Chronological age is a risk factor for SARS-CoV-2 infection and severe COVID-19. Previous findings indicate that epigenetic age could be altered in viral infection. However, the epigenetic aging in COVID-19 has not been well studied. In this study, DNA methylation of the blood samples from 232 healthy individuals and 413 COVID-19 patients is profiled using EPIC methylation array. Epigenetic ages of each individual are determined by applying epigenetic clocks and telomere length estimator to the methylation profile of the individual. Epigenetic age acceleration is calculated and compared between groups. We observe strong correlations between the epigenetic clocks and individual’s chronological age (r > 0.8,p < 0.0001). We also find the increasing acceleration of epigenetic aging and telomere attrition in the sequential blood samples from healthy individuals and infected patients developing non-severe and severe COVID-19. In addition, the longitudinal DNA methylation profiling analysis find that the accumulation of epigenetic aging from COVID-19 syndrome could be partly reversed at late clinic phases in some patients. In conclusion, accelerated epigenetic aging is associated with the risk of SARS-CoV-2 infection and developing severe COVID-19. In addition, the accumulation of epigenetic aging from COVID-19 may contribute to the post-COVID-19 syndrome among survivors.

Background Tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, can be used for predicting immunotherapy responsiveness and survival outcome. However, the evaluation of CD8+ TILs currently relies on histopathological methodology with high variability. We therefore aimed to develop a DNA methylation signature for CD8+ TILs (CD8+ MeTIL) that could evaluate immune response and prognosis in colorectal cancer (CRC). Methods A CD8+ MeTIL signature score was constructed by using CD8+ T cell-specific differentially methylated positions (DMPs) that were identified from Illumina EPIC methylation arrays. Immune cells, colon epithelial cells, and two CRC cohorts (n=282 and 335) were used to develop a PCR-based assay for quantitative analysis of DNA methylation at single-base resolution (QASM) to determine CD8 + MeTIL signature score. Results Three CD8+ T cell-specific DMPs were identified to construct the CD8+ MeTIL signature score, which showed a dramatic discriminability between CD8+ T cells and other cells. The QASM assay we developed for CD8+ MeTIL markers could measure CD8+ TILs distributions in a fully quantitative, accurate, and simple manner. The CD8+ MeTIL score determined by QASM assay showed a strong association with histopathology-based CD8+ TIL counts and a gene expression-based immune marker. Furthermore, the low CD8+ MeTIL score (enriched CD8+ TILs) was associated with MSI-H tumors and predicted better survival in CRC cohorts. Conclusions This study developed a quantitative DNA methylation-based signature that was reliable to evaluate CD8+ TILs and prognosis in CRC. This approach has the potential to be a tool for investigations on CD8+ TILs and a biomarker for therapeutic approaches, including immunotherapy.

Background: Colony-stimulating factor 1 receptor (CSF1R), a classic tyrosine kinase receptor, has been identified as a proto-oncogene in multiple cancers. The CSF1/CSF1R axis is essential for the survival and differentiation of M2-phenotype tumor-associated macrophages (M2 TAMs). However, we found here that the CSF1R expression was abnormally down-regulated in colorectal cancer (CRC), and its biological functions and underlying mechanisms have become elusive in CRC progression. Methods: The expression of class III receptor tyrosine kinases in CRC and normal intestinal mucosa was accessed using The Cancer Genome Atlas and Gene Expression Omnibus datasets and was further validated by our tested cohort. CSF1R was reconstructed in CRC cells to identify its biological functions in vitro and in vivo. We compared CSF1R expression and methylation differences between CRC cells and macrophages. Furthermore, a co-culture system was used to mimic a competitive mechanism between CSF1R-overexpressed CRC cells and M2-like macrophages. We utilized a CSF1R inhibitor PLX3397 to ablate M2 TAMs and evaluated its efficacy on CRC treatment in animal models. Results: We found here that the CSF1R is silenced in CRC, and the reintroduced expression of the receptor in CRC cells can be cleaved by caspases and constrain tumor growth in vitro and in vivo, functioning as a tumor suppressor gene. We further identified CSF1R as a novel dependence receptor, which has the potential to act as either a tumor suppressor gene or an oncogene, depending on its activated state. In CRC tumors, CSF1R expression is enriched in TAMs, and its expression is associated with poor prognosis in patients ith CRC. In a co-culture system, CRC cells expressing CSF1R compete with M2-like macrophages for CSF1R ligands, resulting in a decrease in CSF1R activation and cell proliferation in macrophages. Blocking CSF1R by PLX3397 could deplete M2 TAMs and augments CD8⁺ T cell infiltration, effectively inhibiting tumor growth and metastasis and improving responses to chemotherapy and immunotherapy. Conclusion: Our findings revealed that CSF1R is a novel identified dependence receptor silenced in CRC. The silence abalienates its ligands to stimulate CSF1R expressed on M2 TAMs, which is an appealing therapeutic target for M2 TAM depletion and CRC treatment.

Background: Immune checkpoint inhibitor (ICI) treatment in patients with microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) tumors holds promise in reshaping organ preservation in rectal cancer. However, the benefits are accompanied by distinctive patterns of response, introducing a dilemma in the response evaluation for clinical decision-making. Patients and Methods: Patients with locally advanced rectal cancer with MSI-H/dMMR tumors receiving neoadjuvant ICI (nICI) treatment (n=13) and matched patients receiving neoadjuvant chemoradiotherapy (nCRT; n=13) were included to compare clinical response and histopathologic features. Results: Among the 13 patients receiving nICI treatment, in the final radiologic evaluation prior to surgery (at a median of 103 days after initiation of therapy), progressive disease (n=3), stable disease (n=1), partial response (n=7), and complete response (n=2) were observed. However, these patients were later confirmed as having pathologic complete response, resulting in pseudoprogression and pseudoresidue with incidences of 23.1% (n=3) and 76.9% (n=10), respectively, whereas no pseudoprogression was found in the 13 patients receiving nCRT. We further revealed the histopathologic basis underlying the pseudoprogression and pseudoresidue by discovering the distinctive immune-related regression features after nICI treatment, including fibrogenesis, dense lymphocytes, and plasma cell infiltration. Conclusions: Pseudoprogression and pseudoresidue were unique and prevalent response patterns in MSI-H/dMMR rectal cancer after nICI treatment. Our findings highlight the importance of developing specific strategies for response evaluation in neoadjuvant immunotherapy to identify patients with a good response in whom sphincter/organ-preserving or watch-and-wait strategies may be considered.

More than ten randomized clinical trials are being tested to evaluate the efficacy, effectiveness and safety of a fasting-mimicking diet (FMD) combined with different antitumor agents. UMI-mRNA sequencing, Cell-cycle analysis, Label retention, metabolomics, Multilabeling et al. were used to explore mechanisms. A tandem mRFP-GFP-tagged LC3B, Annexin-V-FITC Apoptosis, TUNEL, H&E, Ki-67 and animal model was used to search for synergistic drugs. Here we showed that fasting or FMD retards tumor growth more effectively but does not increase 5-fluorouracil/oxaliplatin (5-FU/OXA) sensitivity to apoptosis in vitro and in vivo. Mechanistically, we demonstrated that CRC cells would switch from an active proliferative to a slow-cycling state during fasting. Furthermore, metabolomics shows cell proliferation was decreased to survive nutrient stress in vivo, as evidenced by a low level of adenosine and deoxyadenosine monophosphate. CRC cells would decrease proliferation to achieve increased survival and relapse after chemotherapy. In addition, these fasting-induced quiescent cells were more prone to develop drug-tolerant persister (DTP) tumor cells postulated to be responsible for cancer relapse and metastasis. Then, UMI-mRNA sequencing uncovered the ferroptosis pathway as the pathway most influenced by fasting. Combining fasting with ferroptosis inducer treatment leads to tumor inhibition and eradication of quiescent cells by boosting autophagy. Our results suggest that ferroptosis could improve the antitumor activity of FMD + chemotherapy and highlight a potential therapeutic opportunity to avoid DTP cells-driven tumor relapse and therapy failure. A full list of funding bodies can be found in the Acknowledgements section.

Background: N6‐methyladenosine (m6A) modification is an emerging epigenetic regulatory mechanism in tumourigenesis. Considering that AlkB homolog 5 (ALKBH5) is a well‐described m6A demethylase in previous enzyme assays, we aimed to investigate the role of m6A methylation alteration conferred by disturbed ALKBH5 in colorectal cancer (CRC) development.Methods: Expression of ALKBH5 and its correlation with clinicopathological characteristics of CRC were evaluated using the prospectively maintained institutional database. The molecular role and underlying mechanism of ALKBH5 in CRC were explored using in vitro and in vivo experiments with methylated RNA immunoprecipitation sequencing (MeRIP‐seq), RNA‐seq, MeRIP‐qPCR, RIP‐qPCR and luciferase reporter assays.Results: ALKBH5 expression was significantly upregulated in CRC tissues compared to the paired adjacent normal tissues, and higher expression of ALKBH5 was independently associated with worse overall survival in CRC patients. Functionally, ALKBH5 promoted the proliferative, migrative and invasive abilities of CRC cells in vitro and enhanced subcutaneous tumour growth in vivo. Mechanistically, RAB5A was identified as the downstream target of ALKBH5 in CRC development, and ALKBH5 posttranscriptionally activated RAB5A by m6A demethylation, which impeded the YTHDF2‐mediated degradation of RAB5A mRNA. In addition, we demonstrated that dysregulation of the ALKBH5‐RAB5A axis could affect the tumourigenicity of CRC.Conclusions: ALKBH5 facilitates the progression of CRC by augmenting the expression of RAB5A via an m6A‐YTHDF2‐dependent manner. Our findings suggested that ALKBH5‐RAB5A axis might serve as valuable biomarkers and effective therapeutic targets for CRC.