Subject(s)
COVID-19 , Leukocyte L1 Antigen Complex , Biomarkers , Feces , Humans , SARS-CoV-2 , Severity of Illness IndexABSTRACT
BACKGROUND: Tumour microenvironment (TME) of advanced colorectal cancer (CRC) suppresses dendritic cell (DC) maturation. Here, our aim was to determine how the microenvironment of early-stage tumours influences DCs. METHODS: Tumour-conditioned media (TCM) was generated by culturing explant tumour tissue in vitro (n=50). Monocyte-derived DCs (MDDCs) of healthy donors or cancer patients were pretreated with TCM and stimulated with lipopolysaccharide (LPS). DC maturation was assessed by flow cytometry and cytokine production measured by ELISA. RESULTS: TCM from both early- and late-staged tumours abrogated LPS-induction of IL-12p70 secretion, while increasing IL-10. The profile of inflammatory mediators in TCM was similar across stages, and all increased pSTAT3 expression by DCs.CRC patient DCs (n=31) secreted low levels of IL-12p70 and failed to upregulate expression of maturation markers in response to LPS. Furthermore, in vitro culture of autologous DCs with TCM did not change the hypo-responsiveness of patient DCs. CONCLUSION: Our data demonstrates that the TME of all stages of CRC contains inflammatory mediators capable of suppressing local DCs. MDDCs obtained from CRC patients are hyporesponsive to stimuli such as LPS. Measures to reverse the negative influence of the TME on DCs will optimise cancer vaccines in both early- and late-stage CRC.
Subject(s)
Colorectal Neoplasms/immunology , Tumor Microenvironment/immunology , Adult , Aged , Aged, 80 and over , Dendritic Cells/immunology , Female , Humans , Immunosuppression Therapy , Inflammation/immunology , Interleukin-10/immunology , Interleukin-12/immunology , Lipopolysaccharides/immunology , Male , Middle Aged , Monocytes/immunology , STAT3 Transcription Factor/immunologyABSTRACT
BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell's response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel. METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches. RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8 representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2 cells show enhanced migratory ability. At 72 h after paclitaxel, MAD2 cells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2 MCF7 cell line after paclitaxel reflecting the observed increase in senescence. CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.
Subject(s)
Breast Neoplasms/drug therapy , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Cellular Senescence/drug effects , Paclitaxel/pharmacology , Repressor Proteins/metabolism , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/genetics , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/physiology , Cellular Senescence/genetics , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Female , Humans , Mad2 Proteins , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Spindle Apparatus/drug effects , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , TransfectionABSTRACT
Oxidative DNA damage results from DNA adducts such as 8-oxo-7, 8 dihydro-2'-deoxyguanosine (8-oxo-dG), which is a pro-mutagenic lesion. No known association between 8-oxo-dG, disease progression and survival exists in colorectal cancer (CRC). We examined levels of 8-oxo-dG in sporadic CRC to determine its relationship with pathological stage and outcome. A total of 143 CRC patients and 105 non-cancer patients were studied. Nuclear and cytoplasmic 8-oxo-dG was assessed using immunohistochemistry. Double immunofluorescence using 8-oxo-dG and manganese superoxide dismutase (MnSOD) antibodies localised cytoplasmic 8-oxo-dG. Apoptosis was detected using TUNEL. Nuclear staining levels were similar in tumour tissue and matched normal mucosa in both epithelial (P=0.22) and stromal (P=0.85) cells. Epithelial cytoplasmic staining was greater in tumour tissue (P<0.001). Double immunofluorescence localised cytoplasmic 8-oxo-dG to mitochondria. Epithelial and stromal nuclear 8-oxo-dG decreased with local disease spread, but highest levels were found in distant disease (P<0.01). Survival was related to epithelial nuclear and stromal staining in normal mucosa (P<0.001) and tumour (P<0.01) but was unrelated to cytoplasmic staining. Normal control cells in tissue from cancer patients with high levels of 8-oxo-dG failed to undergo cell death. 8-oxo-dG may be an important biomarker of disease risk, progression and survival for CRC patients.
