SPON2 (spondin-2, Mindin, DIL-1) is a member of the F-spondin family of secreted ECM proteins . It is a host innate immune regulator and represents a unique pattern-recognition molecule in the ECM for microbial pathogens . In hepatocellular carcinoma, SPON2 promotes M1-like macrophage recruitment and inhibits tumor metastasis . In contrast, SPON2 is overexpressed in the serum or tissue samples of malignant tumors, such as ovarian cancer  and prostate cancer . Functionally, overexpression of SPON2 in CRC cells increases cell motility and CRC metastasis in mice . 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 ELISA Kit 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.
Colorectal cancer (CRC) is the third most common cancer in the world .
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% . The tumor microenvironment (TME) has been shown to play an essential role in CRC progression and metastasis . 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 .
Infiltration of TAMs is associated with a poor prognosis in cancer patients . However, the role of TAMs in CRC is controversial.
Studies have reported that TAMs are beneficial to the prognosis of patients], 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) and their spatial distribution within tumors . Many recent studies have provided strong evidence that TAMs facilitate CRC growth and progression .
- Most macrophages have the tendency to polarize into an M2-like state in tumors with advanced stages . In addition, most of the macrophages located at the invasive front of advanced CRC tumors display the M2-TAM phenotype . 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 . Monocytes are recruited to tumors by chemokines (e.g., CCL2), cytokines (e.g., colony-stimulating factor-1 (CSF-1)), and their complement cascade . 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) ].
- 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 .
- However, it has been reported to have anti-tumor effects with immune-dependent mechanisms, including activation of CD8 + T cells (CTLs) . 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.
- 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 . Similarly, CSF-1 inhibitory antibodies or pharmacological inhibition of the CSF-1/CSF-1R axis effectively blocked the recruitment of macrophages at tumor sites .
- 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 . 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.
Materials and methods
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 × 105) 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.
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.
Western blotting analysis
Western blotting analysis was performed as previously described  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.
Tumor-associated macrophages (TAMs) are key regulators of the complex interplay between cancer and the immune microenvironment. Tumor cell-derived spondin 2 (SPON2) is an extracellular matrix glycoprotein that has complicated roles in recruitment of macrophages and neutrophils during inflammation. Overexpression of SPON2 has been shown to promote tumor cell migration in colorectal cancer (CRC). However, the mechanism by which SPON2 regulates the accumulation of TAMs in the tumor microenvironment (TME) of CRC is unknown.