Fresh Colorectal Cancer Research Articles

Tissue morphometric measurements do not predict survival following colorectal cancer surgery

BackgroundEx vivo tissue morphometric (TM) measurements have been proposed as a quality marker for colorectal cancer (CRC) surgery. However, their survival associations require clarification. This study aimed to evaluate the feasibility of capturing TM measurements based on ex vivo fresh specimen images and explore the association between these TM measurements and survival outcomes.MethodsA prospective cohort study at Concord Hospital, Sydney was conducted with Stage I to III CRC patients (2009–2019) who underwent an anterior resection (AR) or right hemicolectomy (RH). Using high-resolution digital photographs of fresh CRC specimens, ex vivo tissue morphometric (TM) measurements—resected mesentery area (TM A), distances from high vascular tie to tumour (TM B) and bowel wall (TM C), and bowel length (TM D)—were recorded using Image J. Overall survival (OS) and disease-free survival (DFS) estimates and their associations to clinicopathological variables were investigated with Kaplan–Meier and Cox regression analyses. Linear regression models tested association between TM measurements and lymph node (LN) yield.ResultsOf the 1,425 patients who underwent CRC surgery, TM measurements were performed on 312 patients, with an average age of 69.4 years (SD 12.3), of whom 52.9% were male. The majority had an AR (57.8%). Among AR patients, a 5-year OS rate of 77.4% and a DFS rate of 70.1% were observed, with TM measurements bearing no relationship to survival outcomes. Similarly, RH patients exhibited a 5-year OS rate of 67.2% and a DFS rate of 63.1%, with TM measurements again showing no association with survival. Only TM D (P = 0.02) measurements were associated with the number of LNs examined.ConclusionThis study successfully demonstrates the feasibility of measuring TM measurements on photographs of ex vivo fresh specimens following CRC surgery. The lack of association with survival outcomes questions the utility of TM measurements as a quality metric of CRC surgery.

Iron-(Fe3+)-Dependent Reactivation of Telomerase Drives Colorectal Cancers.

Over-consumption of iron-rich red meat and hereditary or genetic iron overload are associated with an increased risk of colorectal carcinogenesis, yet the mechanistic basis of how metal-mediated signaling leads to oncogenesis remains enigmatic. Using fresh colorectal cancer samples we identify Pirin, an iron sensor, that overcomes a rate-limiting step in oncogenesis, by reactivating the dormant human telomerase reverse transcriptase (hTERT) subunit of the telomerase holoenzyme in an iron-(Fe3+)-dependent manner and thereby drives colorectal cancers. Chemical genetic screens combined with isothermal dose-response fingerprinting and mass spectrometry identified a small molecule SP2509 that specifically inhibits Pirin-mediated hTERT reactivation in colorectal cancers by competing with iron-(Fe3+) binding. Our findings, first to document how metal ions reactivate telomerase, provide a molecular mechanism for the well-known association between red meat and increased incidence of colorectal cancers. Small molecules like SP2509 represent a novel modality to target telomerase that acts as a driver of 90% of human cancers and is yet to be targeted in clinic. Significance: We show how iron-(Fe3+) in collusion with genetic factors reactivates telomerase, providing a molecular mechanism for the association between iron overload and increased incidence of colorectal cancers. Although no enzymatic inhibitors of telomerase have entered the clinic, we identify SP2509, a small molecule that targets telomerase reactivation and function in colorectal cancers.

BRIX1 promotes ribosome synthesis and enhances glycolysis by selected translation of GLUT1 in colorectal cancer.

Ribosome biogenesis protein BRX1 homolog (BRIX1) is critically required for the synthesis of the 60S ribosome subunit. However, the role and mechanism of BRIX1 in colorectal cancer (CRC) remain unclear. Kyoto Encyclopedia of Gene and Genome pathway and Gene Ontology analyses were used for bioinformatics analysis. The rRNA levels were detected in CRC tissues and cells. Nascent RNA synthesis was detected via cellular immunofluorescence. The correlation was analyzed between patient Positron Emission Tomography-Computed Tomography (PET-CT) values and their BRIX1 expression. The extracellular acidification rate (ECAR) and oxygen consumption rate were determined via live metabolic analyses. Polysome fractions were collected for BRIX1 mRNA used in translation. The orthotopic model and Cell Counting Kit-8 (CCK8) assay were used to assess BRIX1 function in CRC. BRIX1 is a core protein involved in ribosome-related pathway changes in CRC. Gene Ontology analysis showed that BRIX1 was primarily enriched in ribosome assembly and ribosome biogenesis pathways. In fresh CRC tissue, rRNA levels (5S, 5.8S, 18S and 28S) were higher in the BRIX1 high-expression group than in the BRIX1 low-expression group. Similarly, BRIX1 knockdown significantly decreased rRNA levels for 5S, 5.8S, 18S and 28S in CRC cells, whereas overexpression of BRIX1 significantly increased these levels. In addition, BRIX1 knockdown inhibited nascent RNA synthesis in CRC cells. In clinical data analysis, BRIX1 expression was related to the glucose uptake in PET-CT. BRIX1 knockdown significantly decreased the ECAR value, glucose uptake and lactic acid production in CRC cells, whereas BRIX1 overexpression significantly increased these. Furthermore, BRIX1 knockdown significantly decreased the protein expression of GLUT1, whereas BRIX1 overexpression significantly increased this; however, expression of BRIX1 mRNA was unaffected in either case. Blocking glycolysis by si-GLUT1 or galactose reversed BRIX1 promotion of glycolysis and cell proliferation in CRC cells.

CCT6A functions as promising diagnostic biomarker and promotes cell proliferation in colorectal cancer.

Background: Chaperonin-containing tailless complex polypeptide 1 subunit 6A (CCT6A) is mainly located in the cytoplasm and considered to be involved in various biological processes in tumors. However, its function and the intrinsic mechanism need to be further elucidated. Methods: Multi-omics analysis was used to evaluate the correlation between CCT6A expression and prognosis of patients, as well as its immune value. CCT6A was knockout by CRISPR-Cas9, and overexpressed by transfecting plasmids in colorectal cancer (CRC) cells. Cell proliferation was analyzed by MTS, EDU staining and colony growth assay, and cell migration was monitored by wound healing assay and Transwell assay. The phosphor-kinase array kit and immunoblotting assay was utilized to explore the potential molecular mechanisms. Results: CCT6A was highly expressed in multiple tumor tissues and significantly correlated with the prognosis of patients. It was also associated with the immune infiltration, immune correlation and prognosis in CRC. CCT6A was highly expressed in CRC biopsies as well as fresh CRC tissues. Meanwhile, knockout of CCT6A reduced cell proliferation, cell cycle and cell migration. On the contrary, overexpression of CCT6A exhibited the opposite phenotypes. Moreover, we identified that HSPD1 and non-phosphorylated P53 were highly increased in CCT6A overexpressed cells, which are involved in regulating tumorigenesis. Conclusions: Therefore, CCT6A positively regulated cell proliferation/migration in CRC cells, and suggesting CCT6A has a high immunological value and is associated with CRC progression, which makes it a potential therapeutic target for CRC.

Abstract 5929: Individual organoid level analyses reveal hidden tumor molecular heterogeneity

Abstract Introduction: Tumor heterogeneity is a major cause of resistance to therapies in cancer patients occurring during disease progression or secondary to therapeutic intervention due to clonal selection. Mapping tumor heterogeneity is crucial for developing therapeutics for cancer patients. Currently there are no reliable models for identifying heterogeneity in vitro. Patient-derived cancer organoids (PDCOs) from tissue samples of patients capture clonal and subclonal mutations. Here we use PDCOs from colorectal cancer (CRC) patients to study molecular heterogeneity across individual organoids and how the heterogeneity changes with passages. Methods: Fresh CRC tissues from 4 patients undergoing endoscopy were obtained following consent on an IRB-approved protocol. PDCOs were cultured using Matrigel and previously published CRC organoid media. Following maturation, individual organoids (spikes) were separated into Matrigel domes and expanded. Parent cultures from high/low passage numbers and expanded spikes were collected for next generation sequencing (NGS). A variant allele frequency (VAF) was determined for each nonsynonymous variant and considered subclonal if the VAF was between 10%-30%. Variants were annotated pathogenic or not using ClinVar. Results: A total of 49 spikes were collected from 4 CRC PDCO lines and compared to the parent cultures using NGS. For low passage parent cultures, a median of 4 (range 3-8) subclonal alterations were identified. At high passages the number of subclonal variants decreased (median: 2 (range 2-4)). The individual organoid spikes maintained the founder (clonal) alterations and most also contained subclonal alterations, though at a lower frequency. Of those subclonal variants in the parent cultures, 31% were found in the spikes and an additional 54% found in the spikes were not identified in the parent. In a high tumor mutation burden line (MTB111), 12/17 spikes showed subclonal alterations in APCA896V, F1515C, Q1544* that were not present in the parent culture. Two PDCO lines (RC46A and RC46B) obtained from distinct sites of the same tumor were generated. One contained a subclonal mutation in MYCN26S in the high passage parent culture and 7/9 spikes. This mutation was not present in any RC46A samples. The high passage RC46A parent culture and 2/7 spikes showed an alteration in ARID1AG1037fs while it was present only in a single spike of RC46B. Conclusion: PDCOs identified subclonal alterations at the single-organoid level and are an exciting tool to study tumor heterogeneity. The spikes presented less subclonal variants than the parent but were largely not clonal. Future implications of using heterogeneity data from single organoids in therapeutic decisions are warranted. Citation Format: Shirsa Udgata, Aishwarya Sunil, Katherine A. Johnson, Cheri A. Pasch, Dustin A. Deming. Individual organoid level analyses reveal hidden tumor molecular heterogeneity [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 5929.

Bromodomain-containing protein 9 activates proliferation and epithelial-mesenchymal transition of colorectal cancer via the estrogen pathway in vivo and in vitro.

Bromodomain-containing protein 9 (BRD9) has been reported to be upregulated in multiple malignancies and facilitate cancer progression. However, there is a paucity of data relating to its expression and biological role in colorectal cancer (CRC). Therefore, this current study examined the prognostic role of BRD9 in CRC and the underlying mechanisms involved. Real-time polymerase chain reaction (PCR) and Western blotting were used to examine the expression of BRD9 in paired fresh CRC and para-tumor tissues from colectomy patients (n=31). Immunohistochemistry (IHC) was performed to assess BRD9 expression in 524 paraffin-embedded archived CRC samples. The clinical variables are including age, sex, carcinoembryonic antigen (CEA), location of tumor, T stage, N stage, and TNM classification. The effect of BRD9 on the prognosis of CRC patients was explored by Kaplan-Meier and Cox regression analyses. Cell counting kit 8 (CCK-8), clone formation assay, transwell assay, and flow cytometry were used to determine CRC cell proliferation, migration, invasion, and apoptosis, respectively. Xenograft models in nude mice were established to investigate the role of the BRD9 in vivo. BRD9 mRNA and protein expression levels were significantly upregulated in CRC cells compared to normal colorectal epithelial cells (P<0.001). IHC analysis of 524 paraffin-embedded archived CRC tissues showed that high BRD9 expression was significantly associated with TNM classifications, CEA, and lymphatic invasion (P<0.01). Univariate and multivariate analyses indicated that BRD9 [hazard ratio (HR): 3.04, 95% confidence interval (CI): 1.78-5.20; P<0.01] expression and sex (HR: 6.39, 95% CI: 3.94-10.37; P<0.01) were independent prognostic factors for overall survival in the entire cohort. Overexpressing BRD9 promoted CRC cell proliferation, while silencing BRD9 inhibited the proliferation of CRC cells. Furthermore, we showed that BRD9 silencing significantly inhibited epithelial-mesenchymal transition (EMT) via the estrogen pathway. Finally, we demonstrated that silencing BRD9 significantly inhibited the proliferation and tumorigenicity of SW480 and HCT116 cells in vitro and in vivo in nude mice (P<0.05). This study demonstrated that BRD9 high could be an independent prognostic risk factor for CRC. Furthermore, the BRD9/estrogen pathway may contribute to the proliferation of CRC cells and EMT, suggesting that BRD9 may be a novel molecular target in the therapeutic treatment of CRC.

A human vascularized microtumor model of patient-derived colorectal cancer recapitulates clinical disease

Accurately modeling tumor biology and testing novel therapies on patient-derived cells is critically important to developing therapeutic regimens personalized to a patient's specific disease. The vascularized microtumor (VMT), or "tumor-on-a-chip," is a physiologic preclinical cancer model that incorporates key features of the native human tumor microenvironment within a transparent microfluidic platform, allowing rapid drug screening in vitro. Herein we optimize methods for generating patient-derived VMT (pVMT) using fresh colorectal cancer (CRC) biopsies and surgical resections to test drug sensitivities at the individual patient level. In response to standard chemotherapy and TGF-βR1 inhibition, we observe heterogeneous responses between pVMT derived from 6 patient biopsies, with the pVMT recapitulating tumor growth, histological features, metabolic heterogeneity, and drug responses of actual CRC tumors. Our results suggest that a translational infrastructure providing rapid information from patient-derived tumor cells in the pVMT, as established in this study, will support efforts to improve patient outcomes.

A novel DNA methylation marker to identify lymph node metastasis of colorectal cancer.

Lymph node metastasis (LNM) of colorectal cancer (CRC) is an important factor for both prognosis and treatment. Given the deficiencies of conventional tests, we aim to discover novel DNA methylation markers to efficiently identify LNM status of CRC. In this study, genome-wide methylation sequencing was performed in a cohort (n=30) using fresh CRC tissue to discover differentially methylated markers. These markers were subsequently validated with fluorescence quantitative PCR in a cohort (n=221), and the optimal marker was compared to conventional diagnostic methods. Meanwhile, immunohistochemistry was used to verify the effectiveness of the antibody corresponding to this marker in a cohort (n=56). LBX2 achieved an AUC of 0.87, specificity of 87.3%, sensitivity of 75.7%, and accuracy of 81.9%, which outperformed conventional methods including imaging (CT, PET-CT) with an AUC of 0.52, CA199 with an AUC of 0.58, CEA with an AUC of 0.56. LBX2 was also superior to clinicopathological indicators including the depth of tumor invasion and lymphatic invasion with an AUC of 0.61and 0.63 respectively. Moreover, the AUC of LBX2 antibody was 0.84, which was also better than these conventional methods. In conclusion, A novel methylation marker LBX2 could be used as a simple, cost-effective, and reliable diagnostic method for LNM of CRC.

Up regulation of long non-coding RNAs BACE1 and down regulation of LINC-PINT are associated with CRC clinicopathological characteristics

BackgroundLong non-coding RNAs (LncRNAs) are known to have regulatory consequences for aberrant gene expression in cancers. The aim of this study was to evaluate the expression levels of long non-encoding RNAs, BACE1 (β-secretase1) and LINC-PINT (Long Intergenic Non-Protein Coding RNA, P53 Induced Transcript), in colorectal cancer (CRC) with clinicopathological parameters.Methods and resultsBioinformatics analysis defining effectual signalling pathways Wnt. A total of 130 tissue samples (50 fresh CRC tissues with parallel adjacent normal tissues (ADJ) accompanied with 30 normal healthy control tissue samples) were collected from the Iranian population. mRNA expression analysis was performed via Real Time Q-PCR. Statistical analysis for comparing CRC expression levels with ADJ and normal healthy tissues were carried out using Kruskal–Wallis tests. The Receiver Operating Characteristic (ROC) curve was plotted for each LNC, separately. We discovered that PINT and BACE1 expression levels were decreased and increased respectively in CRC tumour samples compared with ADJ normal and healthy tissues. Clinicopathological parameter assessment revealed a significant relationship between PINT expression, tumour location, staging and distant metastasis (p < 0.009, p < 0.014, p < 0.008, respectively). Also, BACE1 over expression was significantly associated with tumour site (p < 0.009), metastasis (p < 0.017) and histological differentiation (p < 0.028) and staging (p < 0.017). Furthermore, ROC curve plotting showed LINC-PINT LNC-BACE1 may distinguish between early and late-stage of CRC, highlighting the value of both BACE1 and PINT as CRC progression biomarkers.ConclusionWe investigated two LNCRNAs (PINT and BACE1) as potential CRC prognostic biomarkers, which are imperative for early and effective medical intervention in CRC. Expression levels of PINT and BACE1 in CRC tissue samples may serve to identify metastasis earlier, increasing patient survival rates and expediating clinical treatment options.

High Expression of FCRLB Predicts Poor Prognosis in Patients With Colorectal Cancer.

Background: Mining the prognostic biomarkers of colorectal cancer (CRC) has important clinical and scientific significance. The role of Fc receptor-like B (FCRLB) in solid tumors has never been reported or studied to our knowledge, and the prognostic role of FCRLB in CRC still awaits characterization. Methods: The potential prognostic factor FCRLB was screened out through TCGA database analysis. Then, its expression and associations with clinicopathological variables were assessed in the TCGA CRC cohort. The prognostic value of FCRLB was examined with multiple methods, such as the Kaplan-Meier method, ROC curve, time-dependent ROC analysis, and prediction model nomograms. Then, functional enrichment and annotation among the high and low FCRLB groups were achieved utilizing GO and KEGG analyses and GSEA. Fresh CRC tissue samples obtained clinically were used for the preparation of the tissue microarray and for further validation. Results: FCRLB was highly expressed in CRC tissues compared to normal tissues. Moreover, over-expression of FCRLB correlated with higher CEA levels, advanced T stage, N stage, M stage, AJCC stage, lymphatic invasion, perineural invasion, and incomplete resection (R1 and R2 resection). In addition, high expression of FCRLB was closely correlated to less favorable OS, DSS, and PFI. The analysis of CRC tissue microarray further confirmed the conclusion drawn from the TCGA data analysis. Conclusion: FCRLB is notably up-regulated in CRC tissues and may serve as a potential biomarker of CRC.

A Prognostic Model Using Immune-Related Genes for Colorectal Cancer.

There is evidence suggesting that immune genes play pivotal roles in the development and progression of colorectal cancer (CRC). Colorectal carcinoma patient data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) were randomly classified into a training set, a test set, and an external validation set. Differentially expressed gene (DEG) analyses, univariate Cox regression, and the least absolute shrinkage and selection operator (LASSO) were used to identify survival-associated immune genes and develop a prognosis model. Receiver operating characteristic (ROC) analysis and principal component analysis (PCA) were used to evaluate the discrimination of the risk models. The model genes predicted were verified using the Human Protein Atlas (HPA) databases, colorectal cell lines, and fresh CRC and adjacent tissues. To understand the relationship between IRGs and immune invasion and the TME, we analyzed the content of immune cells and scored the TME using CIBERSORT and ESTIMATE algorithms. Finally, we predicted the potential sensitive chemotherapeutic drugs in different risk score groups by the Genomics of Drug Sensitivity in Cancer (GDSC). A total of 491 IRGs were screened, and 14 IRGs were identified to be significantly related to overall survival (OS) and applied to construct an immune-related gene (IRG) prognostic signature (IRGSig) for CRC patients. Calibration plots showed that nomograms have powerful predictive ability. PCA and ROC analysis further verified the predictive value of this fourteen-gene prognostic model in three independent databases. Furthermore, we discovered that the tumor microenvironment changed significantly during the tumor development process, from early to middle to late stage, which may be an essential factor for tumor deterioration. Finally, we selected six commonly used chemotherapeutic drugs that have the potential to be useful in the treatment of CRC. Altogether, immune genes were used to construct a prognosis model for CRC patients, and a variety of methods were used to test the accuracy of this model. In addition, we explored the immune mechanisms of CRC through immune cell infiltration and TME in CRC. Furthermore, we assessed the therapeutic sensitivity of many commonly used chemotherapeutic medicines in individuals with varying risk factors. Finally, the immune risk model and immune mechanism of CRC were thoroughly investigated in this paper.

High frequency of enterotoxigenic Bacteroides fragilis and Enterococcus faecalis in the paraffin-embedded tissues of Iranian colorectal cancer patients

BackgroundThe association between specific bacteria and colorectal cancer (CRC) has been proposed. Only a few studies have, however, investigated this relationship directly in colorectal tissue with conflicting results. So, we aimed to quantitate Streptococcus gallolyticus, Fusobacterium spp, Enterococcus faecalis and enterotoxigenic Bacteroides fragilis (ETBF) in formalin-fixed and paraffin-embedded (FFPE) colorectal tissue samples of Iranian CRC patients and healthy controls.MethodsA total of 80 FFPE colorectal tissue samples of CRC patients (n = 40) and healthy controls (n = 40) were investigated for the presence and copy number of above bacterial species using quantitative PCR. Relative quantification was determined using ΔΔCT method and expressed as relative fold difference compared to reference gene.ResultsRelative abundance and copy number of E. faecalis and ETBF were significantly higher in CRC samples compared to control group. E. faecalis was more prevalent than ETBF in tumor samples. Frequency of ETBF and E. faecalis in late stages (III/IV) of cancer was significantly higher than early stages (I/II). We did not detect a significant difference in abundance of S. gallolyticus and Fusobacterium spp between two groups.ConclusionOur study revealed the higher concentration of E. faecalis and ETBF in FFPE samples of CRC patients than controls. However, additional investigations on fecal and fresh colorectal cancer tissue samples are required to substantiate this correlation.

NEBL and AKT1 maybe new targets to eliminate the colorectal cancer cells resistance to oncolytic effect of vesicular stomatitis virus M-protein

This study compares the oncolytic effect of vesicular stomatitis virus (VSV) wild type and M51R M-protein on the colorectal tumors of different invasive intensity on SW480 and HCT116 cell lines and 114 fresh colorectal cancer primary cell cultures. Fresh tumor samples were divided into two groups of lower stages (I/II) and higher stages (III/IV) regarding the medical records. The presence of two mutations in the PIK3CA gene and the expression of NEBL and AKT1 genes were evaluated. The cells were transfected with a plasmid encoding VSV wild-type and M51R mutant M-protein. Results showed either wild type or M51R mutant can kill SW480 and stage I/II primary cultures while mutant M-protein had no apoptotic effects on HCT116 cells and stage III/IV primary cultures. NEBL and AKT1 expression were significantly higher in resistant cells. Elevated caspase-9 activity confirmed that the intrinsic apoptosis pathway is the reason for cell death in lower-stage cells. Different tumors from the same cancer exhibit different treatment sensitivity due to genetic difference. NEBL and AKT1 gene expression may be responsible for this difference, which may be the target of future investigations. Therefore, tumor staging should be considered in oncolytic viral treatment as an interfering factor.

Combination of GC-MS based metabolomics analysis with mouse xenograft models reveals a panel of dysregulated circulating metabolites and potential therapeutic targets for colorectal cancer.

BackgroundColorectal cancer (CRC) is a common gastrointestinal tumor with subtle, often undetectable early symptoms, which means that upon diagnosis, patients often present in the middle or late stages of disease. Therefore, the need for an effective biomarker for the early diagnosis and development of novel therapeutic targets is urgent to prolong patient survival time and reduce mortality.MethodsTwenty mice were randomly divided into patient-derived xenograft (PDX) model (transplantation of fresh CRC tumor samples) and control groups (10 mice in each group). All the animals were euthanized using isoflurane at the end of the experiment. Gas chromatography-mass spectrometry (GC-MS)-based metabolomic profiling was performed to investigate the differential metabolites in the serum, and publicly available gene expression data (GSE106582) were analyzed to determine dysregulated metabolic pathways. Joint pathway analysis was used to identify potential metabolic targets. Immunohistochemistry analysis was performed to confirm the presence of the identified targets at the protein level.ResultsA total of 96 differential circulating metabolites were identified, which were predominantly involved in amino acid metabolism. In particular, the serum levels of amino acids such as phenylalanine and aspartic acid were significantly downregulated in the PDX group, suggesting an increased consumption of amino acids in CRC. Moreover, both the mRNA and protein levels of the amino acid transporters, SLC7A5 and SLC1A5, were found to be upregulated in CRC.ConclusionsBy combining GC-MS-based metabolomics profiling with a PDX model of CRC our study successfully identified potential diagnostic circulating metabolites. Dysregulated amino acid metabolism was found to be a significant feature of CRC. The amino acid transporters, SLC7A5 and SLC1A5, were identified as potential metabolic therapeutic targets. This study furthers the understanding of the metabolic processes involved in CRC.

Sensitivity of HER2-amplified colorectal organotypic cancer spheroids at ex vivo resistance to panitumumab and trastuzumab.

68 Background: HER2 amplification is an emerging biomarker in colorectal cancer (CRC) with increased copy number associated with improved clinical outcomes to HER2 targeting. RAS/RAF wildtype CRC also benefit from use of epidermal growth factor receptor inhibition (EGFRi). The sequencing of EGFRi versus HER2 inhibition in low copy number HER2 amplified CRC remains uncertain. Patient-derived cancer organoids (PDCOs) allow an ex vivo method to assess treatment sensitivity. We examined treatment sensitivity of a HER2 amplified PDCO at baseline and following resistance to panitumumab and trastuzumab. Methods: Following IRB-approval, fresh CRC tissue was cultured to maturation. After expansion, subcultures were treated with stepwise (20%) increase to physiologic Cmax of panitumumab (230ug/mL) and trastuzumab (180ug/mL). Threshold for escalation was median relative growth of +20% at 96h. Sensitivity was assessed on primary culture (RC1), panitumumab resistance (RC1-P) and trastuzumab resistance (RC1-T) using 96h of physiologic Cmax panitumumab, trastuzumab, and combination trastuzumab/pertuzumab. Individual sphere response was assessed for change in mean NADH autofluorescence intensity and ratio of NADH/FAD signal. Response was assessed at 96h in comparison to control using effect size of Glass’s Delta (GΔ). Results: Molecular profiling revealed HER2 copy number of 14 with no concurrent alterations in RAS, RAF, or PIK3CA. Time to resistance was similar between panitumumab (55 days) and trastuzumab (51 days). RC1 had baseline growth (+116%) which was reduced with single agent panitumumab (+17%, GΔ=1.40) with intermediate sensitivity to trastuzumab (+48%, GΔ=0.95) and trastuzumab/pertuzumab (46%, GΔ=0.99). Normalized NADH/FAD ratio revealed significant metabolic response to panitumumab (-20%, GΔ=0.66) and trastuzumab/pertuzumab (-35%, GΔ=1.16) with insignificant effect of single agent trastuzumab (-14%, GΔ=0.46). Following resistance to panitumumab, RC1-P had persistent growth with trastuzumab (+68%) which improved in combination trastuzumab/pertuzumab (+34%, GΔ=1.16). Following resistance to trastuzumab, RC1-T was insensitive to EGFRi with panitumumab including persistent growth (+58%, GΔ=0.70) and unchanged metabolism (+2%, GΔ=-0.10). Conclusions: Therapeutic dose escalation in a single PDCO of HER2 amplified CRC suggests improved sensitivity to EGFRi and dual HER2 targeting with trastuzumab/pertuzumab. Resistance to EGFRi resulted in persistent sensitivity to dual HER2 inhibition using trastuzumab/pertuzumab, however resistance to single agent trastuzumab. Resistance to trastuzumab resulted in future insensitivity to EGFRi. Molecular profiling at resistance revealed no pathologic alterations in EGFR or ERBB2 signaling, with ongoing analysis of transcriptional changes by RNAseq.

Reprogramming immunosuppressive myeloid cells by activated T cells promotes the response to anti-PD-1 therapy in colorectal cancer

Overcoming local immunosuppression is critical for immunotherapy to produce robust anti-tumor responses. Myeloid-derived suppressor cells (MDSCs) are key regulators of immunosuppressive networks and promote tumor progression. However, it remains unclear whether and how tumor-infiltrating MDSCs are shaped in response to anti-PD-1 treatment and what their impact on therapeutic efficacy is in colorectal cancer (CRC). In this study, the levels of infiltrating MDSCs were significantly higher in the non-responding organoids and were selectively reduced in the responding group, with MDSCs showing increased apoptosis and attenuated functional activity after anti-PD-1 treatment. A negative correlation between T-cell activation and MDSC function was also observed in fresh human CRC tissues. Mechanistic studies revealed that autocrine IFN-α/β upregulated TRAIL expression on activated T cells to elicit MDSC apoptosis via the TRAIL–DR5 interaction and acted synergistically with TNF-α to inhibit MDSC function of suppressing the T-cell response through the JNK-NMDAR-ARG-1 pathway. Moreover, blockade of IFN-α/β and TNF-α abolished the therapeutic efficacy of anti-PD-1 treatment by preserving the frequency and suppressive activity of infiltrating MDSCs in a CRC mouse model. This result suggested that reprogramming MDSCs by IFN-α/β and TNF-α from activated T cells was necessary for successful anti-PD-1 treatment and might serve as a novel strategy to improve the response and efficacy of anticancer therapy.

Syntaxin 2 promotes colorectal cancer growth by increasing the secretion of exosomes.

Background: Colorectal cancer (CRC) is one of the most common cancers with high mortality worldwide. Uncontrolled growth is an important hallmark of CRC. However, the mechanisms are poorly understood. Methods: Syntaxin 2 (STX2) expression was analyzed in 160 cases of paraffin-embedded CRC tissue by immunohistochemistry, in 10 cases of fresh CRC tissue by western blot, and in 2 public databases. Gain- and loss-of-function analyses were used to investigate the biological function of STX2 in CRC growth. Exosomes isolation, characterization, Co-immunoprecipitation (Co-IP), flow cytometry and fluorescence were conducted to study the molecular mechanism of STX2 in CRC growth. Results: The expression of STX2 was obviously up-regulated in human CRC tissues. Overexpression of STX2 increased the growth of CRC cells in vitro and in vivo. Downregulation of STX2 repressed the growth of CRC. STX2 modulated exosomes secretion of CRC cells which might correlated with Rab8a expression. The secreted exosomes could be ingested by CRC cells, and ultimately promoted the growth of CRC by arresting the tumor cells at S phase. Conclusions: Our data provide evidence that STX2 promotes CRC growth by increasing exosomes secretion of CRC cells; And the modulation of STX2 in exosomes secretion correlates with Rab8a. Thus, our study identified a new mechanism of STX2 in CRC growth and may provide a possible strategy for CRC therapy.

WNT4 secreted by tumor tissues promotes tumor progression in colorectal cancer by activation of the Wnt/\u03b2-catenin signalling pathway

BackgroundWingless and Int-related protein (Wnt) ligands are aberrantly expressed in patients with colorectal cancer (CRC). However, the aberrant level of Wnt ligands in serum have not been explored. Here, we aimed to identify the levels of WNT4 in serum and explored its oncogenic role in CRC.MethodsThe Oncomine database was used to analyze the relationship between WNT4 and the prognosis of CRC. ELISA was performed to measure WNT4 levels in serum and conditioned medium from fresh CRC tissues and adjacent normal tissues. Western blot and immunohistochemistry were carried out to measure the expression of WNT4 in human CRC tissues and adjacent normal tissues. The migration and invasion of CRC cells were determined by trans-well assay, and the effects of WNT4 on CRC invasion and metastasis in vivo were verified by tumor xenograft in nude mice. Cancer-associated fibroblasts (CAFs) and angiogenesis in subcutaneous nodules were detected by immunofluorescence (IF). In addition, the suspended spheres formation and tube formation assay were performed to explore the effects of WNT4 on CAFs and angiogenesis respectively.ResultsWNT4 was significantly upregulated in serum of CRC patients, and CRC tissues were identified as an important source of elevated WNT4 levels in CRC patients. Interestingly, elevated levels of WNT4 in serum were downregulated after tumor resection. Furthermore, we found that WNT4 contributed to epithelial-to-mesenchymal transition (EMT) and activated fibroblasts by activating the WNT4/β-catenin pathway in vitro and in vivo. Moreover, angiogenesis was induced via the WNT4/β-catenin/Ang2 pathway. Those effects could be reversed by ICG-001, a β-catenin/TCF inhibitor.ConclusionOur findings indicated that serum levels of WNT4 may be a potential biomarker for CRC. WNT4 secreted by colorectal cancer tissues promote the progression of CRC by inducing EMT, activate fibroblasts and promote angiogenesis through the canonical Wnt/β-catenin signalling pathway.

Abstract 2701: Patient-derived colorectal cancer organoid therapeutic escalation: Tracking resistance to EGFR inhibition for predicting therapeutic sensitivity

Abstract Background: Precision oncology requires translational tools predictive of clinical benefit. Epidermal growth factor receptor inhibition (EGFRi) yields a survival advantage in RAS/RAF wildtype (wt) colorectal cancer (CRC), however clinical tools do not predict individual patient response or resistance mechanisms. Patient-derived cancer organoids (PDCOs) allow for an ex vivo method to track subclonal response. Here, we examine the development of PDCO EGFRi resistance as a novel biomarker to differentiate treatment sensitivities. Methods: Fresh CRC tissue was obtained following consent to an IRB-approved protocol. Molecular profiling was performed on expanded PDCOs and in parallel to clinical hotspot sequencing. Cultures were treated with stepwise (20%) increases in panitumumab (46ug/mL per escalation) up to the physiologic Cmax (230ug/mL). A HER2 amplified culture was also assessed by escalation of trastuzumab with 20% stepwise increase to physiologic Cmax (180ug/mL). Threshold for escalation to the next dose level was a median relative growth of 20% at 96h. Time to resistance (TTR) was defined from start to persistent PDCO growth at 100% Cmax. Treatment studies assessed the longest diameter of individual spheres, assessed at 48h and compared to control using effect size (Glass's (G) Δ). TTR was compared to molecular profiles of primary resistance and clinical outcomes. Results: CRC PDCOs were derived from 10 patients (5 localized and 5 metastatic samples). PDCOs had diverse molecular profiles: 3 KRAS mutant (mt), 1 PTEN mt, 7 TP53 mt, and 7 RAS/RAF wt. TTR ranged from 26-226 days, with 90% of PDCOs lines achieving resistance. RAS mt PDCOs had a homogeneous TTR of 27.0±1.4 days. RAS wt PDCOs displayed marked variation in therapeutic response with TTR of 51.5±61.7 days (p=0.06, compared to RAS mt). A HER2-amplified PDCO (copy number 14) showed intermediate sensitivity to EGFRi (TTR 55 days), similar to dose escalation of HER2-targeting trastuzumab (TTR of 51 days). This HER2-amplified PDCO showed intermediate sensitivity to single agent trastuzumab (GΔ = 0.56), yet no sensitivity to trastuzumab following EGFRi resistance (GΔ = 0.09). Prospective clinical correlation was possible for 1 PDCO line, a RAS wt PDCO derived prior to EGFRi which showed marked sensitivity with growth and metabolic arrest achieved at 40% CMax consistent with clinical response. Conclusions: We present therapeutic dose escalation using PDCOs as a translational tool to track the clonal dynamics of resistance to EGFRi in CRC. This technique provides an objective measure of TTR to differentiate culture specific sensitivity in a clinically relevant timeframe. Molecular profiling of resistant cultures is ongoing which may provide an ex vivo tool for predicting individual resistance as cancers evade therapeutic pressure of targeted selection. Citation Format: Jeremy D. Kratz, Aishwarya Sunil, Meghan Conroy, Cheri A. Pasch, Nicole Lassen, Hannah Houtler, Kayla K. Lemmon, Linda Clipson, Daniel E. Abbott, Evie H. Carchman, Elise H. Lawson, Noelle K. Loconte, Sam J. Lubner, Kristina A. Matkowskyj, Daniel L. Mulkerin, Nataliya Uboha, Dustin A. Deming. Patient-derived colorectal cancer organoid therapeutic escalation: Tracking resistance to EGFR inhibition for predicting therapeutic sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2701.

Decreased expression of farnesoid X receptor may indicate poor prognosis in patients with colorectal cancer.

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Many studies showed that increased secretion of bile acid may contribute to the pathogenesis of CRC. Farnesoid X receptor (FXR) plays an important role in several physiological functions of bile acid metabolism and circulation, while the diagnostic and prognostic values of FXR remain unknown.MethodsWe used immunohistochemistry (IHC) to examine FXR expression in 132 formalin-fixed paraffin-embedded (FFPE) CRC samples, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also conducted to compare the mRNA expression of FXR between 50 fresh CRC tissues and 50 adjacent normal tissues. Immunohistochemical method has been used to compare the protein expression of FXR in FFPE samples, and the associate between FXR expression and clinical characteristics of the patients were also analyzed. Moreover, RT-qPCR has been applied to quantify the mRNA expression of FXR, and the diagnostic value and prognostic value of FXR were analyzed by receiver operating characteristics (ROC) analysis and Kaplan-Meier survival analysis, respectively.ResultsThe expression of FXR was significantly decreased in CRC tumor samples compared with normal tissues on both protein and mRNA levels, and the expression of FXR was positively correlated with the tumor size, lymph node metathesis and distant metathesis of the patients. Moreover, the area under curve (AUC) of FXR for the diagnosis of CRC was 0.8088, and low expression of FXR was associated with low overall survival (OS) and disease-free survival (DFS) rate.ConclusionsFXR was down-regulated in CRC and may serve as potential diagnostic and prognostic biomarkers.