Low level of stromal lectin‐like oxidized LDL receptor 1 and CD8 + cytotoxic T‐lymphocytes indicate poor prognosis of colorectal cancer

Abstract Background Lectin‐like oxidized LDL receptor‐1 (LOX‐1) has been identified as a new marker for functional myeloid‐derived suppressor cells (MDSCs) that exhibit an immunosuppressive phenotype in the tumor microenvironment (TME). However, the role of LOX‐1+ cells in the TME of colorectal cancer (CRC) remains unknown. Aim This study aimed to determine the expression and significance of LOX‐1 in the TME of clinical CRC specimens. Methods and results We performed immunohistochemical and genetic analyses of LOX‐1, CD8, KRAS, and BRAF in 128 resected CRC specimens and determined the expression of IFN‐γ and IL‐10 using real‐time reverse transcription‐polymerase chain reaction. We analyzed the correlation between LOX‐1, TME factors, gene alteration, clinicopathological factors, and disease prognosis. The co‐expression pattern of LOX‐1, hematopoietic markers, and a fibroblast marker was evaluated using multiplex immunofluorescence staining. Low stromal LOX‐1 expression and low intratumoral CD8+ cytotoxic T‐lymphocyte (CTL) status correlated with poor prognosis. Moreover, stromal LOX‐1‐low/CD8+ CTL‐low status was the most important independent prognostic factor of poor overall survival. Most of the LOX‐1+ stromal cells were positive for CD163+, indicating they were CD163+ M2 macrophages. Conclusions The MDSC marker, LOX‐1, was mainly expressed by M2 macrophages in CRC tissues. LOX‐1+ macrophages and CD8+ CTLs may serve as useful biomarkers for predicting the prognosis of CRC.


| INTRODUCTION
Colorectal cancer (CRC) is the third most common cancer worldwide. 1 The tumor tissue is composed of various cells, including tumor and stromal cells, such as vascular endothelial cells, fibroblasts, inflammatory cells, and immune cells. These cells constitute a complex tumor microenvironment (TME) that facilitates tumor progression, leading to the poor prognosis of CRC. 2 To improve the prognoses of CRC patients, we need to understand the biological significance of the TME, including multiple immune cells present in clinical CRC tissues.
Myeloid-derived suppressor cells (MDSCs) are crucial players in immunosuppression in TME. [3][4][5][6] MDSCs produce immunosuppressive cytokines, such as IL-10 and TGF-β. Moreover, MDSCs inhibit IFN-γ production and proliferation of CD8 + cytotoxic T-lymphocytes (CTLs) and induce the immunosuppressive effects of regulatory T-cells against cancer. 4,7,8 Human MDSCs are characterized by the expression of surface markers, such as CD11b + and CD15 + . 9 However, these markers are also expressed in other immune cells; thus, it is difficult to detect MDSCs using these markers alone. 4 CTLs are crucial players in antitumor immunity owing to their capacity to kill tumor cells in the TME. High intratumoral density of CD8 + CTLs is a good prognostic factor for CRC. 10,11 Moreover, macrophages in the TME play contradictory roles in tumor immunity, that is, tumor preventing (M1 macrophages) and promoting (M2 macrophages) activities. 12,13 Lectin-like oxidized LDL receptor 1 (LOX-1), the main oxidized low-density lipoprotein receptor, is involved in inflammation, atherosclerosis, and reactive oxygen species-and metabolic disordermediated carcinogenesis. 14 LOX-1 is expressed in endothelial cells, smooth muscle cells, macrophages, and tumor cells, including CRC cells. 14,15 LOX-1 was identified as a specific marker for functional MDSCs using flow cytometry and immunohistochemical analysis. 16 Overexpression of LOX-1 induces the differentiation of macrophages into the M2 phenotype. 17 Tumoral LOX-1 increases during transition from normal to neoplastic phenotype in colon adenomas; hence, the expression of LOX-1 is associated with the early stage of the disease. 18 To date, there have been no detailed studies focused on LOX-1 + cells in the TME of CRC.
Here, we investigated the significance of LOX-1 expression in tumor-infiltrating immune cells using clinical CRC specimens. We examined the correlation between LOX-1 expression and TME factors, including cytokines and CD8 + CTLs, changes in CRC-related gene expression, and clinicopathological factors in CRC samples.
Moreover, we analyzed correlation between stromal LOX-1 expression, hematopoietic cells, and cancer-associated fibroblasts. The findings of this study revealed the significance of immunohistochemical and spatial detection of LOX-1 expression in the TME of clinical CRC tissues.

| Sample collection and preparation
The resected CRC specimens were fixed using 10% formaldehyde, embedded in paraffin blocks, and processed as described below. The cancerous tissues were excised and transferred to RNase-free microtubes and immediately frozen in liquid nitrogen and stored at −80 C until RNA extraction.

| Immunohistochemistry
Briefly, 4-μm thick sections were cut from paraffin blocks. The sections were stained using the following primary antibodies: rabbit poly-

| Immunohistochemistry
Each tissue section was evaluated in a blinded fashion by at least two investigators (including one pathologist). In case of discrepancies, both investigators analyzed the slides till they reached a consensus. All sections were examined under a BX43 light microscope (Olympus, Tokyo, Japan).
LOX-1 is expressed on tumor cells 19 and stromal cells 16 ; therefore, we defined the LOX-1 expressed in tumor and stromal cells as tumoral LOX-1 and stromal LOX-1, respectively. Tumoral LOX-1 in CRC tissues was determined using the LOX-1 score calculated based on a semi-quantitative assessment of the presence of LOX-1 and its intensity in accordance with the methodology prescribed in a previous report. 18 Briefly, the intensity of LOX-1 staining was scored as negative/weak (0), moderate (1), and strong (2). LOX-1 + cells were scored in the following manner: <10% LOX-1 expression (0), 10% to 25% LOX-1 expression (1), and >26% (2) LOX-1 expression. The final scores were obtained by adding both the individual scores. According to the final score, the patients were divided into two groups, that is, low (score 0-3) and high (score 4) tumoral LOX-1 groups.
The number of stromal LOX-1 + immune cells and intratumoral CD8 + CTLs was counted in selected five hotspots, using light microscopy (400× magnification; 0.058 mm 2 field area), and density was cal- at 95 C for 10 minutes followed by 45 cycles at 95 C for 15 seconds and 60 C for 10 minutes. The expression of IFN-γ and IL-10 was normalized to that of β-actin and was analyzed using the 2 −ΔΔCT method.

| High-resolution melt curve analysis
We screened the mutations using high-resolution melt curve analysis.  sections were counterstained with DAPI. Images were captured using the BZ-X700 microscope (Keyence). We analyzed 10 cases from the stromal LOX-1 high group using triple-labeled high-power fields.

| Statistical analysis
The χ 2 or Fisher's exact test was used to evaluate the correlation between target protein expression and clinicopathological features.
Correlations were analyzed using nonparametric Spearman's rank 3 | RESULTS

| Distribution of LOX-1 expression and CD8 + CTLs in CRC tissues
CRC patient specimens (n = 128) were subjected to immunohistochemistry. Figure 1 shows representative images of LOX-1 and CD8 expression in CRC tissue. LOX-1 was detected in the cytoplasm of both tumor and stromal cells, with LOX-1 expression being higher in CRC cells than that in noncancerous tissues ( Figure 1A,B  Figure 1C,D).
We also counted the CD8 + CTLs in the tumor tissues, and the  Figure 1E,F). The number of stromal LOX-1 + cells did not correlate with the number of CD8 + CTLs in this CRC cohort (Figure 2).

| Correlation between LOX-1 and CD8 expression and clinicopathological features
The clinicopathological significance of LOX-1 and CD8 expression in the 128 CRC patients is summarized in Table 1 (Table 2).
The association between clinicopathological parameters and 5-year OS was examined for the entire cohort using the Cox proportional hazards model (Table 3). Multivariate analysis revealed that the stromal LOX-1/CD8 + CTL status was the most reliable independent prognostic factor for poor OS (HR: 8.55, 95% CI: 1.92-37.9, P = .004).

| Correlation between stromal LOX-1 + cells, CD8 + CTLs, and cytokines expression in the TME
To determine the significance of stromal LOX-1 expression in the TME, we investigated the correlations between the stromal LOX-1 status and the expression of the CD8-derived cytokine IFN-γ, and the MDSC-derived cytokine IL-10, using real-time RT-PCR. The expression of the two cytokines did not correlate with stromal LOX-1 status ( Figure S2).

| DISCUSSION
To the best of our knowledge, this is the first study to analyze the significance of LOX-1 expression at the immunohistochemistry and spatial levels in the TME of CRC. We showed that CRC patients with low stromal LOX-1 expression and low levels of CD8 + CTL exhibited poor prognosis. Moreover, the combination of low stromal LOX-1 status and low CD8 + CTL counts was the most reliable independent prognostic factor for poor OS. Contrary to previous reports, 25 no correlation was observed between stromal LOX-1 + cells and immunosuppressive conditions, such as IL-10 levels, IFN-γ expression, and density of tumor-infiltrating CD8 + CTLs. Unexpectedly, multiplex fluorescent immunohistochemistry indicated that almost stromal cells expressing LOX-1 in CRC tissues were CD163 + M2 macrophages.
LOX-1, the main oxidized low-density lipoprotein receptor, is involved in inflammation, atherosclerosis, and ROS-and metabolic disorder-associated carcinogenesis. 14 Previous studies reported that LOX-1 is expressed on tumor cells and is highly expressed in progressive CRC. 18 Moreover, a high level of serum LOX-1 is associated with  26 These studies suggest that LOX-1 may be an oncogene. 27 Though these studies involved evaluation of LOX-1 expression in tumor cells and serum, the sample size was limited. In our current study, we evaluated LOX-1 expression in the stroma, and the sample size was relatively large. Moreover, stromal LOX-1-H was related to a good prognosis in our cohort. From these observations, it was inferred that stromal LOX-1 functions as an antitumor suppressive factor in CRC.
MDSCs play a key role in tumor immunosuppression; therefore, an abundance of MDSCs in the blood correlates with cancer progression and poor outcome. 28 F I G U R E 4 Evaluation of LOX-1 + cells using multiplex immunofluorescence in CRC tissues. A, MDSC markers, CD11b (red), CD15 (green), and LOX-1 (magenta). Cells co-expressing CD11b + CD15 + are in yellow. Some LOX-1 + stromal cells partially express CD11b + and CD15 + cells (white arrows). B, LOX-1 + stromal cells expressed CD45 (green) but did not express α-SMA (magenta). C, Almost LOX-1 + cells expressed CD163 (green). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. CRC, clinical colorectal cancer; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; MDSCs, polymorphonuclear MDSCs we evaluated the relationship between the stromal LOX-1/CD8 status and Wnt/β-catenin activation in CRC specimens to understand why the CRC patients with stromal LOX-1-L/CD8+ CTL-L had poorer prognosis compared to other CRC patients. Nuclear accumulation of β-catenin was identified in 60% of the patients with noninflamed phenotype (stromal LOX-1-L/CD8+ CTL-L) and in 40% of the patients with inflamed phenotype (stromal LOX-1-H/CD8+ CTL-H); however, no significant difference was observed between the inflamed and noninflamed phenotypes. Further analyses using larger cohorts are needed to clarify the fundamental role of the stromal LOX-1/CD8 status in clinical CRC samples and its association with poor prognosis.
Our study has some limitations. First, limited number of samples were collected from a single institution. Second, we did not perform a functional analysis of LOX-1 to clarify the correlation between LOX-1, TME, and tumor immunity in CRC tissues and immune cells.
In conclusion, our data showed that CRC patients with LOX-1-L and CD8 + CTL-L phenotypes had a poorer prognosis than patients in other groups. Almost the LOX-1 + cells in CRC stromal tissues were CD163 + M2 macrophages. Furthermore, combined LOX-1/CD8 + CTL status was identified as the most important independent prognostic factor for poor OS. LOX-1 and CD8 status in the tumor stroma may reflect tumor immunogenicity. Accordingly, the combination of LOX-1 + macrophages and CD8 + CTL infiltration may be useful in predicting the prognosis of CRC.