Potential role of M2 TAMs around lymphatic vessels during lymphatic invasion in papillary thyroid carcinoma

The aim of this study was to examine whether lymphatic invasion in papillary thyroid carcinoma (PTC) occurs when tumour-associated macrophages (TAMs) injure lymphatic vessels together with cancer cells. While there was no difference in the lymphatic vessel density in PTC and follicular thyroid carcinoma (FTC), the number of TAMs around the lymphatic vessels was increased in PTC compared to that in FTC. In particular, TAMs were observed together with cancer cells in lymphatic invasive lesions, and the number of M2 cells inside and outside the lymphatic vessels showed a significant correlation. MMP-2 mRNA was expressed in nonneoplastic stromal cells as well as cancer cells, and double immunofluorescence staining confirmed M2 positivity. Consequently, this study reveals that M2 TAMs around lymphatic vessels within the tumour border of PTC may be associated with the lymphatic invasion of cancer cells. This study represents a step forward in elucidating the mechanism of lymphatic invasion.


Comparison of the number of TAMs around the tumour border lymphatic vessels in PTC and FTC. A comparison of the macrophage counts around the lymphatic vessels in PTC and FTC is shown in
Correlation of the number of TAMs inside and outside the areas of lymphatic invasion in PTC. There are two possible origins of macrophages present during the lymphatic invasion of cancer. That is, they may have originally been present in the lymphatic vessels, or they may have invaded with the cancer cells from outside the lymphatic vessels. To show that TAMs in lymphatic invasion may have infiltrated from outside the lymphatic vessels, we examined the correlation of macrophage markers inside and outside the area of lymphatic invasion. The results are shown in Fig. 4. Lymphatic invasion in PTC was identified in 13 of 36 PTCs (Fig. 4a). Macrophages positive for any macrophage marker (PG-M1, KP-1, CD163, CD206, and HO-1) were found inside and outside the areas of lymphatic cancer invasion. The correlation of the number of positive macrophages inside and outside the areas of lymphatic invasion in PTC was as follows: PG-M1 (rs = 0.250, p = 0.409), KP-1 (rs = 0.374, p = 0.208), CD163 (rs = 0.476, p = 0.101), CD206 (rs = 0.806, p = 0.001), and HO-1 (rs = 0.230, p = 0.450) (Fig. 4b). Only the M2 marker CD206 was positively correlated inside and outside the area of lymphatic invasion.

Detection of M2 and non-M2 cells inside and outside the areas of lymphatic invasion by triple IHC.
We compared macrophages inside and outside the area of lymphatic invasion by the detection of an additional different M2 marker from the previous examination. The results are shown in Fig. 4. In 7 specimens in which lymphatic invasion was detected with haematoxylin and eosin staining, the invasion site disappeared after slicing the paraffin-embedding tissue and could not be examined. Finally, lymphatic invasion was detected in 6 specimens (Fig. 5a). There was no difference in the numbers of M2 and non-M2 cells either inside or outside the areas of lymphatic invasion (M2: p = 0.090, non-M2: p = 0.507, Fig. 5b). The number of eligible cases is small and may not be sufficient.

Expression of MMP-2 mRNA according to RT-PCR and in situ RT-PCR and detection of MMP-2 protein in M2 cells by double IHC.
The examination of MMP-2 expression in macrophages is shown in Fig. 6. MMP-2 may be a protein that disrupts the basement membrane of lymphatic vessels. RT-PCR revealed a band corresponding to MMP-2 and GAPDH mRNA in all 5 PTCs (Fig. 6a). Based on the quality of the mRNA in the paraffin blocks, the five most recent cases among the eligible cases were selected. In situ RT-PCR performed on 3 of 5 specimens (cases 1, 2 and 3) revealed positive PTC cells and nonneoplastic cells in the border (Fig. 6b). MMP-2 protein expression in M2 TAMs is shown in Fig. 6c. We performed double IHC to detect MMP-2 and M2 markers in five cases where RT-PCR was performed. Double IHC staining revealed MMP-2 positivity not only in cancer cells involved in lymphatic invasion but also in CD163 + and CD206 + M2 TAMs in all 5 cases.

Discussion
We initially examined the density of lymphatic vessels around the tumour border as a cause of frequent lymph node metastasis in PTC. There was no difference in the density of lymphatic vessels in two areas (the border area and the extratumoural area) between PTC and FTC. Only in the intratumoural area did the number of D2-40 + lymphatic vessels differ. A previous report demonstrated increased lymphatic vessel density in PTC rather than FTC, which is the follicular variant of PTC, and recurrent PTC 14 . Although some of the results of this study were similar, some of the results showed differences. We believe that the difference in the results of the study lies in the difference in the area where the lymphatic vessels were examined. That is, the other study examined intra-and extratumour tissues, whereas we examined the three regions. There was no difference in the number of lymphatic vessels within the tumour, at the tumour border, or outside the tumour between PTCs and FTCs, except for the number of D2-40 + cross sections. We cannot suggest that lymphangiogenesis does not contribute to the frequency of lymph node metastasis, but lymphatic invasion is often identified in the tumour margin on sections. In the present study, there was no difference in the tumour margin, suggesting that factors other than lymphangiogenesis may contribute to the frequency of lymph node metastasis.
In the next step, we focused on TAMs. According to a PubMed search, only one study on the diffuse sclerosing variant of PTC has examined the relationship between lymphatic invasion in PTC and TAMs 10 . A significant increase in TAMs was observed in the tumour border of PTC compared to that of FTC. The expression of the five macrophage markers examined (PG-M1, KP-1, CD163, CD206, and HO-1) in this study was increased in www.nature.com/scientificreports/ www.nature.com/scientificreports/ PTC, considering that both M1 and M2 cells were increased (Fig. 3). In one study, HO-1 was considered a marker of Mox cells (different from M2 cells) 15 , but in another study, M2 cells were reported to express HO-1 16 . It was also shown that the more macrophages there were around the lymphatic vessels, the more likely they developed lymph node metastasis. Although we could not show a difference between M1 and M2 macrophages, this may be one of the reasons why macrophages promote the lymphatic invasion of PTC. In addition, when comparing the positive and negative lymph node cases of PTC, there was an increase in the number of macrophages in the positive lymph node cases. This fact may be another piece of evidence for the contribution of TAMs to lymph node metastasis. Furthermore, because we found a significant difference in the frequency of TAMs in the tumour border of PTC compared to FTC, for the first time, we focused on TAMs and their subtype inside and outside lymphatic vessels with cancer invasion in the border areas and the extratumoural areas in PTC. In lymphatic invasion in PTC, one or more intralymphatic macrophages often coexist with cancer cells (Fig. 4). Intralymphatic M2 cells are frequently observed in areas of lymphatic invasion when there are many perilymphatic TAMs, but the tendency has not been observed in the case of M1 cells. Furthermore, triple immunohistochemistry showed the presence www.nature.com/scientificreports/ of intralymphatic M2 TAMs in areas of lymphatic invasion, although the number of examined specimens was small and was not examined statistically. The two types of intralymphatic macrophages in areas of lymphatic invasion include macrophages already present in the lymphatic vessel and macrophages invading from the perilymphatic stroma in association with lymphatic cancer invasion. The latter may be because only M2 TAMs inside and outside lymphatic vessels were correlated with cancer invasion in this study. M2 TAMs may invade the lymphatic wall together with cancer cells, and they coexist with cancer cells in lymphatic vessels. One study reported that cancer cells infiltrated the collagen membrane when they were cocultured with macrophages in an experimental system using cultured human breast cancer cells, although the cancer cells alone did not infiltrate the collagen membrane 17 . The results of this study are similar, and it is possible that cancer cells infiltrate the basement membrane of lymphatic vessels by coexisting with macrophages. In addition, in Hodgkin lymphoma, the more CD206-positive TAMs are present, the more tumour tissue remodelling advances and then promotes dissemination 18 . Although the M1/M2 polarity of macrophages cannot be simplified, there are many other reports showing that M2 TAMs act as tumour promoters, and we also believe that M2 TAMs have a tumourpromoting effect in PTC.
In this study, the expression of MMP-2 mRNA and protein was found in TAMs together with cancer cells. MMP-2 is also called gelatinase A and is an enzyme that degrades type IV collagen, a component constituting the basement membrane. The expression of MMP-2 has been reported in macrophages in abdominal aortic aneurysms and in alveolar macrophages in pneumothorax 19,20 . On the other hand, Wu et al. 21 demonstrated the increased expression of MMP-2 in PTC cells, and in other carcinomas, the cancer cells themselves secreted and damaged the extracellular matrix. Although there have been few reports of its expression in TAMs thus far, according to the results of this study, both cancer cells and TAMs express MMP-2 and damage the basement membrane of lymphatic vessels, such as macrophages found in inflammatory diseases.
The limitation of this study is that prognostic factors, such as extracapsular invasion and tumour diameter, were not considered. Although there is no specific opinion regarding PTC, the increase in TAMs generally has a poor prognosis in many patients 5,8 and may be confounded with a prognostic factor, such as tumour size 22 .
Consequently, this study revealed for the first time that M2 TAMs around lymphatic vessels within the tumour border of PTC may be associated with lymphatic invasion. This study provides insights into the mechanism of lymphatic invasion, and it may be possible to identify drug targets that inhibit lymph node metastasis in the future.

Methods
Tissue specimens. We examined specimens from 36 patients with PTC diagnosed pathologically between 2010 and 2017 and 10 patients with FTC diagnosed pathologically between 2004 and 2017 at Yamagata University Hospital. We selected tissue sections that included the border between the cancer and the background thyroid gland for PTC and tissue sections exhibiting capsular or vascular invasion for FTC. The brief clinicopathological features are summarized in Table 1. Excised tissues were fixed in 10% buffered formalin for 12-48 h at room temperature and embedded in paraffin.

Ethical issues. This study was approved by the Ethical Review Committee of Yamagata University Faculty
of Medicine (H29-303) and was performed in accordance with the Declaration of Helsinki. The Ethical Review Committee decided that this study did not need written informed consent because information regarding individual features from which others can identify the patient was excluded. Furthermore, we opted out in substitution for written informed consent. All the methods were carried out in accordance with the approved guidelines.
Single IHC. Single IHC was performed as previously described 23 . Three-micrometre-thick sections were deparaffinized. Endogenous peroxidase activity was blocked with methanol containing 0.3% hydrogen peroxide for 30 min on ice. Antigen retrieval was performed using ethylenediaminetetraacetic acid (Antigen Retrieval Solution pH 9; Nichirei Biosciences, Tokyo, Japan) or citric acid (Antigen Retrieval Solution pH 6; Iatron Laboratories, Inc., Tokyo, Japan) in an autoclave (2 atmospheres, 121 °C, 10 min). Sections were incubated with primary antibodies at 4 °C overnight ( Table 2). The labelled streptavidin-biotin peroxidase method (UltraTech HRP Streptavidin-Biotin Detection System, PN IM2391; Immunotech, Marseille, France) and the EnVision+ System HRP-labelled polymer (anti-mouse and anti-rabbit, DAKO, Carpinteria, CA, USA) were used. Positive reactions were detected as brown colouration with 3,3′-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan). Sections were then counterstained with haematoxylin. www.nature.com/scientificreports/ The following items (a, b) were examined: (a) The number of cross-sections of podoplanin (D2-40) + and LYVE1 + lymphatic vessels was counted in three different areas: inside the tumour, on the border, and outside the tumour (for both PTC and FTC). A 0.5 mm width on both sides of the tumour border (total 1 mm width) was defined as the border area. The tumour part inside the border was regarded as inside the tumour, and the nontumourous part outside the border was regarded as outside the tumour. A luminal structure with an endothelial cell lining being immunopositive was defined as a lymphatic vessel. Podoplanin (D2-40) and LYVE1 respond to the lymphatic endothelium but not vascular endothelial cells. The number of cross-sections of the lymphatic vessel was counted manually in 5 fields of a 20 × microscopic power field. (b) The area of lymphatic endothelial cells expressing podoplanin (D2-40) or LYVE1 was calculated.
Double IHC. Double IHC was also performed as previously described 23,24 . The conditions for sections, blocking, antigen retrieval and secondary antibodies were the same as those for single IHC. Positive reactions were detected as brown colouration with 3,3′-diaminobenzidine tetrahydrochloride, the Vulcan Fast Red Chromogen Kit 2 (BIOCARE MEDICAL, Concord, CA, USA) and the HistoGreen Substrate Kit for Peroxidase (Linaris, Heidelberg, Germany). Sections were then counterstained with haematoxylin.
The following three items (c, d) were examined: (c) The number of cells positive for each macrophage marker around the lymphatic vessels in the border area in PTC and FTC was counted in 5 fields obtained at a 40 × microscopic power. (d) The number of cells positive for M1 and M2 markers inside and outside the areas of lymphatic invasion in PTC was counted. In this study, CD68 (PG-M1 or KP-1) + CD163 -CD206cells were defined as M1, and CD163 + CD206 + cells were defined as M2. Specimens in which TTF-1 + PTC cells were observed in D2-40 + lymphatic vessels were designated as containing areas of lymphatic invasion. Furthermore, the intraluminal area of the lymphatic vessel with lymphatic cancer invasion was considered inside the lymphatic wall, and the area surrounding the lymphatic vessel with lymphatic cancer invasion, 100 μm in width, was considered outside the lymphatic wall.
Triple IHC. Triple IHC was also performed as previously described 23,24 . A positive reaction of the first antibody (D2-40) was detected as brown colouration with DAB, and a positive reaction of the second antibody (panmacrophage marker [an antibody cocktail solution for CD68 and CD163]) was detected as light red colouration with Vulcan Fast Red. The HistoGreen Substrate Kit for Peroxidase (Linaris, Heidelberg, Germany) was used for colour detection of the third antibody (c-Maf). Counterstaining was performed with haematoxylin. Triple IHC was used to examine the number of intralymphatic M2 (panmacrophage marker [an antibody cocktail solution for CD68 and CD163] + c-Maf + ) and non-M2 (panmacrophage marker + c-Maf -) cells with lymphatic cancer invasion.

Reverse transcriptase polymerase chain reaction (RT-PCR).
The conditions used for RT-PCR were also as previously described 23 . Formalin-fixed and paraffin-embedded tissues from PTC samples (n = 5) were used for mRNA detection by RT-PCR. mRNA was extracted with a WaxFree RNA Extraction Kit (TrimGen, Sparks, MD, USA) according to the manufacturer's protocol. mRNA (< 5 μg) was used as a template for the synthesis of cDNA (20 μl) with a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, JAPAN). Three microlitres of cDNA was PCR amplified utilizing EmeraldAmp PCR Master Mix (Takara) and specific primers. The primer sequences were 5′-TAC AGG ATC ATT GGC TAC ACACC-3′ (sense) and 5′-GGT CAC ATC GCT CCA GAC T-3′ (antisense) for human MMP-2 (90 bp) and 5′-GCA CCG TCA AGG CTG AGA AC-3′ (sense) and 5′-TGG TGA AGA CGC CAG TGG A-3′ (antisense) for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 138 bp) as the internal control. cDNA was amplified as follows: 94 °C for 10 min; 40 cycles of 94 °C for 30 s, 60 °C for 45 s and 72 °C for 30 s; and 1 cycle of 72 °C for 6 min in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The final PCR products were placed at 4 °C until electrophoresis. We used primers for human MMP-2 designed by PrimerBank. The PCR products were electrophoresed in a 4% agarose gel and stained with ethidium bromide. www.nature.com/scientificreports/ In situ reverse transcriptase polymerase chain reaction (in situ RT-PCR). We examined PTC tissues to determine the localization of MMP-2 mRNA. The conditions used for in situ RT-PCR were as described elsewhere 23,25 . Briefly, dewaxed and rehydrated 5 μm paraffin sections were fixed in 4% paraformaldehyde for 4 h at room temperature. After fixation, the slides were incubated in pepsin (8 mg/ml) for 75 min at 37 °C. After protein digestion, the slides were air-dried and incubated in DNase digestion solution (DNase I recombinant, RNase-free, Roche Diagnostics, Mannheim, Germany) at 37 °C overnight. After DNase digestion, a reverse transcriptase reaction was performed in the solution provided in the Pri-meScript II 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer's protocol. Coverslips were placed over the solutions in the DNase digestion and reverse transcriptase reactions to prevent evaporation. Slide sealers (Takara) were placed on the slides around the tissue prior to PCR.
The in situ RT-PCR mixture consisted of PCR buffer with 15 mM MgCl 2 , PCR digoxigenin (DIG) labelling mix, Taq DNA polymerase and primer pairs for MMP-2 (the same primer pairs used for RT-PCR). A total of 125 μl of PCR mix was applied to the tissue sections, which were then sealed with slide sealers. Slides were placed into a MasterCycler PCR thermal cycler (Eppendorf, Hamburg, Germany) for 1 cycle of 94 °C for 10 min; 30 cycles of 94 °C for 30 s, 60 °C for 45 s and 72 °C for 30 s; and 1 cycle of 72 °C for 6 min. The location of DIG incorporated into PCR amplicons was detected with an alkaline phosphatase assay using anti-DIG-alkaline phosphatase Fab fragments (1:100 dilution) (Roche Diagnostics), which were incubated with the samples for 30 min at 37 °C. The in situ PCR product was detected using Vulcan Fast Red and counterstained with haematoxylin. Tissue sections without reverse transcriptase reactions were used as negative controls.

Statistical analysis.
To calculate the cell count and area in immunohistochemical sections, we used quantification by digital image analysis (HALO imaging analysis software; Indica Labs, Corrales, NM). The Mann-Whitney U test was performed to analyse the difference between the two groups with Microsoft Excel 2013 (Microsoft, Redmond, WA, USA). Spearman's rank correlation coefficient was determined to examine the correlation between the two groups. JMP version 14 (SAS Institute, Tokyo, Japan) was used to plot the individual data. A p value less than 0.05 was considered significant in all tests.

Data availability
The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.