Introduction
Transforming growth factor β (TGFβ) is a multifunctional cytokine, with recent emphasis on its immunoregulatory function [
1]. In cancer, TGFβ could both promote and suppress tumor growth [
2,
3]. Its immunosuppressive function has drawn much attention due to recent progress in cancer immunotherapy [
4]. TGFβ, generally believed to be produced by cancer cells, could suppress the function of tumor-infiltration of both adaptive and innate immune cells (including CD4
+ or CD8
+ T cells, dendritic cells, natural killer cells, neutrophils, and macrophages), and thus cancer tissues are generally under an immunosuppressive microenvironment [
5‐
7].
The present authors have pathologically analyzed human cancer with prominent lymphocytic infiltrate, including gastric [
8,
9] and breast cancers [
10]. Such cancers, associated with an abundance of immune cells, generally show a favorable prognosis (see Supporting Information 1 for histological details). However, occurrence of cancer tissue itself demonstrates that such cancers at the same time exert vigorous immunosuppressive mechanisms to dampen possible immune cell attacks. Infiltration of cancer tissue by regulatory T cells (T
reg cells) is one such mechanism, with their presence related to poor survival of patients [
11,
12]. We analyzed T
reg cells confirming that T
reg cells positively correlated with immune effector cells [
13].
The present study is designed to analyze the in situ localization of TGFβ1 in cancers with prominent lymphocytic infiltrate. As a representative example, here we used lymphocyte-rich gastric cancers (Ly-rich GCs) as a continuation of our study [
8,
9,
13]. We used two sets of control: (1) control/conventional gastric cancers (GCs) without well-formed lymphoid stroma and (2) the secondary lymphoid organs (lymph nodes, Peyer patches, or tonsils). The theoretical basis of the second set of control is that immune responses in cancer tissue may simulate the structure of these secondary lymphoid organs (i.e., tertiary lymphoid tissue) when such immune responses are vigorous [
9]. Herein, we reveal (a) immune cell predominant expression of TGFβ1, (b) the identification of TGFβ
+ immune cell types, and (c) close cell-to-cell contact between TGFβ1
+ dendritic-shaped cells and T
reg cells. Because previous papers on the tissue distribution of TGFβ did not deal with immune responses in gastric cancer tissue [
14‐
16], the present paper describes detailed TGFβ1 localization in immune cells in human cancer tissues for the first time.
Materials and methods
Materials
The present study is a retrospective study using archival materials in the Department of Pathology, mainly Mito Medical Center and partly Mito Saiseikai General Hospital. Ly-rich GCs in this paper include typical lymphoepithelioma-like carcinoma (LELC) (or gastric cancer with the lymphoid stroma), which were characterized by poorly differentiated, solid-type cancer cells surrounded by abundant tumor-infiltrating lymphocytes (TILs), and LELC-like carcinoma showing any type of cancer with TILs in the whole stroma (for details, see Fig. 5 in Appendix
1). We used 23 cases of surgically removed Ly-rich GCs (Epstein-Barr virus [EBV]
+, 14 cases; EBV
−, 9 cases) (median age 65 years, range 47–84, M/F ratio = 16/7) and consecutively sampled 35 lesions of control (i.e., conventional) GCs (all EBV
−) in 32 patients (median age 73 years, range 48–87, M/F ratio = 25/7). The method for the EBV detection was described previously [
9]. Of nine cases of EBV
− Ly-rich GCs, three were considered to be in the microsatellite instability status (Fig. 6 in Appendix
1). Control GCs were associated with either TIL responses along the invasive margin or a general paucity of TILs. Intramucosal carcinomas (pTis or pT1[M]) were not included in this study because typical lymphocyte-rich stroma was observed only when carcinoma cells invade the submucosa. The stage of cancer was classified as described by Tumor-Node-Metastasis (TNM) classification (7th ed.) [
17]. The stage, histological typing, and follow-up analysis are shown in Tables 1 and 2 and Fig. 7 in Appendix
1. For the control of dendritic cells, 25 lesions of surgically resected secondary lymphoid organs from 22 patients were used (8 lymph nodes, 7 tonsils, 4 spleens, 4 appendices, 2 Peyer’s patches) (median age 28, range 5–67, M/F ratio = 10/12). The original diagnosis included tonsillitis, abdominal trauma, and gastrointestinal cancer.
Immunohistochemistry
All histochemical data were obtained using formalin-fixed and paraffin-embedded tissue sections. The primary antibodies used in this study were antigen affinity-purified goat polyclonal antibody to human LAP (TGFβ1) (R&D systems, Minneapolis, MN; no. AF-246-NA; used at 1:250 = 0.4 mg/mL), mouse monoclonal antibodies to human CXCR3 (CD183) (clone 1C6, IgG1; BD, Franklin Lakes, NJ; used at 1:400 = 1.25 μg/mL) and to forkhead box P3 (FoxP3) (clone 236A/E7, IgG1; Abcam, Cambridge, MA; used at 1:100), and antigen affinity-purified rabbit polyclonal antibody to human Smad3C (Immuno-Biological Laboratories Co. [IBL], Fujioka, Japan; no. 28031, used at 1:125 = 0.4 μg/mL). For negative controls, the primary antibodies were replaced by either normal goat- or rabbit IgG (IBL). The incubation time of the primary antibodies was overnight. The immunohistochemical methods were described previously [
9]. In brief, heat antigen retrieval was performed in high pH buffer (S3308, DAKO) at 95 °C for 60 min. The secondary antibodies included horseradish-peroxidase conjugated anti-goat simple stain (Nichirei, Tokyo, Japan), and anti-mouse or anti-rabbit envisions (DAKO). Diaminobenzidine (DAB) (DAKO) was used as the chromogen. Endogenous peroxidase activity was inactivated by immersing tissue sections in 3% H
2O
2 for 5 min after incubation with primary antibodies.
Cell counting
The distribution density of LAP+ immune cells, CXCR3+ cells, and FoxP3+ cells were manually counted as follows: total positively stained cells were counted using an ocular grid (10 × 10 mm lattice) with a × 400 microscopic field (a × 40 objective lens and × 10 ocular lens using a BX51 microscope, Olympus, Tokyo, Japan). The area of one lattice was 0.0625 mm2. At least three areas were counted in each case, and the numbers were averaged. In this analysis, the most densely distributed areas were selected. All statistical analyses were performed using IBM SPSS statistics software, version 21 (IBM Inc., Armonk, NY, USA).
In situ hybridization for TGFβ1 mRNA
TGFβ1 mRNA was detected by RNAscope 2.5 HD Reagent Kit (Advanced Cell Diagnostics, Hayward, CA) according to the manufacturer’s instructions. As a minor modification, an OPAL 520 TSA detection system (PerkinElmer, Waltham, MA) was used for fluorescent labeling instead of chromogenic coloring. As a negative control, a bacterial dapb gene was employed.
Double-labeling immunofluorescence method for LAP and CD83, LAP and CD68, LAP and FoxP3, and LAP and CD3
Formalin-fixed and paraffin-embedded tissue sections were used. Antigen retrieval was performed as described above. The sections were incubated with a mixture of goat anti-human LAP (1:75 = 1.25 μg/mL) and mouse monoclonal anti-human CD83 (1:8; clone 1H4b, Novocastra-Leica Microsystems, Benton Lane, UK), anti-CD68 (1:80; clone PG-M1, DAKO) or anti-CD3 (1:8; clone F7.2.38, DAKO) overnight. Alexa Fluor 488-labeled donkey anti-goat IgG (1:100 = 20 μg/mL, Molecular Probe, Carlsbad, CA) and Alexa Fluor 555-labeled donkey anti-mouse IgG (1:100 = 20 μg/mL) were applied in a mixture for 30 min. After DAPI (Molecular Probe) nuclear staining, specimens were mounted with ProLong Gold (Molecular Probe). Immunofluorescent observation was performed with a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany) or with a Nikon E800 microscope (Nikon, Tokyo, Japan). For negative control, the primary antibodies were replaced by either non-immunized goat IgG (IBL; 1.25 μg/mL) or control mouse IgG1 (DAKO; 1:100 = 4 μg/mL).
Double-labeling chromogenic immunohistochemistry for CD68-LAP, CD83-LAP, and FoxP3-LAP
The immunoperoxidase method for CD68, CD83, and DC-sign was performed as described for single immunohistochemistry. Tissue sections were then re-treated with Tris-EDTA antigen retrieval solution at 95 °C for 20 min to inactivate antibodies and enzymes used in the first step. Then, immunohistochemistry for LAP was performed. The combination of chromogens used was as follows: DAB (brown; DAKO), Vector SG (dark blue/gray; Vector Laboratories, Burlingame, CA) and Vulcan Fast Red (red; Biocare, Concord, CA), DAB (brown; DAKO). For Vulcan Fast Red, we used anti-mouse simple stain conjugated with alkaline phosphatase (Nichirei).
Discussion
The present histopathological study analyzed the in situ expression of TGFβ1 in human Ly-rich GCs to show that its expression is observed mainly in immune cells, but only focally in cancer cells. The number of TGFβ1+ immune cells correlated with those of CXCR3+ cells and Treg cells, which demonstrates more immune-cell responses in Ly-rich GCs than in control GCs. Double staining confirmed TFGβ1 expression in macrophages and/or immature cDCs and mature cDCs, and some T cells. TGFβ1+ dendritic cells harbor lymphocytes including Treg cells in their cytoplasmic processes.
T
reg cells are one of the important immunosuppressive cells. The expression and production of TGFβ in macrophages or immature cDCs is well known, and such cells could induce T
reg cells [
21]. In fact, we have shown here that TGFβ1-expressing and dendritic-shaped cells harbor lymphocytes including T
reg cells along their cytoplasmic processes. These are consistent with observations that T
reg cells are induced in the peripheral tissue from naïve CD4
+ T cells by TGFβ expressed on cDCs. Such T
reg cells also express TGFβ in a mouse model [
19]. In addition, human DCs activated by cancer cells or tumor-associated antigens can induce T
reg cells by producing TGFβ [
22,
23]. Therefore, our morphological data are consistent with close relationship between T
reg cells and LAP (TGFβ1)
+ cDCs. We have shown here that the number of LAP (TGFβ1)
+ immune cells (as a total) correlated with that of T
reg cells, but that of LAP (TGFβ1)
+ cDCs did not. These data would suggest that not a minor part of T
reg cells infiltrates lymphoid stroma probably independently of LAP (TGFβ1)
+ cDCs, and T
reg cells may also be induced in cancer stroma. LAP (TGFβ1)
+ immune cells also include significant number of CD68
+ macrophages. Therefore, relationship between LAP (TGFβ1)
+ CD68
+ cells and T
reg cells is to be analyzed in future studies. Taken together, our data suggest that TGFβ1 could be one of the candidates of immunosuppressive factors in cancers, and that TGFβ1 has a potential to promote cancer growth together with T
reg cells. Functional analyses would be required in future studies.
The distinction between macrophages and DCs is difficult [
24], particularly in inflammatory lesions in the peripheral organs including Ly-rich GCs. Therefore, future multi-color immunohistochemistry will be required for more-detailed in situ characterization of TGFβ1
+ dendritic-shaped cells in cancer tissues.
Next, we need to discuss the differences between the present study and our previous study where we observed TGFβ1 expression in stromal fibroblasts and macrophages in scirrhous gastric carcinoma [
14]. Formerly, we observed TGFβ1 expression in endoplasmic reticulum in spindle-shaped macrophages. Therefore, it is reasonable to speculate that TFGβ1
+ spindle-shaped “fibroblasts” in scirrhous carcinoma in our previous study were in fact spindle-shaped macrophages. Cancer cell expression of TGFβ1 in the previous study is consistent with the present study on control GCs.
We have already observed that the lymphoid stroma in Ly-rich GCs is similar to lymphoid tissue, postulating that the lymphoid stroma corresponds to the tertiary lymphoid tissue [
9]. This concept was later analyzed in details [
25]. Our data here indicate that the lymphoid stroma of cancer are considered to be under more immunosuppressive microenvironment than the secondary lymphoid organs as shown by higher TGFβ expression rate in cDCs. Not only Ly-rich GCs but also control GCs showed a similar distribution of TGFβ1 in the areas with lymphocyte-present stroma, particularly along the invasive margin. This suggests that our data could be widely applicable to various cancers not associated with lymphoid stroma. In conclusion, we would be able to judge the following: (a) TGFβ1 is mainly expressed in immune cells (including macrophages and cDCs) with a close contact to T
reg cells in lymphoid stroma, (b) Ly-rich GCs quantitatively differ from control GCs from the viewpoint of TGFβ expression in immune cells, and (c) the lymphoid stroma of Ly-rich GCs is quantitatively different from the T cell zone of secondary lymphoid organs from the viewpoint of TGFβ expression rate in cDCs. Finally, we need to note that TGFβ could be a target of cancer immunotherapy in combination with, for example, immune checkpoint blockage therapy [
26,
27]. Our study could be a basis of such therapies.
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