Trans‐homophilic interaction of CADM1 promotes organ infiltration of T‐cell lymphoma by adhesion to vascular endothelium

Abstract The initial step of organ infiltration of malignant cells is the interaction with host vascular endothelial cells, which is often mediated by specific combinations of cell adhesion molecules. Cell adhesion molecule 1 (CADM1) is overexpressed in adult T‐cell leukemia/lymphoma (ATL) and provides a cell‐surface diagnostic marker. CADM1 promotes the adhesion of ATL cells to vascular endothelial cells and multiple organ infiltration in mice. However, its binding partner on host cells has not yet been identified. In this study, we show that CADM1 promotes transendothelial migration of ATL cells in addition to the adhesion to vascular endothelial cells. Moreover, CADM1 enhances liver infiltration of mouse T‐cell lymphoma cells, EL4, after tail vein injection, whereas a CADM1 mutant lacking adhesive activity did not. Among the known CADM1‐binding proteins expressed in primary endothelial cells, only CADM1 and CADM4 could induce morphological extension of ATL cells when plated onto glass coated with these proteins. Furthermore, CADM1‐mediated liver infiltration of EL4 cells was canceled in conventional and vascular endothelium‐specific Cadm1 knockout mice, whereas it was not canceled in Cadm4 knockout mice. These results suggest that CADM1 on host vascular endothelial cells is required for organ infiltration of ATL and other T‐cell lymphomas expressing CADM1.


| INTRODUC TI ON
The CADM1 (Cell adhesion molecule 1) gene, which was originally identified as a tumor suppressor in non-small-cell lung cancer, encodes a single-pass transmembrane protein belonging to the immunoglobulin superfamily (IgSF). 1 CADM1 is highly expressed in the brain, testis, and lung in normal tissues, whereas loss of Cadm1 in mice causes autism spectrum disorder, male infertility, and increased tumorigenesis, indicating that CADM1, also known as TSLC1/Necl-2/SynCAM1, plays diverse physiological roles by mediating cell-cell adhesion. [2][3][4][5][6][7] CADM1 was also identified as one of the markedly upregulated genes in ATL cells. 8 ATL is a malignancy of CD4 + T-cells caused by human T-lymphotropic virus type I (HTLV-I) and develops in 3%-6% of HTLV-I carriers with a long latency of about 60 years. 9 ATL is an aggressive form of leukemia with a poor prognosis. It is accompanied by the involvement of bone marrow, lymph nodes, spleen, and extranodal sites such as the skin, liver, central nervous system, and gastrointestinal tract. 10 CADM1 expression is observed in ATL cells and HTLV-I-infected T-cells, but absent in normal and activated CD4 + T-cells. Therefore, CADM1 is used as an established clinical diagnostic marker for ATL cells in combination with CD7, which is gradually downregulated according to the progression of ATL, in a flow cytometric analysis of peripheral blood mononuclear cells. 11 The ectopic expression of CADM1 is caused by constitutive activation of NF-κB due to the autophagic degradation of p47, a negative regulator of NF-κB, in ATL cells. 12 Previous studies have demonstrated that CADM1 promotes organ infiltration of ATL cells. Forced expression of CADM1 in ED-40515(−), an ATL cell line without endogenous expression of CADM1, enhances its adhesion to HUVEC and liver infiltration in NOD-SCID/ γc null (NOG) mice. 8,13 CADM1 induces the invasive phenotype of ATL cells by activating Rac through direct interaction with Tiam1, a Racspecific guanine nucleotide exchange factor. 14 Interestingly, CADM1 expression is also detected in cutaneous T-cell lymphomas such as mycosis fungoides and Sézary syndrome and is associated with unfavorable outcomes, including skin infiltration. [15][16][17] These studies suggested that CADM1 is a potential therapeutic target for organ infiltration of ATL and other T-cell lymphomas.
Although CADM1 is considered to enhance adhesion of T-cell lymphoma cells to vascular endothelial cells, how CADM1 promotes organ infiltration, especially the expression of the homophilic or heterophilic binding partners for CADM1 in host cells, including vascular endothelial cells, is poorly understood. In this study, we focused on the tumor-host interaction mediated by CADM1 and demonstrated that CADM1 expression on host vascular endothelial cells is essential for CADM1-induced organ infiltration of T-cell lymphoma cells.

| Cells
Cells were obtained as listed in Table S1.

| Expression vectors
Human CADM1 with an N-terminal HA tag (HA-CADM1) 19 and its

| Tumor formation assay
Intravenous injection was performed by injecting 5 × 10 4 of MT-2 cells and 1 × 10 6 of EL4 cells into NOG mice and C57BL/6 background mice, respectively. The number of tumor nodules on the liver surface was counted 24 days after injection for MT-2 cells or 14 days for EL4 cells. Subcutaneous injection was performed by injecting 1 × 10 6 of EL4 cells into C57BL/6J mice, and the tumor volumes were measured twice a week.

| Extravasation assay
The extravasation assay was performed as described previously. 22 Briefly, NOG mice were injected with 2 × 10 6 of MT-2 cells into the tail vein and sacrificed 24 h after the injection. The livers were fixed in PBS with 4% formaldehyde/0.3% Triton X-100, washed in 0.3% Triton X-100/PBS, permeabilized with acetone overnight at 4°C, washed again, and soaked in blocking buffer (10% normal goat serum, 1% Triton X-100, 0.3 M glycine in PBS) overnight at 4°C. After washing, the livers were incubated with anti-CD31 antibody (1:50; BD Pharmingen, San Diego, CA, USA) in blocking buffer without glycine overnight at 4°C, then washed and incubated with Alexa Fluor 568-conjugated secondary antibody (Thermo Fisher Scientific) overnight at 4°C. The nuclei were stained using Hoechst 33342 (Nacalai Tesque). Fluorescent signals were obtained by confocal microscopy (Nikon A1+; Nikon, Tokyo, Japan).

| Endothelium adhesion assay
HUVEC were seeded in a 6-well plate precoated with 10 µg/ml fi-

| Production of proteins
The production of the ectodomain of human CADM1-4, NECTIN3, PVR, and CRTAM fused to the Fc region of human IgG 2 has been described previously. 18 The production of CADM1 ectodomain with Cd4-COMP-FLAG-His tag is described in Document S1.

| Surface plasmon resonance imaging
Surface plasmon resonance imaging (SPRi) analyses were performed using an SPRi system OpenPlex (HORIBA France, Palaiseau, France) according to a previous report. 18 Briefly, 10 µM protein samples and normal human IgG (Sigma-Aldrich) in PBS were spotted onto an SPRi-Biochip (HORIBA France) at 10 nl/spot using a DNA Array Spotter (HORIBA Ltd., Kyoto, Japan). The reflectivity of each spot in response to 2.4 µM analyte samples diluted in running buffer (PBS with 0.2% BSA and 0.02% Tween20) was recorded, and the percentage change in reflectivity (%ΔR) was calculated by subtracting the background signal. Data were processed using ScrubberGen software (HORIBA France).

| Cell adhesion assay to Fc-fusion proteins
The coverslips were coated with 140 µg/ml of proteins, and 2 × 10 4 of Jurkat cells were plated onto coverslips in a 24-well plate. After incubation for 2 h at 37°C, the cells were fixed with 4% paraformaldehyde and labeled with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific). The number of cells was counted in randomly selected 20 fields.

| Cell spreading assay
Cell spreading assay was performed as previously described. 18 Briefly, coverslips were coated with 7 µg/ml of proteins, and 1 × 10 4 of ATN-1 cells were plated onto the coverslips in a 24-well plate and incubated for 60 min at 37°C. The cells were fixed in 4% paraformaldehyde and labeled with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific). The cell area was quantified using AxioVision software (Carl Zeiss, Oberkochen, Germany).

| Knockdown of CADM1 attenuates transendothelial migration of ATL cells and HTLV-Itransformed T-cells
One of the most characteristic features of ATL is its infiltration into various organs, including the liver, spleen, and skin. To  Figure 1I). Infiltration of MT-2 cells in the lung and spleen was also attenuated significantly by knockdown of CADM1, and the bone infiltration appeared to be attenuated in the cells with shCADM1 ( Figure S3B). These results suggested that CADM1 would promote the extravasation of ATL and HTLV-I-transformed T-cells in multiple organ infiltration.

| CADM1 promotes liver infiltration of T-cell lymphoma cells through its binding activity to CADM family proteins
Although CADM1 on ATL and HTLV-I-transformed T-cells is shown to be involved in adhesion to vascular endothelial cells, the bind- a previous study had demonstrated that CADM3 with a substitution in the F82 residue, which corresponds to F86 of CADM1, completely lost its trans-homophilic interaction activity. 23 We quantitatively analyzed the interactions of CADM1 or CADM1 F86S with the CADM1-binding proteins using surface plasmon resonance imaging (SPRi). We detected the interactions of CADM1-Fc with CADM1-Fc, CADM2-Fc, CADM3-Fc, CADM4-Fc, and CRTAM-Fc, which is consistent with our previous report. 18 Figure 3B). CADM1 also enhanced the cell aggregation activity of EL4 through trans-homophilic interaction, whereas CADM1 F86S did not ( Figure 3C), showing that CADM1 F86S lacked cell adhesion activity to the CADM family proteins.
Then, we injected EL4 cells expressing CADM1 or CADM1 F86S into the tail vein of C57BL/6 mice, and tumor nodules on the liver surface were examined 2 weeks after the injection. CADM1 promoted liver nodule formation of EL4 cells, whereas CADM1 F86S did not ( Figure 3D,E). Because CADM1 did not affect subcutaneous tumor growth of EL4 cells ( Figure 3F), CADM1 appeared to promote the infiltration step, not the growing step in liver nodule formation after intravenous injection. Therefore, the trans-interaction activity of CADM1 is essential for liver infiltration, and the CADM1 family proteins on vascular endothelial cells may mediate the interaction with CADM1 on T-cell lymphoma cells.

| CADM1 on T-cell lymphoma cells promote liver infiltration through interacting with CADM1 on vascular endothelial cells
To Targeted deletion of Cadm1 in vascular endothelial cells canceled liver infiltration of EL4 cells induced by forced expression of CADM1 ( Figure 5C). These results strongly suggest that CADM1 expression on host vascular endothelial cells is essential for CADM1-induced liver infiltration of T-cell lymphoma cells.
In fact, we have previously reported that CADM1-CADM1 interaction showed higher cell aggregation activity than did the CADM1-CADM4 interaction. 24 Finally, to confirm the difference in the affinity of these interactions, we performed flow cytometry-based binding analysis and showed that CADM1-Fc, but not CADM4-Fc, bound considerable portions of EL4 cells expressing CADM1 ( Figure 5D). Furthermore, we examined quantitative analysis of molecular interaction between CADM1-FLAG and CADM1-Fc or CADM4-Fc using Alpha technology. The binding signal in a unit of alpha counts was much stronger in the CADM1-CADM1 interaction than in the CADM1-CADM4 interaction ( Figure 5E). These findings would provide the molecular basis of the different activity of endothelial CADM1 and CADM4 in organ infiltration of T-cell lymphoma cells (Table S4).   Table S4. At the molecular level, Alpha technology and SPRi analyses demonstrated that the CADM1-CADM1 interaction had higher affinity than the CADM1-CADM4 interaction (Figures 2A and 5E). Flow cytometrybased binding assay also showed that CADM1-Fc protein bound to the surface of EL4 cells expressing CADM1, but CADM4-Fc protein did not ( Figure 5D). Conversely, the adhesion and spreading assays of T-cell lymphoma cells expressing CADM1 on the coverslips coated with CADM1-Fc or CADM4-Fc failed to show a significant difference between CADM1-CADM1 and CADM1-CADM4 interactions ( Figures 3B and 4B), probably because these assays are rather qualitative due to a very high protein concentration. In a physiological condition of cell-cell adhesion activity, we had examined cell aggregation assay in a previous study 24 and shown that the CADM1-CADM1 interaction had a higher cell adhesion activity than the CADM1-CADM4 interaction. Based on these results, we conclude that the CADM1-CADM1 interaction shows higher affinity and biological activity than the CADM1-CADM4 interaction.
In this study, due to the technical limitation of using knockout mice, syngeneic T-lymphoma cells, EL4, were used instead of human ATL or other T-cell lymphoma cells, which are only transplantable to immunocompromised mice. We believe, however, that this finding would be explored in human T-cell lymphomas, providing a new paradigm for understanding the infiltration of T-cell lymphomas.
CADM1 is reported to regulate endothelial progenitor cell migration under the control of the TNFα-NF-κB pathway 25 and may be involved in the vascular endothelial cell barrier. 26 More importantly, blocking the trans-homophilic interaction of CADM1 between Tcell lymphoma cells and vascular endothelial cells could provide a therapeutic target for the organ infiltration of T-cell lymphoma. In fact, a previous study has shown that an anti-CADM1 neutralizing antibody suppressed the adhesion of ATL cells to HUVEC and liver nodule formation of EL4 cells expressing CADM1. 27 Although ATL cells frequently infiltrate into the skin rather than the liver, CADM1 expression on vascular endothelial cells could be a risk factor for the infiltration of T-cell lymphoma cells; therefore, polymorphic variants of the CADM1 gene in host individuals might be associated with the malignant progression of T-cell lymphomas expressing CADM1.
Further studies on CADM1 expression in vascular endothelial cells would lead to a better understanding of the infiltration of ATL and other cutaneous lymphomas such as mycosis fungoides and Sézary syndrome.

ACK N OWLED G M ENTS
The authors thank Drs. Kaoru Uchimaru and Satoru Nagatoishi,