Tumor Microenvironment Of Colorectal Cancer Research Articles

Single-cell transcriptome analysis reveals heterogeneity and convergence of the tumor microenvironment in colorectal cancer.

Colorectal cancer (CRC) ranks second for mortality and third for morbidity among the most commonly diagnosed cancers worldwide. We aimed to investigate the heterogeneity and convergence of tumor microenvironment (TME) in CRC. We analyzed the single-cell RNA sequencing data obtained from the Gene Expression Omnibus (GEO) database and identified 8 major cell types and 25 subgroups derived from tumor, para-tumor and peripheral blood. In this study, we found that there were significant differences in metabolic patterns, immunophenotypes and transcription factor (TF) regulatory patterns among different subgroups of each major cell type. However, subgroups manifested similar lipid metabolic patterns, immunosuppressive functions and TFs module at the end of the differentiation trajectory in CD8+ T cells, myeloid cells and Fibroblasts. Meanwhile, TFs regulated lipid metabolism and immunosuppressive ligand-receptor pairs were detected by tracing the differentiation trajectory. Based on the cell subgroup fractions calculated by CIBERSORTx and bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA), we constructed an immune risk model and clinical risk model of CRC which presented excellent prognostic value. This study identified that the differentiation was accompanied by remodeling of lipid metabolism and suppression of immune function, which suggest that lipid remodeling may be an important trigger of immunosuppression. More importantly, our work provides a new perspective for understanding the heterogeneity and convergence of the TME and will aid the development of prognosis and immunotherapies of CRC patients.

Spatial and single-cell transcriptomics decipher the cellular environment containing HLA-G+ cancer cells and SPP1+ macrophages in colorectal cancer

The cellular interactions in the tumor microenvironment of colorectal cancer (CRC) are poorly understood, hindering patient treatment. In the current study, we investigate whether events occurring at the invasion front are of particular importance for CRC treatment strategies. To this end, we analyze CRC tissues by combining spatial transcriptomics from patients with a public single-cell transcriptomic atlas to determine cell-cell interactions at the invasion front. We show that CRC cells are localized specifically at the invasion front. These cells induce human leukocyte antigen G (HLA-G) to produce secreted phosphoprotein 1 (SPP1)+ macrophages while conferring CRC cells with anti-tumor immunity, as well as proliferative and invasive properties. Taken together, these findings highlight the signaling between CRC cell populations and stromal cell populations at the cellular level.

Apolipoprotein L3 enhances CD8+ T cell antitumor immunity of colorectal cancer by promoting LDHA-mediated ferroptosis.

Aim: Colorectal cancer (CRC) is the leading cause of cancer associated death worldwide and immune checkpoint blockade therapy only benefit a small set of CRC patients. Tumor ferroptosis of CRC reflected immune-activation in our previous findings. Understanding the mechanisms underlying how to bolster CD8+ T cells function through ferroptosis in CRC tumor microenvironment (TME) will greatly benefit cancer immunotherapy. Methods: Genes between ferroptosis and CD8+ T cell function in CRC were screened through Cox, WGCNA and differential expression analysis. Immunohistochemistry and Immunofluorescence analysis were performed. Co-immunoprecipitation were performed to determine protein-protein interaction, mRNA level was determined by qRT-PCR. RSL3 was used to induce ferroptosis, and ferroptosis levels were evaluated by measuring Transmission Electron Microscope analysis, MDA, Fe2+level and cell viability. Results: We screened APOL3 as the significant modulator for ferroptosis-related CD8+ infiltration in CRC. Next, by in vitro and in vivo, we found that increased APOL3 expression was positively correlated with sensitivity to ferroptosis and antitumor ability of CD8+ T cells. Next, we demonstrated that APOL3 can binds LDHA and promote its ubiquitylation-related degradation. Then, based on in vivo analysis and tumor specimen, we discovered the APOL3-LDHA axis can facilitate the tumor ferroptosis and cytotoxic ability of CD8+ T cells through increased IFNγ and decreased lactic acid concentration. Conclusion: The present study demonstrated that APOL3 promotes ferroptosis and immunotherapy in colorectal cancer cells. The present work provides us with a novel target to overcome drug resistance to ferroptosis and immunotherapy.

Impact of Cuproptosis-related markers on clinical status, tumor immune microenvironment and immunotherapy in colorectal cancer: A multi-omic analysis

BackgroundCuproptosis, a novel identified cell death form induced by copper, is characterized by aggregation of lipoylated mitochondrial enzymes and the destabilization of Fe–S cluster proteins. However, the function and potential clinical value of cuproptosis and cuproptosis-related biomarkers in colorectal cancer (CRC) remain largely unknown. MethodsA comprehensive multi-omics (transcriptomics, genomics, and single-cell transcriptome) analysis was performed for identifying the influence of 16 cuproptosis-related markers on clinical status, molecular functions and tumor microenvironment (TME) in CRC. A novel cuproptosis-related scoring system (CuproScore) based on cuproptosis-related markers was also constructed to predict the prognosis of CRC individuals, TME and the response to immunotherapy. In addition, our transcriptome cohort of 15 paired CRC tissue, tissue-array, and various assays in 4 kinds of CRC cell lines in vitro were applied for verification. ResultsCuproptosis-related markers were closely associated with both clinical prognosis and molecular functions. And the cuproptosis-related molecular phenotypes and scoring system (CuproScore) could distinguish and predict the prognosis of CRC patients, TME, and the response to immunotherapy in both public and our transcriptome cohorts. Besides, the expression, function and clinical significance of these markers were also checked and analyzed in CRC cell lines and CRC tissues in our own cohorts. ConclusionsIn conclusion, we indicated that cuproptosis and CPRMs played a significant role in CRC progression and in modeling the TME. Inducing cuproptosis may be a useful tool for tumor therapy in the future.

Applying centrality analysis on a protein interaction network to predict colorectal cancer driver genes

The colorectal cancer tumor microenvironment presents significant genetic heterogeneity with mutations in genes in several signaling pathways. Detecting these driver genes through wet lab experiments is costly and time-consuming. Computational models and bioinformatic tools have become a vital alternative in this effort. One of these novel computational methods, Centrality Analysis, models molecular functions, biological processes and biochemical pathways by creating and analyzing protein-protein interaction networks. Centrality Analysis is an approach to quantify node (in this case, protein) importance in these networks. Essential proteins play critical roles in cell function; therefore, centrality measures serve as a basis to study the relationship between lethality and essentiality by evaluating the topological features of the network. However, there is no established standard to determine the most appropriate centrality measure for analyzing a specific network. The choice of a suitable set is complicated by the impact of network topology because results vary based on network structure, correlation among the selected set of measures, and network data collection methods used. We hypothesized that centrality scores can be used to predict driver genes while statistical and machine learning analyses can identify the relevant centrality features for this task. We proposed different analyses to select a valid set of centrality algorithms to predict driver and non-driver genes. We first recreated a protein-protein interaction network for colorectal cancer featuring known driver and passenger genes, and then compared the centrality scores of eight different algorithms using statistical analysis. We further validated the results by implementing machine learning models. Both analyses identified betweenness and closeness centrality algorithms as most important to predict driver versus non-driver genes.

Unveiling mitophagy-mediated molecular heterogeneity and development of a risk signature model for colorectal cancer by integrated scRNA-seq and bulk RNA-seq analysis.

Accumulating researchers have recognized mitophagy as a key player in tumors, but few studies have investigated its role in the tumor microenvironment (TME). Advances in the technology of single-cell RNA sequencing (scRNA-seq) have allowed unveiling the concealed features of the TME at cellular resolution. This study aimed to elucidate the role of mitophagy within the TME of colorectal cancer (CRC) and to establish a mitophagy-mediated risk model. We assessed mitophagy-related pathway activities at both single-cell and tissue levels. Subsequently, an unsupervised clustering algorithm was employed to identify mitophagy-mediated subtypes. Furthermore, we developed a mitophagy-mediated risk signature (MMRS) using least absolute shrinkage and selection operator (LASSO) Cox analysis and constructed a MMRS model incorporating the risk score and clinical variables. Subsequently, we used quantitative reverse transcription polymerase chain reaction analysis to verify the expression of the screened genes. We retrieved and annotated a total of 14,719 cells from eight samples in the scRNA-seq GSE132465 data set. The activities of mitophagy-related pathways were uniformly upregulated in cancer cells. Integrating with bulk RNA-seq data, we identified two mitophagy-mediated clusters (C1 and C2) with distinct characteristics and prognoses. C2 was identified as a mitophagy-high cluster. Then, we developed a five-gene MMRS via LASSO Cox analysis in The Cancer Genome Atlas (TCGA) cohort. We utilized the GSE39582 cohort to validate the efficacy of our model. The expression of CX3CL1 and INHBB was upregulated in CRC tissues. The present study identified two mitophagy-mediated CRC subtypes with distinct features. Our MMRS may provide potential therapeutic strategies for CRC. The findings of our work offer novel insights into the involvement of mitophagy in CRC.

Resveratrol Loaded Liposomes Disrupt Cancer Associated Fibroblast Communications within the Tumor Microenvironment to Inhibit Colorectal Cancer Aggressiveness

Colorectal cancer (CRC) is a cancer-associated fibroblast, CAF-rich tumor. CAF promotes cancer cell proliferation, metastasis, drug resistance via secretes soluble factors, and extracellular matrices which leads to dense stroma, a major barrier for drug delivery. Resveratrol (RES) is a polyphenolic compound, has several pharmacologic functions including anti-inflammation and anticancer effects. Considering tumor microenvironment of CRC, resveratrol-loaded liposome (L-RES) was synthesized and employed to inhibit CAF functions. The L-RES was synthesized by thin-film hydration method. The cytotoxicity of L-RES was evaluated using MTT assay. Effect of L-RES treated CAF on tumor spheroid growth was performed. Cell invasion was determined using spheroid invasion assay. The effect of L-RES on 5-fluorouracil (5-FU) sensitivity of CRC cells was determined in co-cultured tumor spheroids. Subtoxic dose of L-RES was selected to study possible inhibiting CAF functions. Decreased CAF markers, α-SMA and IL-6 levels, were observed in L-RES treated activated fibroblast. Interestingly, the activated fibroblast promoted invasive ability and drug resistance of CRC cells in co-culture condition of both 2D and 3D cultures and was attenuated by L-RES treatment in the activated fibroblast. Therefore, L-RES provides a promising drug delivery strategy for CRC treatment by disrupting the crosstalk between CRC cells and CAF.

Microenvironmental ammonia enhances T cell exhaustion in colorectal cancer

Effective therapies are lacking for patients with advanced colorectal cancer (CRC). The CRC tumor microenvironment has elevated metabolic waste products due to altered metabolism and proximity to the microbiota. The role of metabolite waste in tumor development, progression, and treatment resistance is unclear. We generated an autochthonous metastatic mouse model of CRC and used unbiased multi-omic analyses to reveal a robust accumulation of tumoral ammonia. The high ammonia levels induce Tcell metabolic reprogramming, increase exhaustion, and decrease proliferation. CRC patients have increased serum ammonia, and the ammonia-related gene signature correlates with altered Tcell response, adverse patient outcomes, and lack of response to immune checkpoint blockade. We demonstrate that enhancing ammonia clearance reactivates Tcells, decreases tumor growth, and extends survival. Moreover, decreasing tumor-associated ammonia enhances anti-PD-L1 efficacy. These findings indicate that enhancing ammonia detoxification can reactivate Tcells, highlighting a new approach to enhance the efficacy of immunotherapies.

41P Single-cell signalling analysis of engineered γδ T cell biotherapeutics for the treatment of colorectal cancer

Colorectal cancer (CRC) is a devastating disease that kills ∼700,000 people worldwide annually, with immunotherapies struggling against the immunosuppressive CRC tumour microenvironment (TME). Tumour infiltrating γδ T cells confer a prognostic benefit to CRC patients and can kill cancer via multiple innate and adaptive mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) against tumour antigens (e.g., B7-H3). We hypothesise that γδ T cells can be exploited as an anti-CRC biotherapeutic but their complex interactions within the CRC TME need to be elucidated.

Abstract PR009: RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition

Abstract Introduction: The RNF43 p.G659fs mutation occurs with a frequency of approximately 8% in colorectal cancer (CRC) and is enriched among microsatellite-instability high tumors. However, despite RNF43 being implicated as a Wnt-signaling tumor suppressor, the function of the RNF43 p.G659fs mutation remains undescribed, with no specific therapies directed against it. We hypothesized that large-scale drug screening coupled with functional validation and testing in CRC pre-clinical models would reveal the functional role and selective vulnerabilities of this alteration, leading to new therapeutic approaches in CRC. Methods: We targeted the RNF43 659 site with CRISPR-Cas9 to generate isogenic RNF43659mut clones of CRC cell lines. We leveraged the Broad Institute’s drug repurposing library of over 5,300 compounds to identify selective hits (z<-3 vs DMSO, >5% selective killing in mutant vs WT) in RNF43659mut isogenic cells. These hits were validated in secondary screens and top drugs were tested in patient-derived organoid and xenograft models of the RNF43 p.G659fs mutation. We assessed the functional role of the mutation using cell viability and colony formation assays, quantitative mass spectrometry proteomics, immunoprecipitation and ubiquitination experiments. The immunomodulatory role of RNF43659mut was assessed through bulk and single-cell RNA Seq analysis of mutant cell lines and patient-derived CRC specimens. Results: Using edited cell line models, we demonstrated that RNF43 p.G659fs has a Wnt-independent oncogenic role in CRC. We performed a comprehensive drug screen in isogenic RNF43659mut HT29 CRC cells with a repurposing library of 5,363 compounds and discovered that RNF43659mut is selectively targeted by PI3K/mTOR inhibitors (p = 1.34 x 10-5, chi-square test). Secondary screens in LS513 RNF43659mut-edited and HCT116 RNF43G659fs-overexpressing cells confirmed these results and we further showed that alpelisib and PF-04691502 (a dual PI3K/mTOR inhibitor) selectively killed patient-derived organoids with the RNF43 p.G659Vfs mutation and reduced tumor growth in RNF43659mut CDX mice (p=0.0005, Student’s t-test). RNF43 p.G659fs increased PI3K/AKT signaling in vitro and in vivo through binding, ubiquitinating and degrading p85 and this was reversed by PI3K/mTOR inhibition, RNF43 knockout or RSPO stimulation. Additionally, in pre-clinical models and single-cell studies of human CRCs (>350,000 single cells), we found that RNF43659mut modulates interferon activity and NK cells in the CRC tumor microenvironment. Conclusions: Through functional and pharmacologic studies in pre-clinical models and human CRCs, we described that RNF43 p.G659fs activates PI3K/AKT signaling and modulates the CRC immune microenvironment. We showed that these effects are reversed with PI3K/mTOR inhibitors and that this class of drugs selectively targets RNF43659mut CRCs. Our results have important therapeutic implications for patients with CRC. Citation Format: Marios Giannakis, Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Xiaozhe Xiong, Carino Gurjao, Maximilien Grandclaudon, Srivatsan Raghavan, Steven M. Corsello, Steven A. Carr, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng. RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR009.

Constructing a Tregs-associated signature to predict the prognosis of colorectal cancer patients: A STROBE-compliant retrospective study.

Colorectal cancer (CRC) ranks as the second leading cause of cancer-related mortality worldwide. Regulatory T cells (Tregs) are a key constituent of immune cells in the tumor microenvironment (TME) and are significantly associated with patient outcomes. Our study aimed to construct a Treg-associated signature to predict the prognosis of CRC patients. The genes' expression values and patients' clinicopathological features were downloaded from TCGA and gene expression omnibus (GEO) databases. The single-cell RNA (scRNA) sequencing data of CRC were analyzed through the Deeply Integrated human Single-Cell Omics database. WGCNA analysis was used to select Tregs-associated genes (TrAGs). The infiltrated levels of immune and stromal cells were accessed through the ESTIMATE algorithm. Cox regression analysis and the LASSO algorithm were implemented to construct prognostic models. Gene set enrichment analysis (GSEA) was performed to annotate enriched gene sets. Based on scRNA sequencing data, our study uncovered that more Tregs were significantly enriched in the TME of CRC. Then we identified 123 differentially expressed TrAGs which mainly participated in immune regulation. Given that CRC patients were reclassified into 2 subgroups with distinct overall survival based on 26 differentially expressed TrAGs with prognostic values, we subsequently constructed a signature for CRC. After training and validating in independent cohorts, we proved that this prognostic model can be well applied to predict the prognosis of CRC patients. Further analysis exhibited that more tumor-suppressing immune cells and higher immune checkpoint genes were enriched in CRC patients with high-risk scores. Moreover, immunohistochemistry analysis validated that the genes in the prognostic model were significantly elevated in CRC tissues. We were the first to construct a prognostic signature for CRC based on TrAGs and further revealed that the poor prognosis of patients was mainly attributed to the tumor-suppressing microenvironment and upregulated immune checkpoint genes in tumor tissues.

Schwann cells promote the migration and invasion of colorectal cancer cells via the activated NF-κB/IL-8 axis in the tumor microenvironment.

Evidence has shown neurons and glial cells were closely related to tumor progression. As the predominant glial cells in the external innervated nerves of the gastrointestinal, the role of Schwann cells (SCs) in colorectal cancer (CRC) has not been well explored. HCT-116 and HT-29 CRC cells were treated with conditioned medium (CM) from SCs, and the cells' proliferative and migrating capacities were examined. Cytokine array analysis was used to identify the tumor-promoting-cytokines from SCs-CM. Molecular changes from SCs after being co-cultured with tumor cells were detected by ELISA and reverse transcription-quantitative PCR. The activation of the nuclear factor kappa B (NF-κB) signaling pathway in SCs was demonstrated by immunofluorescence staining. Neutralizing antibody was used to verify the tumor-promoting effects of key cytokine. Migration and invasion of CRC cells were markedly aided by CM from SCs in vitro. Interleukin-8 (IL-8) was identified as an effective factor. SCs co-cultured with CRC cells upregulated IL-8 expression, which may be related to its activated NF-κB signaling pathway. Neutralization of IL-8 attenuated the tumor-promoting effect of SCs. The present study identified a new mechanism of tumor-neuroglia interaction, enriching the concept of the tumor-neural axis in the tumor microenvironment of CRC, which also inspired potential targets for anti-cancer therapies.

Multiregion single cell analysis reveals a novel subtype of cancer-associated fibroblasts located in the hypoxic tumor microenvironment in colorectal cancer

Background: The tumor microenvironment (TME) plays a critical role in shaping tumor progression and determining the outcome of the therapeutic response. In this study, we aimed to generate a comprehensive cellular landscape of the colorectal cancer (CRC) TME. Methods: We generated a comprehensive single-cell atlas by collecting CRC cases that have been uploaded to the online database and conducting an in-depth secondary analysis. We then carried out spatial transcriptomic sequencing and multiple immunohistochemical analyses to verify the results of the single-cell analysis. Moreover, we applied our findings to the TCGA database and used tissue microarray (TMA) on CRC tissue specimens to validate clinical prognosis. FindingsWe re-analyzed the transcriptomes of 23785 cells, revealing a pattern of cell heterogeneity in the tumor region, leading-edge region, and non-tumor region. A subtype of COL11A1+INHBA+ tumor-resident cancer-associated fibroblasts (CAFs) was identified, and marker genes, transcription factors, and tissue-specific expression differences were noted and suggested to have potential roles in promoting cancer. We further confirmed that COL11A1+INHBA+ tumor-resident CAFs are mainly located in the hypoxic TME and we propose that they interact with CD44+ CRC cells via INHBA. Elevation of INHBA in CRC is associated with a poor prognosis. Interpretation: Our results demonstrated a single cell landscape of CRC in different regions and identified in hypoxic TME a special subtype of CAFs producing INHBA, which promotes CRC development and correlates with poor prognosis. This special subtype of CAFs is a candidate target for translational research.

SLC11A1 associated with tumor microenvironment is a potential biomarker of prognosis and immunotherapy efficacy for colorectal cancer.

Colorectal cancer (CRC) is one of the most lethal cancers of the digestive system. The tumor microenvironment (TME) plays a central role in the initiation and development of CRC. However, little is known about the modulation mechanism of the TME in CRC. In our study, we attempted to identify a biomarker related to the TME modulation that could serve as a potential prognostic biomarker for CRC. We identified differentially expressed genes between the ImmuneScore high/low and StromalScore high/low groups. Using univariate COX regression analysis and hub gene analysis (cytoHubba), SLC11A1 was identified as the only candidate gene for subsequent analysis. CIBERSORT, EPIC, MCPcounter, and immunogenic cell death were performed to evaluate the effect of SLC11A1 on the TME. We also collected samples and performed Real-time quantitative PCR to verify the expression levels of SLC11A1 in CRC and adjacent normal tissues. The IMvigor210 cohort, TIDE score, and immunophenoscore (IPS) were used to analyze the association between SLC11A1 and immunotherapy efficacy. SLC11A1 was highly expressed in CRC tissues compared with its expression in normal colorectal tissues and was associated with poor prognosis and advanced clinicopathological stages. Gene set enrichment analysis showed that TGF-β pathways, JAK-STAT pathways, and angiogenesis were significantly enriched in the high-SLC11A1 group. Single-cell analysis validated the correlation between SLC11A1 and the TME. Using CIBERSORT, EPIC, and MCPcounter algorithms, we found that there was more macrophage and fibroblast infiltration in the SLC11A1 high-expression group. Meanwhile, high-SLC11A1 patients had lower IPS scores, higher TIDE scores, and fewer immunotherapy benefits than those of low-SLC11A1 patients. In conclusion, SLC11A1 plays a crucial role in the TME and could serve as a potential biomarker for poor prognosis and immunotherapy efficacy in CRC.

An analysis of sexual dimorphism in the tumor microenvironment of colorectal cancer.

Women with colorectal cancer (CRC) have survival advantages over men, yet the underlying mechanisms are unclear. T cell infiltration within the CRC tumor microenvironment (TME) correlates strongly with survival. We hypothesized that women with CRC have increased T cell infiltration and differential gene expression in the TME compared to men. Tissue microarrays comprising primary tumor, tumor infiltrated lymph nodes, and uninvolved colon were created from CRC patients. Proportions of CD4 positive (CD4+) and CD8 positive (CD8+) T cells were identified using immunohistochemistry. TME immune- and cancer-related genetic expression from primary and metastatic CRC tumor were also evaluated via the NanoStringIO360 panel and The Cancer Genome Atlas Project database. CD4+ was higher in tumor samples from women compared to men (22.04% vs. 10.26%, p=0.002) and also in lymph node samples (39.54% vs. 8.56%, p=0.001). CD8+ was increased in uninvolved colon from women compared to men (59.40% vs. 43.61%, p=0.015), and in stage I/II tumors compared to III/IV in all patients (37.01% vs. 23.91%, p=0.009). Top CD8+ tertile patients survived longer compared to the bottom (43.9 months vs. 25.3 months, p=0.007). Differential gene expression was observed in pathways related to Treg function, T cell activity, and T cell exhaustion, amongst several others, in women compared to men. Thus, significant sexual dimorphism exists in the TME that could contribute to survival advantages observed in female patients with CRC.

Listeria-Based Vaccines Targeting Interferon-Stimulated Gene 15 (ISG15) for Renal Cell Carcinoma and Colorectal Cancer

<h3>Purpose/Objective(s)</h3> Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been implicated for its central role in innate immunity against intracellular pathogens. Since cancer cells oftentimes have defects in type I IFN signaling pathways, there is a growing body of evidence that demonstrated the role of ISG15 in cancer progression. Here, we investigate to understand if ISG15 could be another novel cancer-associated antigen in renal cell carcinomas (RCC) and colorectal cancer (CRC). Our research objectives include (i) to identify if the elevated expression of ISG15 in RCC and CRC has any prognosis signature, (ii) to explore if ISG15 can be targeted by mean of Listeria-based vaccines, and (iii) to analyze how Listeria-based vaccines targeting ISG15 change the tumor microenvironment of RCC and CRC in syngeneic mouse models. <h3>Materials/Methods</h3> Data on RNA and protein expression of ISG15 and the Kaplan–Meier survival analysis was obtained from Human Protein Atlas, The Cancer Genome Atlas, and from the University of Alabama at Birmingham, Comprehensive Cancer Center (UALCAN). RNA was extracted from tissue or cells and converted to cDNA. The cDNA was then subjected to qPCR analysis with primers specific to ISG15. All mouse experiments were performed in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) at the TTUHSC. <h3>Results</h3> We found that the elevated expression of ISG15 in RCC and CRC is significantly associated with an unfavorable prognosis. Similarly, RCC and CRC murine cell lines also expressed a higher level of ISG15 compared to the normal fibroblast cell line. In subcutaneous syngeneic mouse models of RCC and CRC, vaccination with a Listeria-based vaccine targeting ISG15, designated Lm-LLO-ISG15, significantly controls primary tumor burden and extends median survival compared to control groups. In both models, treatment with Lm-LLO-ISG15 inflames the tumor microenvironment, enhances the influx of immune cells, and induces the expression of programmed cell death protein 1 (PD-1). <h3>Conclusion</h3> ISG15 is a novel cancer-associated antigen and a potential target for active immunotherapies such as Listeria-based vaccines. Treatment with Lm-LLO-ISG15 significantly controls tumor burden in both RCC and CRC syngeneic mouse models and sensitizes the tumors with anti-PD-1 therapies.

Hydrogen sulfide activatable metal-organic frameworks for Fluorescence Imaging-Guided Photodynamic Therapy of colorectal cancer.

Photodynamic therapy (PDT) is a promising alternative and palliative therapeutic strategy for colorectal cancer (CRC). A novel photosensitizer with higher selectivity for CRC and fewer side effects is vital for clinical application. Given that the overexpression of hydrogen sulfide (H2S) in CRC, it is expected to provide a selective stimulus for activatable photosensitizers that in respond to the specific microenvironment. Herein, we report a novel development of metal-organic frameworks (MOFs) composed of meso-Tetra (4-carboxyphenyl) porphine (TCPP) and ferric ion (Fe3+) through a facile one-pot process. Experiments both in vitro and in vivo reveal that the MOF is capable of depredating in response to the high content of H2S in tumor microenvironment of CRC. Accompanying with the degradation and release of TCPP, the fluorescence and photosensitivity effect is switched from “off” to “on”, enabling the MOF to serve as a H2S activatable nano-photosensitizer for real-time fluorescence imaging-guided and targeted PDT of CRC.

Anlotinib Benefits the αPDL1 Immunotherapy by Activating ROS/JNK/AP-1 Pathway to Upregulate PDL1 Expression in Colorectal Cancer.

Colorectal cancer (CRC) is one of the prevalent malignant tumors. This study is aimed at evaluating the mechanism of anlotinib (anlo) on tumor microenvironment (TME) in CRC, and its effects in combination with immune checkpoint inhibitors (ICIs) therapy. Firstly, MC38 and CT26 cells were both exposed to different gradient concentrations of anlo for 72 h, to investigate the cell viability and synergetic therapy efficacy with ICIs by CCK8. The results showed that anlo could obviously inhibit cell growth and showed no synergistic efficacy therapy in combination with αPDL1 in vitro. Then, we found the upregulation of programmed cell death ligand 1(PDL1) expression both in vitro and in vivo after anlo treatment. In vivo, anlo could enhance the percentage of natural killer (NK) cells and M1 macrophage cells and decrease the percentage of M2 macrophage cells in TME. Moreover, we explored the mechanism and we proved that anlo could activate reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) signaling pathway to increase the expression levels of PDL1, IFN-α/β/γ, and CXCL2 in two cell lines in vitro. We also proved that anlo had synergistic effects with ICIs in vivo. Finally, it could also increase the mRNA and protein PDL1 expression levels in human cell lines, which was consistent with mouse CRC cell lines. However, there are still a few limitations. On one hand, the ROS/JNK/AP-1 pathway needs to be proved whether it can be activated in human cell lines. On the other hand, the mechanism behind ROS promoting phosphorylation of JNK needs to be explored.

Traditional Chinese Medicine Regulates the Progression of Inflammatory Factors in the Colorectal Cancer Tumor Microenvironment

Traditional Chinese Medicine Regulates the Progression of Inflammatory Factors in the Colorectal Cancer Tumor Microenvironment

Identification of N6-methylandenosine related lncRNA signatures for predicting the prognosis and therapy response in colorectal cancer patients.

Despite recent advances in surgical and multimodal therapies, the overall survival (OS) of advanced colorectal cancer (CRC) patients remains low. Thus, discerning sensitive prognostic biomarkers to give the optimistic treatment for CRC patients is extremely critical. N6-methyladenosine (m6A) and long noncoding RNAs (lncRNAs) play an important role in CRC progression. Nonetheless, few studies have focused on the impact of m6A-related lncRNAs on the prognosis, tumor microenvironment (TME) and treatment of CRC. In this study, 1707 m6A-related lncRNAs were identified through Pearson correlation analysis and Weighted co-expression network analysis (WGCNA) using The Cancer Genome Atlas (TCGA) cohort. Then, 28 m6A-related prognostic lncRNAs were screened by univariate Cox regression analysis, followed by identifying two clusters by consensus clustering analysis. A prognostic model consisted of 8 lncRNA signatures was constructed by the least absolute shrinkage and selection operator (LASSO). Kaplan–Meier curve analysis and a nomogram were performed to investigate the prognostic ability of this model. The risk score of prognostic model act as an independent risk factor for OS rate. Functional enrichment analysis indicated that lncRNA signatures related tumor immunity. The low-risk group characterized by increased microsatellite instability-high (MSI-H), mutation burden, and immunity activation, indicated favorable odds of OS. Moreover, the lncRNA signatures were significantly associated with the cancer stem cell (CSC) index and drug sensitivity. In addition, 3 common immune genes shared by the lncRNA signatures were screened out. We found that these immune genes were widely distributed in 2 cell types of TME. Finally, a ceRNA network was constructed to identify ZEB1-AS1 regulatory axis in CRC. We found that ZEB1-AS1 was significantly overexpressed in tumor tissues, and was related to the metastasis of EMT and the chemoresistance of 5-Fu in CRC. Therefore, our study demonstrated the important role of m6A-related lncRNAs in TME remodeling. Moreover, these results illustrated the levels of ZEB1-AS1 might be valuable for predicting the progression and prognosis of CRC, and further provided a new target for the diagnosis and treatment of CRC patients.