Heterogeneity Of Colorectal Cancer Research Articles

Uncovering disease-related multicellular pathway modules on large-scale single-cell transcriptomes with scPAFA.

Pathway analysis is a crucial analytical phase in disease research on single-cell RNA sequencing (scRNA-seq) data, offering biological interpretations based on prior knowledge. However, currently available tools for generating cell-level pathway activity scores (PAS) exhibit computational inefficacy in large-scale scRNA-seq datasets. Additionally, disease-related pathways are often identified through cross-condition comparisons within specific cell types, overlooking potential patterns that involve multiple cell types. Here, we present single-cell pathway activity factor analysis (scPAFA), a Python library designed for large-scale single-cell datasets allowing rapid PAS computation and uncovering biologically interpretable disease-related multicellular pathway modules, which are low-dimensional representations of disease-related PAS alterations in multiple cell types. Application on colorectal cancer (CRC) datasets and large-scale lupus atlas over 1.2 million cells demonstrated that scPAFA can achieve over 40-fold reductions in the runtime of PAS computation and further identified reliable and interpretable multicellular pathway modules that capture the heterogeneity of CRC and transcriptional abnormalities in lupus patients, respectively. Overall, scPAFA presents a valuable addition to existing research tools in disease research, with the potential to reveal complex disease mechanisms and support biomarker discovery at the pathway level.

GPA33 expression in colorectal cancer can be induced by WNT inhibition and targeted by cellular therapy.

GPA33 is a promising surface antigen for targeted therapy in colorectal cancer (CRC). It is expressed almost exclusively in CRC and intestinal epithelia. However, previous clinical studies have not achieved expected response rates. We investigated GPA33 expression and regulation in CRC and developed a GPA33-targeted cellular therapy. We examined GPA33 expression in CRC cohorts using immunohistochemistry and immunofluorescence. We analyzed GPA33 regulation by interference with oncogenic signaling in vitro and in vivo using inhibitors and conditional inducible regulators. Furthermore, we engineered anti-GPA33-CAR T cells and assessed their activity in vitro and in vivo. GPA33 expression showed consistent intratumoral heterogeneity in CRC with antigen loss at the infiltrative tumor edge. This pattern was preserved at metastatic sites. GPA33-positive cells had a differentiated phenotype and low WNT activity. Low GPA33 expression levels were linked to tumor progression in patients with CRC. Downregulation of WNT activity induced GPA33 expression in vitro and in GPA33-negative tumor cell subpopulations in xenografts. GPA33-CAR T cells were activated in response to GPA33 and reduced xenograft growth in mice after intratumoral application. GPA33-targeted therapy may be improved by simultaneous WNT inhibition to enhance GPA33 expression. Furthermore, GPA33 is a promising target for cellular immunotherapy in CRC.

The RAS/BRAF genes status in patients with colorectal cancer (review)

Colorectal cancer (CRC) is the third in prevalence among oncological diseases worldwide and second in the structure of oncological mortality. Genetic assessment of CRC is a necessary stage during selecting further treatment for patients. Many studies demonstrate a diverse distribution of mutations in the KRAS, NRAS, and BRAF genes in CRC. A critical literature review was conducted in order to systematize data on the mutational profile and genetic heterogeneity of these driver mutations in Russian patients with CRC. Articles were searched for in open databases. Totally 17 Russian studies and 3 English meta-analyses were analyzed for comparison with Russian data. Mutations in the KRAS, NRAS, and BRAF genes, according to Russian and international studies, are found in 40 %, 4 %, and 7 % in CRC patients, respectively. The frequency and specific localization of mutations may depend on the geographical location and nationality of the cohort. High intertumoral and intratumoral heterogeneity in CRC, especially in KRAS gene mutations, significantly influences the choice of further therapy and underscores the need for more detailed study of the mutational profile of the primary tumor, affected lymph nodes, and distant metastases. In Russia, several molecular genetic methods are used to determine somatic mutations in CRC with different sensitivity and specificity, the most common is real-time PCR. More accurate diagnostic methods include digital droplet PCR, Sanger sequencing, and next-generation sequencing, but each method has its limitations that must be considered when planning diagnostics and research. The promising directions in personalized oncology is the study of gene copy number variations, which may contribute to the development of new methods for treating CRC in the future. Despite the large number of studies, some aspects of the mutational profile of CRC in Russian studies remain poorly understood, which is why further research is needed on patients with colorectal cancer in Russia.

Investigating the role of senescence biomarkers in colorectal cancer heterogeneity by bulk and single-cell RNA sequencing

Colorectal cancer (CRC) is one of most common tumors worldwide, causing a prominent global health burden. Cell senescence is a complex physiological state, characterized by proliferation arrest. Here, we investigated the role of cellular senescence in the heterogeneity of CRC. Based on senescence-associated genes, CRC samples were classified into different senescence patterns with different survival, cancer-related biological processes and immune cell infiltrations. A senescence-related model was then developed to calculate the senescence-related score to comprehensively explore the heterogeneity of each CRC sample such as stromal activities, immunoreactivities and drug sensitivity. Single-cell analysis revealed there were different immune cell infiltrations between low and high senescence-related model genes enrichment groups, which was confirmed by multiplex immunofluorescence staining. Pseudotime analysis indicated model genes play a pivotal role in the evolution of B cells. Besides, intercellular communication modeled by NicheNet showed tumor cells with higher enrichment of senescence-related model genes highly expressed CXCL2/3 and CCL3/4, which attracted immunosuppressive cell infiltration and promoted tumor metastasis. Finally, top 6 hub genes were identified from senescence-related model genes by PPI analysis. And RT-qPCR revealed the expression differences of hub genes between normal and CRC cell lines, indicating to some extent the clinical practicability of senescence-related model. To sum up, our study explores the impact of cellular senescence on the prognosis, TME and treatment of CRC based on senescence patterns. This provides a new perspective for CRC treatment.

Molecular subtypes of colorectal cancer in the era of precision oncotherapy: Current inspirations and future challenges.

Colorectal cancer (CRC) is among the most hackneyed malignancies. Even patients with identical clinical symptoms and the same TNM stage still exhibit radically different clinical outcomes after receiving equivalent treatment regimens, indicating extensive heterogeneity of CRC. Myriad molecular subtypes of CRC have been exploited for decades, including the most compelling consensus molecular subtype (CMS) classification that has been broadly applied for patient stratification and biomarker-drug combination formulation. Encountering barriers to clinical translation, however, CMS classification fails to fully reflect inter- or intra-tumor heterogeneity of CRC. As a consequence, addressing heterogeneity and precisely managing CRC patients with unique characteristics remain arduous tasks for clinicians. In this review, we systematically summarize molecular subtypes of CRC and further elaborate on their clinical applications, limitations, and future orientations. In recent years, exploration of subtypes through cell lines, animal models, patient-derived xenografts (PDXs), organoids, and clinical trials contributes to refining biological insights and unraveling subtype-specific therapies in CRC. Therapeutic interventions including nanotechnology, clustered regulatory interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9), gut microbiome, and liquid biopsy are powerful tools with the possibility to shift the immunologic landscape and outlook for CRC precise medicine.

Integrated analysis of tumor-associated macrophages and M2 macrophages in CRC: unraveling molecular heterogeneity and developing a novel risk signature.

Emerging investigations have increasingly highlighted the critical role of tumor-associated macrophages (TAMs) and M2 macrophages in cancer development, progression, and metastasis, marking them as potential targets in various cancer types. The main objective of this research is to discover new biomarkers associated with TAM-M2 macrophages in colorectal cancer (CRC) and to dissect the molecular heterogeneity of CRC by combining single-cell RNA sequencing and bulk RNA-seq data. By utilizing weighted gene co-expression network analysis (WGCNA), we acquired TAM-M2-associated genes by intersecting TAM marker genes obtained from scRNA-seq data with module genes of M2 macrophages derived from bulk RNA-seq data. We employed least absolute shrinkage and selection operator (LASSO) Cox analysis to select predictive biomarkers from these TAM-M2-related genes. Quantitative polymerase chain reaction (qPCR) was employed to validate the mRNA expression levels of the genes identified in the screening. This led to the development of the TAM-M2-related signature (TAMM2RS). We also conducted functional and immune landscape analyses of different risk groups. The combination of scRNA-seq and bulk RNA-seq analyses yielded 377 TAM-M2-related genes. DAPK1, NAGK, and TRAF1 emerged as key prognostic genes in CRC, which were identified through LASSO Cox analysis. Utilizing these genes, we constructed and validated the TAMM2RS, demonstrating its effectiveness in predicting survival in CRC patients. Our research offers a thorough investigation into the molecular mechanisms associated with TAM-M2 macrophages in CRC and unveils potential therapeutic targets, offering new insights for treatment strategies in colorectal cancer.

An inflammation-related subtype classification for analyzing tumor microenvironment and clinical prognosis in colorectal cancer.

The inflammatory response plays an essential role in the tumor microenvironment (TME) of colorectal cancer (CRC) by modulating tumor growth, progression, and response to therapy through the recruitment of immune cells, production of cytokines, and activation of signaling pathways. However, the molecular subtypes and risk score prognostic model based on inflammatory response remain to be further explored. Inflammation-related genes were collected from the molecular signature database and molecular subtypes were identified using nonnegative matrix factorization based on the TCGA cohort. We compared the clinicopathological features, immune infiltration, somatic mutation profile, survival prognosis, and drug sensitivity between the subtypes. The risk score model was developed using LASSO and multivariate Cox regression in the TCGA cohort. The above results were independently validated in the GEO cohort. Moreover, we explored the biological functions of the hub gene, receptor interacting protein kinase 2 (RIPK2), leveraging proteomics data, in vivo, and in vitro experiments. We identified two inflammation-related subtypes (inflammation-low and inflammation-high) and have excellent internal consistency and stability. Inflammation-high subtype showed higher immune cell infiltration and increased sensitivity to common chemotherapeutic drugs, while inflammation-low subtype may be more suitable for immunotherapy. Besides, the two subtypes differ significantly in pathway enrichment and biological functions. In addition, the 11-gene signature prognostic model constructed from inflammation-related genes showed strong prognostic assessment power and could serve as a novel prognostic marker to predict the survival of CRC patients. Finally, RIPK2 plays a crucial role in promoting malignant proliferation of CRC cell validated by experiment. This study provides new insights into the heterogeneity of CRC and provides novel opportunities for treatment development and clinical decision making.

Abstract 1150: Unveiling the spatial landscape of tumor and stroma heterogeneity in colorectal cancer with spatial transcriptomics

Abstract Heterogeneity in colorectal cancer (CRC) manifests in the diversity and interactions among tumor cells, support stroma, and immune components, critically impacting patient prognosis and therapeutic efficacy. In this study, spatial transcriptomics (ST, 10x Genomics Visium Spatial Gene Expression) was performed on 40 retrospectively collected CRC samples, along with detailed clinicopathological parameters and long-term clinical outcomes, with the goal of unraveling the molecular topography of the tumor and surrounding stromal compartments, and associating their spatial interactions to clinical outcomes. Five primary RNA groups were identified through unsupervised clustering on ST data, correlating with tissue classes detected in H&E-stained images via a pre-trained deep learning model. Markers and pathways specific to these tissues were identified. Furthermore, immune cell infiltration in these regions was deconvoluted using reference single-cell RNA sequencing (scRNA-seq) data. Sub-clustering within the tumor groups revealed five major tumor subtypes, each characterized by a distinct set of differentially expressed marker genes. Enrichment analysis across these subtypes highlighted distinct activation and suppression in pathways including KRAS, p53, MAPK, and Integrin. Tumors exhibited intra- and inter-tumoral heterogeneity. Location-specific tumor subtypes were observed, with those in right-sided CRC displaying increased immune cell infiltration, linked to enriched pathways such as Type II Interferon Signaling and Cytokine Signaling. Conversely, left-sided CRC subtypes had reduced immune cell presence, and were characterized by enhanced pathways related to vascular development, Hypoxia, and p53 Signaling. Inferred clonal lineages from copy number variation (CNV) using ST suggested a spatially structured phylogenetic organization corresponding to transcriptional clusters. Additionally, the ST data unveiled distinct spatial distribution patterns across various cancer-associated fibroblast (CAF) phenotypes, such as matrix, inflammatory, vessel-associated, tumor-promoting, and antigen-presenting CAFs. Transcriptional patterns uncovered through scRNA-seq, including epithelial-mesenchymal transition (EMT) signatures and consensus molecular subtypes (CMS), displayed unique spatial layouts within the tumor microenvironment. To our knowledge, this is the most comprehensive study to date on spatial transcriptomic heterogeneity within CRC patients, revealing distinct tumor, stroma and immune compositions and spatial structures. This spatially detailed map provides novel insights into CRC heterogeneity, critical for advancing personalized treatment. Citation Format: Jie Zhou, Todd B. Sheridan, Sergii Domanskyi, Stephanie L. Cowles, Sunghee Park, Ilham Putra, Olga Anczukow, Jeffrey H. Chuang, Jill C. Rubinstein. Unveiling the spatial landscape of tumor and stroma heterogeneity in colorectal cancer with spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1150.

Abstract 881: Cell-type-specific gene programs suggest immune and stromal drivers of relapse in colorectal cancer

Abstract Background: Molecular profiling of colorectal cancer (CRC) has demonstrated marked heterogeneity between tumors that appear histologically similar. Although existing classification systems such as the Consensus Molecular Subtypes (CMS) have shown clinical utility, these bulk sequencing-based methods fail to adequately address the intra-tumoral heterogeneity of CRC. To disentangle the complex landscape and identify clinically-relevant properties of CRC we generated a single cell sequencing (scRNASeq) database from 225 tumor samples derived from resection of primary colon or liver metastases. Our analysis defines cell-type-specific gene programs significantly associated with relapse and survival and suggests interventions which may abrogate their effect. Methods: Utilizing a bio-repository of CRC specimens with an average of 5 years of clinical follow-up, we performed scRNASeq on 225 patient samples comprising ~ 1,000,000 total cells and 2.4 billion gene expression measurements. Following traditional clustering approaches, we then inferred gene expression programs through consensus non-negative matrix factorization (cNMF) to characterize cell activities. Gene program activity was explored on a cell-type-specific level and associated with clinical outcomes such as relapse. Treatment effects were additionally explored. Results: We identified 56 cell-type specific gene programs significantly associated with relapse in adult CRC patients. Interestingly these programs spanned all cellular compartments with the majority found in stromal or immune, as opposed to tumor cells. Program activity such as the non-canonical Granulocyte Chemotaxis program in B Cells was protective of relapse (relapse median expression 0% activity vs non-relapse activity 40% pAdj <.05 × 1050) whereas the Epithelial Mesenchymal Transition (EMT) program in Cancer Associated Fibroblasts (CAF) was strongly associated with relapse (relapse median expression 16% activity vs non-relapse activity 0% pAdj <.05 × 1010). Cumulative program usage enabled stratification on a patient level to provide an estimate of comparative disease free survival (DFS) and overall survival (OS); gene-programs were significantly associated with outcomes such as the CAF-EMT program which was associated with a DFS HR of 3 (pAdj <.0005). Responses to chemotherapy and treatment effect were also explored; prior treatment with Bevacizumab was associated with a significant reduction in the CAF-EMT program (11% activity in non-exposed patients versus 0% activity in exposed patients pAdj <.05 × 1010). Conclusions: Analysis of the largest CRC scRNASeq cohort to date identifies a number of previously undescribed cell-type specific gene programs significantly associate with relapse after surgery. These programs provide insights into the underlying mechanisms and suggest that the TME plays an important role in relapse. Citation Format: Nicholas J. Hornstein, Mingshuang Wang, Abdelrahman Yousef, Saikat Chowdhury, Fadl Zeineddine, Mohammad Zeineddine, Mahmoud Yousef, Shuangjie You, Betul Gunes, David Menter, Scott Kopetz, John Paul Shen. Cell-type-specific gene programs suggest immune and stromal drivers of relapse in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 881.

PI3K/Akt/mTOR Signaling Pathway as a Target for Colorectal Cancer Treatment.

In the last decade, pathway-specific targeted therapy has revolutionized colorectal cancer (CRC) treatment strategies. This type of therapy targets a tumor-vulnerable spot formed primarily due to an alteration in an oncogene and/or a tumor suppressor gene. However, tumor heterogeneity in CRC frequently results in treatment resistance, underscoring the need to understand the molecular mechanisms involved in CRC for the development of novel targeted therapies. The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of the rapamycin (PI3K/Akt/mTOR) signaling pathway axis is a major pathway altered in CRC. The aberrant activation of this pathway is associated with CRC initiation, progression, and metastasis and is critical for the development of drug resistance in CRC. Several drugs target PI3K/Akt/mTOR in clinical trials, alone or in combination, for the treatment of CRC. This review aims to provide an overview of the role of the PI3K/Akt/mTOR signaling pathway axis in driving CRC, existing PI3K/Akt/mTOR-targeted agents against CRC, their limitations, and future trends.

Identification and validation of mitophagy-related signatures as a novel prognostic model for colorectal cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Mitophagy is associated with tumorigenesis and development of malignancy. However, the specific role of mitophagy has yet not been systematically explored in CRC. The RNA-sequencing dataset of CRC from The Cancer Genome Atlas (TCGA) and microarray data of gene expression profiles of CRC from Gene Expression Omnibus (GEO) were downloaded. Mitophagy-related gene sets were obtained from the Pathway Unification database. The package "limma" was used for differential gene expression analysis. Kaplan-Meier (KM) survival analyses were utilized to evaluate the prognostic value of the mitophagy regulators. Single-sample gene set enrichment analysis (ssGSEA) was used to estimate the infiltrating immune cells and the activity of immune response. The ConsensusClusterPlus algorithm was used to determine mitophagy-related subtypes. Principal component analysis (PCA) was used to create composite measurement of mitophagy scores. The R packages "survminer" and "ReGlot" were used to plot the nomogram and calibration curves. Integrated analysis of the GEO and TCGA databases revealed some common differentially expressed genes (DEGs) in CRC. MFN2, UBB, PINK1, and PRKN were significantly downregulated in CRC samples as compared to normal samples, and other genes were significantly upregulated in CRC samples. KM survival analyses showed that high expression of ATG12 and MAP1LC3B predicted a poor prognosis, whereas high expression of TOMM22 and TOMM40 predicted a better prognosis. Mitophagy showed significant correlation with immune-related pathways in CRC samples. We identified 2 distinct CRC subtypes with different mitophagy accumulation, of which subtype B had better prognosis and immune activity. The mitophagy score may be employed as a new and efficient clinical predictor in conjunction with other clinical indicators to predict the prognosis of CRC patients. We systematically investigated the CRC heterogeneity with reference to mitophagy based on bioinformatics analyses, and the findings of this study might provide some guidance for future research into potential biomarkers for diagnosis and prognosis prediction of CRC patients.

DNA methylation modulates epigenetic regulation in colorectal cancer diagnosis, prognosis and precision medicine.

Colorectal cancer (CRC) is a multifaceted disease influenced by the interplay of genetic and environmental factors. The clinical heterogeneity of CRC cannot be attributed exclusively to genetic diversity and environmental exposures, and epigenetic markers, especially DNA methylation, play a critical role as key molecular markers of cancer. This review compiles a comprehensive body of evidence underscoring the significant involvement of DNA methylation modifications in the pathogenesis of CRC. Moreover, this review explores the potential utility of DNA methylation in cancer diagnosis, prognostics, assessment of disease activity, and prediction of drug responses. Recognizing the impact of DNA methylation will enhance the ability to identify distinct CRC subtypes, paving the way for personalized treatment strategies and advancing precision medicine in the management of CRC.

Host genetics-associated mechanisms in colorectal cancer.

Colorectal cancer (CRC) represents the second leading cause of cancer incidence and the third leading cause of cancer deaths worldwide. There is currently a lack of understanding of the onset of CRC, hindering the development of effective prevention strategies, early detection methods and the selection of appropriate therapies. This article outlines the key aspects of host genetics currently known about the origin and development of CRC. The organisation of the colonic crypts is described. It discusses how the transformation of a normal cell to a cancer cell occurs and how that malignant cell can populate an entire colonic crypt, promoting colorectal carcinogenesis. Current knowledge about the cell of origin of CRC is discussed, and the two morphological pathways that can give rise to CRC, the classical and alternative pathways, are presented. Due to the molecular heterogeneity of CRC, each of these pathways has been associated with different molecular mechanisms, including chromosomal and microsatellite genetic instability, as well as the CpG island methylator phenotype. Finally, different CRC classification systems are described based on genetic, epigenetic and transcriptomic alterations, allowing diagnosis and treatment personalisation.

Molecular Complexity of Colorectal Cancer: Pathways, Biomarkers, and Therapeutic Strategies.

Colorectal cancer (CRC) is a diverse disease entity and a leading cause of cancer-related mortality worldwide. CRC results from the accumulation of multiple genetic and epigenetic alterations. This heterogeneity of CRC underscores the significance of understanding its molecular landscape, as variations in tumor genetics can greatly influence both patient prognosis and therapeutic response. The molecular complexity of CRC is defined by three major carcinogenesis pathways: chromosomal instability (CIN), microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP). These pathways contribute to the onset and progression of CRC through mutations, epigenetic modifications, and dysregulated cellular signalling networks. The heterogeneous nature of CRC continues to pose challenges in identifying universally effective treatments, highlighting the need for personalized approaches. Hence, the present review aims at unravelling the molecular complexity of CRC that is essential for improving diagnosis, prognostication, and treatment. We detail on the current understanding of the molecular framework of CRC, central signalling pathways of CRC associated with its initiation to a malignant phenotype, further invasion, progression, metastases, and response to therapy. Continued research into CRC's pathways and biomarkers will pave the way for the development of more precise and effective therapeutic strategies, ultimately improving patient outcomes.

Deciphering the Tumor Microenvironment of Colorectal Cancer and Guiding Clinical Treatment With Patient-Derived Organoid Technology: Progress and Challenges.

Colorectal cancer (CRC) is one of the most prevalent malignant tumors of the digestive tract worldwide. Despite notable advancements in CRC treatment, there is an urgent requirement for preclinical model systems capable of accurately predicting drug efficacy in CRC patients, to identify more effective therapeutic options. In recent years, substantial strides have been made in the field of organoid technology, patient-derived organoid models can phenotypically replicate the original intra-tumor and inter-tumor heterogeneity of CRC, reflecting cellular interactions of the tumor microenvironment. Patient-derived organoid models have become an indispensable tool for investigating the pathogenesis of CRC and facilitating translational research. This review focuses on the application of organoid technology in CRC modeling, tumor microenvironment, and guiding clinical treatment, particularly in drug screening and personalized medicine. It also examines the existing challenges encountered in clinical organoid research and provides a prospective outlook on the future development directions of clinical organoid research, encompassing the standardization of organoid culture technology and the application of tissue engineering technology.

Host-microbiota interactions contributing to the heterogeneous tumor microenvironment in colorectal cancer.

Colorectal cancer (CRC) exhibits pronounced heterogeneity and is categorized into four widely accepted consensus molecular subtypes (CMSs) with unique tumor microenvironments (TMEs). However, the intricate landscape of the microbiota and host-microbiota interactions within these TMEs remains elusive. Using RNA-sequencing data from The Cancer Genome Atlas, we analyzed the host transcriptomes and intratumoral microbiome profiles of CRC samples. Distinct host genes and microbial genera were identified among the CMSs. Immune microenvironments were evaluated using CIBERSORTx and ESTIMATE, and microbial coabundance patterns were assessed with FastSpar. Through LASSO penalized regression, we explored host-microbiota associations for each CMS. Our analysis revealed distinct host gene signatures within the CMSs, which encompassed ferroptosis-related genes and specific immune microenvironments. Moreover, we identified 293, 153, 66, and 109 intratumoral microbial genera with differential abundance, and host-microbiota associations contributed to distinct TMEs, characterized by 829, 1,270, 634, and 1,882 robust gene-microbe associations for each CMS in CMS1-CMS4, respectively. CMS1 featured inflammation-related HSF1 activation and gene interactions within the endothelin pathway and Flammeovirga. Integrin-related genes displayed positive correlations with Sutterella in CMS2, whereas CMS3 spotlighted microbial associations with biosynthetic and metabolic pathways. In CMS4, genes involved in collagen biosynthesis showed positive associations with Sutterella, contributing to disruptions in homeostasis. Notably, immune-rich subtypes exhibited pronounced ferroptosis dysregulation, potentially linked to tissue microbial colonization. This comprehensive investigation delineates the diverse landscapes of the TME within each CMS, incorporating host genes, intratumoral microbiota, and their complex interactions. These findings shed light on previously uncharted mechanisms underpinning CRC heterogeneity and suggest potential therapeutic targets.NEW & NOTEWORTHY This study determined the following: 1) providing a comprehensive landscape of consensus molecular subtype (CMS)-specific tumor microenvironments (TMEs); 2) constructing CMS-specific networks, including host genes, intratumoral microbiota, and enriched pathways, analyzing their associations to uncover unique patterns that demonstrate the intricate interplay within the TME; and 3) revealing a connection between immune-rich subtypes and ferroptosis activation, suggesting a potential regulatory role of the microbiota in ferroptosis dysregulation of the colorectal cancer TME.

Notch receptor/ligand diversity: contribution to colorectal cancer stem cell heterogeneity.

Cancer cell heterogeneity is a key contributor to therapeutic failure and post-treatment recurrence. Targeting cell subpopulations responsible for chemoresistance and recurrence seems to be an attractive approach to improve treatment outcome in cancer patients. However, this remains challenging due to the complexity and incomplete characterization of tumor cell subpopulations. The heterogeneity of cells exhibiting stemness-related features, such as self-renewal and chemoresistance, fuels this complexity. Notch signaling is a known regulator of cancer stem cell (CSC) features in colorectal cancer (CRC), though the effects of its heterogenous signaling on CRC cell stemness are only just emerging. In this review, we discuss how Notch ligand-receptor specificity contributes to regulating stemness, self-renewal, chemoresistance and cancer stem cells heterogeneity in CRC.

Luciferase Expressing Preclinical Model Systems Representing the Different Molecular Subtypes of Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose-response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications.

Unravelling the Complexity of Colorectal Cancer: Heterogeneity, Clonal Evolution, and Clinical Implications.

Colorectal cancer (CRC) is a global health concern and a leading cause of death worldwide. The disease's course and response to treatment are significantly influenced by its heterogeneity, both within a single lesion and between primary and metastatic sites. Biomarkers, such as mutations in KRAS, NRAS, and BRAF, provide valuable guidance for treatment decisions in patients with metastatic CRC. While high concordance exists between mutational status in primary and metastatic lesions, some heterogeneity may be present. Circulating tumor DNA (ctDNA) analysis has proven invaluable in identifying genetic heterogeneity and predicting prognosis in RAS-mutated metastatic CRC patients. Tumor heterogeneity can arise from genetic and non-genetic factors, affecting tumor development and response to therapy. To comprehend and address clonal evolution and intratumoral heterogeneity, comprehensive genomic studies employing techniques such as next-generation sequencing and computational analysis are essential. Liquid biopsy, notably through analysis of ctDNA, enables real-time clonal evolution and treatment response monitoring. However, challenges remain in standardizing procedures and accurately characterizing tumor subpopulations. Various models elucidate the origin of CRC heterogeneity, highlighting the intricate molecular pathways involved. This review focuses on intrapatient cancer heterogeneity and genetic clonal evolution in metastatic CRC, with an emphasis on clinical applications.

Colorectal cancer and therapy response: a focus on the main mechanisms involved.

The latest GLOBOCAN 2021 reports that colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Most CRC cases are sporadic and associated with several risk factors, including lifestyle habits, gut dysbiosis, chronic inflammation, and oxidative stress. To summarize the biology of CRC and discuss current therapeutic interventions designed to counteract CRC development and to overcome chemoresistance. Literature searches were conducted using PubMed and focusing the attention on the keywords such as "Current treatment of CRC" or "chemoresistance and CRC" or "oxidative stress and CRC" or "novel drug delivery approaches in cancer" or "immunotherapy in CRC" or "gut microbiota in CRC" or "systematic review and meta-analysis of randomized controlled trials" or "CSCs and CRC". The citations included in the search ranged from September 1988 to December 2022. An additional search was carried out using the clinical trial database. Rounds of adjuvant therapies, including radiotherapy, chemotherapy, and immunotherapy are commonly planned to reduce cancer recurrence after surgery (stage II and stage III CRC patients) and to improve overall survival (stage IV). 5-fluorouracil-based chemotherapy in combination with other cytotoxic drugs, is the mainstay to treat CRC. However, the onset of the inherent or acquired resistance and the presence of chemoresistant cancer stem cells drastically reduce the efficacy. On the other hand, the genetic-molecular heterogeneity of CRC often precludes also the efficacy of new therapeutic approaches such as immunotherapies. Therefore, the CRC complexity made of natural or acquired multidrug resistance has made it necessary the search for new druggable targets and new delivery systems. Further knowledge of the underlying CRC mechanisms and a comprehensive overview of current therapeutic opportunities can provide the basis for identifying pharmacological and biological barriers that render therapies ineffective and for identifying new potential biomarkers and therapeutic targets for advanced and aggressive CRC.