Gene signatures derived from transcriptomic-causal networks stratify colorectal cancer patients for effective targeted therapy

Colorectal cancer (CRC) is the third most commonly diagnosed cancer with 1.9 million new cases and the second leading cause of cancer-related deaths, with 935, 000 deaths reported in 2023. Despite advances in therapy, the 5-year survival rate for metastatic CRC remains as low as 14%, emphasizing the need for novel treatment strategies. Previously, we have validated that Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical alfa kinase as a potential molecular target that is commonly expressed in highly aggressive solid cancers such as triple-negative breast cancer, pancreatic, ovarian, and lung cancers and demonstrated that it acts as a key player in the survival, proliferation, invasion, and tumor growth of cancer cells and promoting cell. In this study, we investigated the role of eEF2K in colon cancer tumorigenesis and progression and its potential as a therapeutic target using a novel eEF2K small molecule inhibitor and siRNA-based knockdown studies. We found that eEF2K expression is highly expressed in CRC cell lines and eEF2K is associated with shorter patient survival and poor prognostic marker in CRC patients (NCI-TCGA patient database), inhibition of eEF2K in various CRC cell lines (HCT116, RKO, COLO320, and with different genetic alterations leads to suppression on cell proliferation, migration, and invasion. Western blot analysis confirmed that treatment with the siRNA and the eEF2K inhibitor reduced the activity of clinically significant signaling molecules including Src, FAK, and Cyclin D1 which are crucial for tumor growth and progression. In vivo, targeting of eEF2K by eEF2K siRNA encapsulated in iron oxide nanoparticles (IONPS) and our novel water soluble small-molecule eEF2K inhibitor suppressed CRC tumor growth of HTC116 and RKO tumors (flank) CRC tumor models in nude mice, with no observed toxicity. Analysis of tumor tissues by immunohistochemistry showed significant inhibition of the Ki-67 proliferation marker and induction of apoptosis by TUNEL staining. Our study provides the first evidence that eEF2K is a clinically significant potential molecular target and contributes to tumorigenesis and progression of CRC. Its effective inhibition is safe and can suppress tumor growth in preclinical CRC models. These promising results lay the foundation for further developing eEF2K-targeted therapies, offering a potential new approach for treating CRC. Citation Format: Nermin Kahraman, Ezgi Biltekin, Ogun Ali Gul, Goknur Kara, Izabela Fokt, M. Akay Yasemin, Waldemar Priebe, Metin Akay, Bulent Ozpolat. Inhibition of Eukaryotic elongation factor 2 kinase (eEF2K) disrupts key processes in colorectal cancer progression and tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6953.

Aspirin could reduce the risk of cancer metastasis. Circulating tumor cells (CTCs) are a key factor of cancer metastasis, but no evidence has revealed how aspirin affects CTCs and its epithelial-mesenchymal transition (EMT). Here, we conducted a clinical trial to investigate how aspirin affects CTCs in metastatic colorectal cancer (MCC) and breast cancer patients (MBC). The trial is retrospective registered at clinicaltrials.gov (NCT02602938). The eligible patients are given 100mg aspirin q.d. for 8weeks, and CTCs are evaluated at baseline, 4 and 8weeks for absolute number, phenotype (epithelial type, E+, mesenchymal type, M+, and biophenotypic type, B+), and vimentin expression. Data on 21 MCC and 19 MBC patients are analyzed, and it revealed that the CTC numbers decreased with aspirin treatment in MCC (p<0.001) but not MBC (p=0.0532); besides, ratio of E+ CTCs increased (p=0.037) and M+ CTCs decreased at 2months in MCC (p=0.013), but neither the ratio of E+ or M+ CTCs changes significantly in MBC; vimentin expression of M+ CTCs is higher than E+ and B+ CTCs either in MBC or MCC patients at baseline (p<0.01); and aspirin suppresses the vimentin expression in M+ (p=0.002)and B+ (p=0.006) CTCs of MCC and M+ CTCs of MBC (p=0.004); besides it find vimentin expression in B+ (p=0.004) or M+ (p<0.001), CTCs are markedly decreased in patients with total CTC numbers declined. Aspirin could decrease CTCs numbers and block EMT transition in MCC patients and part of MBC patients.

P-269 - Correlation between the prognostic value of tumor-infiltrating lymphocytes (TILS) and sidedness in colorectal cancer (CC) patients (pts)

Long-term prognosis remains poor for colorectal cancer (CRC) patients with advanced disease due to treatment resistance. The identification of novel targets is essential for the development of new therapeutic approaches. GPR56, an adhesion GPCR, is highly expressed in CRC tumours and correlates with poor survival. Here, we describe the generation and preclinical evaluation of a novel ADC consisting of an anti-GPR56 antibody (10C7) conjugated with the DNA-damaging payload duocarmycin. RNA-seq dataset analysis was performed to determine GPR56 expression in CRC subtypes. The specificity of binding, epitope mapping, and internalisation of 10C7 was examined. 10C7 was conjugated to payload and ADC cytotoxicity was assessed against a panel of CRC cell lines and tumour organoids. Antitumour efficacy was evaluated in xenograft models of CRC cell lines and patient-derived tumours. High GPR56 was shown to be associated with the microsatellite stable (MSS) subtype that accounts for 80-85% of CRC. GPR56 ADC selectively induced cytotoxicity in CRC cells and tumour organoids at low nanomolar potency in a GPR56-dependent manner and showed significant antitumour efficacy against GPR56-expressing xenograft models. This study provides the rationale for the future development of a GPR56-targeted ADC approach to potentially treat a large fraction of MSS CRC patients.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer

Previous work suggests that the long noncoding RNA HCC associated long non-coding RNA (HANR) is associated with hepatocellular carcinoma (HCC) progression, but its significance in the context of colorectal cancer (CRC) remains to be determined. Therefore, in this study we assessed the prognostic and diagnostic value of HANR in patients suffering from CRC.The HANR expression in 165 pairs of CRC cancer and adjacent non-cancerous prostate tissues was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Student t test was conducted for intergroup comparison. Pearson correlation test was used for correlation analysis. Survival curves were carried out by the Kaplan-Meier method and evaluated using the log-rank test. Multivariable Cox proportional hazard risk regression model was performed to screen the independent factor affected the prognosis of CRC patients.In this study, levels of HANR were significantly higher in CRC tumor samples relative to adjacent normal tissue samples (P < .001). A ROC analysis suggested HANR expression could be reliably used to differentiate between normal and CRC tumor tissue. In addition, elevated HANR expression was positively correlated with more advanced and aggressive CRC features, such as a larger tumor size (P = .003), increased invasion depth (P = .012), and more advanced TNM stage (P = .011). Survival analyses revealed that elevated HANR expression was correlated with worse overall survival (P = .002) and disease-free survival (P = .003). A multivariate analysis further confirmed the relevance of HANR as an independent predictor of CRC patient outcomes.In summary, these results indicate that the lncRNA HANR is a promising prognostic indicator in CRC patients.

Purpose. Despite the recent progress in diagnosis and treatment for colorectal cancer (CRC), prognosis of CRC patients still remains poor. Metastatic recurrence is the major causes of cancer-related death in CRC patients, and recent progress in treatment options, especially technical advances in invasive treatment for metastatic lesion, have improved the prognosis of CRC patients with metastasis. Furthermore, almost half of patients that receive chemotherapy derive no clinical benefit, though all are exposed to these severe toxic and expensive therapeutic regimens in patients with metastatic CRC patients. In view of these underlying issues, for improvement of patient's prognosis, there is a keen interest in developing robust biomarkers that can identify the subset of patients who can benefit from intensive post-treatment surveillance protocols for early detection of recurrence, and prompt decision of appropriate treatment protocol. Herein, we systemically and comprehensively evaluated differentially levels of serum cytokines using array-based techniques to identify novel and reliable serum biomarker to predict metastasis and poor outcome in CRC. Methods. The study design included an initial dual screening phases, and followed by a subsequent clinical validation phase in this study.In discovery phase, we examined cytokine profiling using preoperative serum from two different CRC cohorts (n = 30) to identify differentially expressed serum cytokines in CRC patients with metastasis. In validation phase, serum levels of MCP-4 were assessed in 194 CRC patients by enzyme-linked immunosorbent assay, and their relationships with clinicopathological findings, including recurrence and survival, were investigated. Results. In discovery phase, three cytokines (elevated: MCP-4, ENA-78; decreased:Ckb8-1) were overlapped as a differentially expressed cytokines in serum from CRC patients with metastasis compared those without metastasis. High serum MCP-4 was significantly associated with older age (p = 0.033), advanced T stage (p = 0.029), distant metastasis (p = 0.011) and UICC stage classification (p = 0.006). Cox regression analysis showed that elevated MCP-4 was a significant and independent prognostic factor of disease free survival (HR:2.6, 95%CI:1.0-6.7) and overall survival (HR:2.7, 95%CI:1.3-5.9) in all CRC patients. Furthermore, logistic regression analysis revealed that high serum MCP-4 level was an independent marker in predicting distant metastasis in CRC (OR:3.8, 95%CI:1.3-10.9). Conclusion. Our comprehensive study highlights the clinical feasibility of serum MCP-4 as a prognostic and predictive biomarker for distant metastasis and recurrence in CRC patients. Quantification of serum MCP-4 concentration might support the early detection/prediction of recurrence and may contribute to the prediction of clinical outcomes in CRC. Citation Format: Yoshinaga Okugawa, Yuji Toiyama, Koji Tanaka, Mikio Kawamura, Takahito Kitajima, Tadanobu Shimura, Hiroki Imaoka, Hiroyuki Fujikawa, Susumu Saigusa, Yasuhiro Inoue, Motoyoshi Tanaka, Yasuhiko Mohri, Chikao Miki, Ajay Goel, Masato Kusunoki. Elevated serum monocyte chemotactic protein 4 (MCP4) as a novel noninvasive prognostic and predictive biomarker for detection of metastasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5016.

Prognosis in colorectal cancer (CRC) correlates closely with the density of tumor infiltrating T lymphocytes (TIL) in the primary tumor, suggesting that the adaptive cellular immune system can influence the natural history of this disease. However, it is unclear if TIL in CRC comprise a non-specific inflammatory response, or if T cells in TIL are specifically reactive with antigens expressed on CRC tumor cells. To date the study of TIL interaction with autologous CRC cells has been hampered by the small numbers of tumor cells and TIL that can be obtained from CRC surgical samples. We have developed reliable methods for expanding primary human CRC tumor cells as xenografts in immune-deficient mice. Moreover, we have adapted next generation DNA sequencing of T cell receptor β chain CDR3 regions to define the clonal composition of CRC TIL with exquisite precision and depth. In combination with standard procedures for expanding T cells in vitro, we are using these techniques to dissect TIL-CRC tumor cell interactions at the cellular and molecular level. The CD4+/CD8+ surface phenotype and TCRβ CDR3 sequence repertoire of TIL from 7 CRC tumor samples (a primary colon tumor from one patient, an ovarian metastasis from a second patient, and 4 non-contiguous hepatic metastases from a third patient), and from T cells in blood or normal adjacent tissue was evaluated by flow cytometry and next-generation sequencing, respectively. Flow cytometry revealed that CD4+ T cells comprised the majority of TIL expanded from the tumors from all 3 patients. Comparison of the TCRβ CDR3 sequence repertoires in the tumor and normal tissues demonstrated that the TIL were clonally distinct from the T cells present in blood or normal adjacent tissue. Moreover, extensive overlap in the repertoire of TIL isolated from the 4 hepatic metastases in patient 3 was observed, suggesting the presence in all 4 metastases of a population of T cells with similar antigenic specificity. We designed TCRβ CDR3 sequence-specific PCR primers to determine whether specific TCRβ sequences identified by next-generation sequencing were primarily found in the CD4+ or CD8+ subsets of TIL. The most common CDR3 sequences observed in TIL from the primary colon tumor of patient 1 were primarily found in the CD4+ subset, while the most common CDR3 sequences in the ovarian metastasis from patient 2 and the hepatic metastases from patient 3 were primarily found in the CD8+ subset. The reactivity of CD8+ and CD4+ T cells expanded from CRC tumor, tumor-associated ascites fluid, normal adjacent tissue, and blood against autologous CRC tumor cells (freshly harvested from xenografts) and autologous dermal fibroblasts was assessed for in a series of 5 patients, including patients 1-3. T cells expanded from the 4 tissue compartments were evaluated by interferon-γ (IFN-γ) ELISPOT for reactivity with xenograft-derived autologous tumor cells or autologous dermal fibroblasts. No significant IFN-γ reactivity was seen when T cells from any compartment were cultured with autologous dermal fibroblasts. In all 5 patients, IFN-γ reactivity with autologous human epithelial cell adhesion molecule (EpCAM) sorted tumor cells was observed. This reactivity was more prominent in purified CD4+ T cells than in CD8+ T cells. No significant difference in the reactivity of T cells from various compartments against autologous tumor cells was observed, but T cells expanded from tumor associated ascites fluid showed overall lower levels of reactivity against all target cells. Collectively, these results demonstrate that CRC TIL comprise a clonally distinct population of T cells containing both CD4+ and CD8+ T cells that are specifically reactive with autologous CRC tumor cells. However, T cells reactive with autologous tumor cells can also be isolated from other compartments such as normal adjacent tissue and peripheral blood. Citation Format: Jeffrey Chou, Christie-Lynn L. Mortales, Sheila Ojeaburu, Raymond SW Yeung, Edus H. Warren. Autologous T cell responses against patient-derived colorectal cancer xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B49.

Tumor infiltrating immune cells often show a skewed phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. Natural Killer (NK) cells are effectors lymphocytes of innate immunity, primarily involved in immunosurvelliance against tumors through their cytotoxic activity. We previously reported that NKs from Non Small Cell Lung Cancer (NSCLC) show the decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNγlow phenotype [Bruno et al, Neoplasia, 2014; Bruno et al, JNCI, 2014]. Here we investigated whether tumor associated (TANKs) and tumor infiltrating (TINKs) NK cells undergo an angiogenic-switch in colorectal cancer (CRC). NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling, by antibody membrane array or angiogenesis functional assay on human umbilical endothelial vein cells (HUVECs) We found that CD56brightCD16− NK cells predominate in CRC adjacent and tumor tissues, produce VEGF, PlGF, IL-8 and show impaired cytotoxicity. Further, TINK/TANKs from CRC patients express the decidual NK markers CD9 and CD49a. Secretomic analysis on CRC peripheral blood NKs revealed up regulation for several pro angiogenic factors, including Angiogenin, Angiopoietin-1/2, TIMP-1/2, Tie-2, MMP1, MMP9. Media conditioned by FACS sorted NK cells from peripheral blood and tumor tissue of CRC patients were able to induce HUVEC proliferation, migration and the formation of capillary like structures. Our data demonstrate that TINK/TANKS from CRC patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. We propose that TINK/TANKs could represent the hallmark for a new paradigm in CRC inflammation. 1. Bruno A, Focaccetti C, Pagani A, Imperatori AS, Spagnoletti M, Rotolo N, Cantelmo AR, Franzi F, Capella C, Ferlazzo G, Mortara L, Albini A, Noonan DM. The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer. Neopla sia. 2013 Feb;15(2):133-42. 2. Bruno A, Ferlazzo G, Albini A, Noonan DM. A think tank of TINK/TANKs: tumor infiltrating/tumor-associated natural killer cells in tumor progression and angiogenesis. J Natl Cancer Inst. 2014 Aug;106(8):dju200 Citation Format: Antonino Bruno, Barbara Bassani, Davide Giuseppe D’Urso, Silvia Zanellato, Elisabetta Gini, Elisa Cassinotti, Luigi Boni, Lorenzo Mortara, Adriana Albini, Douglas M. Noonan. Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3244.

BackgroundAlthough postoperative chemotherapy is widely accepted as the standard modality for Dukes' stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous in vitro and clinical investigations reported that CRC patients with wild-type p53 gene (TP53)-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated TP53-tumors do not. However, these studies evaluated the mutation-status of TP53 by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined.MethodsTo evaluate the significance of the TP53 mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy, we examined the mutation by direct sequencing from fresh CRC tumors and evaluated the R72P polymorphism of the mutated TP53 by a combined mutant allele- and polymorphic allele-specific polymerase chain reaction (PCR).ResultsThe TP53 mutation occurred in 147 (70%) of 211 Japanese CRC tumors. The mutation was observed in 93 (63%) tumors on the R72 allele and in 54 (37%) tumors on the P72 allele. Although the alterations to TP53 have no prognostic significance for CRC patients overall, we found that Dukes' stage C CRC patients who did not receive postoperative chemotherapy and carried the mutated TP53-R72 showed significantly longer survival times than those with the mutated TP53-P72 when evaluated by overall survival (p = 0.012).ConclusionUsing a combined mutant allele- and polymorphic allele-specific PCR, we defined the codon 72 polymorphic status of the TP53 mutated allele in Japanese CRC patients. We raised a possibility that Dukes' stage C colorectal cancer patients with tumors carrying TP53 mutation, especially the P72 allele, benefited from 5-FU based postoperative chemotherapy.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in western countries. Approximately one-quarter of newly diagnosed patients for CRC have metastases, and a further 40%-50% experience disease recurrence or develop metastases after all standard therapies. Therefore, understanding the molecular mechanisms involved in the progression of CRC and subsequently developing novel therapeutic targets is crucial to improve management of CRC and patients' long-term survival. Several tyrosine kinase receptors have been implicated in CRC development, progression and metastasis, including epidermal growth factor receptor (EGFR) and vascular EGFR. Recently, tropomyosin-related kinase B (TrkB), a tyrosine kinase receptor, has been reported in CRC and found to clearly exert several biological and clinical features, such as tumor cell growth and survival in vitro and in vivo, metastasis formation and poor prognosis. Here we review the significance of TrkB and its ligand brain derived-neurotrophic factor in CRC. We focus on their expression in CRC tumor samples, and their functional roles in CRC cell lines and in in vivo models. Finally we discuss therapeutic approaches that can lead to the development of novel therapeutic agents for treating TrkB-expressing CRC tumors.

BackgroundHistone epigenetic modification disorder is an important predisposing factor for the occurrence and development of many cancers, including colorectal cancer (CRC). The role of MYSM1, a metalloprotease that deubiquitinates monoubiquitinated histone H2A, in colorectal cancer was identified to evaluate its potential clinical application value.MethodsMYSM1 expression levels in CRC cell lines and tumor tissues were detected, and their associations with patient survival rate and clinical stage were analyzed using databases and tissue microarrays. Gain- and loss-of-function studies were performed to identify the roles of MYSM1 in CRC cell proliferation, apoptosis, cell cycle progression, epithelial-mesenchymal transition (EMT) and metastasis in vitro and in vivo. ChIP, rescue assays and signal pathway verification were conducted for mechanistic study. Immunohistochemistry (IHC) was used to further assess the relationship of MYSM1 with CRC diagnosis and prognosis.ResultsMYSM1 was significantly downregulated and was related to the overall survival (OS) of CRC patients. MYSM1 served as a CRC suppressor by inducing apoptosis and inhibiting cell proliferation, EMT, tumorigenic potential and metastasis. Mechanistically, MYSM1 directly bound to the promoter region of miR-200/CDH1, impaired the enrichment of repressive H2AK119ub1 modification and epigenetically enhanced miR-200/CDH1 expression. Testing of paired CRC patient samples confirmed the positive regulatory relationship between MYSM1 and miR-200/CDH1. Furthermore, silencing MYSM1 stimulated PI3K/AKT signaling and promoted EMT in CRC cells. More importantly, a positive association existed between MYSM1 expression and a favorable CRC prognosis.ConclusionsMYSM1 plays essential suppressive roles in CRC tumorigenesis and is a potential target for reducing CRC progression and distant metastasis.

Background: Colorectal cancer (CRC) is currently the third most prevalent cancer worldwide and ranks as the second deadliest. Colon cancer is characterized by disruption in cellular structure and polarity, processes that are commonly regulated by cell traffcking. The molecular motor Myosin 5b (Myo5b) is a key component of intracellular traffcking in the intestinal epithelium. Myo5b moves along F-actin tracks to transport cargo to and from the apical membrane. When Myo5b function is impaired, it leads to significant changes in gastrointestinal epithelial cells, including hyper-proliferation, alterations in tight junction proteins, and reduced differentiation. For Myo5b to function properly it must be properly folded; a process regulated by the myosin chaperone Unc45a. Recent work demonstrates that Myo5b levels are decreased in gastric cancer and during the progression of colorectal tumors. However, the mechanism driving this altered Myo5b expression in colorectal cancer remains unknown. We hypothesize that microRNAs regulate the expression of Unc45a, which subsequently impacts Myo5b function, promoting colorectal cancer. Methods and Results: Analysis of high-throughput RNA-sequencing data from the Cancer Genome Atlas (TCGA) revealed hyper-methylation of the Myo5b gene in CRC tumors (n=286) compared to controls (n=41). Findings in gastric cancer have shown that hyper-methylation leads to the silencing of Myo5b gene expression. Consistent with these findings, we observe a significant reduction in Myo5b gene expression in CRC tumors. Immunostaining of CRC tumor arrays and analysis from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) demonstrated a corresponding decrease in Myo5b protein levels. For comparison, we examined Myo5b levels in normal human colonic organoids and the colon cancer cell lines T84,HT-29, HT29-MTX, HCT-8 and HCT-116. Using qPCR, we confirmed a significant reduction in Myo5b gene expression in cancer cells compared to human colonic organoids. We postulated that even if Myo5b was expressed in CRC, it might not function properly if Unc45a function was perturbed. Using the same databases, we found that while Unc45a gene expression levels remain unchanged, its protein levels were significantly reduced. Analysis of the miRMap data reveled multiple microRNAs that could target Unc45a. The top 10 Unc45a-targeting microRNAs were all found to be elevated in CRC compared to normal control tissue, suggesting that microRNAs may lead to reduced Unc45a protein levels. The functional importance of decreased Myo5b in CRC was underscored by the observation that CRC patients with lower levels of Myo5b had reduced survival compared to those CRC patients with higher levels of Myo5b. Additionally, the loss of Myo5b correlated with increased ribosome biogenesis, a key driver of tumor proliferation. Conclusions: These findings indicate that reduced Myo5b in CRC may be attributed to gene methylation and impaired protein folding by Unc45a. The loss of Myo5b holds significance, as it is associated with poorer prognosis and linked with cancer progression. National Diabetes and Digestive and Kidney Diseases Grant R43 DK109820, RO1 DK048370, KO1 DK121869, MUSC start up funds, Histochemical 2022 Cornerstone Grant, and NIDDK T32. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mounting evidence suggests that a prominent T cell response in the colorectal cancer (CRC) tumor microenvironment is a valuable prognostic indicator, independent of stage at diagnosis. However, the strength and quality of host immune responses are highly variable across patients, and the factors that influence this variability are not well understood. The unique microbial communities present in the CRC tumor microenvironment may contribute to the modulation of local host immune responses, but prior investigations have been limited and focused on candidate microbes. Here, we investigated the association between the CRC tumor bacterial microbiome and the quantity and clonality of the T cell infiltrate. This investigation included 51 CRC from the Molecular Epidemiology of Colorectal Cancer (MECC) study, a population-based case-control study in northern Israel. Genomic DNA was extracted from fresh frozen tumors using microbiome-optimized techniques and sequenced using a PCR amplicon-mediated workflow on the Illumina MiSeq. Sequences from the V1-V3 hypervariable regions of the bacterial 16S rRNA gene region were clustered into operational taxonomic units (OTUs), a proxy for species, and taxonomic identification was determined using the Ribosomal Database Project. In addition, T cell repertoires were measured using the DNA-based immunoSEQ assay (Adaptive Biotechnologies), and T cell fraction (i.e. proportion of rearranged T cells relative to all nucleated cells) and clonality (i.e. T cell receptor diversity) were calculated. Bacterial sequencing was successful in 48 (94%) of samples, with a total of 868 OTUs identified. Klebsiella and Escherichia/Shigella were the most prevalent genera, detected in approximately 90% of tumors, followed by Propionibacterium, Pelomonas, and Veillonella, with each detected in approximately 50% of tumors. Fusobacterium, a genus of oral anaerobic bacteria believed to accelerate tumor progression and enable immune evasion in CRC, was detected in 20% of tumors. When present, Bacteroides and Fusobacterium were the most abundant, comprising 10-15% of all bacteria within each tumor, on average. ImmunoSEQ analysis identified a mean productive T cell fraction of 0.08 (SD: 0.16) and a mean productive clonality of 0.14 (SD: 0.06). Analysis of molecular variance (AMOVA) of the Theta YC measure of dissimilarity showed a statistically significant difference in bacterial community structure (i.e. composition and relative abundance) between samples in the highest versus lower three quartiles of T cell fraction (P=0.03) and clonality (P=0.01). These preliminary findings suggest a potential relationship between microbial community structure and the quantity and diversity of the CRC T cell infiltrate. Additional analyses are underway to better understand this association as well as the independent contributions of the microbiome to CRC prognosis. Citation Format: Christine M. Pierce, Bo-young Hong, Hannah J. Hoehn, Maria F. Gomez, Marilena Melas, Kevin McDonnell, Youngchul Kim, Erica Sodergren, George Weinstock, Hedy S. Rennert, Thomas Giordano, Joel Greenson, Gad Rennert, Stephen B. Gruber, Stephanie L. Schmit. Microbes in the tumor microenvironment: Bacterial influences on host immunity in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4746.

Colorectal cancer (CRC) is an aggressive disease and the leading cause of cancer death, characterised by high heterogeneity and various risk factors related to its etiology (1-3). During carcinogenesis, adenomatous polyps, which represent most premalignant lesions (85-90% of sporadic CRC), can develop into CRC (4-6). About 20-25% of CRC patients are diagnosed with metastatic disease, which is associated with poorer survival rates. CRC can spread to various tissues, including lymph nodes, liver, lungs, peritoneum, bones, and the central nervous system (7). The liver is the most common site for detecting metastatic CRC and is involved in 25% to 50% of cases. However, a rare form of CRC with bone metastasis occurs in 3% to 7% of patients; these individuals often have worse survival outcomes, with shorter survival times and limited treatment options (8-10). TGF-β and BMP signalling pathways are crucial mechanisms in tissue homeostasis, promoting cell proliferation and differentiation during crypt formation in the intestine (11-15). Dysregulation of these pathways in intestinal cells can impair their tumour-suppressing functions and facilitate tumour development (16-18). In particular, mutations in TGFBR2, BMPR1A, and SMAD4 have been identified as contributing to CRC carcinogenesis (2, 3, 19-23). Patients with consensus molecular subtype (CMS) 4, categorised based on gene expression signature, are associated with prominent activation of TGF-β, along with stromal infiltration, epithelial to mesenchymal transition (EMT), and angiogenesis, leading to poorer survival outcomes (19). In silico analysis of gene expression levels of TGF-β and BMP signalling components across CRC cell lines using the DepMap database, along with examination of key proteins and their phosphorylation statuses through Western blots, revealed that ligand activation occurs at various levels, with notable activation of upstream receptors such as TGFBR2, ACVR1B, and BMPR1A. Increased expression of signal transduction genes, including SMAD1, SMAD2, SMAD3, and SMAD5, was also observed. Interestingly, although phosphorylation of SMAD1/5/8 was detected in SW620 cell lines (a metastatic CRC cell line), SMAD4, a central element in signal transduction, was found to be downregulated. Apart from the differential expression of TGF-β and BMP signalling in CRC, the investigation of gene expression in these pathways within disease-free bone marrow (BM) cells using the Stemformatics database was also investigated to provide some insight into whether this morphogenic pathway is involved in metastatic spread to the bone. Here we demonstrated high levels of TGF-β and BMP signalling pathways in the bone marrow associated cells, indicating maintenance of homeostasis and serving as a baseline reference for further research. Assessment of epithelial SMAD4 expression in colorectal polyps using immunohistochemistry (IHC) and digital weighted histoscoring with QuPath revealed that low SMAD4 levels in adenomatous polyps correlated with higher grades of dysplasia, different histological subtypes, the presence of metachronous polyps, and served as a prognostic marker. This marker indicated an increased risk of developing metachronous polyps, particularly in the tubulovillous polyp subtype. Additionally, transcriptomic analysis of tubulovillous polyps showed that upregulation of genes involved in protein deubiquitination occurs in polyps with low SMAD4 levels, along with a likely enrichment in tyrosine metabolism, PPAR signalling, arginine and proline metabolism, leukocyte transendothelial migration, and basal cell carcinoma. In CRC, lower epithelial SMAD4 expression was strongly associated with higher tumour stages and increased tumour stroma. Moreover, lower SMAD4 expression in CRC tumours had prognostic significance, predicting decreased cancer-specific survival in CRC patients, especially in right-sided tumours. A combination of tumour SMAD4 levels and stroma percentages suggested that the worst survival outcome was in patients with low SMAD4 expression in the tumour and high stroma content. Transcriptomic analysis also identified downregulation of SOD3 and enrichment of aminopeptidase activity in this group. Differential expression patterns at gene and protein levels, along with phosphorylation activity, were observed across the complex crosstalk of MAPK/ERK, WNT/β-catenin, TGF-β/BMP, and PI3K signalling pathways in CRC cell lines. However, the mechanisms regulating SMAD4 activity in CRC remain unclear.Testing the TGF-β and BMP signalling inhibitor (LDN-212854) in combination with the standard chemotherapy (Fluoropyrimidine; 5-FU) showed synergistic effects on CRC cell viability, cell cycle arrest, cell proliferation, and cell recovery in an in vitro 2D study. Conditioned media from BM and hepatic cells influence changes in CRC behaviour. The 3D bioprinted SW620 spheroids in 2% alginate and 8% gelatin hydrogel supported physiological interactions, spheroid survival and growth, and were used for drug screening, demonstrating efficacy of the LDN-212854/5-FU combination in a more mechanophysical 3D system. Further development of this in vitro 3D model to become multicell by incorporating metastatic CRC culture with BM and hepatic niches would enhancefuture CRC research and drug discovery.