Background
Colorectal cancer (CRC) is the third most common cancer in the world [
1]. Metastasis is the major cause of cancer morbidity and mortality in CRC. Although novel therapeutic choices have been improved, the five-year survival of patients with metastatic CRC remains only approximately 14% [
2]. The tumor microenvironment (TME) has been shown to play an essential role in CRC progression and metastasis [
3]. Understanding the components of the TME and their interplay with tumor cells is helpful for developing new strategies against metastatic CRC.
Tumor-associated macrophages (TAMs) are prominent tumor-infiltrating immune cells in the TME that suppress antitumor immunity and foster tumor progression [
4‐
6]. Infiltration of TAMs is associated with a poor prognosis in cancer patients [
7,
8]. However, the role of TAMs in CRC is controversial. Studies have reported that TAMs are beneficial to the prognosis of patients [
9,
10], and most of these studies analyzed TAMs without considering the heterogeneity of these macrophages (e.g., distinct pro- or anti-inflammatory subpopulations (M1-TAMs and M2-TAMs) [
11] and their spatial distribution within tumors [
12]. Many recent studies have provided strong evidence that TAMs facilitate CRC growth and progression [
13‐
17]. Most macrophages have the tendency to polarize into an M2-like state in tumors with advanced stages [
18,
19]. In addition, most of the macrophages located at the invasive front of advanced CRC tumors display the M2-TAM phenotype [
12]. However, how tumor cells affect TAM accumulation and their pro-tumoral phenotype in invasive CRC has not yet been well established.
TAMs are classically thought to be derived from peripheral blood monocytes [
20‐
22]. Monocytes are recruited to tumors by chemokines (e.g., CCL2), cytokines (e.g., colony-stimulating factor-1 (CSF-1)), and their complement cascade [
23,
24]. They extravasate from the peripheral circulation and differentiate into TAMs in the TME and are polarized into M2 macrophages by cytokines (e.g., CCL2, CSF1, IL10 and CCL5) [
25‐
27]. IL10 has been detected in the tumor microenvironment of many cancer types and has been thought to promote tumor immune escape by polarizing TAMs to the M2 phenotype and inhibiting the functions of antigen presenting cells [
28‐
30]. However, it has been reported to have anti-tumor effects with immune-dependent mechanisms, including activation of CD8 + T cells (CTLs) [
31]. Therefore, it will be necessary to evaluate a potential therapeutic intervention by either inhibiting or promoting the IL10 pathway on a case-by-case basis in specific cancer types and patient subpopulations. Inhibition of CCL2/CCR2 signaling blocks the recruitment of inflammatory monocytes and reduces metastasis in mouse models of breast cancer, hepatocellular cancer, and prostate cancer [
24,
32‐
34]. However, suppression of CCL2 expression only leads to a transient reduction in myeloid cell recruitment and a temporary delay in metastatic tumor growth in a mouse model of CRC [
35]. Similarly, CSF-1 inhibitory antibodies or pharmacological inhibition of the CSF-1/CSF-1R axis effectively blocked the recruitment of macrophages at tumor sites [
36]. However, the degree of CSF1R signaling dependency of macrophages at different locations is unclear and the clinical translation of depleting TAMs by targeting CSF-1/CSF1R is limited [
37]. Therefore, the regulation of TAM accumulation and their function in invasive CRC must be comprehensively understood and the efficiency of current therapies must be improved.
SPON2 (spondin-2, Mindin, DIL-1) is a member of the F-spondin family of secreted ECM proteins [
38]. It is a host innate immune regulator and represents a unique pattern-recognition molecule in the ECM for microbial pathogens [
39]. In hepatocellular carcinoma, SPON2 promotes M1-like macrophage recruitment and inhibits tumor metastasis [
40]. In contrast, SPON2 is overexpressed in the serum or tissue samples of malignant tumors, such as ovarian cancer [
41] and prostate cancer [
42]. Functionally, overexpression of SPON2 in CRC cells increases cell motility and CRC metastasis in mice [
43]. The distinct effect of SPON2 on metastasis in hepatocellular carcinoma and in CRC is probably due to the discrepancy in macrophage infiltration in the two types of tumors. In the current study, we demonstrated that tumor cell-derived SPON2 promotes the infiltration of TAMs with an M2-like phenotype and tumor metastasis in CRC. Mechanistically, SPON2 induces transendothelial migration of monocytes by activating PYK2.
Materials and methods
Cell lines
The human cell lines SW620, SW480, HCT116, DLD1, RKO, HUVEC and THP-1 and the mouse cell lines CT26, CMT93, MC38, RAW264.7 and C166 were preserved in the Department of Pathology, Southern Medical University, China. SW620 and SW480 cells were cultured in Leiboviz’s L-15 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). HCT116 cells were cultured in McCoy’s 5A medium (Gibco) with 10% FBS. RKO and HUVEC cells were cultured in F-12 K medium (Gibco) with 10% FBS. DLD1, THP-1, CT26 and MC38 cells were cultured in RPMI 1640 medium (Gibco) with 10% FBS (Gibco). CMT93, RAW264.7 and C166 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% FBS.
Plasmids and generation of stably transfected cell lines
The SPON2 plasmid was generated by cloning PCR-amplified full-length human SPON2 cDNA into pCDH. For deletion of SPON2, 4 short hairpin RNA (shRNA) sequences were separately cloned into a pLKO.1 vector. The vectors pCDH and pLKO.1 were purchased from Addgene Inc. Transfection of plasmids was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. Cells (2 × 10
5) were seeded and infected by lentivirus generated by pCDH-SPON2-puro and pLKO.1-SPON2-shRNA-puro for 3 days. Stable cell lines expressing SPON2 and SPON2-shRNAs were selected with 1 µg/mL puromycin for 5 days. The primer sequences are provided in Supplementary Table S
1.
RNA isolation, reverse transcription (RT) and real-time PCR
Total RNA samples were extracted from the cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Gene expression was analyzed by Q-RT-PCR using an ABI PRISM 7500 sequence detection system (Applied Biosystems, USA). For cDNA synthesis, 1 µg RNA was reverse transcribed using a reverse transcriptase kit (Vazyme). PCR amplification was performed using SYBR Green (Vazyme) in a total volume of 25 µl. The primer sequences used for amplification are provided in Supplementary Table S
1.
Western blotting analysis
Western blotting analysis was performed as previously described [
44] using anti-SPON2 (A12077, ABclonal), anti-PYK2 (bs-3357R, Bioss), anti-p-PYK2 (bs-3400R, Bioss), anti-FAK (bs-1340R, Bioss), anti-Zyxin (60,254–1-Ig, Proteintech), anti-RhoA (HPA062346, Sigma-Aldrich), anti-cortactin (11,381–1-AP, Proteintech), anti-IL10 (20,850–1-AP, Proteintech), anti-CCL2 (66,272–1-Ig, Proteintech), and anti-CSF1 (14,779–1-AP, Proteintech) antibodies. Mouse monoclonal anti-α-tubulin antibody (RM2007, RayAntibody) was used as the internal control.
Double immunohistochemistry staining and immunohistochemistry
Double immunohistochemistry staining was performed as previously described using SPON2 (Bioss, bs-11064R) and CD68 antibodies (ab213363, Abcam) [
45]. Immunohistochemistry (IHC) staining was performed as previously described using SPON2 (bs-11064R, Bioss) and CD163 (ZM-0428, ZSGB-BIO) [
46]. The degree of CD68 and CD163 IHC staining was reviewed and scored based on the proportion of positively stained stromal cells. The degree of SPON2 IHC staining was reviewed and scored independently by two observers based on both the proportion of positively stained tumor cells and the intensity of staining. The proportion of tumor cells was scored as follows: 0 (no positive tumor cells), 1 (< 10% positive tumor cells), 2 (10–50% positive tumor cells), and 3 (> 50% positive tumor cells). The intensity of staining was graded according to the following criteria: 0 (no staining); 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). The staining index was calculated as the staining intensity score × the proportion of positive tumor cells. Using this method of assessment, we evaluated the expression of SPON2 in benign colon epithelium and malignant lesions by determining the staining index based on scores of 0, 1, 2, 3, 4, 6, and 9. Cutoff values for SPON2 were selected on the basis of a measure of heterogeneity with the log-rank statistical test with respect to overall survival. Optimal cutoff values were identified: a staining index ≥ 4 was used to define tumors with high SPON2 expression, and an index ≤ 3 was used to define tumors with low SPON2 expression.
Bone Marrow-Derived Macrophages (BMDM)
Bone marrow was isolated from femurs and tibias from C57 BL/6 mice using a 23- gauge needle. Cells were then cultured in RPMI 1640 supplemented with 10% FBS, 2.0mML-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, non-essential amino acids, 14.2 mM b-mercaptoethanol, to which was added murine recombinant CSF1 (10 ng/ml; PeproTech, 351–02). Differentiating macrophages were cultured by transferring non-adherent cells 24 h after bone-marrow cells isolation and replenishing the medium with a fresh one every 48 h. BMDM differentiation was confirmed by flow cytometric evaluation of F4/80 expression. M0 macrophages (M0/M) were obtained by treating cultured cells with 10 ng/mL CSF1 for 5 days. Macrophages were then polarized to alternatively activated (M2/M) using 10 ng/mL IL4 (Peprotech, 214–14).
Collection of conditioned media
To collect condition media (supernatants), SW480/Vector, SW480/SPON2, SW620/Scramble, SW620/shSPON2#1, SW620/shSPON2#2, MC38/Vector, MC38/SPON2, MC38/Scramble, MC38/shSpon2#1 and MC38/shSpon2#2 cells (5 × 106 /100 mm dish) were incubated for 24 h. Media were removed and replaced with 8 ml serum-free L-15 or RPMI 1640. Supernatants were collected 24 h later with any floating cells removed by 0.45 mm filter. The cell suspensions collected in ultrafiltration centrifuge tube were centrifuged for 10 min, and the collected supernatants were stored at − 80℃ until further use.
ELISA
The amounts of IL10, CCL2, and CSF1 protein in the supernatant were respectively determined using human IL10, CCL2, CSF1 specific ELISA kits (DAKEWE, 1,111,002; MEIMIAN, MM-2022H2; MEIMIAN, MM-0081H2). All experiments were performed according to the manufacturer’s instructions.
Transendothelial migration assay (iTEM)
HUVEC monolayers or C166 cells on Transwell inserts were treated with F-12 K or DMEM media containing 10% FBS for 4 h at 37 °C. Inserts were placed over 24-well plates coated with a thin layer of Matrigel (354,262, Corning). DIL-labeled THP-1 cells were added to the top chamber with HUVECs and allowed to transmigrate through HUVECs. DIL-labeled mouse M0 macrophages were added to the top chamber with C166 and allowed to transmigrate through C166 cells. After 48 h of migration, the top chamber was removed and the cell number in the bottom chamber was fixed and stained. Transwell inserts were counterstained with DAPI and observed by fluorescence microscopy. The results were quantified by counting the number of THP-1 cells and mouse M0 macrophages passing through the endothelium in the same field (20 ×) and expressed as standardized values for at least three separate experiments. The quantification of the assay was carried out in at least three separate experiments, with each Transwell counting 5 fields.
Cell adhesion assay
HUVECs (2 × 105) were cultured on coverslips. Prior to the cell adhesion assay, green fluorescent protein (GFP)-labeled HUVECs were pretreated with conditioned medium for 2 h. Then, DIL-labeled THP-1 cells were cocultured with THP-1 cells for 2 h. Subsequently, the cultured cells were gently washed 3 times with PBS to remove the non-adhered THP-1 cells. The DIL-labeled THP-1 cells adhered to the HUVEC monolayer from ten random fields were counted under a fluorescence microscope.
Immunofluorescence staining
For immunofluorescence staining, 4 μm sections were cut from the paraffin-embedded blocks. Antigen retrieval was performed in a pressure cooker (95 °C for 30 min). Slices were blocked with PBS containing bovine serum albumin at room temperature for 1 h. The slides were placed in primary antibodies and incubated at 4 °C overnight. Primary antibodies against PYK2 (bs-3357R, Bioss), Zyxin (60,254–1-Ig, Proteintech), Mac2 (125,402, BioLegend), and ZO-1 (66,452–1-Ig, Proteintech) were used. Then, the slides were incubated with a mixture of two secondary antibodies for 1 h in a dark room. The following secondary antibodies were used: Alexa Fluor 488-labeled anti-rabbit (A23220, Abbkine), Alexa Fluor 488-labeled anti-rat (A23240, Abbkine), Alexa Fluor 594-labeled anti-rat (A23440, Abbkine), Alexa Fluor 488-labeled anti-mouse (A23210, Abbkine), Alexa Fluor 594-labeled anti-mouse (A23410, Abbkine), and DyLight488 Phalloidin (12935S, Cell Signaling Technology). Slides were counterstained with DAPI and observed by fluorescence microscopy.
Migration assay
An 8-μm-pore filter membrane of the Boyden chamber was used for the migration assay. RAW264.7 cells (1 × 105) were seeded in the upper chamber, and 300 μL of conditioned medium was added to the lower chamber as an inducer. After incubation for 36 h, the filter was removed and fixed with neutral formaldehyde and then stained with hematoxylin. Three independent experiments were performed.
Tumor models and treatments
Female C57 BL/6 mice (4–6 weeks old) obtained from the Animal Center of Southern Medical University, Guangzhou, China, were injected in the right flank with MC38/Vector, MC38/SPON2, MC38/Scramble, MC38/shSpon2#1 and MC38/shSpon2#2 cells (1 × 106 cells/mouse) on day 0. A CSF1R-specific inhibitor (BLZ945) (T6119, TOPSCIENCE) and DMSO control were administered on days 6, 7, 8, 9, 10, 11 and 12 after tumor inoculation through intragastric administration at a dosage of 200 mg/kg. When the average volume of tumors was over 200 mm3, anti-mouse IL10 neutralizing monoclonal antibodies (504,908, Biolegend; 500ug/kg, qd), anti-mouse integrin β1 neutralizing monoclonal antibodies (102,202, Biolegend; 1 mg/kg, qd), defactinib (T1996, TOPSCIENCE; 10 mg/kg, qd) or normal saline (NS) (equal volume/qd, control) were injected intraperitoneally daily for one week respectively. Tumors were measured every two days. Mice were generally sacrificed when tumors became necrotic or their volume reached 2500 mm3, which was recorded as death for the tumor growth curve. Surgical orthotopic injection of CRC cells (1 × 106 per mouse) onto the mesentery of the cecum were performed in C57BL/6 mice after anesthesia was administered. The mice were euthanized 30 days after surgery, the individual organs were excised, and metastases were observed by histological analysis.
Flow cytometry
CRC tumor single cells were isolated using a mouse tumor dissociation kit (130–096-730, Miltenyi Biotec). Single tumor cells were stained with the indicated antibodies for 30 min on ice. Antibodies against M2-TAMs: CD45 (APC-Cy7, Cat# 103,116, BioLegend), CD11b (BV605, Cat# 101,237, BioLegend), F4/80 (BV421, Cat# 123,137, BioLegend), and CD206 (APC, Cat# 141,708, BioLegend). After two washes, the cell pellet was resuspended and analyzed by flow cytometry.
Computational Analysis
To quantify the immune infiltration for each sample, the gene expression data of 433 TCGA Colon (COAD) and Rectum (READ) cancer samples were downloaded from GDC database, which is the normalized RSEM expression. Single sample gene set enrichment analysis (ssGSEA) was applied via the “GSVA” package based on 28 immune cell gene sets (Supplementary Table S
2) that retrieved from previous study [
47]. For the analysis of correlation between expression of SPON2 and expression of M2-TAM signature, the 433 TCGA colorectal samples were first clustered using M2-TAM signatures adapted as previously described (Supplementary Table S
3) [
48] into M2-Low, M2-Medium and M2-High (distance between pairs of samples was measured by Manhattan distance and clustering was then performed using Ward’s method hierarchical clustering). Then expression of SPON2 in each sample of each cluster was analyzed.
Statistical analysis
SPSS 20.0 was used for all statistical analyses. Pearson correlation analysis was used for expression correlation analysis. The Kaplan–Meier method was used to analyze the survival rate. Two-tailed independent Student’s t-test was used to analyze two groups. A value of p < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Discussion
Dysregulation of SPON2 expression has been documented in several human cancers, including prostate cancer [
42,
52], gastric cancer [
53] and ovarian cancer [
41]. High expression levels of SPON2 mRNA and protein predicted poor prognosis of CRC patients [
54]. In the current study, we demonstrated the critical roles of the tumor-derived SPON2 in the infiltration of M2-TAMs and tumor progression in advanced CRC. High SPON2 protein levels are correlated with more infiltrated M2-TAMs and poor prognosis of patients with advanced CRC. Mechanistically, SPON2 activates the integrin-PYK2 pathway in mononuclear cells/macrophages to promote their transendothelial migration and infiltration into CRC. In contrast to our findings, SPON2 not only promotes infiltration of M1-like macrophages but also inhibits tumor cell migration and tumor metastasis in hepatocellular carcinoma [
40]. The distinct effect of SPON2 on metastasis in hepatocellular carcinoma and in CRC is probably due to the discrepancy in the cell biology and TME in the two types of malignancies. For example, TAMs infiltrated in CRC frequently showed inflammatory and angiogenic features (M2-TAMs) [
55]. On the contrary, SPON2 prevented F-actin assembly, thereby inhibiting hepatocellular carcinoma cell migration by α5β1 integrin inactivated RhoA [
55]. Since SPON2 regulates Rho GTPase expression in DCs by interacting with the integrins α4β1 and α5β1 [
56], different expression and activity of integrins in CRC and hepatocellular carcinoma may also contribute to the functional contradiction. Although integrin α5β1 is overexpressed in hepatocellular carcinoma [
40], it is frequently lost in colorectal cancer cells compared with normal intestinal epithelium [
57].
It has been documented that SPON2 directly binds to bacterial and viral pathogens to initiate innate immune responses and functions as an opsonin for macrophage phagocytosis [
39,
58]. The SPON2/integrin β1/Rac signaling process plays a critical role in DC priming of T lymphocytes in a murine inflammation model [
56]. These studies suggest an essential role of SPON2 in the function of myeloid cells during infection and inflammation processes. However, the roles of SPON2 in recruiting and function of myeloid cells in the circumstances of malignant are largely unclear. Our findings demonstrate a positive correlation between high SPON2 protein levels and greater M2-TAM infiltration in the intratumoral area in CRC patients. We also show that SPON2-driven M2-TAM infiltration plays an important role in tumor invasion and metastasis in CRC. In detail, SPON2 promotes monocytes/macrophage migration by activation PYK2 signaling in these cells. TAMs are classically thought to be derived from peripheral blood monocytes [
59‐
61]. A multistep cascade of capture, rolling, slow rolling, firm adhesion, adhesion strengthening, and intraluminal crawling precedes the transendothelial migration of monocytes [
62,
63]. Transendothelial migration is controlled by complicated signaling and requires the coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. FAK and PYK2 are members of the FAK family [
64,
65], which plays a critical role in modulating the cell cytoskeleton and structures to regulate cell migration [
66,
67]. Actin-binding proteins, such as paxillin, vinculin and zyxin, are recruited by the activated members of the FAK family and assemble the focal adhesion complex to regulate cell migration [
49,
50,
68]. This is the first report identifying SPON2 promote the transendothelial migration of monocytes to maintain the infiltration of TAMs. We confirmed that SPON2-induced monocyte transendothelial migration is regulated by the focal adhesion signaling pathway. SPON2 can activate PYK2 in macrophages and monocytes by interaction with integrin β1. Activated PYK2 recruits more zyxin to promote the formation of focal adhesion complexes involved in stress fiber maintenance of migrating cells. In addition, SPON2 increases the activity and expression of RhoA and cortactin by activating PYK2, which promotes cytoskeletal remodeling and transendothelial migration.
Tumor cells secrete significant amounts of cytokines to promote M2 phenotype polarization in tumor microenvironment. IL10 is highly expressed in colorectal cancer cells, and polarizes TAMs to the M2 phenotype, which promotes cancer cell migration and metastasis.[
28,
69]. Inhibition of CCL2/CCR2 or CSF1/CSF1R signaling pathway can reduce the intratumoral infiltration of M2-TAM and inhibit tumor growth and metastasis [
26,
70]. Besides the effects on promoting cell migration, SPON2 also helps M2-polarization of the macrophages during their recruitment into the TME by upregulating the expression and production of cytokines, including IL10, CCL2 and CSF1. Blocking IL10 abrogates the SPON2-mediated intratumoral M2-TAM enrichment into the tumor and tumor growth. In addition, AP1, which activated by ERK1/2 pathway was reported as upstream regulator of cytokines IL10, CCL2 and CSF1 [
28,
71‐
75]. It has been documented that SPON2 protein in the ECM interacts with integrin β1 and activates focal adhesion pathway and ERK1/2 pathway [
76,
77]. Therefore, we speculated that SPON2 may activate ERK/AP1 pathways to upregulate the expression of IL10, CCL2 and CSF1 by binding integrin β1. Furthermore, it has been reported that CCL2, IL10, and CSF1 not only expressed in tumor cells, but also in macrophages [
30]. It is interesting to determine whether SPON2 affects this cytokines in macrophages. Overall, we uncovered a novel role for SPON2 in the regulation of macrophage polarization in colorectal cancer. However, how SPON2 regulates cytokine expression remains to be explored.
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