metastatic sites can become activated to myofibroblasts prior to the arrival of the metastatic deposits.23 Alternatively, CAFs may have arrived at the metastatic site as a co-migrator with OTSCC, referred to as the collective pathway of invasion.14 Clusters of carcinoma cells, including tumor cells with EMT-derived migratory potential, associate with CAFs, and move in concert to protrude into the adjacent tissues driven by the leading “pathfinders” (i.e., CAFs), towards lymphovascular pathways. Experimental migratory clusters of tumor cells were identified 30 years ago within the lymphatic vessels.41 Recently, circulating tumor fragments enveloped by blood vessel wall elements were also reported in human tumors.42 This route could also be appropriate for CAFs.

Either or both pathways (CAF recruitment to the metastatic site, and CAF co-invasion) are probably operational for metastatic OTSCC, although this study cannot address this question. Of note, studying IHC markers like syndecan-1 and SPARC, gave the remarkable sense of continuity between the tumor and microenvironment. One could not pinpoint the delineation between tumor and supporting microenvironment. This suggests that some invading OTSCC do not invade through the supporting tissues but rather bring the specialized microenvironment along as a retinue during the invasion.

In summary, this is the first demonstration of CAFs within metastatic RLNs in OTSCC patients. We believe that CAFs are an important facilitator of invasion and metastasis, along with EMT. Our findings support the idea that carcinoma and microenvironment co-evolve, share and maintain a similar invasive profile at both the primary site and RLNs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Hana Vered for technical assistance and Esther Eshkol for editorial assistance. This study was supported by the Dave and Sarah Babish Fund (M.V.) and the Academy of Finland (T.S.).

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References