Cell Infiltration In Colorectal Cancer Research Articles

RNA demethylase ALKBH5 suppresses tumorigenesis via inhibiting proliferation and invasion and promoting CD8+ T cell infiltration in colorectal cancer

ALKBH5 belongs to the ALKB family consists of a Fe (II) and a-ketoglutarate-dependent dioxygenase. ALKBH5 directly catalyzes the oxidative demethylation of m6A-methylated adenosine. ALKBH5 involves in tumorigenesis and tumor progression, and is often dysregulated in a wide range of cancers, including colorectal cancer. Emerging evidence indicates that the expression of ALKBH5 is associated with the abundance of infiltrating immune cells in the microenvironment. However, how ALKBH5 affects immune cell infiltration in the microenvironment in colorectal cancer (CRC) has not been reported. The aim of this study was to identify how the expression of ALKBH5 affects the biological behaviors of CRC cell lines and regulates the effects on infiltrating CD8+ T cells in CRC microenvironment with its specific mechanism. Firstly, the transcriptional expression profiles of CRC were downloaded from TCGA database and integrated via R software (4.1.2). Between CRC and normal colorectal tissues, ALKBH5 mRNA expressions were compared (Wilcoxon rank-sum). We further identified the expression levels of ALKBH5 in CRC tissues and cell lines through quantitative PCR, western blot, and immunohistochemistry. Then, how ALKBH5 affects the biological behaviors of CRC cells were confirmed by gain- and loss-of-function analysis. Furthermore, the relationship between ALKBH5 level and 22 tumor-infiltrating immune cells was examined through CIBERSORT in R software. Furthermore, we explored the correlation between ALKBH5 expression and tumor-infiltrated CD8+, CD4+ and regulatory T cells by utilizing the TIMER database. Finally, the association between chemokines and CD8+ T cells infiltration in CRC was analyzed using GEPIA online database. qRT-PCR, WB and IHC were used to further determine the effect of ALKBH5 on NF-κB-CCL5 signaling axis and CD8+ T cells infiltration. Clinically, ALKBH5 expression was downregulated in CRC and low levels of ALKBH5 expression were correlated with poor overall survival (OS). Functionally, overexpression of ALKBH5 reduced the proliferation, migration and invasion of CRC cells, and vice versa. Overexpression of ALKBH5 suppresses NF-κB pathway, thus reduces CCL5 expression and promotes CD8+ T cells infiltration in CRC microenvironment. ALKBH5 is poorly expressed in CRC, and overexpression of ALKBH5 attenuates CRC malignant progression by inhibiting CRC cell proliferation, migration, invasion and promoting CD8+ T cells infiltration in the tumor microenvironment through NF-κB-CCL5 axis.

Correlation between SMADs and Colorectal Cancer Expression, Prognosis, and Immune Infiltrates.

In recent years, the incidence and mortality of colorectal cancer (CRC) are increasing, and the 5-year survival rate of advanced metastatic CRC is poor. Small mothers against decapentaplegic (SMAD) superfamily are intracellular signal transduction proteins associated with the development and prognosis of a variety of tumors. At present, no study has systematically analysed the relationship between SMADs and CRC. Here, R3.6.3 was used to analyse the expression of SMADs in pan-cancer and CRC. Protein expression of SMADs were analysed by Human Protein Atlas (HPA). Gene expression profiling interactive analysis (GEPIA) was used to evaluate the correlation between SMADs and tumor stage in CRC. The effect of R language and GEPIA on prognosis was analysed. Mutation rates of SMADs in CRC were determined by cBioPortal, and potentially related genes were predicted using GeneMANIA. R analysis was used to correlate immune cell infiltration in CRC. Both SMAD1 and SMAD2 were found to be weakly expressed in CRC and correlated with the immune invasion level. SMAD1 was correlated with patient prognosis, and SMAD2 was correlated with tumor stage. SMAD3, SMAD4, and SMAD7 were all expressed at low levels in CRC and associated with a variety of immune cells. SMAD3 and SMAD4 proteins were also expressed at low levels, and SMAD4 had the highest mutation rate. SMAD5 and SMAD6 were overexpressed in CRC, and SMAD6 was also associated with patient overall survival (OS) and CD8+ T cells, macrophages, and neutrophils. Our results reveal innovative and strong evidence that SMADs can be used as biomarkers for the treatment and prognosis of CRC.

USP20 is a predictor of poor prognosis in colorectal cancer and associated with lymph node metastasis, immune infiltration and chemotherapy resistance.

Colorectal cancer (CRC) is a highly prevalent malignancy with a poor prognosis. USP20 can support progression of variety of tumors. USP20 was shown to promote breast tumor metastasis, and proliferation of oral squamous carcinoma cells. However, the role of USP20 in CRC remains unclear. We used bioinformatics to analyze the expression and prognosis of USP20 in pan-cancer and explore the relationship between USP20 expression and immune infiltration, immune checkpoints, and chemotherapy resistance in CRC. The differential expression and prognostic role of USP20 in CRC was validated by qRT-PCR and immunohistochemistry. Cox univariate and multivariate analyses were performed to assess risk factors for poor prognosis of CRC, and new prognostic prediction models were constructed and evaluated by decision curve analysis (ROC) and receiver operating characteristic (DCA). USP20 was overexpressed in CRC cell lines to explore the effect of USP20 on the functionalities of CRC cells. Enrichment analyses were used to explore the possible mechanism of USP20 in CRC. The expression of USP20 was lower in CRC tissues than adjacent normal tissues. Compared with low USP20 expression patients, CRC patients with high USP20 expression level had shorter OS. Correlation analysis showed that USP20 expression was associated with lymph node metastasis. Cox regression analysis revealed USP20 as an independent risk factor for poor prognosis in CRC patients. ROC and DCA analyses showed that the performance of the newly constructed prediction model was better than the traditional TNM model. Immune infiltration analysis shown that USP20 expression is closely associated with T cell infiltration in CRC. A co-expression analysis showed that USP20 expression was positively correlated with several immune checkpoint genes including ADORA2A, CD160, CD27 and TNFRSF25 genes and positively associated with multiple multi-drug resistance genes such as MRP1, MRP3, and MRP5 genes. USP20 expression positively correlated with the sensitivity of cells to multiple anticancer drugs. Overexpression of USP20 enhanced the migration and invasive ability of CRC cells. Enrichment pathway analyses showed the USP20 may play a role via the Notch pathway, Hedgehog pathway and beta-catenin pathway. USP20 is downregulated in CRC and associated with prognosis in CRC. USP20 enhances CRC cells metastasis and is associated with immune infiltration, immune checkpoints, and chemotherapy resistance.

MXRA8 is an immune-relative prognostic biomarker associated with metastasis and CD8+ T cell infiltration in colorectal cancer.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. Tumor metastasis and CD8+ T cell infiltration play a crucial role in CRC patient survival. It is important to determine the etiology and mechanism of the malignant progression of CRC to develop more effective treatment strategies. We conducted weighted gene co-expression network analysis (WGCNA) to explore vital modules of tumor metastasis and CD8+ T cell infiltration, then with hub gene selection and survival analysis. Multi-omics analysis is used to explore the expression pattern, immunity, and prognostic effect of MXRA8. The molecular and immune characteristics of MXRA8 are analyzed in independent cohorts, clinical specimens, and in vitro. MXRA8 expression was strongly correlated with tumor malignancy, metastasis, recurrence, and immunosuppressive microenvironment. Furthermore, MXRA8 expression predicts poor prognosis and is an independent prognostic factor for OS in CRC. MXRA8 may be a potential immunotherapeutic and prognostic biomarker for CRC.

M5C regulator-mediated modification patterns and tumor microenvironment infiltration characterization in colorectal cancer: One step closer to precision medicine.

The RNA modification 5-methylcytosine (m5C) is one of the most prevalent post-transcriptional modifications, with increasing evidence demonstrating its extensive involvement in the tumorigenesis and progression of various cancers. Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related deaths worldwide. However, the role of m5C modulators in shaping tumor microenvironment (TME) heterogeneity and regulating immune cell infiltration in CRC requires further clarification. The transcriptomic sequencing data of 18 m5C regulators and clinical data of patients with CRC were obtained from The Cancer Genome Atlas (TCGA) and systematically evaluated. We found that 16 m5C regulators were differentially expressed between CRC and normal tissues. Unsupervised cluster analysis was then performed and revealed two distinct m5C modification patterns that yielded different clinical prognoses and biological functions in CRC. We demonstrated that the m5C score constructed from eight m5C-related genes showed excellent prognostic performance, with a subsequent independent analysis confirming its predictive ability in the CRC cohort. Then we developed a nomogram containing five clinical risk factors and the m5C risk score and found that the m5C score exhibited high prognostic prediction accuracy and favorable clinical applicability. Moreover, the CRC patients with low m5C score were characterized by "hot" TME exhibiting increased immune cell infiltration and higher immune checkpoint expression. These characteristics were highlighted as potential identifiers of suitable candidates for anticancer immunotherapy. Although the high m5C score represented the non-inflammatory phenotype, the CRC patients in this group exhibited high level of sensitivity to molecular-targeted therapy. Our comprehensive analysis indicated that the novel m5C clusters and scoring system accurately reflected the distinct prognostic signature, clinicopathological characteristics, immunological phenotypes, and stratifying therapeutic opportunities of CRC. Our findings, therefore, offer valuable insights into factors that may be targeted in the development of precision medicine-based therapeutic strategies for CRC.

Identification and Validation of the lncRNA MYOSLID as a Regulating Factor of Necroptosis and Immune Cell Infiltration in Colorectal Cancer following Necroptosis-Related LncRNA Model Establishment

Simple SummaryColorectal cancer is one of the most common cancers and the second leading cause of deaths due to cancer. In this study, we developed a neural model based on only four lncRNAs to predict the overall survival rate of colorectal cancer patients. Moreover, we validated the value of analysing the lncRNA MYOSLID, one of the hub lncRNAs in our model, which promotes colorectal cancer by regulating necroptosis. Our study offered some essential insights into predicting the prognosis of colorectal cancer patients and may help to assist diagnosis and treatment in the future.Necroptosis is a newly defined form of programmed cell death that plays an important role in cancers. However, necroptosis-related lncRNAs (NRLs) involved in colorectal cancer (CRC) have not yet been thoroughly studied. Methods: In this study, a 4-NRL model was developed based on the least absolute shrinkage and selection operator (LASSO) algorithm. A series of informatic, in vitro and in vivo analyses were applied to validate the prognostic value of the model and the potential function of the hub lncRNA MYOSLID. Results: The model exhibited an excellent capacity for the prediction of overall survival and other clinicopathological features of CRC patients using Kaplan–Meier (K–M) survival curves and receiver operating characteristic (ROC) curves. Furthermore, a significant difference in the levels of immune cells, such as CD4 memory T cells and activated mast cells, between two risk groups was observed. The low-risk patients had a higher expression of immune checkpoints, such as PDCD1 (PD-1) and CD274 (PD-L1). The levels of MYOSLID, a hub lncRNA in our model, were higher in CRC tissues than in normal tissues. Knockdown of MYOSLID induced necroptosis and inhibited the proliferation of CRC cells in vitro and in vivo. Interestingly, knockdown of MYOSLID also increased the percentage of CD4+ and CD8+ T cells in subcutaneously transplanted tumours. Conclusion: Our model is a promising biomarker that can be used to predict clinical outcomes in CRC patients, and MYOSLID plays an important role in regulating necroptosis and immune cell infiltration in CRC.

Integrated analysis of the functions and clinical implications of exosome circRNAs in colorectal cancer.

BackgroundExosome circRNAs (Exo-circRNAs) regulate cancer progression and intercellular crosstalk in the tumor microenvironment. However, their biological functions and potential clinical importance in colorectal cancer (CRC) remain unknown.MethodsWe used exoRBase 2.0 data to identify significant differentially expressed Exo-circRNAs (Exo-DEcircRNAs) in CRC patients and healthy individuals. The least absolute shrinkage and selector operation algorithm, support vector machine-recursive feature elimination, and multivariate Cox regression analyses were used to select candidate Exo-circRNAs and constructed a diagnostic model. Quantitative reverse transcription-polymerase chain reaction analysis was performed to confirm the expression of Exo-circRNAs in the serum samples of patients. Furthermore, we constructed an exosome circRNA-miRNA-mRNA network for CRC. Upregulated target mRNAs in the exosome competing endogenous RNA (Exo-ceRNA) network were used for functional and pathway enrichment analyses. We identified 22 immune cell types in CRC patients using CIBERSORT. Correlation analysis revealed the relationship between Exo-ceRNA networks and immune-infiltrating cells. The relationship between target mRNAs and immunotherapeutic response was also explored. Finally, using the Kaplan–Meier survival curve, a prognostic upregulated target mRNA was screened. We constructed a survival-related Exo-ceRNA subnetwork and explored the correlation between the Exo-ceRNA subnetwork and immune-infiltrating cells.ResultsThe constructed diagnostic model had a high area under the curve (AUC) value in both the training and validation sets (AUC = 0.744 and AUC = 0.741, respectively). qRT-PCR confirmed that the Exo-circRNAs were differentially expressed in CRC serum samples. We constructed Exo-ceRNA networks based on the interactions among seven upregulated Exo-DEcircRNAs, eight differentially expressed miRNAs, and twenty-two differentially expressed mRNAs in CRC. Functional enrichment analysis revealed that the upregulated target mRNAs were significantly enriched in cytoskeletal motor activity and the PI3K-Akt signaling pathway. Co-expression analysis showed a significant correlation between the Exo-ceRNA networks and immune cells. The significant correlation was observed between target mRNAs and the immunotherapeutic response. Additionally, based on the prognostic upregulated target gene (RGS2), we constructed a survival-related Exo-ceRNA subnetwork (Exosome hsa_circ_0050334-hsa_miR_182_5p-RGS2). CIBERSORT results revealed that the Exo-ceRNA subnetwork correlated with M2 macrophages (P = 4.6e-07, R = 0.31).ConclusionsOur study identified an Exo-diagnostic model, established Exo-ceRNA networks, and explored the correlation between Exo-ceRNA networks and immune cell infiltration in CRC. These findings elucidated the biological functions of Exo-circRNAs and their potential clinical implications.

Abstract 5552: Novel set of OX40 targeted antibodies and VHHs include agonists and antagonists for future development

Abstract Immunotherapy is one of the most effective modern treatments for cancer. While immunotherapy operates through a broad class of mechanisms, modulating immune checkpoints have provided some of the most effective approaches, including inhibition of PD-1/PD-L1 pathway, CTLA-4, LAG-3, OX40, Tim-3, KIR and TIGIT to reverse tumor immune escape.OX40, also known as CD134 or TNFRSF4, is a membrane protein expressed in CD4+ and CD8+ T cells, neutrophils and NK cells. In the process of T cell activation, OX40 plays a synergistic stimulating function by binding with ligand OX40L. OX40 agonist immunotherapy has been previously shown to improve the survival rate of glioblastoma in mice, prevent tumor growth in ovarian cancer, increase the prognostic significance of CD8+ cell infiltration in colorectal cancer, and eliminate the inhibitory function of Foxp3+ Treg cells that constitutively Express OX40. On the contrary, blocking OX40 axis is a promising strategy to treat T cell hyperactivation in autoimmune diseases. As recently reported in the literature, a two-armed, random, double blinded phase 2b clinical trial demonstrated that telazorlimab, an antagonist OX40 antibody developed by Ichnos Sciences, met primary efficacy endpoints in treating atopic dermatitis.In this research, we developed a set of novel antibodies to OX40 using a hybridoma and VHH synthetic library; the set of antibodies includes both agonists and antagonists.We developed one hybridoma origin mAb into a human framework. Agonist activity was equal to MOXR0916 in NF- κB reporter-Jurkat assay, dependent on coupling by proteinA/cell-bound CD32b, non-competing with OX40L, and epitope mapped to domain 2+3.We identified dozens of binders from our synthetic single domain VH library. After construction into a IgG1Fc fusion form, 4 VHHs were obtained with significant agonistic activity, dependent on coupling by proteinA/cell bound CD32b. Another 5 VHHs were also obtained with significant antagonistic activity without coupling by proteinA/cell-bound CD32b. The 4 agonistic VHHs were confirmed with binding to domain 1+2, competitive to OX40L’s binding.These VHHs constructs represent a promising set of novel biologics to modulate OX40—as agonists or antagonists—and serve as the basis for development of potential new therapies in the clinic. Citation Format: Chenguang Cai, Xiang Li, Shin-Chen Hou, Guoqian Sun, Ying Li, Ting Yang, Fengzhi Zhang, Qi Sun, Shiying Fu, Shuang Zhao, You'you Lin, Feizheng Xue. Novel set of OX40 targeted antibodies and VHHs include agonists and antagonists for future development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5552.

The role of miR-4469 as a tumor suppressor regulating inflammatory cell infiltration in colorectal cancer

BackgroundMicroRNA (miRNA) regulates gene expression posttranscriptionally, and some of them function in tumor suppression and can be used in drug development. As a result, identifying and screening miRNAs that suppress tumors would be a significant addition to tumor treatment. MethodsIn this study, we analyzed the miRNA expression profile of colorectal cancer (CRC), constructed a negative regulatory network of the miRNA-target genes, and identified miR-4469 as one of the key tumor suppressors miRNAs. We analyzed the expression and survival of miR-4469 in pan-cancer, experimentally verified the expression level of miR-4469 in CRC cells and the effect on CRC cell proliferation and migration. We screened miR-4469 target genes for enrichment analysis and immune cell infiltration analysis and validated target gene expression to clarify the regulatory mechanisms involved in miR-4469. ResultsmiR-4469 was more highly expressed in normal colorectum tissues compared to CRC tissues and correlated with survival time in patients with multiple cancers. It was shown that miR-4469 was highly expressed in normal colon cells and miR-4469 expression could inhibit the proliferation and migration of CRC cells. In addition, studies on the mechanism showed that miR-4469 function is mainly related to the regulation of inflammatory cell infiltration, and the key target genes of miR-4469 in this process are SLC2A3, FGR, PLEKHO2, and MYO1F. ConclusionmiR-4469 is a tumor suppressor in CRC, and its regulatory mechanism mainly affects the infiltration of inflammatory cells in the cancer tissue.

Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer.

Growing evidence has revealed that the E2F family of transcription factor 2 (E2F2) participates in the tumorigenesis and progression of various tumors, but its role in colorectal cancer (CRC) remains largely unknown. Herein, the aim of our study was to investigate the exact role of E2F2 in CRC. The expression levels of E2F2 in CRC were appraised based on the Tumor Immune Estimate Resource (TIMER), Oncomine, The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) database. The results were further confirmed using CRC tumor tissues and normal controls by experimental assays including immunohistochemistry, qRT-PCR and western blot. The survival analysis of E2F2 in CRC was analyzed using PrognoScan database and TCGA data sets. In addition, the functional roles of E2F2 were examined by Gene Set Enrichment Analysis (GSEA) and immune infiltration analysis. Our results illustrated that E2F2 was significantly downregulated in CRC samples. The low E2F2 expression in CRC was prominently correlated with N, M stage and pathological stage. Decreased E2F2 expression had an unfavorable overall survivial (OS), disease free survival (DFS), disease specific survival (DSS) and progress free interval (PFI). Multivariate cox regression showed E2F2 could be an independent prognostic factors of OS in CRC. Receiver operating characteristic (ROC) analysis showed that E2F2 may serve as a potential diagnostic biomarker for CRC patients. GSEA disclosed that E2F2 was probably involved in several pathways, including ATR pathway, ATM signalling pathway, mismatch repair, base excision repair, homologous recomibination, Fanconi Anemia pathway, multicancer invasiveness signature, and cancer stem cells. Moreover, E2F2 was significantly correlated with the infiltration level of Th2, aDC, Th17, NK CD56dim, T helper and pDC cells. The current study demonstrates that decreased E2F2 expression is closely associated with poor prognosis and immune cell infiltration in CRC, which can be a promising independent prognostic biomarker and potential treatment target for CRC.

Characteristics of CD8+ T cell infiltration in colorectal cancer and their correlation with prognosis

Objective: As cytotoxic T cells, CD8+ T lymphocytes can kill tumor cells by releasing perforin and other effector molecules, but the correlation between their infiltration level and the prognosis of colorectal cancer varies in previous studies. This study aims to explore the distribution of CD8+T cells in tumor center and invasive margin of colorectal cancer, and to analyze their correlation with the prognosis of patients. Methods: A retrospective cohort study was used to analyze the clinicopathological features of 221 patients with colorectal cancer from the colorectal cancer pathological database of the Sixth Affiliated Hospital of Sun Yat-sen University between 2009 and 2012. Case inclusion criteria: (1) colorectal cancers confirmed by postoperative pathology; (2) patients with follow-up data. Exclusion criteria: (1) multiple primary cancers; (2) inflammatory bowel disease, Lynch syndrome or familial adenomatous polyposis; (3) no available paraffin slides; (4) patients receiving preoperative radiotherapy or chemotherapy. A total of 221 patients met the criteria. Immunohistochemical staining was used to count the CD8+ T cells in tumor center and invasive margin in the paraffin slides. Meanwhile the relative expression of CD8B gene in 22 fresh freeze samples of colorectal cancer was detected. Then the correlation of the expression with CD8+T cell density was examined. The patients were divided into high and low infiltration groups according to the level of CD8+T cells. Log-rank test was applied to compare the overall survival of the two groups of patients, and Cox regression analysis was used to adjust the prognostic significance of CD8+T cell infiltration. Results: There were 118 males and 103 females. In 221 slides, CD8+T cells infiltrating in invasive margin were more than those in tumor center [median (range): 37(0-141) / field vs. 14(0-106) / field, Z=-11.985, P<0.001], and the number of CD8+T cell in the tumor center was positively correlated with those in invasive margin (r=0.610, P<0.001). The number of CD8+ T cell in tumor center was positively correlated with the relative expression of CD8B gene (r=0.524, P=0.012). Survival analysis showed that the overall survival of the high infiltration group was better than that of the low infiltration group both in tumor center and invasive margin (median survival: 84.1 months vs. 73.5 months, P<0.001; 84.2 months vs. 75.9 months, P=0.002). Cox regression analysis revealed that high CD8+T cell infiltration in tumor center was an independent protective factor of overall survival (HR=0.369, 95% CI: 0.168-0.812, P=0.013). Conclusions: The infiltration level of CD8+T cells in tumor center is lower than that in invasive margin, and they are positively correlated. The level of CD8+ T cell infiltration in tumor center is related to overall survival and can be used as a potential pronostic marker.

CRYAB predicts clinical prognosis and is associated with immunocyte infiltration in colorectal cancer.

BackgroundαB-Crystallin (CRYAB) is differentially expressed in various tumors. However, the correlation between CRYAB and immune cell infiltration in colorectal cancer (CRC) remains unclear.Materials & MethodsKaplan–Meier survival curves in The Cancer Genome Atlas (TCGA) were used to evaluate the relationship between CRYAB expression and both overall survival and progression-free survival. The relationships between CRYAB expression and infiltrating immune cells and their corresponding gene marker sets were examined using the TIMER database.ResultsThe expression of CRYAB was lower in CRC tumor tissues than in normal tissues (P < 0.05). High CRYAB gene expression and high levels of CRYAB gene methylation were correlated with high-grade malignant tumors and more advanced tumor, nodes and metastasis (TNM) cancer stages. In addition, in colorectal cancer, there was a positive correlation between CRYAB expression and immune infiltrating cells including neutrophils, macrophages, CD8 + T cells, and CD4 + T cells, as well as immune-related genes including CD2, CD3D, and CD3E. Methylation sites such as cg13084335, cg15545878, cg13210534, and cg15318568 were positively correlated with low expression of CRYAB.ConclusionBecause CRYAB likely plays an important role in immune cell infiltration, it may be a potential tumor-suppressor gene in CRC and a potential novel therapeutic target and predictive biomarker for colorectal cancer (CRC).

SH2D4A downregulation due to loss of chromosome 8p is associated with poor prognosis and low T cell infiltration in colorectal cancer.

Colorectal cancer (CRC) develops through chromosomal instability (CIN) or microsatellite instability (MSI) due to deficient mismatch-repair (dMMR). We aimed to characterise novel cancer-associated genes that are downregulated upon malignant transformation in microsatellite stable (MSS) CRCs, which typically exhibit CIN with proficient mismatch-repair (pMMR). Comprehensive screening was conducted on adenomas, MSI/MSS CRCs and cell lines, followed by copy number analysis, and their genetic and prognostic relevance was confirmed in microarray and RNA-seq cohorts (n = 3262, in total). Immunohistochemistry for SH2D4A was performed in 524 specimens of adenoma, carcinoma in situ and dMMR/pMMR CRC. The functional role of SH2D4A was investigated using CRC cell lines. A set of 11 genes, including SH2D4A, was downregulated during the adenoma-carcinoma sequence in MSS/CIN CRCs, mainly due to chromosome 8p deletions, and their negative prognostic impact was validated in independent cohorts. All adenomas were SH2D4A positive, but a subset of CRCs (5.3%) lacked SH2D4A immunohistochemical staining, correlating with poor prognosis and scarce T cell infiltration. SH2D4A depletion did not affect cell proliferation or IL-6-induced STAT3 phosphorylation. Our findings suggest that downregulation of multiple genes on chromosome 8p, including SH2D4A, cooperatively contribute to tumorigenesis, resulting in the immune cold tumour microenvironment and poor prognosis.

Construction and Comprehensive Prognostic Analysis of a lncRNA-miRNA-mRNA Regulatory Network and Tumor Immune Cell Infiltration in Colorectal Cancer.

Colorectal cancer (CRC) is a malignant tumor with high morbidity and mortality worldwide. Recent studies have shown that long noncoding RNAs (lncRNAs) play an important role in almost all human tumors, including CRC. Competitive endogenous RNA (ceRNA) regulatory networks have become hot topics in cancer research. Tumor-infiltrating immune cells (TICs) have also been reported to be closely related to the survival and prognosis of CRC patients. In this study, we used the lncRNA–miRNA–mRNA regulatory network combined with tumor immune cell infiltration to predict the survival and prognosis of 598 CRC patients. First, we downloaded the lncRNA, mRNA, and miRNA transcriptome data of CRC patients from The Cancer Genome Atlas (TCGA) database and identified differentially expressed genes through “limma” package of R software. The ceRNA regulatory network was established by using the “GDCRNATools” R package. Then, univariate Cox analysis and least absolute shrinkage and selection operator analysis were performed to identify the optimal prognostic network nodes, including SRPX, UST, H19, SNHG7, hsa-miR-29b-3p, and TTYH3. Next, we analyzed the differences in 22 types of TICs between 58 normal subjects and 206 CRC patients and included memory CD4 T cells, dendritic cells and neutrophils in the construction of a prognostic model. Finally, we identified the relationship between the ceRNA prognostic model and the infiltrating immune cell prognostic model. In conclusion, we constructed two prognostic models that provide insights on the prognosis and treatment strategy of CRC.

TGF-β2 is a Prognostic Biomarker Correlated with Immune Cell Infiltration in Colorectal Cancer: A STROBE-compliant article.

Transforming growth factor-beta (TGF-β2) is an important cytokine regulating immune cell function. However, whether TGF-β2 controls the invasion of colorectal cancer (CRC) by immune cells is unknown. Therefore, we evaluated the expression of TGF-β2 using multiple databases and determined the relationship between TGF-β2 expression and tumor immune infiltration defined by a set of genetic markers. The analysis demonstrated that the expression of TGF-β2 is closely related to the outcome of many cancers, and this correlation was particularly strong in CRC. In addition, the increased expression of TGF-β2 was significantly associated with the expression of various markers of specific immune cell subpopulations, and overexpression of TGF-β2 was closely related to the prognosis of colon cancer patients. Moreover, TGF-β2 was related to the prognosis and infiltration of the tumor by immune cells in CRC patients. The obtained results indicate that TGF-β2 is a critical factor regulating the recruitment of immune cells and controls their infiltration into colorectal tumors. Thus, high expression of TGF-β2 not only facilitates the prognosis in CRC patients, but also may provide a new target for the treatment of CRC.

Infiltration by IL22-Producing T Cells Promotes Neutrophil Recruitment and Predicts Favorable Clinical Outcome in Human Colorectal Cancer.

Immune cell infiltration in colorectal cancer effectively predicts clinical outcome. IL22, produced by immune cells, plays an important role in inflammatory bowel disease, but its relevance in colorectal cancer remains unclear. Here, we addressed the prognostic significance of IL22+ cell infiltration in colorectal cancer and its effects on the composition of tumor microenvironment. Tissue microarrays (TMA) were stained with an IL22-specific mAb, and positive immune cells were counted by expert pathologists. Results were correlated with clinicopathologic data and overall survival (OS). Phenotypes of IL22-producing cells were assessed by flow cytometry on cell suspensions from digested specimens. Chemokine production was evaluated in vitro upon colorectal cancer cell exposure to IL22, and culture supernatants were used to assess neutrophil migration in vitro Evaluation of a testing (n = 425) and a validation TMA (n = 89) revealed that high numbers of IL22 tumor-infiltrating immune cells were associated with improved OS in colorectal cancer. Ex vivo analysis indicated that IL22 was produced by CD4+ and CD8+ polyfunctional T cells, which also produced IL17 and IFNγ. Exposure of colorectal cancer cells to IL22 promoted the release of the neutrophil-recruiting chemokines CXCL1, CXCL2, and CXCL3 and enhanced neutrophil migration in vitro Combined survival analysis revealed that the favorable prognostic significance of IL22 in colorectal cancer relied on the presence of neutrophils and was enhanced by T-cell infiltration. Altogether, colorectal cancer-infiltrating IL22-producing T cells promoted a favorable clinical outcome by recruiting beneficial neutrophils capable of enhancing T-cell responses.

Identification of prognosis-associated immune genes and exploration of immune cell infiltration in colorectal cancer.

Aim: To identify prognosis-related immune genes (PRIGs) and construct a prognosis model of colorectal cancer (CRC) patients for clinical use. Materials & methods: Expression profiles were obtained from The Cancer Genome Atlas database and identified differentially expressed PRIGs of CRC. Results: A prognostic model was conducted based on nine PRIGs. The risk score, based on prognosis model, was an independent prognostic predictor. Five PRIGs and risk score were significantly associated with the clinical stage of CRC and five immune cells related to the risk score. Conclusion: The risk score was an independent prognostic biomarker for CRC patients. The research excavated immune genes that were associated with survival and that could be potential biomarkers for prognosis and treatment for CRC patients.

Correlation of clinico-pathologic data with inflammatory cells infiltration in colorectal cancer

Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide. At every phase of cancer development, the inflammatory process has an important impact. Accurate assessment inflammatory cells in the tumour environment in conjunction with clinico-pathologic features can be a relevant prognostic or predictive parameter. Purpose: To analyse inflammatory cell infiltration in CRC tumour mass and correlate with chosen clinico-pathologic parameters. Materials and methods: The study group consisted of 160 patients (64 women, 96 men) diagnosed with colorectal cancer who underwent surgery. Tissue material obtained from routine histopathological diagnosis was stained with H&amp;E and used to assess the type of inflammatory cells in the invasive front and centre of the tumour. Results were subjected to statistical analysis with the age and gender of patients, tumour localization, tumour growth and size, TNM stage, adenocarcinoma type, fibrosis, necrosis, metastasis and tumour invasion (by the Spearman’s correlation coefficient test). Results: The presence of neutrophils in the invasive front of tumour mass was associated with fibrosis and inflammatory cell infiltration in the invasive front of tumour. Macrophages in the invasive front of tumour were found to correlate with tumour growth (expanding and infiltrate). Macrophages and eosinophils were associated with inflammatory cell infiltration in the invasive front and in the centre of tumour. Conclusions: The type of inflammatory cells in the invasive front or centre of the tumour may be useful to prognoses clinical features of colorectal cancer

Tumor Infiltration by OX40+ Cells Enhances the Prognostic Significance of CD16+ Cell Infiltration in Colorectal Cancer.

Objectives:Analysis of tumor immune infiltration has been suggested to outperform tumor, node, metastasis staging in predicting clinical course of colorectal cancer (CRC). Infiltration by cells expressing OX40, a member of the tumor necrosis factor receptor family, or CD16, expressed by natural killer cells, monocytes, and dendritic cells, has been associated with favorable prognosis in patients with CRC. We hypothesized that assessment of CRC infiltration by both OX40+ and CD16+ cells might result in enhanced prognostic significance.Methods:Colorectal cancer infiltration by OX40 and CD16 expressing cells was investigated in 441 primary CRCs using tissue microarrays and specific antibodies, by immunohistochemistry. Patients’ survival was evaluated by Kaplan-Meier and log-rank tests. Multivariate Cox regression analysis, hazard ratios, and 95% confidence intervals were also used to evaluate prognostic significance of OX40+ and CD16+ cell infiltration.Results:Colorectal cancer infiltration by OX40+ and CD16+ cells was subclassified into 4 groups with high or low infiltration levels in all possible combinations. High levels of infiltration by both OX40+ and CD16+ cells were associated with lower pT stage, absence of peritumoral lymphocytic (PTL) inflammation, and a positive prognostic impact. Patients bearing tumors with high infiltration by CD16+ and OX40+ cells were also characterized by significantly longer overall survival, as compared with the other groups. These results were confirmed by analyzing an independent validation cohort.Conclusions:Combined infiltration by OX40+ and CD16+ immune cells is an independent favorable prognostic marker in CRC. The prognostic value of CD16+ immune cell infiltration is significantly improved by the combined analysis with OX40+ cell infiltration.

Combined prognostic value of CD274 (PD-L1)/PDCDI (PD-1) expression and immune cell infiltration in colorectal cancer as per mismatch repair status

The CD274 (programmed cell death ligand-1, PD-L1)/PDCD1 (programmed cell death-1, PD-1) pathway is crucial suppressor of the cytotoxic immune response. Antibodies targeting CD274 or PDCD1 have been revealed to be effective in several malignancies. In colorectal cancer, the response to CD274/PDCD1 blockage is associated with microsatellite instability. However, the value of CD274/PDCD1 for predicting response to treatment or survival benefit is still unclear. The aims of the study were (1) to clarify differences in immune microenvironment and expression of checkpoint proteins (CD274/PDCD1) in DNA mismatch repair-proficient, mismatch repair-deficient, and hereditary Lynch syndrome-associated colorectal cancer, and (2) to assess the prognostic value of these factors and their combinations. Ninety-four mismatch repair-deficient colorectal cancers, 100 age, sex, and AJCC/UICC stage-matched mismatch repair-proficient colorectal cancers, and 48 Lynch syndrome-associated colorectal cancers were analyzed. Using whole section samples, detailed analysis of immune cell score, PDCD1, and CD274 expression was performed. Overlapping of CD274 expression in tumor and immune cells was almost complete (95%). Immune cell score and CD274/PDCD1 positivity were significantly more frequent in mismatch repair-deficient than in mismatch repair-proficient colorectal cancers (70% vs. 41% (high immune cell score); 81% vs. 49% (PDCD1high), 23% vs. 1% (CD274 on tumor cells) and 68% vs. 30% (CD274 on immune cells), P < 0.001), and were associated strongly with each other. Although the independent impact of immune cell score, PDCD1, and CD274 on immune cells was moderate, the immunoprofile parameter combining the above three factors appeared to be a strong independent prognostic factor for disease-specific survival and overall survival (P = 0.001) and had suggestive impact on disease-free survival (P = 0.011). Our results encourage the use of immune cell score analysis together with PDCD1 and CD274 detection to improve the prognostic evaluation of colorectal cancer patients. Particularly, the analyses from whole tissue sections are encouraged to allow reliable and cell-specific analyses of CD274 expression.