Differential Immunohistochemical Expression Profiles of Perlecan

Pathology – Research and Practice 212 (2016) 426–436

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Pathology – Research and Practice

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Original article

Differential immunohistochemical expression proﬁles of

perlecan-binding growth factors in epithelial dysplasia, carcinoma

in situ, and squamous cell carcinoma of the oral mucosa

a,b a c a

Mayumi Hasegawa , Jun Cheng , Satoshi Maruyama , Manabu Yamazaki ,

a,c a b a,c,∗

Tatsuya Abé , Hamzah Babkair , Chikara Saito , Takashi Saku

Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences,

Niigata, Japan

Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate

School of Medical and Dental Sciences, Niigata, Japan

Oral Pathology Section, Department of Surgical Pathology, Niigata University Hospital, Niigata, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The intercellular deposit of perlecan, a basement-membrane type heparan sulfate proteoglycan, is con-

Received 7 October 2015

sidered to function as a growth factor reservoir and is enhanced in oral epithelial dysplasia and carcinoma

Received in revised form 15 January 2016

in situ (CIS). However, it remains unknown which types of growth factors function in these perlecan-

Accepted 14 February 2016

enriched epithelial conditions. The aim of this study was to determine immunohistochemically which

growth factors were associated with perlecan in normal oral epithelia and in different epithelial lesions

Keywords:

from dysplasia and CIS to squamous cell carcinoma (SCC). Eighty-one surgical tissue specimens of oral SCC

Perlecan

containing different precancerous stages, along with ten of normal mucosa, were examined by immuno-

Perlecan-binding growth factors

histochemistry for growth factors. In normal epithelia, perlecan and growth factors were not deﬁnitely

Oral squamous cell carcinoma

expressed. In epithelial dysplasia, VEGF, SHH, KGF, Flt-1, and Flk-1were localized in the lower half of rete

Carcinoma in situ

Epithelial dysplasia ridges (in concordance with perlecan, 33–100%), in which Ki-67 positive cells were densely packed. In

Cell proliferating zone CIS, perlecan and those growth factors/receptors were more strongly expressed in the cell proliferating

zone (63–100%). In SCC, perlecan and KGF disappeared from carcinoma cells but emerged in the stromal

space (65–100%), while VEGF, SHH, and VEGF receptors remained positive in SCC cells (0%). Immuno-

ﬂuorescence showed that the four growth factors were shown to be produced by three oral SCC cell

lines and that their signals were partially overlapped with perlecan signals. The results indicate that per-

lecan and its binding growth factors are differentially expressed and function in speciﬁc manners before

(dysplasia/CIS) and after (SCC) invasion of dysplasia/carcinoma cells.

1. Introduction two-phase appearance, which results from a sharp and contrastive

layering of the upper keratinized cell layer and the lower half

It remains a challenge to make objective histopathological diag- basaloid cells, is recognized in some particular histological types

noses of oral borderline malignancies from epithelial dysplasia and of epithelial dysplasia or CIS [4–12], and it could be an impor-

carcinoma in situ (CIS) to microinvasive squamous cell carcinomas tant histopathological hallmark of potentially malignant epithelial

(SCC) only on hematoxylin and eosin (HE) stained sections, as the lesions even on HE sections. In the lower half of the two-phase

conventional grading systems are too heavily dependent on the epithelial dysplasia, composed of basaloid cells which are immuno-

subjectivity of pathologists, which leads to considerable disagree- histochemically positive for Ki-67 [5,6] as well as podoplanin

ment [1–3]. Recently, we have proposed that the characteristic [12,13], there are enriched intercellular deposits of extracellular

matrix (ECM) molecules such as perlecan, a basement-membrane

type heparan sulfate proteoglycan [14–18]. In addition, the basaloid

∗ cells in the lower half showed simultaneous loss of E-cadherin and

Corresponding author at: Division of Oral Pathology, Department of Tissue

nuclear translocation of ␤-catenin from the cell membrane, which

Regeneration and Reconstruction, Niigata University Graduate School of Medical

indicates that those basaloid cells form a cell proliferating center

and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan.

E-mail address: [email protected] (T. Saku). in the lower half [6].

http://dx.doi.org/10.1016/j.prp.2016.02.016

M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436 427

To further conﬁrm our hypothesis that the lower half of the [13]. SCC cells were cultured in Dulbecco’s modiﬁed Eagle medium

two-phase epithelial dysplasia is a cell proliferation center and (DMEM) (Gibco, Invitrogen, Thermo Fisher Scientiﬁc, Waltham,

that its histopathological recognition is of considerable help for MA, USA), which contained 10% fetal bovine serum (FBS) (Gibco),

the objective differential diagnosis of oral borderline malignancies, 50 ␮g/ml streptomycin, and 50 IU/ml penicillin (Gibco). They were

◦

we now consider it necessary to investigate the expression pro- incubated at 37 C in a humidiﬁed 5% carbon dioxide/95% air atmo-

ﬁles of perlecan-binding growth factors in oral epithelial dysplasia sphere.

and CIS comparatively in normal epithelia and SCC because per-

lecan has been known to be an important extracellular reservoir 2.3. Antibodies

for several kinds of growth factors or cytokines [19] including vas-

cular endothelial growth factor (VEGF) [20], sonic hedgehog (SHH) Polyclonal antibodies against the mouse basement membrane-

[21], or keratinocyte growth factor (KGF) [22,23]. VEGF, which acts type perlecan core protein were raised in rabbits as described

on endothelial cells to promote angiogenesis, is also required for elsewhere (diluted at 50 ␮g/ml) [14,16]. Mouse monoclonal anti-

tumor cells to proliferate in a cell-autonomous and angiogenesis- bodies against VEGF (clone C-1, IgG2a, 1:200), Flk-1 (A-3, IgG1,

independent manner [24]. It is known that the SHH signaling 1:300) and rabbit polyclonal antibodies against KGF (IgG, 1:50)

pathway regulates cell migration, proliferation, and apoptosis in were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,

several cancer cells from the skin, oral cavity, gastrointestinal CA, USA). Rabbit polyclonal antibodies against Flt-1 (IgG, 1:2000)

tracts, urinary bladder, and lung [25]. KGF has been recognized as were obtained from Oncogene Research Products (La Jolla, CA, USA)

a mesenchymal cell-derived paracrine mediator of epithelial cell and those against SHH (IgG, 1:100) were obtained from Abcam Inc.

growth [26], but it is also known to stimulate various carcinoma (Cambridge, UK). A mouse monoclonal antibody against human

cells from the biliary tract [27] and breast [28], though it has not Ki-67 (MIB-1, IgG1, 1:50) was obtained from Dako (Glostrup,

been immunolocalized in SCC of the head and neck [29]. Thus, Denmark).

the expression modes of these molecules in oral SCC are some-

what controversial. Their pathophysiological functions also remain 2.4. Immunohistochemistry

totally unknown during the oral precancerous stages, though per-

lecan has been suggested to function in epithelial dysplasia and CIS Parafﬁn sections were subjected to immunohistochemical stain-

[6,9,14,16]. ings for perlecan core protein, VEGF, KGF, SHH, Flt-1, Flk-1, and

In this study, our aim was to determine comparative immuno- Ki-67 by using the Envision+/HRP system (Dako). For VEGF, sec-

histochemical proﬁles in oral mucosal epithelia ranging from tions were treated with 0.15% trypsin (type II, Sigma Chemical

normal to SCC among the following molecules: perlecan; Ki-67, a Co., St Louis, MO, USA) in 10 mM Tris–HCl (pH 7.6) for 30 min at

◦

cell cycle marker; such perlecan-binding factors as KGF, SHH and 37 C. For SHH, Flt-1, Flk-1 and Ki-67, sections were autoclaved

◦

VEGF; as well as VEGF receptors Flt-1 and Flk-1. in citric acid buffer (pH 6.0) at 120 C for 10 min. After that, the

sections were rinsed in 0.01 M PBS containing 0.5% milk pro-

tein (Morinaga Milk Industry Co. Ltd., Tokyo, Japan) and 0.05%

2. Materials and methods

Triton X-100 (T-PBS) and treated with 0.3% hydrogen peroxide

in methanol for 30 min at room temperature to block endoge-

2.1. Tissue materials

nous peroxidase activities. After rinsing in T-PBS, sections were

incubated with 5% milk protein in T-PBS for 1 h at room temper-

Eighty-one surgical specimens of SCC or CIS and 10 biopsy

ature to block non-speciﬁc protein-binding sites. They were then

specimens of epulis of the oral mucosa were selected from the ◦

incubated with the primary antibodies overnight at 4 C. After incu-

surgical pathology ﬁles in the Division of Oral Pathology, Niigata

bation, the sections were rinsed in T-PBS and incubated with the

University Graduate School of Medical and Dental Sciences. Each

polymer-immune complexes (EnVision+peroxidase, rabbit/mouse,

specimen simultaneously contained histopathologically different

Dako) for 1 h at room temperature. After rinsing with T-PBS, they

lesions ranging from frankly invasive and well-differentiated SCC

were treated with 0.02% 3,3 -diaminobenzimine (Dohjindo Lab-

foci and foci of CIS, epithelial dysplasia, and epithelial hyperpla-

oratories, Kumamoto, Japan) in 0.05 M Tris–HCl buffer (pH 7.6)

sia to deﬁnitely normal epithelial parts. From these specimens,

containing 0.005% hydrogen peroxide to visualize the reaction

we selected 30 foci of normal and hyperplastic epithelia, 50 of

products. Finally, the sections were counterstained with hema-

moderate epithelial dysplasia with the characteristic two-phase

toxylin. For control studies on antibodies, the primary antibodies

appearance [4–6], 45 of CIS, and 30 of SCC, all of which were diag-

were replaced with pre-immune rabbit IgG or mouse IgG subclasses

nosed on hematoxylin and eosin (HE) stained sections as well as on

(Dako).

their immunohistochemically stained sections for keratin 13 (K13),

Following HE staining and immunohistochemistry examina-

a prickle cell marker; K19, a basal cell marker; Ki-67, a cell prolif-

tions for K13, K19, K17, K16, and Ki-67, performed as described

eration marker; and K17/K16, carcinoma cell markers, as we have

elsewhere [5–12], all of the focus samples were classiﬁed as (i)

described elsewhere [5–9]. The diagnostic criteria used in this study

normal or hyperplastic epithelia, (ii) mild and moderate epithe-

are described in a separate section. All the specimens were rou-

lial dysplasia, (iii) CIS, or (iv) SCC. We did not use the category of

tinely ﬁxed in 10% formalin and embedded in parafﬁn. Serial 3-␮m

severe dysplasia because we considered that there was no objective

sections were cut from parafﬁn blocks, and one set of the sections

distinction between so-called severe dysplasia and CIS [5].

was stained with HE while the other sets were used for immuno-

histochemistry. The experimental protocol for analyzing surgical

2.5. Immunohistochemical evaluation

materials was reviewed and approved by the Ethical Board of the

Niigata University Graduate School of Medical and Dental Sciences

Foci of SCC, CIS, dysplasia, and normal epithelial parts were

(Oral Life Science).

evaluated by extension and intensity of the immunohistochemi-

cal reactions for the three perlecan-binding molecules, VEGF, SHH,

2.2. Cells and KGF, and compared with those for perlecan. The staining was

evaluated in four epithelial zones—basal, parabasal, lower prickle,

SCC cell systems (ZK-1, ZK-2, and MK-1) were established from and upper prickle layers as indicated in Figs. 1–3—for positive

SCC arising in the tongue (ZK-1 and ZK-2) and gingiva (MK-1) ratios. Each layer was considered positive (+) or not positive (−)

428 M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436

Fig. 1. Normal oral squamous epithelium. (a) Hematoxylin and eosin (HE) stain; (b) immunoperoxidase stain for extracellular matrix protein perlecan; (c) cell proliferation

marker Ki-67; perlecan-binding factors: (d) KGF; (e) VEGF; (f) SHH; and VEGF receptors: (g) Flt-1; (h) Flk-1, hematoxylin counterstain. (A–H) ×160. In normal (a) or hyperplastic

epithelia, perlecan was localized faintly in the parabasal cells layer (b) in which Ki-67 positive (+) cells were located (c). While KGF was not positive in the epithelial layer

(d), VEGF was deﬁnitely positive in the cytoplasm of basal or parabasal cells, in addition to vascular endothelial cells and other stromal cells in the lamina propria (e). SHH

was faintly positive in nuclei of epithelial cells from the basal to lower prickle cell layers as well as in round-shaped stromal cells (f). Flt-1 and Flk-1 were mainly localized

within the nuclei of epithelial cells from the basal to lower prickle cell layers in addition to vascular endothelial cells (g, h).

when a particular molecule was or was not expressed in epithe- test for independence. Differences with P < 0.05 were considered

lial cells located in those layers without consideration of staining signiﬁcant.

intensities or extensions. In terms of SCC foci, the four layers

were basically separated corresponding to normal ones within 2.6. Immunoﬂuorescence

the range from the periphery (basal) to the center (keratinized).

In the epithelial zone, both nuclear and cytoplasmic stainings Immunoﬂuorescence experiments were performed using

TM TM

−

were equally counted positive. The +/ judgments were agreed Nunc Lab-Tek II Chamber Slide System (Thermo Fisher).

upon by three examiners who were experienced pathologists. Cells were plated at the concentration at 1.2 × 10 cells/well and

Results were expressed as the ratio of positive foci to all the cultivated for 7 days. The cells were washed with PBS and ﬁxed

examined ones. In addition, rates of immunolocalization in con- with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for

cordance between the three growth factors and perlecan were 30 min on ice. To prevent non-speciﬁc protein binding, they were

calculated for better understanding their colocalization. Statistical incubated with 5% milk protein in PBS containing 0.05% Triton

◦

differences were determined by a Student’s t-test or a chi-square X-100 overnight at 4 C. The cells were then incubated with the

M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436 429

Fig. 2. Oral epithelial dysplasia with a characteristic two-phase appearance. (a) HE stain; immunoperoxidase stain for perlecan (b), Ki-67 (c), KGF (d), VEGF (e), SHH (f), Flt-1

(g), and Flk-1 (h), hematoxylin counterstain. (a–h) ×260. In two-phase dysplasia (a), perlecan was positive mainly on the cell border of basaloid cells densely packed in the

lower half of the epithelial layer (b) in which Ki-67+ cells were stratiﬁed from the ﬁrst basal layer (c). KGF was faintly positive on the cell border and in the cytoplasm of the

lower half (d), while VEGF was strongly positive in the cytoplasm in the lower half (e). SHH was mainly positive in nuclei in the lower half (f). Flt-1 (g) and Flk-1 (h) were

localized mainly in the nuclei of the lower-half cells.

primary antibodies (perlecan, diluted at 50 ␮g/ml in PBS; VEGF, 3. Results

1:100; KGF, 1:50; SHH, 1:50) and further with secondary anti-

bodies. For double immunoﬂuorescence, cells were ﬁrstly stained Immunohistochemical staining results are separately described

for perlecan using secondary goat antibodies against rabbit IgG in each category as follows. Table 1 summarizes the ratios of pos-

TM TM

conjugated with Alex Fluor 488 (Molecular Probes , Thermo itive foci by layers for VEGF, SHH and KGF (upper row) and their

Fisher), and then sequentially for VEGF using a secondary goat IgG concordance rates with perlecan (lower row) in each category.

against mouse IgG conjugated with Alexa Fluor 568 (Thermo

Fisher). When secondarily stained for SHH, or KGF, the rabbit

3.1. Normal/hyperplastic epithelia

antibodies against SHH and KGF were directly labeled with Alexa

Fluor 568 without using dye-conjugated secondary antibodies.

In normal and hyperplastic epithelia from SCC/CIS or epulis

Finally, cells were counterstained with Cellstain Hoechst-33258

specimens (Fig. 1a), perlecan was faintly positive in and above the

solution (Dojindo) diluted at 1:100 in PBS. For control studies,

parabasal cells layer (Fig. 1b), where Ki-67 positive (+) cells were

the primary antibodies were replaced with pre-immune rabbit or

sporadically located (Fig. 1c). Perlecan was occasionally positive in

mouse IgGs.

the lamina propria connective tissue. Such immunohistochemical

430 M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436

Fig. 3. Oral carcinoma in situ (CIS). (a) HE stain; immunoperoxidase stain for perlecan (b), Ki-67 (c), KGF (d), VEGF (e), SHH (f), Flt-1 (g), and Flk-1 (h), hematoxylin counterstain.

(a–h) ×240. In CIS (a), perlecan was localized on the cell border in the whole epithelial layer except for a few cell layers just beneath the surface keratinized layer (b). Ki-67+

cells were spread in the perlecan+ rete ridge area (c). KGF was positive on the cell border as well as in the cytoplasm in the perlecan+ area (d). VEGF was expressed mainly

in the cytoplasm in the perlecan+ area (e). SHH+ areas were nearly the same as the four molecules mentioned above, though SHH was localized in the nuclei (f). Flt-1 was

localized in the nuclei and cytoplasm in the perlecan+ area including surface keratinized layer (g), while Flk-1 was mainly positive in the nuclei of the lower half zone of the

rete ridges (h).

proﬁles were stably observed even in hyperplastic epithelia cover- because perlecan was not deﬁnitely expressed either. It was thus

ing the epulis (not shown). While KGF was not deﬁnitely positive in suggested that VEGF and SHH signals mainly expressed in the basal

the epithelial layer (Fig. 1d), VEGF was positive in the cytoplasm of and parabasal layers of normal/hyperplastic epithelia were medi-

basal or parabasal cells, in addition to vascular endothelial cells and ated by ligands other than perlecan.

other stromal cells in the lamina propria (Fig. 1e). SHH was faintly

positive in nuclei of epithelial cells from the basal to lower prickle 3.2. Epithelial dysplasia

cell layers as well as in round-shaped stromal cells in the lamina

propria (Fig. 1f). Flt-1 and Flk-1, VEGF-receptors, were mainly local- In epithelial dysplasia with the characteristic two-phase appear-

ized within the nuclei of epithelial cells from the basal to lower ance (Fig. 2a), perlecan was positive mainly on the cell border (in

prickle cell layers, and Flt-1 was also localized in vascular endothe- the intercellular space) of the lower half of the epithelial layer, in

lial cells (Fig. 1g, h). The immunohistochemical positivities for the addition to the subepithelial connective tissue (Fig. 2b). Ki-67+ cells

three growth factors were compared with those of perlecan by four were stratiﬁed up to the ﬁfth layers from the bottom (Fig. 2c).

epithelial layers in every category of epithelial lesions as shown The perlecan localization showed a meshwork-like appearance.

in Table 1. Their colocalizations with perlecan were not observed In the same manner, KGF was faintly positive on the cell border

M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436 431

of the lower half (Fig. 2d). VEGF was more strongly positive in

the lower half (Fig. 2e), in which SHH was mainly positive in the

0 0

nuclei of epithelial cells (Fig. 2f). Flt-1 (Fig. 2g) and Flk-1 (Fig. 2h) 88 93

Upper prickle

were localized mainly in the nuclei of the lower half. The rates of

immunolocalization in concordance between the three growth fac-

tors and perlecan were 33–100% in the lower two layers, though

0 8

Lower prickle 96 98

those in the upper prickle cell layer were not so stable between

the growth factors (Table 1). The differences on the positive ratios

for the three molecules in epithelial dysplasia were signiﬁcantly

higher than normal/hyperplastic epithelia (P < 0.001). 0 8 (%) Parabasal 96 98

3.3. CIS

0 0 Perlecan 96 98

Basal

In CIS (Fig. 3a), perlecan was localized on the cell border in the

whole epithelial layer except for a few cell layers just beneath the

(100) (71) (100) (100) keratinized surface layer, in addition to the subepithelial connec-

0 0 Upper prickle

62 95

tive tissue (Fig. 3b). The meshwork-like appearance of perlecan was

more intensive and expansive than that in dysplasia. Since Ki-67+

cells were spread in perlecan+ rete ridge parts in CIS (Fig. 3c), it (–) (65) (97) (100)

was conﬁrmed that perlecan was expressed in the cell proliferat- 0 0 Lower prickle

62 95

ing zone in every category of oral squamous epithelia from normal parentheses)

and dysplasia up to CIS. KGF was positive on the cell border as

well as in the cytoplasm in the perlecan+ area (Fig. 3d). VEGF was (–) (65) (97) (100)

right, lesions.

0 0

Parabasal similarly expressed mainly in the cytoplasm in the perlecan+ area 62 95 (%,

(Fig. 3e). Although SHH was localized in the nuclei, SHH+ areas

were nearly the same as the areas positive for the four molecules (100) (65) (97) (100)

epithelial

0 0

KGF Basal mentioned above (Fig. 3f). Flt-1 was localized in both the nuclei

62 95

localization and cytoplasm in the perlecan+ area including the keratinized sur-

face layer (Fig. 3g), while Flk-1 was mainly positive in nuclei of the (100) (33) (98) (–) squamous

0 lower half of rete ridges (Fig. 3h). VEGF, its receptors, and SHH were 29 Upper prickle perlecan 100

oral also localized in vascular endothelial cells in the stroma (Fig. 3e–h).

The concordance rates between the three growth factors and per- with

lecan were 63–100% in the whole layers (Table 1). The concordances (100) (74) (96)(–) 91

layers

12 71 rates for KGF and SHH in CIS were signiﬁcantly higher than those Lower prickle

100

in epithelial dysplasia, and the differences were statistically sig-

concordance niﬁcant in each layer (P < 0.001), while there were no signiﬁcant

epithelial

(100) (90) (100)(–) 94

for differences for VEGF.

41 86 four Parabasal

100 ratio by

3.4. SCC

their (–) (93) (100) 100 (–)

factors

In SCC (Fig. 4a), perlecan (Fig. 4b) and KGF (Fig. 4d) were focally 65 89 SHH Basal and 100

localized in the stromal connective tissue space around invading

left)

SCC foci but not in SCC cells (Fig. 4b), most of which were posi- growth

(%, (–) (41) (100) 100 (–)

tive for Ki-67 (Fig. 4c). In contrast, VEGF was strongly positive in 3 36 97

Upper prickle

the cytoplasm of SCC cells and of stromal cells, including vascular binding layers

endothelial cells (Fig. 4e). SHH was also positive in the nuclei and

its

in the cytoplasm of SCC cells as well as in those of stromal cells vs. (100) (90) (63)(–) 98

(Fig. 4f). Flt-1 was positive in the cytoplasm of SCC cells (Fig. 4g), 13 86 Lower prickle epithelial 100

and Flk-1 was localized both in the nuclei and cytoplasm of SCC

b 100%). four

perlecan cells (Fig. 4h). Both of the VEGF receptors were positive in vas-

for cular endothelial cells in the stroma (Fig. 4g, h). The concordance (100) (100) (63)(–) 62

value:

40 rates between VEGF/SHH and perlecan were nearly 0% in the whole Parabasal 100 100 ratios

factors

layers of SCC foci, while those between KGF and perlecan were

65–100% not only within SCC foci but also in the stromal space (–) (100) (100)(–) 62

(Table 1). (maximum growth

positive

Basal VEGF

100 100 100

3.5. Immunoﬂuorescence in oral SCC cells in culture localization.

localization 30 30 50

The immunoﬂuorescence signals for perlecan and three growth

Focus number 155

factors were compared in the three SCC cell systems. At day 3 after perlecan

perlecan-binding

seeding, when ZK-1 cells formed small colonies (Fig. 5a–c), per- of

perlecan situ 45

immunohistochemical in

to lecan was localized both in the perinuclear zone in the cytoplasm cell

ratios

as well as in the peripheral cell border of external ends (Fig. 5a).

identical

number

KGF showed almost similar localizations in the perinuclear zone as 1

carcinoma epithelia dysplasia, moderate

Not Identical

well as in the external cell border (Fig. 5b). Merged images for the a Positive Lesions Normal/hyperplastic Carcinoma Squamous Epithelial Total b Table

Comparative

two molecules obviously showed their colocalizations especially in

432 M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436

Fig. 4. Oral squamous cell carcinoma (SCC). (a) HE stain; immunoperoxidase stain for perlecan (b), Ki-67 (c), KGF (d), VEGF (e), SHH (f), Flt-1 (g), and Flk-1 (h), hematoxylin

counterstain. (a–h) ×160. In invading fronts of SCC (a), perlecan (b) and KGF (d) were localized in the stromal connective tissue space but not in trabecular SCC cell nests,

which were packed with Ki-67+ SCC cells (c). In contrast, VEGF was strongly positive in SCC cells, in addition to stromal cells including vascular endothelial cells (e). SHH was

also positive in SCC cells (f). Flt-1 was weakly positive in the cytoplasm of SCC cells (g), and Flk-1 was apparently localized in both the nuclei and cytoplasm of SCC cells (h).

the external ends (Fig. 5c, arrows). Signals for VEGF were also colo- for those four molecules at day 5 when cells reached their conﬂu-

calized with those for perlecan (Fig. 5d, f) in the perinuclear space ency (not shown). Thus, different from immunoperoxidase staining

as well as in the nuclei in addition to on the cell border (Fig. 5e, results in tissue sections, the biosynthesis of KGF, VEGF, and SHH

f, arrow, external end; arrowhead, intercellular). Similar colocal- were conﬁrmed in all of the three oral SCC cell types, and their

ization patterns including those in the external ends suggestive of immunoﬂuorescence signals were focally colocalized with perlecan

such cellular processes as ﬁlopodia or lamellipodia were obtained signals.

between perlecan (Fig. 5g, i) and SHH (Fig. 5h, i). In MK-1 cells,

which were taller than ZK-1 and formed more condensed aggrega- 4. Discussion

tion within colonies, thick dot-like signals for perlecan (Fig. 5j) and

KGF (Fig. 5 k) were colocalized in the perinuclear to intercellular We have for the ﬁrst time demonstrated the immunohis-

cell border (Fig. 5l, arrowhead). Similar tendencies in colocalization tochemical proﬁles of perlecan-binding growth factors in the

between perlecan (Fig. 5m) and VEGF (Fig. 5n) or between perlecan developmental process of oral epithelial malignancies from epithe-

(Fig. 5p) and SHH (Fig. 5q) were observed in the central zone of lial dysplasia to SCC. Before invasion or up to the stage of CIS,

the colonies (Fig. 5o, r, arrowheads). ZK-2 showed signal patterns the expressions of perlecan and perlecan-binding growth fac-

similar to ZK-1 (not shown). The three cell types showed signals tors were overlapped, and they spread within the epithelial

M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436 433

Fig. 5. Double immunoﬂuorescence for perlecan and its binding growth factors in oral SCC cells in culture. (a–i) ZK-1 cells, (j–r) MK-1 cells, (a, d, g, j, m, p) perlecan, (b, k) KGF,

(e, n) VEGF, (h, q) SHH, (c, f, i, l, o, r) merges of perlecan and KGF, VEGF, and SHH, nuclear counterstain with Hoechst-33258, no counterstain in single immunoﬂuorescence,

(a–r) ×680. At day 3 after seeding, when ZK-1 cells formed small colonies (a–c), perlecan was localized both in the perinuclear zone in the cytoplasm as well as in the

peripheral cell border of external ends (a). KGF showed almost similar localizations in the perinuclear zone as well as in the external cell border (b). Merged images for the

two molecules obviously showed their colocalizations especially in the external ends (c, arrows). VEGF signals were also colocalized with those for perlecan in the perinuclear

space (e) as well as in the nuclei in addition to on the cell border (f, arrow, external end; arrowhead, intercellular). Similar colocalization patterns were obtained between

perlecan (g) and SHH (h, i, arrow). In MK-1 cells, thick dot-like signals for perlecan (j, arrow) and KGF (k, arrow) were colocalized in the perinuclear to intercellular cell border

(l, arrowhead). Similar tendencies in colocalization between perlecan (m, arrow) and VEGF (n, arrow) or between perlecan (p, arrow) and SHH (q, arrow) were observed in

the central zone of the colonies (o, r, arrowheads).

layer, though the growth factors were conﬁned to the basal only in the SCC foci, as these three growth factors were con-

zone in normal epithelia. Once carcinoma cells started to invade, ﬁrmed to be biosynthesized in oral SCC cells in culture. The

the expressions of perlecan and KGF were converted from car- present histological study indicates that perlecan plays important

cinoma cells to stromal cells, while VEGF and SHH remained roles in oral epithelial dysplasia, CIS, and SCC by differentially

434 M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436

modulating the perlecan-bound growth signals before and after their biosynthesis switching from carcinoma cells to stromal cells

invasion. on invasion is not so surprising, though it is unknown at present

In the present study, the upper half and the lower half of two- what sorts of molecular mechanisms regulate VEGF and SHH to

phase dysplasia were well contrasted by the presence of VEGF, remain in carcinoma cells, or KGF to move to stromal cells after

SHH, and KGF in the lower half, which was in accordance with invasion.

the intercellular deposit of perlecan, and by their absence in the As to VEGF, Iozzo and his group have shown that angiogenesis

upper half. The results clearly indicate that the perlecan-enriched in the zebraﬁsh embryonic development as well as prolifera-

lower half is also enriched with perlecan-binding growth factors, tion of human endothelial cells, both of which were dependent

and that the lower half is therefore optimized for proliferation of on VEGF-VEGF-receptors, were modulated by perlecan [46]. In

Ki-67+ basaloid cells [4,5,11]. In other words, the lower half of prostate carcinomas, heparin-binding growth factors, including

the two-phase appearance, which is characterized by the simul- VEGF or FGF-2, are reduced in the absence of perlecan, suggest-

taneous loss of E-cadherin and nuclear translocation of ␤-catenin ing that the VEGF signaling is controlled by perlecan [47]. In

from the cell membrane [6], can be regarded as a distinct cell pro- the present study, the immunohistochemical expressions of Flt-

liferating center in epithelial dysplasia. These molecular devices 1 and Flk-1 were similarly related to VEGF/perlecan expressions

for cellular proliferation seem to be correlated from each other in oral epithelial dysplasia, CIS, and SCC. There have been sev-

under the circumstance of the intercellular deposits of perlecan. eral studies reporting differential expressions between these two

Based on such molecular crosstalk via perlecan, we have proposed VEGF receptors in malignancies, in addition to angiogenetic func-

the concept of the intraepithelial stroma [30] not only in oral tions [48]. An autocrine manner of VEGF signaling via Flt-1 has

epithelial lesions but also in odontogenic organs [31] or tumors already been shown to function in carcinogenesis and prolifera-

[32]. With the increase in dysplastic grades, the Ki-67+/perlecan+ tion of epidermal tumor cells [23], in proliferation of pleomorphic

area expanded together with the areas which were also positive adenoma cells [49], or in migration and invasion of pancreatic

for VEGF, SHH and KGF. Finally in the stage of CIS, the whole carcinoma cells [50]. Since Flk-1 and VEGF were found in dys-

epithelial layer became positive for Ki-67, perlecan, and the growth plastic nodules of the liver [51], the Flk-1 expressions could be

factors. related to cell proliferation. Thus, the VEGF signaling via the two

The roles of perlecan in cancer cell growth, invasion, metasta- receptors may be different from tumor to tumor or from organ

sis and angiogenesis have been well documented in various types to organ.

of tumors including human oral [33], salivary [34], breast [35], and From the present results, it is now obvious that varieties of per-

liver [36] carcinomas or melanoma [37]. However, most of the stud- lecan signaling play important roles in the SCC growth both before

ies on the function of perlecan were performed in single cell culture and after invasion. Since the functional modes are differentially reg-

systems, namely in circumstances in which cancer cells are iso- ulated before and after invasion at the tissue level, it is necessary

lated from or not in contact with any other types of cells, including to conﬁrm the phenomena in in vitro studies using co-culture sys-

stromal ﬁbroblasts. Therefore, these experimental conditions must tems before the whole molecular mechanism of oral SCC invasion

correspond at tissue levels with CIS in which carcinoma cells are mediated by perlecan is fully understood.

not exposed to stromal cells [14,16,38]. When perlecan is secreted

by parenchymal (carcinoma) cells before invasion [16,17,33], it is

5. Conclusions

reasonable to expect perlecan-binding molecules function at the

same time within the parenchymal space. A variety of perlecan-

The present study demonstrated the signiﬁcance of intercellu-

binding molecules, including VEGF, SHH, FGF, EGF, PDGF, and

lar deposit of perlecan, a basement-membrane type heparan sulfate

TGF-␤, are basically regarded as tumor cell growth factors, though

proteoglycan in oral precancerous lesions and SCC. Before invasion,

their perlecan-biding modes are different from each other [39,40].

namely in oral epithelial dysplasia and CIS, VEGF, SHH, KGF, Flt-1,

Perlecan consists of a core protein with a molecular mass of approx-

and Flk-1were colocalized in the lower half of rete ridges where

imately 500 kDa and three major heparan sulfate (HS) chains which

perlecan is enriched and Ki-67+ proliferating cells are condensed.

are attached to domain I of the core protein [18,41]. VEGF binds

After invasion, perlecan and KGF disappeared from SCC cells but

to HS chains [19], while SHH binds to both HS chains and the

emerged in the stromal space, while VEGF, SHH, and VEGF recep-

core protein [20], and KGF binds to domains III and V of the core

tors remained in SCC cells. Since we have reported that perlecan

protein of perlecan [21]. However, their perlecan-association situa-

biosynthesis was switched from CIS cells to stromal ﬁbroblasts on

tions have never been investigated at the tissue levels. The present

and after invasion of SCC [14,17,33], and that the switching was

study has revealed that perlecan and its binding growth factors

differentially correlated with that of such perlecan receptors as dys-

are at least colocalized in epithelial dysplasia and CIS foci before

troglycan and integrin ␤1 [16]. Immunohistochemistry must be one

invasion, but after that, those growth factors do not always behave

and only tool to demonstrate such switching phenomena in tissue

together with carcinoma cells because the biosynthesis of per-

samples. It is now reasonably explained that perlecan is required

lecan has been demonstrated to be switched over from carcinoma

for recruiting growth factors to oral SCC/CIS/dysplasia cells for their

cells to stromal cells in co-culture experiments [33]. Similar to

proliferation and invasion.

the perlecan-binding growth factors, perlecan receptors have been

shown to switch from ␣-dystroglycan to integrin ␤1 on invasion of

oral SCC [16]. In prostate carcinomas, KGF and VEGF of stromal ori-

6. Conﬂicts of interest

gin have been shown to mediate interactions between tumor cells

and stromal cells to induce secretion of ECM-degrading enzymes

We declare that we have no conﬂicts of interest.

for invasion [37].

Similar to our present results, the absence of KGF expressions in

Acknowledgments

head and neck SCC cells and their presence in stromal ﬁbroblasts

has already been reported [27]. We have also demonstrated that

This work was supported in part by Grants-in-Aid for Scientiﬁc

KGF is enriched in the stromal space with spindle cells, which are

Research from the Japan Society for the Promotion of Science (JSPS

active in proliferation, in the invading front of salivary pleomor-

KAKENHI grant nos. 25305035 and 25462849 to J.C.; 23406038,

phic adenomas [42]. Since KGF [43], VEGF [44], and SHH [45] are

26305032, and 15K15693 to T.S.).

known to be produced by both epithelial and mesenchymal cells,

M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436 435

References [23] M. Mongiat, J. Otto, R. Oldershaw, F. Ferrer, J.D. Sato, R.V. Iozzo, Fibroblast

growth factor-binding protein is a novel partner for perlecan protein core, J.

[1] J.J. Pindborg, P.A. Reichart, C.J. Smith, I. van der Waal, Precancerous lesions, in: Biol. Chem. 276 (2001) 10263–10271.

J.J. Pindborg, P.A. Reichart, C.J. Smith, I. van der Waal (Eds.), Histological [24] B.M. Lichtenberger, P.K. Tan, H. Niederleithner, N. Ferrara, P. Petzelbauer, M.

Typing of Cancer and Precancer of the Oral Mucosa, World Health Sibilia, Autocrine vegf signaling synergizes with EGFR in tumor cells to

Organization International Histological Classiﬁcation of Tumours, 2nd ed., promote epithelial cancer development, Cell 140 (2010) 268–279.

Springer-Verlag, Berlin, 1997, pp. 24–26. [25] P.A. Beachy, S.S. Karhadkar, D.M. Berman, Tissue repair and stem cell renewal

[2] I.R. Kramer, R.B. Lucas, J.J. Pindborg, L.H. Sobin, Deﬁnition of leukoplakia and in carcinogenesis, Nature 432 (2004) 324–331.

related lesions: an aid to studies on oral precancer, Oral Surg. Oral Med. Oral [26] T. Yamayoshi, T. Nagayasu, K. Matsumoto, T. Abo, Y. Hishikawa, T. Koji,

Pathol. 46 (1978) 518–539. Expression of keratinocyte growth factor/ﬁbroblast growth factor-7 and its

[3] N. Gale, B.Z. Pilch, D. Sidransky, A. El-Naggar, W. Westra, J. Califano, N. receptor in human lung cancer: correlation with tumour proliferative activity

Johnson, D.G. MacDonald, Epithelial precursor lesions, in: L. Barnes, J.W. and patient prognosis, J. Pathol. 204 (2004) 110–118.

Eveson, P. Reichart, D. Sidransky (Eds.), World Health Organization [27] R. Amano, N. Yamada, Y. Doi, M. Yashiro, M. Ohira, A. Miwa, K. Hirakawa,

Classiﬁcation of Tumours, Pathology & Genetics, Head and Neck Tumours, Signiﬁcance of keratinocyte growth factor receptor in the proliferation of

IARC, Lyon, 2005, pp. 177–179. biliary tract cancer, Anticancer Res. 30 (2010) 4115–4121.

[4] M. Syafriadi, J. Cheng, K.Y. Jen, H. Ida-Yonemochi, M. Suzuki, T. Saku, [28] Y. Hishikawa, N. Tamaru, K. Ejima, T. Hayashi, T. Koji, Expression of

Two-phase appearance of oral epithelial dysplasia resulting from focal keratinocyte growth factor and its receptor in human breast cancer: its

proliferation of parabasal cells and apoptosis of prickle cells, J. Oral Pathol. inhibitory role in the induction of apoptosis possibly through the

Med. 34 (2005) 140–149. overexpression of bcl-2, Arch. Histol. Cytol. 67 (2004) 455–464.

[5] T. Kobayashi, S. Maruyama, J. Cheng, H. Ida-Yonemochi, M. Yagi, R. Takagi, T. [29] B. Knerer, M. Formanek, B. Schickinger, H. Martinek, J. Kornfehl, Different KGF

Saku, Histopathological varieties of oral carcinoma in situ: diagnosis aided by expression in squamous cell carcinomas of the head and neck and in normal

immunohistochemistry dealing with the second basal cell layer as the mucosa, Acta Otolaryngol. 118 (1998) 438–442.

proliferating center of oral mucosal epithelia, Pathol. Int. 60 (2010) 156–166. [30] H. Ida-Yonemochi, M. Nakajima, T. Saku, Heparanase, heparan sulfate and

[6] C.G. Alvarado, S. Maruyama, J. Cheng, H. Ida-Yonemochi, T. Kobayashi, M. perlecan distribution along with the vascular penetration during stellate

Yamazaki, R. Takagi, T. Saku, Nuclear translocation of ␤-catenin synchronized reticulum retraction in the mouse enamel organ, Arch. Oral Biol. 55 (2010)

with loss of E-cadherin in oral epithelial dysplasia with a characteristic 778–787.

two-phase appearance, Histopathology 59 (2011) 283–291. [31] H. Ida-Yonemochi, K. Ohshiro, W. Swelam, H. Metwaly, T. Saku, Perlecan, a

[7] T. Mikami, J. Cheng, S. Maruyama, T. Kobayashi, A. Funayama, M. Yamazaki, basement membrane-type heparan sulfate proteoglycan, in the enamel

H.A. Adeola, L. Wu, S. Shingaki, C. Saito, T. Saku, Emergence of keratin 17 vs. organ: its intraepithelial localization in the stellate reticulum, J. Histochem.

loss of keratin 13: their reciprocal immunohistochemical proﬁles in oral Cytochem. 53 (2005) 763–772.

carcinoma in situ, Oral Oncol. 47 (2011) 497–503. [32] H. Ida-Yonemochi, M.S. Ahsan, T. Saku, Differential expression proﬁles

␣

␤

[8] A. Funayama, J. Cheng, S. Maruyama, M. Yamazaki, T. Kobayashi, M. Syafriadi, between -dystroglycan and integrin 1 in ameloblastoma: two possible

S. Kundu, S. Shingaki, C. Saito, T. Saku, Enhanced expression of podoplanin in perlecan signalling pathways for cellular growth and differentiation,

oral carcinomas in situ and squamous cell carcinomas, Pathobiology 78 Histopathology 58 (2011) 234–245.

(2011) 171–180. [33] H. Metwaly, S. Maruyama, M. Yamazaki, M. Tsuneki, T. Abe, K.Y. Jen, J. Cheng,

[9] H. Ida-Yonemochi, S. Maruyama, T. Kobayashi, M. Yamazaki, J. Cheng, T. Saku, T. Saku, Parenchymal-stromal switching for extracellular matrix production

Loss of keratin 13 in oral carcinoma in-situ: a comparative study of protein on invasion of oral squamous cell carcinoma, Hum. Pathol. 43 (2012)

and gene expression levels using parafﬁn sections, Mod. Pathol. 25 (2012) 1973–1981.

784–794. [34] S. Kimura, J. Cheng, K. Toyoshima, K. Oda, T. Saku, Basement membrane

[10] A. Funayama, S. Maruyama, M. Yamazaki, K. Al-Eryani, S. Shingaki, C. Saito, J. heparan sulfate proteoglycan (perlecan) synthesized by ACC3, adenoid cystic

Cheng, T. Saku, Intraepithelially entrapped blood vessels in oral carcinoma carcinoma cells of human salivary gland origin, J. Biochem. 125 (1999)

in-situ, Virchows Arch. 460 (2012) 473–480. 406–413.

[11] T. Kobayashi, S. Maruyama, T. Abe, J. Cheng, R. Takagi, C. Saito, T. Saku, Keratin [35] V.I. Guelstein, T.A. Tchypysheva, V.D. Ermilova, A.V. Ljubimov, Myoepithelial

10-positive orthokeratotic dysplasia: a new leucoplakia-type precancerous and basement membrane antigens in benign and malignant human breast

entity of the oral mucosa, Histopathology 61 (2012) 910–920. tumors, Int. J. Cancer 53 (1993) 269–277.

[12] T. Mikami, S. Maruyama, T. Abe, T. Kobayashi, M. Yamazaki, A. Funayama, S. [36] T. Roskams, R. De Vos, G. David, B. Van Damme, V. Desmet, Heparan sulphate

Shingaki, T. Kobayashi, J. Cheng, T. Saku, Keratin 17 is co-expressed with proteoglycan expression in human primary liver tumours, J. Pathol. 185

14-3-3 sigma in oral carcinoma in-situ and squamous cell carcinoma and (1998) 290–297.

modulates cell proliferation and size but not cell migration, Virchows Arch. [37] I.R. Cohen, A.D. Murdoch, M.F. Naso, D. Marchetti, D. Berd, R.V. Iozzo,

466 (2015) 559–569. Abnormal expression of perlecan proteoglycan in metastatic melanomas,

[13] M. Tsuneki, M. Yamazaki, S. Maruyama, J. Cheng, T. Saku, Cancer Res. 54 (1994) 5771–5774.

Podoplanin-mediated cell adhesion through extracellular matrix in oral [38] A. Kaminski, J.C. Hahne, el-M. Haddouti, A. Florin, A. Wellmann, N. Wernert,

squamous cell carcinoma, Lab. Invest. 93 (2013) 921–932. Tumour-stroma interactions between metastatic prostate cancer cells and

[14] T. Ikarashi, H. Ida-Yonemochi, K. Ohshiro, J. Cheng, T. Saku, Intraepithelial ﬁbroblasts, Int. J. Mol. Med. 18 (2006) 941–950.

expression of perlecan, a basement membrane-type heparan sulfate [39] H. Ida-Yonemochi, I. Satokata, H. Ohshima, T. Sato, M. Yokoyama, Y. Yamada,

proteoglycan reﬂects dysplastic changes of the oral mucosal epithelium, J. T. Saku, Morphogenetic roles of perlecan in the tooth enamel organ: an

Oral Pathol. Med. 33 (2004) 87–95. analysis of overexpression using transgenic mice, Matrix Biol. 30 (2011)

[15] W.M. Tilakaratne, T. Kobayashi, H. Ida-Yonemochi, W. Swelam, M. Yamazaki, 379–388.

T. Mikami, C.G. Alvarado, A.M. Shahidul, S. Maruyama, J. Cheng, T. Saku, [40] J. Kruegel, N. Miosge, Basement membrane components are key players

Matrix metalloproteinase 7 and perlecan in oral epithelial dysplasia and in specialized extracellular matrices, Cell. Mol. Life Sci. 67 (2010)

carcinoma in situ: an aid for histopathologic recognition of their cell 2879–2895.

proliferation centers, J. Oral Pathol. Med. 38 (2009) 348–355. [41] R.V. Iozzo, I.R. Cohen, S. Grassel, A.D. Murdoch, The biology of perlecan: the

[16] M.S. Ahsan, M. Yamazaki, S. Maruyama, T. Kobayashi, H. Ida-Yonemochi, M. multifaceted heparan sulphate proteoglycan of basement membranes and

Hasegawa, A.H. Ademola, J. Cheng, T. Saku, Differential expression of perlecan pericellular matrices, Biochem. J. 302 (Pt. 3) (1994) 625–639.

receptors, ␣-dystroglycan and integrin ␤1, before and after invasion of oral [42] S. Maruyama, J. Cheng, M. Yamazaki, A. Liu, T. Saku, Keratinocyte growth

squamous cell carcinoma, J. Oral Pathol. Med. 40 (2011) 552–559. factor colocalized with perlecan at the site of capsular invasion and vascular

[17] S. Maruyama, Y. Shimazu, T. Kudo, K. Sato, M. Yamazaki, T. Abé, H. Babkair, J. involvement in salivary pleomorphic adenomas, J. Oral Pathol. Med. 38 (2009)

Cheng, T. Aoba, T. Saku, Three-dimensional visualization of perlecan-rich 377–385.

neoplastic stroma induced concurrently with the invasion of oral squamous [43] J.S. Rubin, D.P. Bottaro, M. Chedid, T. Miki, D. Ron, G.R. Cunha, P.W. Finch,

cell carcinoma, J. Oral Pathol. Med. 43 (2014) 627–636. Keratinocyte growth factor as a cytokine that mediates

[18] S. Maruyama, M. Itagaki, H. Ida-Yonemochi, T. Kubota, M. Yamazaki, T. Abé, H. mesenchymal–epithelial interaction, EXS 74 (1995) 191–214.

Yoshie, J. Cheng, T. Saku, Perlecan-enriched intercellular space of junctional [44] M. Klagsbrun, P.A. D’Amore, Vascular endothelial growth factor and its

epithelium provides primary infrastructure for leukocyte migration through receptors, Cytokine Growth Factor Rev. 7 (1996) 259–270.

squamous epithelial cells, Histochem. Cell Biol. 142 (2014) 297–305. [45] J. Mao, B.M. Kim, M. Rajurkar, R.A. Shivdasani, A.P. McMahon, Hedgehog

[19] J.M. Whitelock, J. Melrose, R.V. Iozzo, Diverse cell signaling events modulated signaling controls mesenchymal growth in the developing mammalian

by perlecan, Biochemistry 47 (2008) 11174–11183. digestive tract, Development 137 (2010) 1721–1729.

[20] A. Muthusamy, C.R. Cooper, R.R. Gomes Jr., Soluble perlecan domain I [46] J.J. Zoeller, J.M. Whitelock, R.V. Iozzo, Perlecan regulates developmental

enhances vascular endothelial growth factor-165 activity and receptor angiogenesis by modulating the vegf-vegfr2 axis, Matrix Biol. 28 (2009)

phosphorylation in human bone marrow endothelial cells, BMC Biochem. 11 284–291.

(2010) 43. [47] C. Savorè, C. Zhang, C. Muir, R. Liu, J. Wyrwa, J. Shu, H.E. Zhau,

[21] S. Datta, M. Pierce, M.W. Datta, Perlecan signaling: helping hedgehog L.W. Chung, D.D. Carson, M.C. Farach-Carson, Perlecan knockdown in

stimulate prostate cancer growth, Int. J. Biochem. Cell Biol. 38 (2006) metastatic prostate cancer cells reduces heparin-binding growth factor

1855–1861. responses in vitro and tumor growth in vivo, Clin. Exp. Metastasis 22 (2005)

[22] M. Mongiat, K. Taylor, J. Otto, S. Aho, J. Uitto, J.M. Whitelock, R.V. Iozzo, The 377–390.

protein core of the proteoglycan perlecan binds speciﬁcally to ﬁbroblast [48] G. Neufeld, T. Cohen, S. Gengrinovitch, Z. Poltorak, Vascular endothelial

growth factor-7, J. Biol. Chem. 275 (2000) 7095–7100. growth factor (VEGF) and its receptors, FASEB J. 13 (1999) 9–22.

436 M. Hasegawa et al. / Pathology – Research and Practice 212 (2016) 426–436

[49] W. Swelam, H. Ida-Yonemochi, S. Maruyama, K. Ohshiro, J. Cheng, T. Saku, receptor-1 promotes migration and invasion in pancreatic carcinoma cell

Vascular endothelial growth factor in salivary pleomorphic adenomas: one of lines, Cancer 104 (2005) 427–438.

the reasons for their poorly vascularized stroma, Virchows Arch. 446 (2005) [51] K. Nakamura, Y. Zen, Y. Sato, K. Kozaka, O. Matsui, K. Harada, Y. Nakanuma,

653–662. Vascular endothelial growth factor, its receptor ﬂk-1, and hypoxia inducible

[50] J.S. Wey, F. Fan, M.J. Gray, T.W. Bauer, M.F. McCarty, R. Somcio, W. Liu, D.B. factor-1alpha are involved in malignant transformation in dysplastic nodules

Evans, Y. Wu, D.J. Hicklin, L.M. Ellis, Vascular endothelial growth factor of the liver, Hum. Pathol. 38 (2007) 1532–1546.