Colorectal Cancer Cell Tissues Research Articles

Abstract LB349: Cysteine restriction enhances antitumor immunity in colorectal cancer

Abstract Background: Colon cancer is the second leading cause of cancer mortality worldwide, however, there are still issues with multidrug resistance and severe adverse reactions. Previous studies have shown that sulfur-containing amino acids are involved in cancer development, drug resistance, and immune evasion. Here, Our experimental findings suggested that restricting the intake of sulfur-containing amino acids may present a novel strategy for treating colorectal cancer. Methods: We assessed the impact of sulfur-containing amino acid restriction (SAR) on distinct cellular subpopulations within the tumor microenvironment through single-cell sequencing on colon carcinogenesis induced by azoxymethane/dextran sodium sulfate (AOM/DSS). Employing our custom-produced neutralizing antibodies against SLC7A11 to restrict cysteine intake, we examined the therapeutic implications and underlying molecular mechanisms by RNA-seq, Multiplexed immunofluorescence staining and Western blotting. Results: In the AOM/DSS model, SAR led to a slight reduction in mouse body weight compared to the normal diet. However, this dietary restriction significantly enhanced the survival rate of mice and effectively suppressed tumor growth. The single-cell sequencing results indicated a significant increase in the proportion of T cells within the tumor microenvironment in response to SAR. This suggested that restricting the intake of sulfur-containing amino acids may be a promising anti-tumor strategy. Previous studies have indicated high expression of the SLC7A11, a cysteine transporter protein, in various cancers, including colorectal cancer. To further investigate the role of cysteine in colorectal cancer, we developed the neutralizing antibody 1A4 against SLC7A11. Our results indicated that 1A4 markedly inhibited colorectal cancer development in mice. As known, 85% of colorectal cancers are microsatellite stable, which hardly benefit from treatment with immune checkpoint inhibitors (ICIs). Compared to anti-PD-1 and anti-CTLA4, 1A4 significantly inhibited the growth of CT26 in vivo. Multiplexed immunofluorescence staining results demonstrated that 1A4 significantly increased the proportion of CD8+ T cells in colorectal cancer tissues. Conclusion: Limiting cysteine intake significantly inhibits the growth of colorectal cancer, increases the proportion of CD8+ T cells, and enhances anti-tumor immunity. Citation Format: Jichang Li, Kang Xia, Zeruo Yang, Xiaoxue Pan, Xiaojing Yang, Pengyuan Wang, Shanwen Chen. Cysteine restriction enhances antitumor immunity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB349.

Precursor exhausted CD8+T cells in colorectal cancer tissues associated with patient's survival and immunotherapy responsiveness.

Exhausted CD8+T cells represent a distinct cellular lineage that emerges during both chronic infections and cancers. Recent studies have shown that persistent antigen exposure can drive the differentiation of precursor exhausted CD8+T cells, termed Tpex cells, which are characterized as TCF-1+PD-1+CD8+T cells. Elevated Tpex cell frequencies in the tumor microenvironment (TME) are associated with improved overall survival (OS) in cancer patients and heightened responsiveness to anti-PD-1 therapy. In our present study, we utilized multi-color immunohistochemistry (mIHC) to determine the localization and clinical implications of tumor-infiltrating Tpex cells within the TME of human colorectal cancer (CRC) tissues. We also conducted a multi-omics integrative analysis using single-cell RNA sequencing (scRNA-seq) data derived from both the murine MC38 tumor model and human CRC tissues. This analysis helped delineate the transcriptional and functional attributes of Tpex cells within the CRC TME. Furthermore, we employed spatial transcriptome sequencing data from CRC patients to investigate the interactions between Tpex cells and other immune cell subsets within the TME. In conclusion, our study not only established a method for Tpex cell detection using mIHC technology but also confirmed that assessing Tpex cells within the CRC TME could be indicative of patients' survival. We further uncovered the transcriptional and functional characteristics of Tpex cells in the TME and ascertained their pivotal role in the efficacy of immunotherapy against CRC.

The Mechanistic Target of Rapamycin Complex 1 Pathway Contributes to the Anti-Tumor Effect of Granulocyte-Macrophage-Colony-Stimulating Factor-Producing T Helper Cells in Mouse Colorectal Cancer

ABSTRACT Introduction The role of granulocyte-macrophage-colony-stimulating factor-producing T helper (ThGM) cells in colorectal cancer (CRC) development remains unclear. This study characterizes the function of ThGM cells in mouse CRC. Methods Mouse CRC was induced by administrating azoxymethane and dextran sulfate sodium. The presence of ThGM cells in CRC tissues and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in ThGM cells was detected by flow cytometry. The impact of mTORC1 signaling on ThGM cell function was determined by in vitro culture. The effect of ThGM cells on CRC development was evaluated by adoptive transfer assays. Results ThGM cells, which expressed granulocyte-macrophage-colony-stimulating factor (GM-CSF), accumulated in CRC tissues. mTORC1 signaling is activated in CRC ThGM cells. mTORC1 inhibition by rapamycin suppressed ThGM cell differentiation and proliferation and resulted in the death of differentiating ThGM cells. mTORC1 inhibition in already differentiated ThGM cells did not induce significant cell death but decreased the expression of GM-CSF, interleukin-2, and tumor necrosis factor-alpha while impeding cell proliferation. Furthermore, mTORC1 inhibition diminished the effect of ThGM cells on driving macrophage polarization toward the M1 type, as evidenced by lower expression of pro-inflammatory cytokines, major histocompatibility complex class II molecule, and CD80 in macrophages after co-culture with rapamycin-treated ThGM cells. Lentivirus-mediated knockdown/overexpression of regulatory-associated protein of mTOR (Raptor) confirmed the essential role of mTORC1 in ThGM cell differentiation and function. Adoptively transferred ThGM cells suppressed CRC growth whereas mTORC1 inhibition abolished this effect. Conclusion mTORC1 is essential for the anti-CRC activity of ThGM cells.

Comprehensive analysis of transcriptome data and experimental identification show that solute carrier 35 member A2 (SLC35A2) is a prognostic marker of colorectal cancer.

Colorectal cancer (CRC) is a solid tumor with high morbidity and mortality rates. Accumulating evidence shows that the soluble carrier family 35 member A2 (SLC35A2), a nucleotide sugar transporter, plays a key role in the pathogenesis of various tumors. However, its expression and function in CRC has not been fully elucidated. The prognosis-related gene SLC35A2 was obtained using differential analysis, prognosis correlation analysis, and LASSO regression screening. Its expression levels in CRC tissues were analyzed, and so was the relationship of this expression with clinical characteristics of patients. Subsequently, the expression levels were correlated with clinicopathological parameters using immunohistochemical analysis. Analysis based on GO/KEGG databases was used to reveal the potential mechanisms of SLC35A2. Next, we explored the relationship between SLC35A2 and immune cells in CRC tissues. A nomogram was created to help understand the prognosis of CRC patients. Finally, western blotting and qRT-PCR reaction were used to verify the expression of SLC35A2 in CRC cell lines. SLC35A2 expression was upregulated and related to tumor pathological stage and lymph node metastasis, indicating that SLC35A2 is an independent prognostic factor and a potential diagnostic marker for CRC. We verified by IHC, WB and PCR that the expression of SLC35A2 was up-regulated in colorectal cancer tissues and cell lines, and its high expression was related to the tumor pathological stage of CRC clinical samples. Our study found that SLC35A2 can be used as a biomarker for the diagnosis and prognosis of CRC, providing motivation for further study.

HER-2 Expression in Colorectal Cancer and Its Correlation with Immune Cell Infiltration.

This study aimed to investigate the effect of increased HER-2 expression on tumor-infiltrating lymphocytes (TILs) and determine its impact on the prognosis of colorectal cancer (CRC) patients; Methods: HER-2, CD4, CD8, CD19, LY6G, CD56, CD68, CD11b, and EpCam expression in CRC tissues and adjacent paracancerous tissues were assessed using multiplex fluorescence immunohistochemical staining. The correlation between HER-2 expression and the number of TILs in CRC tissues was analyzed. Kaplan-Meier and Cox proportional hazards models were used to analyze survival outcomes; Results: The expression of HER-2 in tumor tissues was higher than that in paracancerous tissues (1.31 ± 0.45 vs. 0.86 ± 0.20, p < 0.05). Additionally, there was an increase in the numbers of CD4+, CD8+, CD19+, and CD68+ cells in CRC tissues (14.11 ± 1.10 vs. 3.40 ± 0.18, p < 0.005; 0.16 ± 0.12 vs. 0.04 ± 0.04, p < 0.005; 0.71 ± 0.46 vs. 0.25 ± 0.13, p < 0.0005; 0.27 ± 0.24 vs. 0.03 ± 0.11, p < 0.05). An increase in HER-2 expression was positively correlated with an increase in CD4, CD8, and CD19 (p < 0.0001). In HER-2-positive CRC tissues, CD68 expression was increased (0.80 ± 0.55 vs. 0.25 ± 0.22, p < 0.05). In HER-2-upregulated CRC tissues, CD4, CD8, CD19, CD68, CD11b, Ly6G, and CD56 expressions were elevated (0.70 ± 0.37 vs. 0.32 ± 0.17, p = 0.03; 0.22 ± 0.13 vs. 0.09 ± 0.06, p = 0.03; 0.31 ± 0.19 vs. 0.12 ± 0.08, p = 0.02; 1.05 ± 0.62 vs. 0.43 ± 0.21, p < 0.01; 1.34 ± 0.81 vs. 0.53 ± 0.23, p < 0.01; 0.50 ± 0.31 vs. 0.19 ± 0.10, p < 0.01; 1.26 ± 0.74 vs. 0.52 ± 0.24, p < 0.01). Furthermore, increased HER-2 expression was an independent risk factor for recurrence-free survival (RFS) in patients (p < 0.01, HR = 3.421); Conclusions: The increased expression of HER-2 and its relationship with immune cells will provide new insights for immunotherapy in CRC patients.

Abstract 3019: Density of T cell subsets in colorectal cancer in relation to disease-specific survival

Abstract Background. Prior studies have demonstrated that the overall density of T cells in colorectal tumors is favorably associated with colorectal cancer (CRC) survival; however, few studies have considered the potentially distinct roles of heterogeneous T cell subsets in different tissue regions in relation to CRC outcomes. Methods. We used data and colorectal tumor specimens from a subset of participants in three studies (total N=878). We profiled the in situ T cell landscape of CRC using digital imaging, machine learning, and a customized 9-plex multiplexed immunofluoresence panel with antibodies directed against CD3, CD4, CD8, CD45RA, CD45RO, FOXP3, and MKI67. This allowed us to determine the density of T cell subsets defined according to marker co-localization and according to tissue region (i.e., epithelial, stromal). We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of T cell subset densities in CRC with disease-specific survival, adjusting for age, sex, tumor site, microsatellite instability status, CpG island methylator phenotype status, status of somatic mutations in BRAF and KRAS, stage, and study. Results. Overall, the densities of CD3+CD4+ helper T cells and CD3+CD8+ cytotoxic T cells in CRC were favorably associated with CRC-specific survival. However, in analyses stratified by tissue region, the association of CD3+CD4+ helper T cell density with survival was limited to density in stromal regions (HR for highest vs. lowest density quartile=0.40, 95% CI: 0.26-0.63, ptrend&amp;lt;0.001), whereas the association with CD3+CD8+ cytotoxic T cells was limited to density in epithelial regions (HR=0.48, 95% CI: 0.31-0.76, ptrend=0.001). These significant associations persisted after mutually adjusting region-specific CD3+CD4+ and CD3+CD8+ T cell densities for each other. Associations of memory T cell subsets (i.e., CD3+CD4+CD45RO+ and CD3+CD8+ CD45RO+ T cells) with survival were more modest than associations with overall CD3+CD4+ and CD3+CD8+ T cell densities, and were similar in magnitude across stromal and epithelial tissue regions. Conclusions. The density of T cells in CRC tissue is significantly associated with CRC survival, independent of stage at diagnosis and other evaluated tumor markers; however, the T cell subsets that are associated with CRC outcomes differ according to tissue region. These findings highlight the differing roles of distinct T cell subsets in CRC with regard to cancer outcomes, and support the need for additional work identifying factors that contribute to T cell response profiles in CRC. Citation Format: Amanda I. Phipps, Jonathan Nowak, Yasutoshi Takashima, Daniel Buchanan, Andressa Dias Costa, Steven Gallinger, Jeroen Huyghe, Li Hsu, Conghui Qu, Claire Thomas, Sushma Thomas, Tomotaka Ugai, Ulrike Peters, Shuji Ogino. Density of T cell subsets in colorectal cancer in relation to disease-specific survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3019.

Identification and analysis of a CD8+ T cell-related prognostic signature for colorectal cancer based on bulk RNA sequencing and scRNA sequencing data: A STROBE-compliant retrospective study.

Colorectal cancer (CRC) is one of the most common malignancies worldwide, leading to a large number of cancer-related mortalities. Aberrant CD8+ T cell infiltration plays a critical role in tumor progression and patient prognosis. This study aimed to identify a prognostic model for CRC based on CD8+ T cell-related genes. The infiltration levels of immune cells in CRC tissues were accessed by the ESTIMATE algorithm. Weighted gene co-expression network analysis (WGCNA) analysis was used to select CD8+ T cell-related genes. Prognostic genes were identified using Cox regression analysis and Kaplan–Meier curves. The least absolute shrinkage and selection operator (LASSO) algorithm was used to construct prognostic models. Gene set enrichment analysis (GSEA) was performed to annotate enriched gene sets. Single-cell RNA (scRNA) sequencing analysis was used to examine gene expression in different cell types. We found that the downregulated infiltration level of CD8+ T cells was an independent prognostic factor for CRC and selected a cluster of differentially expressed genes correlated with CD8+ T cell infiltration (CD8TDEGs). Subsequently, we identified 18 prognostic CD8TDEGs, according to which patients were reclassified into two clusters with distinct overall survival. Seven prognostic CD8TDEGs were selected to calculate the constructed prognostic model’s risk scores. Interestingly, although CRC tissues with higher risk scores had higher infiltration levels of CD8+ T cells, the level of immune checkpoint genes was also high. Moreover, the scRNA-sequencing analysis showed that the expression levels of CD8TDEGs in the prognostic model varied among different types of cells. This study constructed a novel prognostic model for CRC and provided a foundation for targeting CD8+ T cell infiltration to improve the survival of CRC patients.

AHCYL1 Is a Novel Biomarker for Predicting Prognosis and Immunotherapy Response in Colorectal Cancer.

Background Colorectal cancer (CRC) is the third most frequent cancer worldwide. The AHCYL1 gene is required for CNV and has a close association with the tumor immune microenvironment. However, the predictive value of the AHCYL1 gene in patients with CRC remains unknown. Methods AHCYL1 gene with prognostic potential was comprehensively analyzed. Next, using LASSO Cox regression, we fully examined and integrated the AHCYL1 and AHCYL1-related genes from TCGA database. Meanwhile, TCGA database was used to study the connection between AHCYL1 and the tumor immune microenvironment and tumor mutation burden (TMB) in CRC. The influence of AHCYL1 in tumor growth and the recruiting ability of CD8+ T cells were verified, respectively, in vivo and in tissues. To ascertain the connection between AHCYL1 and AHCYL1-related genes and the prognosis of CRC, a prognostic model was created and validated. Result We demonstrated that AHCYL1 has a differential expression and patients with AHCYL1 deletion get shorter survival in CRC. Additionally, the tissues without AHCYL1 have a weaker ability to recruit the natural killer (NK) cell, CD8+ T cells, and tumor-infiltrating lymphocytes (TILs) and response to immunotherapy. Additionally, knockdown of AHCYL1 promoted tumor growth in the CRC mouse model and recruited lower CD8+ T cells in CRC tissues. TCGA database was used to classify patients into low- and high-risk categories based on the expression of four genes. Meanwhile, we discovered an association between the low-risk group and a lower TMB and a higher response to immunotherapy. Finally, a predictive nomogram based on these genes was developed and verified, yielding a C-index of 0.74. Conclusion For CRC patients, the prognostic model based on AHCYL1 and AHCYL1-related genes showed a high predictive performance in terms of prognosis and immunotherapy response.

Siglec-7 is a predictive biomarker for the efficacy of cancer vaccination against metastatic colorectal cancer.

Cancer immunotherapy, including vaccination, is considered a major scientific and medical breakthrough. However, cancer immunotherapy does not result in durable objective responses against colorectal cancer (CRC). To improve the efficacy of immunotherapy, the present study investigated several biomarkers for selecting patients who were expected to respond well to immunotherapy. Firstly, a comprehensive proteomic analysis was performed using tumor tissue lysates from patients enrolled in a phase II study, in which five human leukocyte antigen (HLA)-A*24:02-restricted peptides were administered. Sialic acid-binding immunoglobulin type lectin (Siglec)-7 was identified as a potential predictive biomarker. Subsequently, this biomarker was validated using western blot analysis, and immunofluorescence using tissue samples from the patients enrolled in the phase II study. The expression levels of Siglec-7 detected by immunofluorescence were quantified and their association with overall survival (OS) in patients treated with the peptide vaccine was examined. Furthermore, considering the important role of tumor-infiltrating lymphocytes (TILs) for CRC prognosis, the densities of CD3+, CD4+, CD8+ and forkhead box P3 (FOXP3)+ T cells in CRC tissues were examined and compared with Siglec-7 expression. The mean expression levels of Siglec-7 were significantly higher in patients with poor prognosis, with an OS of ≤2 years, as shown in comprehensive proteomic analysis (P=0.016) and western blot analysis (P=0.025). Immunofluorescence analysis demonstrated that Siglec-7 was expressed in intratumoral macrophages. The OS in patients with high Siglec-7 expression was significantly shorter than in that in patients with low Siglec-7 expression (P=0.017) in the HLA-A*24:02-matched patients. However, this difference was not observed in the HLA-unmatched patients. There was no significant difference in OS between patients according to the numbers of TILs, nor significant correlation between TILs and Siglec-7 expression. In conclusion, Siglec-7 expression in macrophages in tumor tissue may be a novel predictive biomarker for the efficacy of immunotherapy against metastatic CRC.

Characterization of natural killer cells in colorectal tumor tissue of rats fed a control diet or a high-fat diet

BackgroundObesity is a major public health problem with an increasing prevalence reaching pandemic levels. The incidence and mortality for colorectal cancer is augmented in overweight and obese individuals. Previous studies demonstrated an impaired number, phenotype and functionality of natural killer (NK) cells under obese conditions. So far, the influence of obesity on NK cells in colorectal cancer tissue remained unclear. Therefore, the aim of the study was to investigate the occurrence and localization of NK cells in colorectal tumors of normal weight and diet-induced obese rats. MethodsWistar rats were fed a normal-fat diet (control) or a high-fat diet (HFD) to induce obesity. In half of the experimental groups azoxymethane (AOM) was injected to induce colorectal cancer. Tumors in colon and rectum were histopathologically classified in adenomas and adenocarcinomas and immunohistologically stained with the rat NK cell marker CD161. Occurrence and localization of NK cells were analyzed and quantified in the tunica mucosa and tunica submucosa of colorectal adenomas and the tunica submucosa of colorectal adenocarcinomas. ResultsNK cells are localized in the tunica mucosa and the tunica submucosa of colorectal tumors with NK cell accumulations as follicle-like aggregates especially in regions of the lamina muscularis mucosae and the lamina propria mucosae of the tunica mucosa as well as in regions of the tunica submucosa adjacent to the lamina muscularis mucosae. Although not statistically significant, the CD161 staining was clearly reduced in the tunica mucosa of colorectal tumors of rats fed a HFD compared to rats fed a control diet. Moreover, the CD161 staining in the tunica mucosa was positively correlated with the final body weight of AOM-treated rats independent of the supplied diet. DiscussionFor the first time, these results provide information about the localization and quantity of NK cells in colorectal tumor tissue of rats fed a control diet or high-fat diet. The slight reduction of NK cell number in colorectal tissue of rats fed a high-fat diet may contribute to an impaired tumor defense and the increased colorectal tumor outcome in diet-induced obese rats.

Calculation of immune cell proportion from batch tumor gene expression profile based on support vector regression

In addition to tumor cells, a large number of immune cells are found in the tumor microenvironment (TME) of cancer patients. Tumor-infiltrating immune cells play an important role in tumor progression and patient outcome. We improved the relative proportion estimation algorithm of immune cells based on RNA-seq gene expression profiling and solved the multiple linear regression model by support vector regression ([Formula: see text]-SVR). These steps resulted in increased robustness of the algorithm and more accurate calculation of the relative proportion of different immune cells in cancer tissues. This method was applied to the analysis of infiltrating immune cells based on 41 pairs of colorectal cancer tissues and normal solid tissues. Specifically, we compared the relative fractions of six types of immune cells in colorectal cancer tissues to those found in normal solid tissue samples. We found that tumor tissues contained a higher proportion of CD8 T cells and neutrophils, while B cells and monocytes were relatively low. Our pipeline for calculating immune cell proportion using gene expression profile data can be freely accessed from GitHub at https://github.com/gutmicrobes/EICS.git.

Upregulation of adipocyte enhancer-binding protein 1 in endothelial cells promotes tumor angiogenesis in colorectal cancer.

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer‐binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT‐PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target.

Regulatory T cells specifically suppress conventional CD8\u03b1\u03b2 T cells in intestinal tumors of APCMin/+ mice

The presence of activated T cells in colorectal cancer tissues is a strong predictor of patient survival. Our previous studies have shown that regulatory T cells (Treg) are able to reduce T cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in the murine APCMin/+ model for microsatellite stable intestinal tumors. In this study, we investigated the effect of Treg depletion on the density and effector functions of different TCRαβ+ and TCRγδ+ T cell populations in intestinal tumors. We used the APCMin/+\\DEREG mouse model, which harbor a diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the density of conventional TCRαβ+CD8αβ+ T cells was significantly increased in Treg-depleted tumors in comparison with Treg-proficient tumors. Furthermore, TCRαβ+CD8αβ+ T cells showed increased proliferation and activation as well as increased Granzyme B and IFN-γ production in Treg-depleted tumors. In sharp contrast, the densities and effector functions of TCRαβ+CD8αα+ T cells and TCRγδ+ T cells remained unchanged by Treg depletion. We also documented a distinct population of IL-17A+TNF+ TCRγδ+CD8− T cells in tumors, which were not affected by Treg depletion. We conclude that Treg depletion affects only conventional TCRαβ+CD8αβ+ T cells in intestinal tumors, while unconventional T cells and T cells in unaffected tissue are not altered. Immunotherapies aimed at depleting Treg from tumors may thus be a viable option for reinvigoration of conventional cytotoxic T cells with a Th1 cytokine profile.

Targeting Colorectal Cancer Stem Cells as an Effective Treatment for Colorectal Cancer.

As one of the common cancers that threaten human life, the recurrence and metastasis of colorectal cancer seriously affect the prognosis of patients. Although new drugs and comprehensive treatments have been adopted, the current treatment effect on this tumor, especially in advanced colorectal cancer, is still not satisfactory. More and more evidence shows that tumors are likely to be a stem cell disease. In recent years, the rise of cancer stem cell theory has provided a new way for cancer treatment. Studies have found that a small number of special cells in colorectal cancer tissues that induce tumorigenesis, proliferation, and promote tumor migration and metastasis, namely, colorectal cancer stem cells. Colorectal cancer stem cells are defined with a group of cell-surface markers, such as CD44, CD133, CD24, epithelial cell adhesion factor molecule, LGR5, and acetaldehyde dehydrogenase. They are highly tumorigenic, aggressive, and chemoresistant and thus are critical in the metastasis and recurrence of colorectal cancer. Therefore, targeting colorectal cancer stem cells may become an important research direction for the future cure of colorectal cancer.

Distribution of Neuroendocrine Marker-Positive Cells in Colorectal Cancer Tissue and Normal Mucosal Tissue: Consideration of Histogenesis of Neuroendocrine Cancer

Background: Colorectal neuroendocrine carcinoma (NEC) is a rare disease, and mixed cases with colorectal adenocarcinoma also exist. The histogenesis of this disease remains unclear. We studied the numbers of neuroendocrine marker-positive cells in adenocarcinoma tissue and in normal ­mucosal tissue to investigate the relation between adenocarcinoma and NEC and to discuss the histogenesis of NEC. Methods: We studied a total of 354 curatively resected cases of stage II or III colon cancer and 36 cases of rectal cancer treated at the Tokai University Hospital between 2007 and 2012. Adenocarcinoma tissue and normal mucosal tissue were immunohistochemically stained with chromogranin A, synaptophysin, and CD56. Cases in which neuroendocrine marker-positive cells were found in cancer tissue were defined as positive. In normal mucosa, the numbers of positive cells per 15 high-power fields (HPF) were counted. Results: Among the 390 cases, 181 cases had right sided colon cancer, 173 cases had left sided colon cancer, and 36 cases had rectal cancer. The rates of positive staining for chromogranin A, synaptophysin, and CD56 were significantly higher in the right sided colon than in the left sided colon, consistent with the preferred sites of NEC as reported previously. Cells positive for chromogranin A and synaptophysin in normal mucosa were significantly more common in the rectum and the left sided colon than in the right sided colon. No site-specific differences were found for CD56. Conclusions: Neuroendocrine marker-positive cells in colorectal cancer tissue are more common in the right sided colon, whereas neuroendocrine marker-positive cells in normal mucosa are more common in the rectum. These results suggest that NEC may arise from preceding adenocarcinomas.

Prognostic significance of resident CD103+CD8+T cells in human colorectal cancer tissues

The prognostic significance and clinical implications of resident CD103+CD8+T cells in human colorectal cancer tissues still remains largely unexplored. In our present study, we aimed to characterize the resident CD8+T cells in human colorectal cancer tissues by using double staining of CD103 and CD8, and further evaluated the prognostic significance of resident CD8+T cells in colorectal cancer. We found that the OS rate of the colorectal cancer patients with higher infiltration of CD8+T cells, or with higher numbers of resident CD103+CD8+T cells, or with higher ratio of CD103+CD8+T cells over total CD8+T cells in cancer tissues was significantly better than that of the patients with lower infiltration of CD8+T cells, or with lower numbers of resident CD103+CD8+T cells, or with higher ratio of CD103+CD8+T cells over total CD8+T cells in cancer tissues, respectively. Moreover, higher infiltration of CD8+T cells in colorectal cancer tissues was significantly and inversely correlated with advanced TNM stage. Higher numbers of resident CD103+CD8+T cells in colorectal cancer tissues were significantly and inversely correlated with distant metastasis status. Higher ratio of CD103+CD8+T cells over total CD8+T cells in colorectal cancer tissues was significantly and inversely correlated with age status. The COX model analysis demonstrated that higher infiltration of CD8+T cells, higher numbers of resident CD103+CD8+T cells, or higher ratio of CD103+CD8+T cells over total CD8+T cells in colorectal cancer tissues, could serve as independent prognostic predictors for colorectal cancer patients. Taken together, our present study demonstrated the density of tumor infiltrating CD8+T cells or the numbers of resident CD103+CD8+T cells in colorectal tissues could be used as an important prognostic predictor for this malignancy.

Distribution of neuroendocrine marker-positive cells in colorectal cancer tissue and adjacent mucosa.

617 Background: Neuroendocrine carcinoma (NEC) is a rare disease and has been reported to most frequently arise in the right side of the colon. In the 2010 WHO classification, mixed adenoneuroendocrine carcinoma (MANEC) was defined as a neoplasm consisting of NEC and adenocarcinoma components. To clarify the histogenesis of NEC, we attempted to detect neuroendocrine marker-positive cells in cancer tissue and in the adjacent mucosa in patients with adenocarcinoma. Methods: The study group comprised 390 patients with Stage II or III colorectal adenocarcinoma between 2007 and 2012. Immunostaining was performed with anti chromogranin A, synaptophysin, and CD56 antibodies. Cases with positively stained cells in cancer tissue were defined as positive. In the adjacent mucosa, at least 5 cm from the tumor, the numbers of positive cells per 15 HPF were measured. Results: Tumor location was right side in 181 patients, left side in 173, and the rectum in 36 patients. Positive rates of Chromogranin A in cancer tissues were 23.7% in the right colon, 13.2% in the left colon, and 19.4% in the rectum. Those of synaptopysin were 35.3%, 21.9%, and 30.6%, respectively. Those of CD56 were 22.6%, 8.0%, and 16.7%, respectively. Positive rates of these three markers in right colon were significantly higher than those in left colon and rectum. (p = 0.0115, p = 0.0054, p = 0.0062). In the adjacent mucosa, the mean numbers of positive cells for chromogranin A were 62.2 ± 20.5 in the right colon, 131.9 ± 44.7 in the left colon, and 243.7 ± 60.2 in the rectum (p &lt; 0.001). Those for synaptophysin were 47.7 ± 23.5, 95.3 ± 35.1, and 156.9 ± 56.8, respectively. (p &lt; 0.001). There were no significant differences in the number of positive cells for CD56 among the sites (p = 0.295). Conclusions: In cancer tissue, the rate of positive staining for neuroendocrine marker-positive cells was higher in the right side of the colon, whereas in normal mucosa the rates of positive staining for these cells were higher in the sigmoid colon and the rectum. These results suggest that neuroendocrine marker-positive cells are an acquired characteristic of cancer tissue.

Impact of pigment epithelium-derived factor on colorectal cancer in vitro and in vivo.

Pigment epithelial derived factor (PEDF) is a secreted glycoprotein that is a non-inhibitory member of the serine protease inhibitor (serpin) family. PEDF exhibits multiple biological properties including neuroprotective, anti-angiogenic, and immune-modulating. Interestingly, PEDF exerts the inhibitory effects in cancers derived from certain tissues, including prostatic, ovarian, and pancreatic carcinomas. The current study aimed to elucidate its role in colorectal cancer development. PEDF expression in human colorectal cancer tissue was assessed using quantitative polymerase chain reaction (qPCR) and immunohistochemical staining (IHC). The effect of treatment with recombinant PEDF on cellular function was examined using in vitro functional assays. PEDF expression was downregulated in colorectal cancer cell tissue. Treatment with recombinant PEDF resulted in significant decreases in the rate of colorectal cancer cell migration and invasion and an increase in cellular adhesion in colorectal cancer cell lines examined. These results indicate that upregulation of PEDF expression may serve as a new strategy for further investigation of therapeutic relevance to the prevention of the metastatic spread of colorectal cancer.

Osteopontin-integrin engagement induces HIF-1α-TCF12-mediated endothelial-mesenchymal transition to exacerbate colorectal cancer.

Osteopontin (OPN) is a multi-functional phospho-glycoprotein that can stimulate angiogenesis through acting on endothelial cells. As angiogenic sprouting involves endothelial-to-mesenchymal transition (EndoMT), we are intrigued to know whether OPN exerts an effect on EndoMT. Clinically, we indeed detected EndoMT-derived cells next to OPN-expressing cells in colorectal cancer tissues. Furthermore, we treated OPN to primary cultures of endothelial cells to investigate the EndoMT-inducing activity and the underlying mechanisms. Integrin αVβ3 rather than CD44 is involved in OPN-induced EndoMT. OPN-integrin αVβ3 engagement induces HIF-1α expression through a PI3K/Akt/TSC2-mediated and mTORC1-dependent protein synthesis pathway, which in turn trans-activates TCF12 gene expression. TCF12 further interacts with EZH2 and histone deacetylases to transcriptionally repress VE-cadherin gene and thus facilitates EndoMT. Like cancer-associated fibroblasts, EndoMT-derived cells promote tumor growth and metastasis by secreting certain proteins. Secreted HSP90α is a candidate suggested by microwestern array assay, and is herein verified to induce stemness properties in colorectal cancer cells. As OPN is overexpressed in human cancers, OPN-induced EndoMT and EndoMT-derived cells can be potentially taken as cancer therapeutic targets.

Expression of P4H9-detecting molecule on spindle-shaped fibroblasts to indicate malignant phenotype of colorectal cancer.

539 Background: Cancer cells and fibroblasts are coordinated for cancer progression. Our previous study has shown that P4H9, produced by epitope for b2 integrin, detected a molecule on fibroblasts in mammary fat pad mouse model in response to Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressing cancer cells. P4H9 detecting molecule (PDM) expression appeared to be associated with myofibroblast differentiation. The aim of the present study is to investigate whether PDM express on fibroblast and cancer cells in clinical tissue samples, and whether PDM expressing cells in colorectal cancer tissue are correlated with clinicopathological features of patients with colorectal cancer. Methods: Immunohistochemistry were conducted with P4H9 on clinical tissue samples from 156 patients with colorectal cancer. The risk factors for metastases and survival were calculated for clinical implication of PDM expressing spindle shaped fibroblasts. Immunofluorescence with P4H9 were performed on CCD-18Co fibroblasts and colorectal cancer cell lines, HT29 and HCT116. Results: Multivariate analysis showed that PDM expressing spindle shaped fibroblasts was an independent risk factor for lymph node metastasis, hematogenous metastasis, and short survival. Kaplan-Meier survival curve presented that PDM expressing spindle shaped fibroblasts was associated with shorter survival time (p&lt; 0.0001). PDM expressing spindle shaped fibroblasts is associated with metastasis and shorter survival of the patients with colorectal cancer. Immunofluorescence showed that PDM expressed on CCD-18Co fibroblasts and colorectal cancer cell lines, HCT116 with fibroblast like morphology. Conclusions: PDM expressing spindle shaped fibroblasts may play a role to bring out malignant phenotype of colorectal cancer. PDM may be a new biomarker and a novel therapeutic target for patients with colorectal cancer.