PET imaging of colon cancer CD73 expression using cysteine site-specific 89Zr-labeled anti-CD73 antibody.

CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety-specific conjugation with deferoxamine-maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P < 0.001). Immunoblotting and immunohistochemistry confirmed strong CD73 expression in CT26/CD73 tumors and low expression in CT26 tumors. 4T1.2 tumor mice also showed clear 89Zr-CD73 IgG accumulation at 8 days (3.75 ± 0.70%ID/g) with high tumor-to-blood ratio compared to 89Zr-isotype-IgG (4.91 ± 1.74 vs. 1.20 ± 0.28; P < 0.005). 89Zr-CD73 IgG specifically targeted CD73 on high expressing cancer cells in vitro and tumors in vivo. Thus, 89Zr-CD73 IgG immuno-PET may be useful for the non-invasive monitoring of CD73 expression in tumors of living subjects.

DEPDC1B: A novel tumor suppressor gene associated with immune infiltration in colon adenocarcinoma.

BACKGROUND: Recent research indicates a positive correlation between DEP structural domain-containing 1B (DEPDC1B) and the cell cycle in various tumors. However, the role of DEPDC1B in the infiltration of the tumor immune microenvironment (TIME) remains unexplored. METHODS: We analyzed the differential expression and prognostic significance of DEPDC1B in colon adenocarcinoma (COAD) using the R package "limma" and the Gene Expression Profiling Interactive Analysis (GEPIA) website. Gene set enrichment analysis (GSEA) was employed to investigate the functions and interactions of DEPDC1B expression in COAD. Cell Counting Kit-8 (CCK-8) assays and colony formation assays were utilized to assess the proliferative function of DEPDC1B. Correlations between DEPDC1B expression and tumor-infiltrating immune cells, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI) status were examined using Spearman correlation analysis and CIBERSORT. RESULTS: DEPDC1B was highly expressed in COAD. Elevated DEPDC1B expression was associated with lower epithelial-to-mesenchymal transition (EMT) and TNM stages, leading to a favorable prognosis. DEPDC1B mRNA was prominently expressed in COAD cell lines. CCK-8 and colony formation assays demonstrated that DEPDC1B inhibited the proliferation of COAD cells. Analysis using the CIBERSORT database and Spearman correlation revealed that DEPDC1B correlated with four types of tumor-infiltrating immune cells. Furthermore, high DEPDC1B expression was linked to the expression of PD-L1, CTLA4, SIGLEC15, PD-L2, TMB, and MSI-H. High DEPDC1B expression also indicated responsiveness to anti-PD-L1 immunotherapy. CONCLUSIONS: DEPDC1B inhibits the proliferation of COAD cells and positively regulates the cell cycle, showing a positive correlation with CCNB1 and PBK expression. DEPDC1B expression in COAD is associated with tumor-infiltrating immune cells, immune checkpoints, TMB, and MSI-H in the tumor immune microenvironment. This suggests that DEPDC1B may serve as a novel prognostic marker and a potential target for immunotherapy in COAD.

γδ T cells in human colon adenocarcinomas comprise mainly Vδ1, Vδ2, and Vδ3 cells with distinct phenotype and function.

Γδ T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor (TCR) Vδ usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed γδ T cell profiling in colon tumours, using mass and flow cytometry, mRNA quantification, and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vδ1 Vδ2 cells. Sequencing of the Vδ complementarity-determining region 3 showed that almost all non-Vδ1 Vδ2 cells used Vδ3 and that tumour-infiltrating γδ clonotypes were unique for every patient. Non-Vδ1Vδ2 cells from colon tumours expressed several activation markers but few NK cell receptors and exhaustion markers. In addition, mRNA analyses showed that non-Vδ1 Vδ2 cells expressed several genes for proteins with tumour-promoting functions, such as neutrophil-recruiting chemokines, Galectin 3, and transforming growth factor-beta induced. In summary, our results show a large variation in γδ T cell subsets between individual tumours, and that Vδ3 cells make up a substantial proportion of γδ T cells in colon tumours. We suggest that individual γδ T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.

Association of CD8+TILs co-expressing granzyme A and interferon-γ with colon cancer cells in the tumor microenvironment.

CD8+T cells secreting granzyme A (GZMA) can induce pyroptosis in tumor cells by effectively cleaving gasdermin B (GSDMB), which is stimulated by interferon-γ (IFN-γ). However, the interaction between GZMA-expressing CD8+T cells and GSDMB-expressing tumor cells in colon cancer remains poorly understood. Our research employed multi-color immunohistochemistry (mIHC) staining and integrated clinical data to explore the spatial distribution and clinical relevance of GZMA- and IFN-γ-expressing CD8+ tumor-infiltrating lymphocytes (TILs), as well as GSDMB-expressing CK+ cells, within the tumor microenvironment (TME) of human colon cancer samples. Additionally, we utilizing single-cell RNA sequencing (scRNA-seq) data to examine the functional dynamics and interactions among these cell populations. scRNA-seq analysis of colorectal cancer (CRC) tissues revealed that CD8+TILs co-expressed GZMA and IFN-γ, but not other cell types. Our mIHC staining results indicated that a significant reduction in the infiltration of GZMA+IFN-γ+CD8+TILs in colon cancer patients (P < 0.01). Functional analysis results indicated that GZMA+IFN-γ+CD8+TILs demonstrated enhanced activation and effector functions compared to other CD8+TIL subsets. Furthermore, GSDMB-expressing CK+ cells exhibited augmented immunogenicity. Correlation analysis highlighted a positive association between GSDMB+CK+ cells and GZMA+IFN-γ+CD8+TILs (r = 0.221, P = 0.033). Analysis of cell-cell interactions further showed that these interactions were mediated by IFN-γ and transforming growth factor-ß (TGF-ß), the co-stimulatory molecule ICOS, and immune checkpoint molecules TIGIT and TIM-3. These findings suggested that GZMA+IFN-γ+CD8+TILs modulating GSDMB-expressing tumor cells, significantly impacted the immune microenvironment and patients' prognosis in colon cancer. By elucidating these mechanisms, our present study aims to provide novel insights for the advancement of immunotherapeutic strategies in colon cancer.

MS-20 enhances the gut microbiota-associated antitumor effects of anti-PD1 antibody.

Cancer immunotherapy has been regarded as a promising strategy for cancer therapy by blocking immune checkpoints and evoking immunity to fight cancer, but its efficacy seems to be heterogeneous among patients. Manipulating the gut microbiota is a potential strategy for enhancing the efficacy of immunotherapy. Here, we report that MS-20, also known as "Symbiota®", a postbiotic that comprises abundant microbial metabolites generated from a soybean-based medium fermented with multiple strains of probiotics and yeast, inhibited colon and lung cancer growth in combination with an anti-programmed cell death 1 (PD1) antibody in xenograft mouse models. Mechanistically, MS-20 remodeled the immunological tumor microenvironment by increasing effector CD8+ T cells and downregulating PD1 expression, which were mediated by the gut microbiota. Fecal microbiota transplantation (FMT) from mice receiving MS-20 treatment to recipient mice increased CD8+ T-cell infiltration into the tumor microenvironment and significantly improved antitumor activity when combined with anti-PD1 therapy. Notably, the abundance of Ruminococcus bromii, which increased following MS-20 treatment, was positively associated with a reduced tumor burden and CD8+ T-cell infiltration in vivo. Furthermore, an ex vivo study revealed that MS-20 could alter the composition of the microbiota in cancer patients, resulting in distinct metabolic pathways associated with favorable responses to immunotherapy. Overall, MS-20 could act as a promising adjuvant agent for enhancing the efficacy of immune checkpoint-mediated antitumor therapy.

The intestinal flora and nutritional status and immune function characteristics of obese colon cancer patients.

BACKGROUND: The research aims to explore the characteristics of intestinal flora, nutritional status and immune function in patients with different types of obese colon cancer. METHODS: A retrospective analysis is conducted on 64 cases of obese colon cancer diagnosed from June 2018 to January 2020. According to the histological staging of the cancer, they are classified into adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma, with corresponding cases of 24, 22 and 18, respectively. The intestinal flora (Bifidobacterium, Lactobacillus, Enterococcus faecalis, Escherichia coli, and yeast), nutritional status (Hb, Alb, PA, TFN, and PNI), immune function (IgG, IgM, IgA, CD4+, CD8+, and CD4+/CD8+) are analyzed in the different groups of patients. Survival curves are evaluated by Kaplan-Meier method and log-rank test for tumour death, local recurrence, and distant metastasis. RESULTS: There were no statistically significant differences in intestinal flora (Bifidobacterium, Lactobacillus, Enterococcus faecalis, Escherichia coli, and yeast), nutritional status (Hb, Alb, PA, TFN, and PNI) and immune function (IgG, IgM, IgA, CD4+, CD8+, and CD4+/CD8+) between different groups. There was a significant correlation between intestinal flora, nutritional status and immune function for all three. The survival curves of tumour death, local recurrence and distant metastasis in different groups of obese colon cancer patients were statistically significant. The tumor mortality rate, local recurrence, and distant metastasis rate in adenocarcinoma were 78.65%, 54.25% and 48.26% respectively. CONCLUSION: There are differences in intestinal flora, nutritional status and immune function among different types of obese colon cancer patients, but adenocarcinoma has the least benefit in intestinal flora, poor nutritional status, and weakest immune function.

A combined radio-immunotherapy regimen eradicates late-stage tumors in mice.

Background: The majority of experimental approaches for cancer immunotherapy are tested against relatively small tumors in tumor-bearing mice, because in most cases advanced cancers are resistant to the treatments. In this study, we asked if even late-stage mouse tumors can be eradicated by a rationally designed combined radio-immunotherapy (CRI) regimen. Methods: CRI consisted of local radiotherapy, intratumoral IL-12, slow-release systemic IL-2 and anti- CTLA-4 antibody. Therapeutic effects of CRI against several weakly immunogenic and immunogenic mouse tumors including B78 melanoma, MC38 and CT26 colon carcinomas and 9464D neuroblastoma were evaluated. Immune cell depletion and flow cytometric analysis were performed to determine the mechanisms of the antitumor effects. Results: Tumors with volumes of 2,000 mm3 or larger were eradicated by CRI. Flow analyses of the tumors revealed reduction of T regulatory (Treg) cells and increase of CD8/Treg ratios following CRI. Rapid shrinkage of the treated tumors did not require T cells, whereas T cells were involved in the systemic effect against the distant tumors. Cured mice developed immunological memory. Conclusions: These findings underscore that rationally designed combination immunotherapy regimens can be effective even against large, late-stage tumors.

Establishing a model composed of immune-related gene-modules to predict tumor immunotherapy response.

At present, tumor immunotherapy has been widely applied to treat various cancers. However, the accuracy of predicting treatment efficacy has not yet achieved a significant breakthrough. This study aimed to construct a prediction model based on the modified WGCNA algorithm to precisely judge the anti-tumor immune response. First, we used a murine colon cancer model to screen corresponding DEGs according to different groups. GSEA was used to analyze the potential mechanisms of the immune-related DEGs (irDEGs) in each group. Subsequently, the intersection of the irDEGs in every group was acquired, and 7 gene-modules were mapped. Finally, 4 gene-modules including cogenes, antiPD-1 immu-genes, chemo immu-genes and comb immu-genes, were selected for subsequent study. Furthermore, a clinical dataset of gastric cancer patients receiving immunotherapy was enrolled, and the irDEGs were identified. A total of 34 vital irDEGs were obtained from the intersections of the vital irDEGs and the four gene-modules. Next, the vital irDEGs were analyzed by the modified WGCNA algorithm, and the correlation coefficients between the 4 gene-modules and the response status to immunotherapy were calculated. Thus, a prediction model based on correlation coefficients was built, and the corresponding model scores were acquired. The AUC calculated according to the model score was 0.727, which was non-inferior to that of the ESTIMATE score and the TIDE score. Meanwhile, the AUC calculated according to the classification of the model scores was 0.705, which was non-inferior to that of the ESTIMATE classification and the TIDE classification. The prediction accuracy of the model was validated in clinical datasets of other cancers.

Chemical conjugation mitigates immunotoxicity of chemotherapy via reducing receptor-mediated drug leakage from lipid nanoparticles.

Immunotoxicity remains a major hindrance to chemotherapy in cancer therapy. Nanocarriers may alleviate the immunotoxicity, but the optimal design remains unclear. Here, we created two variants of maytansine (DM1)-loaded synthetic high-density lipoproteins (D-sHDL) with either physically entrapped (ED-sHDL) or chemically conjugated (CD-sHDL) DM1. We found that CD-sHDL showed less accumulation in the tumor draining lymph nodes (DLNs) and femur, resulting in a lower toxicity against myeloid cells than ED-sHDL via avoiding scavenger receptor class B type 1 (SR-B1)-mediated DM1 transportation into the granulocyte-monocyte progenitors and dendritic cells. Therefore, higher densities of lymphocytes in the tumors, DLNs, and blood were recorded in mice receiving CD-sHDL, leading to a better efficacy and immune memory of CD-sHDL against colon cancer. Furthermore, liposomes with conjugated DM1 (CD-Lipo) showed lower immunotoxicity than those with entrapped drug (ED-Lipo) through the same mechanism after apolipoprotein opsonization. Our findings highlight the critical role of drug loading patterns in dictating the biological fate and activity of nanomedicine.

Parabacteroides distasonis-Derived Outer Membrane Vesicles Enhance Antitumor Immunity Against Colon Tumors by Modulating CXCL10 and CD8+ T Cells.

Purpose: Given the potent immunostimulatory effects of bacterial outer membrane vesicles (OMVs) and the significant anti-colon tumor properties of Parabacteroides distasonis (Pd), this study aimed to elucidate the role and potential mechanisms of Pd-derived OMVs (Pd-OMVs) against colon cancer. Methods: This study isolated and purified Pd-OMVs from Pd cultures and assessed their characteristics. The effects of Pd-OMVs on CT26 cell uptake, proliferation, and invasion were investigated in vitro. In vivo, a CT26 colon tumor model was used to investigate the anti-colon tumor effects and underlying mechanisms of Pd-OMVs. Finally, we evaluated the biosafety of Pd-OMVs. Results: Purified Pd-OMVs had a uniform cup-shaped structure with an average size of 165.5 nm and a zeta potential of approximately -9.56 mV, and their proteins were associated with pathways related to immunity and apoptosis. In vitro experiments demonstrated that CT26 cells internalized the Pd-OMVs, resulting in a significant decrease in their proliferation and invasion abilities. Further in vivo studies confirmed the accumulation of Pd-OMVs in tumor tissues, which significantly inhibited the growth of colon tumors. Mechanistically, Pd-OMVs increased the expression of CXCL10, promoting infiltration of CD8+ T cells into tumor tissues and expression of pro-inflammatory factors TNF-α, IL-1ß, and IL-6. Notably, Pd-OMVs demonstrated a high level of biosafety. Conclusion: This paper elucidates that Pd-OMVs can exert significant anti-colon tumor effects by upregulating the expression of the chemokine CXCL10, thereby increasing the infiltration of CD8+ T cells into tumors and enhancing antitumor immune responses. This suggests that Pd-OMVs may be developed as a novel nanoscale potent immunostimulant with great potential for application in tumor immunotherapy. As well as developed as a novel nano-delivery carrier for combination with other antitumor drugs.

PROGNOSTIC VALUE OF THE DENSITY OF TUMOR-INFILTRATING LYMPHOCYTES AND ITS ASSOCIATION WITH CLINICAL-MORPHOLOGICAL FEATURES OF COLON ADENOCARCINOMAS.

AIM: To study the prognostic value of the density of tumor-infiltrating lymphocytes (TILs) and its association with other clinical-morphological parameters in colon adenocarcinomas (CAC). MATERIALS AND METHODS: 236 CAC samples were examined. TILs density was estimated as the percentage of tumor stromal area occupied by TILs. By the index of TILs density, the patients were divided into 3 groups: TILs 0-9% (n = 88); TILs 10-39% (n = 106); TILs > 40% (n = 42). Dependent on this index, their overall survival (OS) was analyzed. RESULTS: Kaplan - Meier curves revealed a significant (p < 0.001) difference in the OS for patients with different TILs infiltration intensities. Multivariate Cox's proportional hazard regression model analysis has confirmed that patients with moderate TILs density (HR 0.57, 95% CI 0.34-0.96, p = 0.035) had better OS rates compared to low TILs density. TILs were associated with the stage (p < 0.001), lymph node metastasis pN (p < 0.001), distant metastasis M (p < 0.001), and the patient's outcome (p < 0.001). CONCLUSION: TILs can be considered an additional prognostic tool during regular histological examination and are strongly associated with the most significant clinical-morphological features of CAC.

Machine learning evaluation of immune infiltrate through digital tumour score allows prediction of survival outcome in a pooled analysis of three international stage III colon cancer cohorts.

BACKGROUND: T-cell immune infiltrates are robust prognostic variables in localised colon cancer. Evaluation of prognosis using artificial intelligence is an emerging field. We evaluated whether machine learning analysis improved prediction of patient outcome in comparison with analysis of T cell infiltrate only or in association with clinical variables. METHODS: We used data from two phase III clinical trials (Prodige-13 and PETACC08) and one retrospective Italian cohort (HARMONY). Cohorts were split into training (N = 692), internal validation (N = 297) and external validation (N = 672) sets. Tumour slides were stained with CD3mAb. CD3 Machine Learning (CD3ML) score was computed using graphical parameters within the tumour tiles obtained from CD3 slides. CD3 infiltrates in tumour core and invasive margin were automatically detected. Associations of CD3 infiltrates and CD3ML with 5-year Disease-Free Survival (DFS) were examined using univariate and multivariable survival models by Cox regression. FINDINGS: CD3 density both in the invasive margin and the tumour core were significantly associated with DFS in the different sets. Similarly, CD3ML score was significantly associated with DFS in all sets. CD3 assessment did not provide added value on top of CD3ML assessment (Likelihood Ratio Test (LRT), p = 0.13). In contrast, CD3ML improved prediction of DFS when combined with a clinical risk stage (LRT, p = 0.001). Stratified by clinical risk score (High or Low), patients with low CD3ML score had better DFS. INTERPRETATION: In all tested sets, machine learning analysis of tumour cells improved prediction of prognosis compared to clinical parameters. Adding tumour-infiltrating lymphocytes assessment did not improve prognostic determination. FUNDING: This research received no external funding.

Resiquimod-loaded cationic liposomes cure mice with peritoneal carcinomatosis and induce specific anti-tumor immunity.

Peritoneal carcinomatosis (PC) is characterized by a high recurrence rate and mortality following cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC), primarily due to incomplete cancer elimination. To enhance the standard of care for PC, we developed two cationic liposomal formulations aimed at localizing a toll-like receptor agonist, resiquimod (R848), in the peritoneal cavity to activate the immune system locally to specifically eradicate residual tumor cells. These formulations effectively extended R848 retention in the peritoneum by >10-fold, resulting in up to a 2-fold increase in interferon α (IFN-α) induction in the peritoneal fluid, without increasing the plasma levels. In a CT26 colon cancer model with peritoneal metastases, these liposomal R848 formulations, when combined with oxaliplatin (OXA)-an agent used in HIPEC that induces immunogenic cell death-increased tumor infiltration of effector immune cells, including DCs, CD4, and CD8 T cells. This led to the complete elimination of PC in 60-70% of the mice, while the control mice reached humane endpoints by 30 days. The cured mice developed specific antitumor immunity, as re-challenging them with the same tumor cells did not result in tumor establishment. However, inoculation with a different tumor line led to tumor development. Additionally, exposing CT26 tumor antigens to the splenocytes isolated from the cured mice induced the expansion of CD4 and CD8 T cells and the release of IFN-γ, demonstrating long-term immune memory to the specific tumor. The anti-tumor efficacy of these liposomal R848 formulations was mediated via CD8 T cells with different levels of involvement of CD4 and B cells, and the combination with an anti-PD-1 antibody achieved a cure rate of 90%.

Constructing a prognostic model for colon cancer: insights from immunity-related genes.

BACKGROUND: Colon cancer (CC) is a malignancy associated with significant morbidity and mortality within the gastrointestinal tract. Recurrence and metastasis are the main factors affecting the prognosis of CC patients undergoing radical surgery; consequently, we attempted to determine the impact of immunity-related genes. RESULT: We constructed a CC risk model based on ZG16, MPC1, RBM47, SMOX, CPM and DNASE1L3. Consistently, we found that a significant association was found between the expression of most characteristic genes and tumor mutation burden (TMB), microsatellite instability (MSI) and neoantigen (NEO). Additionally, a notable decrease in RBM47 expression was observed in CC tissues compared with that in normal tissues. Moreover, RBM47 expression was correlated with clinicopathological characteristics and improved disease-free survival (DFS) and overall survival (OS) among patients with CC. Lastly, immunohistochemistry and co-immunofluorescence staining revealed a clear positive correlation between RBM47 and CXCL13 in mature tertiary lymphoid structures (TLS) region. CONCLUSION: We conclude that RBM47 was identified as a prognostic-related gene, which was of great significance to the prognosis evaluation of patients with CC and was correlated with CXCL13 in the TLS region.

MC1R regulates T regulatory cell differentiation through metabolic reprogramming to promote colon cancer.

BACKGROUND: Until 2021, colon cancer was a leading cancer globally. Early detection improves outcomes; however, advanced cases still having poor prognosis. Therefore, an understanding of associated molecular mechanisms is crucial for developing new preventive and therapeutic strategies for colon cancer. METHODS: The TCGA database was analyzed to assess melanocortin 1receptor (MC1R) expression in colon cancer and its link with patient prognosis. Further, models and diverse experimental techniques were employed to investigate the impact of MC1R on colon cancer progression and its underlying mechanism was elucidated. RESULTS: In a follow-up study of clinical patients, the important role of MC1R was identified in the development of colon cancer. First, MC1R was expressed more highly in colon tumor tissues than in adjacent tissues. In addition, MC1R was associated with colon cancer prognosis, and higher expression of MC1R tended to predict a worse prognosis. This conclusion was verified in MC1R-/- mice, which showed a greater resistance to tumor growth than wild-type mice, as expected. Further investigation revealed a significant change in the portion of Tregs in MC1R-/- mice, while the portion of CD4 + and CD8 + T cells remained unchanged. The in vitro experiments revealed a weaker ability of the MC1R-/- T cells to differentiate into Tregs. Previous studies report that the functional integrity of Tregs is interwoven with cellular metabolism. Therefore, MC1R was deduced to regulate the differentiation of Tregs by reprogramming the metabolism. As expected, MC1R-/- T cells exhibited weaker mitochondrial function and a lower aerobic oxidation capacity. Concurrently, the MC1R-/- T cells had stronger limiting effects on colon cancer cells. According to these results, the MC1R inhibitor was hypothesized as a potential therapeutic agent to suppress colon cancer. The results showed that upon MC1R suppression, the tumors in the mice developed more slowly, and the mice survived longer, potentially providing a novel strategy to treat clinical colon cancer. CONCLUSION: By regulating Tregs differentiation, MC1R overexpression in colon cancer correlates with poor prognosis, while MC1R inhibition shows potential as a therapeutic approach to slow tumor growth and enhance survival.

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

Principles of risk assessment in colon cancer: immunity is key.

In clinical practice, the administration of adjuvant chemotherapy (ACT) following tumor surgical resection raises a critical dilemma for stage II colon cancer (CC) patients. The prognostic features used to identify high-risk CC patients rely on the pathological assessment of tumor cells. Currently, these factors are considered for stratifying patients who may benefit from ACT at early CC stages. However, the extent to which these factors predict clinical outcomes (i.e. recurrence, survival) remains highly controversial, also uncertainty persists regarding patients' response to treatment, necessitating further investigation. Therefore, an imperious need is to explore novel biomarkers that can reliably stratify patients at risk, to optimize adjuvant treatment decisions. Recently, we evaluated the prognostic and predictive value of Immunoscore (IS), an immune digital-pathology assay, in stage II CC patients. IS emerged as the sole significant parameter for predicting disease-free survival (DFS) in high-risk patients. Moreover, IS effectively stratified patients who would benefit most from ACT based on their risk of recurrence, thus predicting their outcomes. Notably, our findings revealed that digital IS outperformed the visual quantitative assessment of the immune response conducted by expert pathologists. The latest edition of the WHO classification for digestive tumor has introduced the evaluation of the immune response, as assessed by IS, as desirable and essential diagnostic criterion. This supports the revision of current cancer guidelines and strongly recommends the implementation of IS into clinical practice as a patient stratification tool, to guide CC treatment decisions. This approach may provide appropriate personalized therapeutic decisions that could critically impact early-stage CC patient care.

Enhanced systemic antitumor efficacy of PD-1/PD-L1 blockade with immunological response induced by photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is an antitumor therapy and has traditionally been regarded as a localized therapy in itself. However, recent reports have shown that it not only exerts a direct cytotoxic effect on cancer cells but also enhances body's tumor immunity. We hypothesized that the immunological response induced by PDT could potentially enhance the efficacy of programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) blockade. METHODS: The cytotoxic effects of PDT on colon 26 cells were investigated in vitro using the WST assay. We investigated whether the antitumor effect of anti-PD-1 antibodies could be amplified by the addition of PDT. We performed combination therapy by randomly allocating tumor-bearing mice to four treatment groups: control, anti-PD-1 antibodies, PDT, and a combination of anti-PD-1 antibodies and PDT. To analyze the tumor microenvironment after treatment, the tumors were resected and pathologically evaluated. RESULTS: The viability rate of colon 26 cells decreased proportionally with the laser dose. In vivo experiments for combined PDT and anti-PD-1 antibody treatment, combination therapy showed an enhanced antitumor effect compared with the control. Immunohistochemical findings of the tumor microenvironment 10 days after PDT indicated that the number of CD8+ cells, the area of Iba-1+ cells and the area expressing PD-L1 were significantly higher in tumors treated with combination therapy than in tumors treated with anti-PD-1 antibody alone, PDT alone, or the control. CONCLUSIONS: PDT increased immune cell infiltration into the tumor microenvironment. The immunological response induced by PDT may enhance the efficacy of PD-1/PD-L1 blockade.

Analyzing the Functional Roles and Immunological Features of Chemokines in COAD.

Chemokines are key proteins that regulate cell migration and immune responses and are essential for modulating the tumor microenvironment. Despite their close association with colon cancer, the expression patterns, prognosis, immunity, and specific roles of chemokines in colon cancer are still not fully understood. In this study, we investigated the mutational features, differential expression, and immunological characteristics of chemokines in colon cancer (COAD) by analyzing the Tumor Genome Atlas (TCGA) database. We clarified the biological functions of these chemokines using Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. By univariate and multivariate COX regression analyses, we developed chemokine-based prognostic risk models. In addition, using Gene Set Enrichment Analysis (GSEA) and Gene Set Variant Analysis (GSVA), we analyzed the differences in immune responses and signaling pathways among different risk groups. The results showed that the mutation rate of chemokines was low in COAD, but 25 chemokines were significantly differentially expressed. These chemokines function in several immune-related biological processes and play key roles in signaling pathways including cytokine-cytokine receptor interactions, NF-kappa B, and IL-17. Prognostic risk models based on CCL22, CXCL1, CXCL8, CXCL9, and CXCL11 performed well. GSEA and GSVA analyses showed significant differences in immune responses and signaling pathways across risk groups. In conclusion, this study reveals the potential molecular mechanisms of chemokines in COAD and proposes a new prognostic risk model based on these insights.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.