CtBP1 promotes tumour‐associated macrophage infiltration and progression in non–small‐cell lung cancer

Abstract The progression of lung cancer is majorly facilitated by TAMs (tumour‐associated macrophages). However, how the TAMs infiltrate the NSCLC microenvironment and the associated biochemical are not fully elaborated. Research has revealed that changes in CtBP1 modulates innate immunity. Here, we investigated if CtBP1 facilitates infiltration of TAM and the subsequent progression of NSCLC. Immunohistochemical analysis was carried out in 96 NSCLC patients to estimate the clinicopathological importance of CtBP1 in the disease. CtBP1 overexpression and knockdown were carried out to assess the activity of CtBP1 in NSCLC cells. Elevated expression of CtBP1 correlated positively with TAMs infiltration into NSCLC tissues, induced EMT (epithelial‐mesenchymal transition) in NSCLC cells and modulated the activated NF‐κB signalling pathway leading to increase in CCL2 secretion from NSCLC cells, thus promoting TAM recruitment and polarization. TAM induction and polarization reduced significantly on exhausting p65 in NSCLC cells with CtBP1. Moreover, infiltration of TMAs was reduced remarkably on antagonist‐mediated blocking of CCR2 and impeded the progression of NSCLC in a mouse model. These findings thus show a novel insight into the process of CtBP1‐regulated TAM infiltration in NSCLC.

are discerned in the tumour microenvironment as alternatively activated macrophages, also known as M2, which are characteristically weak in antigen presentation, marked cytokine and chemokine (such as TGF-β, interleukin-10, CCL17 and CCL22) expression. 11 TAMs promote immune suppression angiogenesis, and metastasis in cancers via the release of chemokines, cytokines, matrix metalloproteases and growth factors. Enhanced infiltration of TAM is related to a poor prognosis in NSCLC. 12,13 Earlier research has revealed that STAT-3, NF-κB (nuclear factor-κB) and hypoxia-inducible factor-1 signalling pathways play a role in recruitment and polarization of TAMs. 14,15 However, the process involved in TAMs infiltration of the NSCLC cells has not been well elucidated.
Various important cellular processes are modulated by CtBP family proteins. 16,17 While only a single CtBP protein is expressed in the invertebrate genomes, two related proteins CtBP1 and CtBP2 are coded by the vertebrate genomes, which carry out genetically concomitant and specific roles through the course of development. 18 CtBP1 and CtBP2 in their primary splice forms act as transcriptional corepressors, whereas the other splice variants have diverse roles in the cytosol. 17,19 Previous study has shown that CtBP1 interacts with SOX2 to promote the growth, migration and invasion of lung adenocarcinoma. 20 However, the role of CtBP1 in NSCLC remains to be explored.
In the current study, we evaluated the effect of CtBP1 on recruitment as well as polarization of TAMs by in vitro cell coculture assay and through in vivo animal model studies. The revelations of this study confer new insight into the processes involved in the crosstalk between TAMs and NSCLC cells.

| Patients and tissue samples
Human NSCLC tissues (n = 96) and paired normal lung tissues ( Table 1.
A549 and H1299 cells which stably overexpressed CtBP1, and A549 and H1299 cells with CtBP1 knockdown were confirmed. To develop NSCLC CM, NSCLC cells at 5 × 10 6 /mL with varying treatments were given three washes with serum-free RPMI1640 and further maintained in serum-free RPMI-1640 for next three full days.
The supernatant was collected after filtering through 0.22 μm filters and kept at 4℃ until further studies.

| Immunohistochemistry
EnVision™ Flex + System (Dako, Santa Clara, CA, USA) was used to stain the lung cancer sections that were fixed in paraformalin and embedded in paraffin. EnVision™ Flex target retrieval solution (same brand) was employed to deparaffinize and unmask epitopes in the samples. Endogenous peroxidase was blocked by adding 0.3% hydrogen peroxide for 5 min. This was followed by the addition of primary antibody at 4°C overnight. Subsequently, the following treatments were done: 15  at × 400 magnification in a minimum of five areas and based on the scores were categorized per specimen as: score 0, negative; score 1, <25% positive cells; score 2, 25%-50% positive cells; score 3, 50%-75% positive cells; and score 4, >75% positive cells. This was followed by scoring of immunostaining intensity as follows: 1+, weak; 2+, moderate; and 3+, intense. A total score was obtained by multiplying the two above-mentioned values. A low expression was indicated by a total score ≤4 and >4 indicated a high expression. Three experienced investigators blinded to the patient conditions evaluated the immunostaining independently.

| Enzyme-linked immunosorbent assay
After various treatments, the NSCLC cells were kept in a medium without serum for two full days and the culture supernatant was collected to measure CCL2 concentration. Next, incubation of THP-1 macrophages was done with CM for two full days to determine the concentration of IL-10 as well as CCL17 and CCL22. After giving thrice wash with PBS, the cells were kept in medium with no serum for two full days and the culture supernatant was collected to determine CCL2, IL-10, CCL17 and CCL22 concentration using ELISA Kit as per instructions.

| Real-time RT-PCR
The real-time PCR was performed as previous studies. 21

| Western blotting
The Western blotting was performed as previous studies. 23,24 Briefly, cell samples were mixed with lysis buffer (0.01 M Tris-HCl,

| Flow cytometry assay
Post-incubation with different CM for two full days, collection of THP-1 macrophages was done. The cells were given cold PBS (containing 5% human serum and 0.1% NaN 3 ) wash thrice, kept in CD163 antibody conjugated with PE (Phycoerythrin) for 60 min and evaluated through Cytomics FC500 from Beckman Coulter (Fullerton, CA).

| Mouse model
The

| Statistical studies
GraphPad Prism 6.0 from GraphPad Software was used for statistical analyses. The results were expressed as the mean ± SD (standard deviation). Two-tailed independent Student's t tests and ANOVA (analysis of variance) were used to analyse quantitative data. Fisher's exact tests and chi-square tests were used to compare categorical variables. Clinical correlations were analysed using chi-square (χ 2 ) tests, and survival analysis was conducted by the Kaplan-Meier method along with log-rank tests.
Statistically significant differences were considered when P values were < 0.05.

| CtBP1 levels increased and associated with a poor prognosis in NSCLC patients
To investigate the role of CtBP1 in NSCLC, the clinical role of CtBP1 was explored in NSCLC samples. Firstly, we evaluated CtBP1 expression using immunohistochemistry (IHC), western blotting, and real-time PCR assays in NSCLC and paired normal tissues. CtBP1 expression enhanced significantly in NSCLC tissues than in matched, normal tissues, as observed by IHC assay (Figure 1A,B). This up-regulation was confirmed further in eight paired NSCLC tissues and normal tissues at the transcript and protein levels ( Figure 1C,D). We also investigated if there was any correlation between CtBP1 expression with clinical and pathological characteristics and human NSCLC prognosis. Notably, enhanced CtBP1 expression is associated remarkably with TNM in its advanced stage, metastasis of the lymph node, and a poor differentiation of tumour in NSCLC (Table 1).
Furthermore, patients with high expression of CtBP1 exhibited a shorter OS (overall survival) than those with low CtBP1, as assessed by Kaplan-Meier analysis ( Figure 1E). Cox's multivariate analysis indicated that this enhanced expression of CtBP1 was deemed an independent risk factor for the prognosis of OS in NSCLC patients (Table 3). Thus, CtBP1 levels upregulate frequently in human NSCLC and is related relatively poor survival.

TA B L E 2 Primers list
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| CtBP1 promoted the proliferation, migration and invasion of NSCLC cells
To  with the control knockdown group, knockdown CtBP1 decreased cell proliferation, invasion and migration in A549 and H1299 cells ( Figure 2D, E, H, I and K). Thus, our finding demonstrated that CtBP1 could promote in vitro proliferation, invasion and migration of tumour cells.

| Elevated CtBP1 levels promote CCL2 secretion from NSCLC cells
Earlier studies report that cytokines play a vital part in remodelling TME and promoting EMT. 25,26 The cytokines CCL (C-C motif chemokine ligand)-2 and −5 promote TAMs recruitment, as well as IL-8, which is a ligand for C-X-C motif CXCR (chemokine receptor)-1 and −2, are expressed highly on M2 macrophage surface, and TAMs exhibit phenotype like that of M2. 26 Notwithstanding, alterations induced by CtBP1 in cytokines are not yet extensively understood.
The major cytokines regulated by CtBP1 were found on exploring a series of cytokines and by investigating if CtBP1 could induce the secretion of cytokines to affect the TME ( Figure 3A). Indeed, the expression of CCL2 elevated in overexpressing CtBP1 in NSCLC cells ( Figure 3B). However, the level of CCL2 decreased in CtBP1 knockdown cells ( Figure 3C). Furthermore, ELISA was used for CCL2 secretion analysis. Our findings demonstrated that CtBP1 overexpression promotes CCL2 level, while knockdown CtBP1 decreases CCL2 level (Figurer 3D and 3E). Therefore, the above data indicate that CtBP1 regulates CCL2 expression and secretion in NSCLC cells.

| NF-κB mediated CtBP1-induced CCL2 upregulation
Then, the process of CtBP1-mediated CCL2 induction in NSCLC cells was analysed. Studies have revealed that CtBP1 promotes cytokine  Figure 4C and 4D). Moreover, the Western blotting assay revealed that overexpression CtBP1 led to p65 nuclear translocation in A549 and H1299 cells ( Figure 4E and 4F).

| CtBP1 promotes recruitment and polarization of macrophages by CCL2 in NSCLC cells
Several stromal components, including macrophages, play vital parts in progression of a tumour and in TME remodelling. 28  with those in the control group ( Figure 5D,E). Thus, the above data shown that CtBP1 possibly promotes macrophage recruitment and polarization by CCL2 in NSCLC.

| TAM is required for CtBP1-induced NSCLC progression
Finally, the syngeneic NSCLC mice model was used to determine the role of TAM on CtBP1-mediated NSCLC progression. CtBP1 tumourbearing mice were treated with sc-202525 or Clodronate-liposome, the tumour growth curve and growth were evaluated. Compared with the control group, overexpression of CtBP1 promotes tumour growth, which was attenuated by sc-202525 or Clodronateliposome treatment ( Figure 6A-6C). To assess the infiltration of TAM, IHC staining was done using human-specific monoclonal antibody CD163 (10D6). As shown in Figure 6D-6E, TAM infiltration in tumour microenvironment increased significantly due to overexpression of CtBP1, which could be abrogated by treating with sc-202525 or Clodronate-liposome treatment. Thus, these outcomes confirmed that CtBP1 promoted TAM infiltration and progression of NSCLC.

| D ISCUSS I ON
The microenvironment of tumour consists of cytokines, immunocytes and chemokines. The infiltration of lymphocyte happens in glioma and the clinical outcome is predicted by the presence of TILs (tumour-infiltrating lymphocytes). 29 Nearly 30% of brain tumour immunity and facilitate metastasis. 13 We observed that elevated levels of CtBP1 in NSCLC cells promoted TAM recruitment and polarization, in accordance with above-mentioned findings. Our study also showed that CCL2 secretion is induced by enhanced CtBP1, the most important chemoattractant for TAM recruitment and polarization, consistent with previous findings. Being a factor with several functions, CCL2 takes part in diverse aspects of liver disease, such as cirrhosis and hepatocarcinogenesis. 41 Research shows that the polarization and accumulation of macrophages through CCR2 receptor signalling pathway are aided by CCL2. 42 Additionally, targeting signalling of CCL2/CCR2 with a CCR2 antagonist was found to remarkably reduce TAM recruitment and polarization, thus enhancing the immunotherapeutic effect of tumour. 42 Accordingly, our study showed CCL2 as a vital mediator to link CtBP1 and TAM infiltration.
This finding was further confirmed in an orthotopic nude NSCLC mice model by injecting antagonist of CCR2.
Being a prototypical inflammatory and immune signalling pathway, NF-κB pathway is a primary modulator of innate immunity and inflammation. 43 A NF-κB subunit, p65, is normally localized by its inhibitor IκB to the cytoplasm. 43 Translocation of NF-κB p65 to the nucleus occurs after stimulation and functions as a transcription factor following dissociation of IκB from NF-κB. 44 We found in this study that the result of CtBP1 overexpression is CCL2 production and significantly suppressed phosphorylated p65 nuclear translocation as a result of treatment with NF-κB inhibitor. These observations thus implicate the involvement of signalling pathway for NF-κB in CCL2 secretion and production induced by CtBP1.
In conclusion, increased CtBP1 level was observed in NSCLC cells, which up-regulated CCL2 secretion, to aid in TAM recruitment and polarization and subsequently facilitated progression of NSCLC. The outcomes show overexpressed CtBP1-mediated interaction between NSCLC cells and TAMs. The current study further enhances our knowledge of the process involving function of CtBP1 in NSCLC progression.

ACK N OWLED G EM ENTS
This study was supported by grant from Science and Technology Department of Jilin Province (No.20200708109YY).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.