Tissues Of Colorectal Cancer Patients Research Articles

LncRNA VLDLR-AS1 Gene Expression in Colorectal Cancer in Patients from East Azerbaijan Province, Iran

Background: Colorectal cancer (CRC) is the third most common cancer that frequently spreads to other parts of the body, with a low chance of recovery and a high mortality rate. Long non-coding RNAs are considered significant prognostic and diagnostic indicators because they are dysregulated in various cancers and have distinctive expression patterns and high tissue- and cell-specificity. VLDLR-AS1 lncRNA deregulation has been associated with several malignancies. Objectives: This study aimed to evaluate the expression levels of VLDLR-AS1 in CRC. It was the first time this assessment was conducted. Methods: We studied 188 samples, including 94 tumor samples and 94 paired adjacent non-tumor tissues. Total RNA was extracted, and its quantity and purity were assessed. TaKaRa PrimeScript 1st Strand cDNA Synthesis Kit (Kusatsu, Japan) was used for the reaction. The StepOnePlus Real-Time PCR System (Applied Biosystems) was set up to assess the relative expression of VLDLR-AS1. Results: According to the qRT-PCR data, the VLDLR-AS1 expression levels in CRC tissues were significantly lower than in tumor margins (P < 0.0001). In addition, receiver operating characteristic curve analysis demonstrated that VLDLR-AS1 expression can discriminate between tumor and non-tumor samples with the sensitivity and specificity of 72.34% and 51.06%, respectively (P = 0.03, AUC = 0.6274). However, no significant association was found between the expression levels of VLDLR-AS1 and clinicopathological features in CRC patients. Conclusions: The results of this study indicated that VLDLR-AS1 lncRNA is significantly downregulated in the tumor tissues of CRC patients compared to healthy tumor margin tissues. This evidence shows the potential of this gene as a promising biomarker for the early detection of CRC development.

Impaired Expression of the Salvador Homolog-1 Gene Is Associated with the Development and Progression of Colorectal Cancer.

Salvador homolog-1 (SAV1) is a component of the Hippo pathway that regulates tissue growth and homeostasis by affecting diverse cell processes, including apoptosis, cell division, and differentiation. The aberrant expression of Hippo pathway components has been observed in various human cancers. This study aimed to examine the expression level of the SAV1 gene in colorectal cancer (CRC) and its prognostic value and associations with tumor progression. We obtained matched pairs of tumor tissue and non-cancerous mucosa of the large intestine from 94 CRC patients as well as 40 colon biopsies of healthy subjects collected during screening colonoscopy. The tissue samples and CRC cell lines were quantified for SAV1 mRNA levels using the quantitative polymerase chain reaction method, while SAV1 protein expression was estimated in the paired tissues of CRC patients using immunohistochemistry. The average level of SAV1 mRNA was decreased in 93.6% of the tumor tissues compared to the corresponding non-cancerous tissues and biopsies of healthy colon mucosa. A downregulated expression of SAV1 mRNA was also noted in the CRC cell lines. Although the average SAV1 immunoreactivity was increased in the CRC samples compared to the non-cancerous tissues, a decreased immunoreactivity of the SAV1 protein in the tumor specimens was associated with lymph node involvement and higher TNM disease stage and histological grade. The results of our study suggest that the impaired expression of SAV1 is involved in CRC progression.

The Role of CPNE7 (Copine-7) in Colorectal Cancer Prognosis and Metastasis.

Colorectal cancer (CRC) is one of the most common and deadly cancers in the world. However, no effective treatment for the disease has yet been found. For this reason, several studies are being carried out on the treatment of CRC. Currently, there is limited understanding of the role of CPNE7 (copine-7) in CRC progression and metastasis. The results of this study show that CPNE7 exerts an oncogenic effect in CRC. First, CPNE7 was shown to be significantly up-regulated in CRC patient tissues and CRC cell lines compared to normal tissues according to IHC staining, qRT-PCR, and western blotting. Next, this study used both systems of siRNA and shRNA to suppress CPNE7 gene expression to check the CPNE7 mechanism in CRC. The suppressed CPNE7 significantly inhibited the growth of CRC cells in in vitro experiments, including migration, invasion, and semisolid agar colony-forming assay. Moreover, the modified expression of CPNE7 led to a decrease in the levels of genes associated with epithelial-mesenchymal transition (EMT). The epithelial genes E-cadherin (CDH1) and Collagen A1 were upregulated, and the levels of mesenchymal genes such as N-cadherin (CDH2), ZEB1, ZEB2, and SNAIL (SNAL1) were downregulated after CPNE7 inhibition. This study suggests that CPNE7 may serve as a potential diagnostic biomarker for CRC patients.

Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer

BackgroundCancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.MethodsSpheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.ResultsCircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.ConclusionsCircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

SARS‐CoV‐2 virus associated angiotensin converting enzyme 2 expression modulation in colorectal cancer: Insights from mRNA and protein analysis COVID-19 associated (ACE2) expression in colorectal cancer

The SARS‐CoV‐2 virus gains entry into human cells by exploiting the angiotensin‐converting enzyme 2 (ACE2), a key component known as the spike protein (S), as a point of entry. Initially, SARS‐CoV‐2 suppresses the natural function of ACE2, leading to a gradual decline in cell health. Additionally, individuals with cancer are considered more susceptible to COVID-19. This study investigates the expression patterns of ACE2 in colorectal cancer (CRC) patients with and without a history of COVID-19 infection. RT-PCR was used to analyze samples from both cancerous and adjacent non-affected colorectal tissues of 47 CRC patients, comprising two groups: 24 CRC patients with no history of COVID-19 and 23 CRC patients with a recent history of COVID-19 infection. Epithelial CR cells were isolated from both types of tissues and cultured to evaluate cell adhesion. Immunohistochemistry analyses were conducted to examine ACE2 protein expression using various ACE2 antibodies for both cell types. The study revealed ACE2 mRNA expression in all CRC tissues of patients with and without a history of COVID-19. ACE2 expression was significantly higher in CRC patients without a history of COVID-19. Notably, the non-affected colorectal cancer (NACRC) tissues of patients without a history of COVID-19 also showed ACE2 expression, whereas no ACE2 expression was detected in the biopsies of CRC patients with a positive COVID-19 history. ACE2 antibodies were employed to validate ACE2 protein expression at the mRNA level. COVID-19 appears to downregulate ACE2 expression in both CRC and NACRC tissues of CRC patients with a positive history of COVID-19 infection.

Bifidobacterium adolescentis induces Decorin+ macrophages via TLR2 to suppress colorectal carcinogenesis

BackgroundThe interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is largely unknown. Here, we elucidated the functional role of B. adolescentis and its possible mechanism on the manipulation of Decorin+ macrophages in colorectal cancer.MethodsThe relative abundance of B. adolescentis in tumor or para-tumor tissue of CRC patients was analyzed. The role of B. adolescentis was explored in the CRC animal models. The single cell-RNA sequencing (scRNA-seq) was used to investigate the myeloid cells subsets in TME. The expression level of TLR2/YAP axis and its downstream Decorin in macrophages were tested by Western blot and qRT-PCR. Knockdown of Decorin in Raw264.7 was performed to investigate the effect of Decorin+ macrophages on subcutaneous tumor formation. Multi-immunofluorescence assay examined the number of Decorin+ macrophages on the CRC tissue.ResultsWe found that the abundance of B. adolescentis was significantly reduced in tumor tissue of CRC patients. Supplementation with B. adolescentis suppressed AOM/DSS-induced tumorigenesis in mice. ScRNA-seq and animal experiment revealed that B. adolescentis increased Decorin+ macrophages. Mechanically, Decorin was activated by TLR2/YAP axis in macrophages. The abundance of B. adolescentis was correlated with the number of Decorin+ macrophages and the expression level of TLR2 in tumor tissue of CRC patients.ConclusionsThese results highlight that B. adolescentis induced Decorin+ macrophages and provide a novel therapeutic target for probiotic-based modulation of immune microenvironment in CRC.

Prognostic value of the levels of CTLA-4 and its ligand B7.2 in patients with colorectal cancer

Aim. To develop a computer program to determine the probability of colorectal cancer based on the assessment of the levels of CTLA-4 and its ligand B7.2.Materials and methods. The study included 44 patients with colorectal cancer (CRC) and 25 patients with benign tumors of the colon. The control group consisted of 25 individuals who had been operated for colon injury. We determined the levels of CTLA-4 and B7.2 in the blood serum and in the supernatants of tumor tissue and lymph node homogenates using flow cytofluorometry.Results. We found that the level of CTLA-4 in the blood serum increased by 2.77 times in CRC patients compared to the control group (p < 0.001). The concentration of CTLA-4 in the tumor tissue in patients with CRC was 2.34 times higher than in the control group (p = 0.007). The concentration of the B7.2 ligand in the blood serum of patients with CRC exceeded this parameter in the control group by 2.51 times (p = 0.002). The concentration of B7.2 in the tumor tissue of CRC patients was 1.68 times higher (p = 0.004) than in the control group. The analysis of the obtained data determined the parameters that have prognostic value in the structure of the diagnostic model. Using these parameters, we developed a computer program to determine the probability of CRC in the patient.Conclusion. The data obtained demonstrate an increase in the levels of CTLA-4 and its ligand B7.2 in the serum and tumor tissue of patients with CRC.

MicroOrganoSpheres as a clinically applicable precision oncology platform for the discovery of novel therapies in colorectal cancer.

3592 Background: Patient-derived models of cancer, such as cell lines, patient-derived organoids, and patient-derived xenografts, are useful models of patient response in the clinic. However, these models are often not clinically applicable within the time periods necessary to inform clinical decision making, as they can take weeks to months to develop. An ideal platform using patient-derived models would be generated from a core biopsy with a subsequent drug screen within 10-14 days to minimize delay in therapy. We recently reported the development of MicroOrganoSpheres (MOS) that can be used in drug screens within 14 days of obtaining a biopsy. In the current study, we use this MOS system as precision oncology platform in colorectal cancer (CRC) to identify new therapies and predict response to therapy. Methods: CRC patient tissue samples were collected under a Duke Institutional Review Board approved protocol (Pro00089222). Resections or biopsies were mechanically and enzymatically digested to obtain a single cell suspension. Cells were then plated in Matrigel at a ratio of 20,000 cells:5 µL Matrigel to establish “mini-bulk” organoid cultures. After establishment for 5-7 days, cultures were harvested with subsequent generation of MOS at a ratio of 50 cells per MOS. After growing for 3-4 days, MOS were used for dose-response curves using oxaliplatin, SN38, and 5-Fluorouracil (5-FU) as well as high-throughput drug screens with the NCI Approved Oncology Drugs Set VI library. Results: We developed and optimized a MOS pipeline on over 50 CRC specimens, including 9 primary rectal, 35 primary CRC, 12 CRC liver metastasis, and 1 CRC lung metastasis lines with a success rate of 80% and an average of 10-21days from biopsy to MOS generation. The high success of generating CRC MOS in a clinically applicable time frame led to the next phase of the project where a total of 10 CRC MOS were tested against standard of care chemotherapy agents used in CRC (oxaliplatin, irinotecan and 5-FU) as well as the NCI Approved Oncology Drugs Set VI within 14-21days of establishment. We noted a range of sensitivity of approximately 100-fold for standard of care agents. The most sensitive drugs found in the high-throughput screen were Bortezomib, Carfilzomib, and Panobinostat and the most resistant were Gefitinib, Chlorambucil, and Procarbazine hydrochloride. Conclusions: These results demonstrate that our MOS pipeline can be used as a precision oncology platform within a clinically applicable time frame to potentially guide therapy. We are now in the process of correlating drug response in MOS to patient outcome data and these findings will be presented at the annual meeting.

KLF2 inhibits colorectal cancer progression and metastasis by inducing ferroptosis via the PI3K/AKT signaling pathway.

Krüppel-like factor 2 (KLF2) belongs to the zinc finger family and is thought to be a tumor suppressor gene due to its low expression in various cancer types. However, its functional role and molecular pathway involvement in colorectal cancer (CRC) are not well defined. Herein, we investigated the potential mechanism of KLF2 in CRC cell invasion, migration, and epithelial-mesenchymal transition (EMT). We utilized the TCGA and GEPIA databases to analyze the expression of KLF2 in CRC patients and its correlation with different CRC stages and CRC prognosis. RT-PCR, western blot, and immunohistochemistry assays were used to measure KLF2 expression. Gain-of-function assays were performed to evaluate the role of KLF2 in CRC progression. Moreover, mechanistic experiments were conducted to investigate the molecular mechanism and involved signaling pathways regulated by KLF2. Additionally, we also conducted a xenograft tumor assay to evaluate the role of KLF2 in tumorigenesis. KLF2 expression was low in CRC patient tissues and cell lines, and low expression of KLF2 was associated with poor CRC prognosis. Remarkably, overexpressing KLF2 significantly inhibited the invasion, migration, and EMT capabilities of CRC cells, and tumor growth in xenografts. Mechanistically, KLF2 overexpression induced ferroptosis in CRC cells by regulating glutathione peroxidase 4 expression. Moreover, this KLF2-dependent ferroptosis in CRC cells was mediated by inhibiting the PI3K/AKT signaling pathway that resulted in the suppression of invasion, migration, and EMT of CRC cells. We report for the first time that KLF2 acts as a tumor suppressor in CRC by inducing ferroptosis via inhibiting the PI3K/AKT signaling pathway, thus providing a new direction for CRC prognosis assessment and targeted therapy.

ATG4B and pS383/392-ATG4B serve as potential biomarkers and therapeutic targets of colorectal cancer

BackgroundAutophagy related protease 4B (ATG4B) is a protease required for autophagy processing, which is strongly implicated in cancer progression. Phosphorylation of ATG4B is crucial for activation of its protease activity. However, little is known about the relationship of ATG4B and its phosphorylated form at Ser 383 and 392 sites (pS383/392-ATG4B), with clinical outcomes, particularly in colorectal cancer (CRC).MethodsThe ATG4B gene expression in CRC patients was obtained from The Cancer Genome Atlas (TCGA) database to analyze its clinical relevance. Tissue microarrays composed of 118 CRC patient specimens were used to determine the associations of ATG4B and pS383/392-ATG4B protein levels with prognosis. The biological functions of ATG4B in CRC cells were inspected with cell proliferation, mobility and spheroid culture assays.ResultsATG4B gene expression was elevated in tumor tissues of CRC patients compared to that in adjacent normal tissues and high level of ATG4B expression was associated with poor survival. Similarly, protein levels of ATG4B and pS383/392-ATG4B were highly correlated with worse overall survival and disease-free survival. Stratification analysis results showed that high level of ATG4B had significantly higher risk of mortality in males and elderly patients compared to those female patients and patients 60 years or younger. In contrast, multivariate Cox’s regression analysis indicated that high level of pS383/392-ATG4B was significantly linked to unfavorable overall survival and disease-free survival of males and elderly patients, whereas, it had no correlation with female patients and patients 60 years or younger. Moreover, high level of ATG4B was positively associated with increased mortality risk in patients with advanced AJCC stages (III and IV) and lymph node invasion (N1 and N2) for both overall survival and disease-free survival. Nevertheless, high level of pS383/392-ATG4B was positively correlated with increased mortality risk in patients with early AJCC stages (I and II) and without lymph node invasion (N0). In addition, silencing ATG4B attenuated migration, invasion, and further enhanced the cytotoxic effects of chemotherapeutic drugs in two and three-dimensional cultures of CRC cells.ConclusionsOur results suggest that ATG4B and pS383/392-ATG4B might be suitable biomarkers and therapeutic targets for CRC.

Abstract 2120: Exploring the genomic landscape of colorectal cancer for enabling clinical genomics informed and real-world data based clinical development

Abstract Background: Activation of the MAPK-signaling by RAS- (KRAS or NRAS) alterations is an important driver event in colorectal cancer (CRC) tumorigenesis that determines treatment selection (1).Real-world data (RWD) mining is an important tool to inform patient selection and stratification (2,3). Here, RWD genomics data mining was conducted using the Foundation Medicine database to explore RAS and BRAF-alterations in CRC patients and to investigate the general alterations’ landscape - including rare alterations. The prevalence of microsatellite instability (MSI) and tumor mutational burden (TMB) were also explored. Methods: The prevalence of gene alterations and their co-occurrence with RAS/BRAF-alterations in tumor tissue of CRC patients (N=42800) were investigated using the FoundationInsightsTM web platform, which includes harmonized results from the FoundationOneCDx, FoundationOneHeme, and PD-L1 assays from 2012 to March 2022. Data was collected from FMI solid and heme tests from 2012 to March 2022. Alterations that render a particular gene ‘altered’ included single nucleotide variants (SNVs) or insertion-deletion mutations (Indels), collectively referred to as short variants (SVs), rearrangements (RE) and copy number events (CN). TMB (cut-off 10 mut/MB) and MSI status were also characterized. Results: Altogether, 36.9% of CRC samples were RAS/BRAF-WT and 63.1% had a known or likely functional RAS/BRAF-alteration, respectively. Of all KRAS and NRAS alterations (SV, CN and RE regardless of annotation as functional or presence in a hotspot), SVs in exons 2, 3 and 4 constituted 96.1% and 94.2% of all alterations, respectively. Relative to all samples the potentially targetable KRAS p.G12C, p.G12D and p.G12V mutations were present in 3.6%, 15.1% and 10.2% of samples, respectively. The p.G12E variant was present in less than ten samples. The five most frequently altered potentially targetable genes were PIK3CA (19.6%), PTEN (8.7%), ERBB2 (4.7%), EGFR (2.5%) and FGFR1 (2.0%). NTRK-alterations (NTRK1/2/3) were present in 0.9%, ROS1-alterations in 0.4%, RET-alterations in 0.6% and ALK-alterations in 0.5% of samples, respectively. The prevalence of immuno-oncology biomarkers was also explored; overall, 9.6% of samples were TMB-high, 6.1% MSI-high and 0.6% POLE-altered. MSI-high or TMB-high samples were significantly more abundant in the RAS/BRAF-altered subset. In the RAS/BRAF-WT subset 4.6% were MSI-high and 7.9% TMB-high, whereas in the RAS/BRAF-altered subset 7.1% were MSI-high and 10.6% TMB-high. Conclusion: RWD based clinical genomic profiling confirms that the vast majority of RAS-alterations in CRC are SVs in exons 2, 3 and 4. Both TMB-high and MSI-high samples are significantly more abundant in the RAS/BRAF-altered cohort. RWD confirms that PI3K-, ERBB- and FGFR-signaling pathways are frequently altered in CRC. RWD based clinical genomic profiling has the potential to support comprehensive clinical study design as well as patient selection/stratification. Citation Format: Markus Schulze, XiaoZhe (Janet) Wang, Jawad Hamad, Julia F. Hopkins, Julia F. Hopkins, Jürgen Scheuenpflug, Zheng Feng. Exploring the genomic landscape of colorectal cancer for enabling clinical genomics informed and real-world data based clinical development [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 2120.

Abstract 4322: AI assisted detection and characterization of tertiary lymphoid structures in patients with colorectal cancer

Abstract Tertiary Lymphoid Structures (TLSs) are ectopic immune cell aggregates that form in response to abnormal physiology and inflammation. TLS presence in the tumor environment has been associated with positive clinical outcomes and response to immune checkpoint inhibition therapy. Using a fluorescent multiplex panel consisting of Pan-cytokeratin (PanCK), Programmed Death Ligand-1 (PD-L1), cluster of differentiation (CD) 8, CD3, and CD68 we developed a HALO-based artificial intelligence (AI) classifier to detect TLSs in colorectal cancer (CRC) tissue. In conjunction with cell-type and spatial analyses, we used the AI TLS classifier to characterize several features of the TLS and peri-TLS environment. We trained the AI classifier using 51 examples of TLSs from 8 patients, and applied it to images of 21 CRC patient tissues. TLSs were detected in 9/21 patient samples. We then used cellular detection and spatial analysis to characterize features of the TLS, including the germinal center (GC), T cell zone and peri-TLS area. We detected a higher cell density (Mean=12250 ±2039 SD; cells/mm2) within the interior of AI-detected GCs relative to the 1st 200 μm outside of the AI-defined GC border (8005±1870). In addition, we examined the spatial distribution of CD3+ and CD8+ T cells relative to the TLS GC. We found elevated CD3+ cell density in the interior of the GC (5784±1649) and within 200 μm of the GC border (5126±2239) which decreased at distances >200 μm from the GC border. We found CD8+ density to be elevated within the 1st 200 μm of the GC border (1903±1182) and declined 400 μm away from the GC border (963±828). The spatial distribution of T cells surrounding an area of high cell density is indicative of a T cell zone surrounding germinal center. Further analysis of the AI-detected TLS GCs indicated that PD-L1+ cell density was highest within the germinal center (355± 534;9) relative to the exterior of the germinal center (1st 150μm outside germinal center:276± 441). We created an AI classifier that can detect TLS(s) within patient tissue in the absence of a B-cell marker and confirmed that our AI classifier was detecting TLSs by characterizing canonical features of TLSs. Further exploration of the TLS and peri-TLS area may offer insights into immune-tumor interaction within the tumor microenvironment, and paired detection of TLSs in a clinical cohort with outcome data may predict patient response to immune checkpoint inhibitor therapy. Citation Format: Samantha Burg, Nathaniel Hart, Astin Powers, Wenjun Zhang. AI assisted detection and characterization of tertiary lymphoid structures in patients with colorectal cancer. [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 4322.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer

BackgroundAdenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition.MethodsThis study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients’ data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used.ResultsBy TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells’ migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells’ migration.ConclusionsAct1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.

Bovine meat and milk factor protein expression in tumor-free mucosa of colorectal cancer patients coincides with macrophages and might interfere with patient survival.

Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n= 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68+/CD163+ macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC.

Identification and characterization of four bacteriome- and mycobiome-derived subtypes in tumour and adjacent mucosa tissue of colorectal cancer patients.

214 Background: The bacteriome and mycobiome, collectively referred to as the microbiome, is a key player in CRC pathogenesis, progression and response to chemotherapy, radiotherapy and immunotherapy. Microbial modulation and exacerbation in CRC is likely attributable to alterations of composition and diversity of the microbiome rather than to a single microbial species. We therefore systematically investigated alterations in the bacteriome and mycobiome of CRC patients in tumours and matched adjacent mucosa resulting in the identification of microbiome-based subtypes associated with host clinico-pathological and molecular characteristics. Methods: We estimated bacterial and fungal composition from RNA sequencing experiments using a validated subtractive method ( PathSeq) in in-house and publicly available cohorts. We evaluated the quality of the microbial estimates with two sets of orthogonal analysis determining i) the agreement with composition from an independent method and ii) retrieval of expected clinically-relevant microbial signatures. Diversity and composition of bacteriome and mycobiome of tumour and adjacent mucosa, and resulting subtypes were computationally deconvoluted using > 10,000 samples. Results: The bacteriome of tumours had higher dominance and lower ɑ-diversity compared to matched adjacent local and distant mucosa. Tumours were enriched with Proteobacteria (Gammaproteobacteria class), Fusobacteria (including Fusobacterium Nucleatum species) and Basidiomycota fungi (Malasseziaceae family). Tumours were depleted of Bacteroidetes (Bacteroidia class), Firmicutes (Clostridia class) and Ascomycota (Sordariomycetes and Saccharomycotina). Tumours and adjacent mucosa samples were classified into 4 microbial subtypes, termed C1 to C4, based on the bacteriome and mycobiome composition. The bacterial Propionibacteriaceae, Enterobacteriaceae, Fusobacteriaceae, Bacteroidaceae and Ruminococcaceae along with the fungal Malasseziaceae, Saccharomycetaceae and Aspergillaceae were among the key families driving the microbial subtyping. Microbial subtypes were associated with distinct tumour histology and patient phenotypes and served as independent prognostic marker for disease-free survival. Key associations between microbial subtypes and alterations in host immune response and signalling pathways were validated in the TCGA pan-cancer cohort. The microbial subtyping demonstrated stratification value in the pan-cancer settings beyond merely representing differences in survival by cancer type. Conclusions: This study demonstrates the translational potential of microbial subtyping in CRC patient stratification, and provides avenues to design tailored microbiota modulation therapy to further precision oncology.

MicroRNA-455-3p accelerate malignant progression of tumor by targeting H2AFZ in colorectal cancer

ABSTRACT Colorectal cancer (CRC) becomes the second leading cause of cancer-related deaths in 2020. Emerging studies have indicated that microRNAs (miRNAs) play a key role in tumorigenesis and progression. The dysfunctions of miR-455-3p are observed in many cancers. However, its biological function in CRC remains to be confirmed. By sequencing serum sample, miR-455-3p was found to be up-regulated in CRC patients. RT-qPCR demonstrated that the miR-455-3p expression was both higher in the serum and tumor tissues of CRC patients. Furthermore, it indicated that miR-455-3p had the ability in promoting cell proliferation, suppressing cell apoptosis, and stimulating cell migration. In vivo experiments also showed that miR-455-3p promoted tumor growth. Additionally, H2AFZ was proved as the direct gene target of miR-455-3p by dual-luciferase assay. Taken together, miR-455-3p functioned as a tumor promoter in CRC development by regulating H2AFZ directly. Thus, it has enormous potential as a biomarker in the diagnosis of CRC.

Oxidized low-density lipoprotein stimulates CD206 positive macrophages upregulating CD44 and CD133 expression in colorectal cancer with high-fat diet.

BACKGROUNDOxidized low-density lipoprotein (ox-LDL), which is abnormally increased in the serum of colorectal cancer (CRC) patients consuming a high-fat diet (HFD), may be one of the risk factors for the development of CRC. Ox-LDL exerts a regulatory effect on macrophages and may influence CRC through the tumor microenvironment. The role of ox-LDL in CRC remains unclear.AIMTo investigate the role of ox-LDL through macrophages in HFD associated CRC.METHODSThe expression of ox-LDL and CD206 was detected in colorectal tissues of CRC patients with hyperlipidemia and HFD-fed mice by immunofluorescence. We stimulated the macrophages with 20 μg/mL ox-LDL and assessed the expression levels of CD206 and the cytokines by cell fluorescence and quantitative polymerase chain reaction. We further knocked down LOX-1, the surface receptor of ox-LDL, to confirm the function of ox-LDL in macrophages. Then, LoVo cells were co-cultured with the stimulated macrophages to analyze the CD44 and CD133 expression by western blot.RESULTSThe expression of ox-LDL and the CD206 was significantly increased in the stroma of colorectal tissues of CRC patients with hyperlipidemia, and also upregulated in the HFD-fed mice. Moreover, an increased level of CD206 and decreased level of inducible nitric oxide synthase were observed in macrophages after ox-LDL continuous stimulation. Such effects were inhibited when the surface receptor LOX-1 was knocked down in macrophages. Ox-LDL could induce CD206+ macrophages, which resulted in high expression of CD44 and CD133 in co-cultured LoVo cells.CONCLUSIONOx-LDL stimulates CD206+ macrophages to upregulate CD44 and CD133 expression in HFD related CRC.

A novel long noncoding RNA SP100-AS1 induces radioresistance of colorectal cancer via sponging miR-622 and stabilizing ATG3

Although radiotherapy is an essential modality in the treatment of colorectal cancer (CRC), the incidence of radioresistance remains high clinically. Long noncoding RNAs (lncRNAs) reportedly play critical roles in CRC radioresistance by regulating genes or proteins at the transcriptional or post-translational levels. This study aimed to identify novel lncRNAs involved in radioresistance. We found that SP100-AS1 (lncRNA targeting antisense sequence of SP100 gene) was upregulated in radioresistant CRC patient tissues using RNA-seq analysis. Importantly, knockdown of SP100-AS1 significantly reduced radioresistance, cell proliferation, and tumor formation in vitro and in vivo. Mechanistically, mass spectrometry and bioinformatics analyses were used to identify the interacting proteins and microRNAs of SP100-AS1, respectively. Moreover, SP100-AS1 was found to interact with and stabilize ATG3 protein through the ubiquitination-dependent proteasome pathway. In addition, it could serve as a sponge for miR-622, which targeted ATG3 mRNA and affected autophagic activity. Thus, lncRNA SP100-AS1 could act as a radioresistance factor in CRC patients via RNA sponging and protein stabilizing mechanisms. In conclusion, the present study indicates that SP100-AS1/miR-622/ATG3 axis contributes to radioresistance and autophagic activity in CRC patients, suggesting it has huge prospects as a therapeutic target for improving CRC response to radiation therapy.

Parvimonas micra promotes colorectal tumorigenesis and is associated with prognosis of colorectal cancer patients

Large-scale fecal shotgun metagenomic sequencing revealed the high abundance of Parvimonas micra in colorectal cancer (CRC) patients. We investigated the role and clinical significance of P. micra in colorectal tumorigenesis. The abundance of P. micra was examined in 309 fecal samples and 165 colon biopsy tissues of CRC patients and healthy subjects. P. micra was significantly enriched in fecal samples from 128 CRC patients compared to 181 healthy subjects (P < 0.0001); and in colon tissue biopsies from 52 CRC patients compared to 61 healthy subjects (P < 0.0001). Multivariate analysis showed that P. micra is an independent risk factor of poor survival in CRC patients (Hazard Ratio: 1.93). P. micra strain was isolated from feces of a CRC patient. Apcmin/+ mice gavaged with P. micra showed significantly higher tumor burden and tumor load (both P < 0.01). Consistently, gavage of P. micra significantly promoted colonocyte proliferation in conventional mice, which was further confirmed by germ-free mice. P. micra colonization up-regulated genes involved in cell proliferation, stemness, angiogenesis and invasiveness/metastasis; and enhanced Th17 cells infiltration and expression of Th17 cells-secreted cytokines (Il-17, Il-22, and Il-23) in the colon of Apcmin/+, conventional and germ-free mice. P. micra-conditioned medium significantly promoted the differentiation of CD4+ T cells to Th17 cells (IL-17+CD4+ phenotype) and enhanced the oncogenic Wnt signaling pathway. In conclusion, P. micra promoted colorectal tumorigenesis in mice by inducing colonocyte proliferation and altering Th17 immune response. P. micra may act as a prognostic biomarker for poor survival of CRC patients.