Integrative analysis of single-cell and bulk RNA seq to reveal the prognostic model and tumor microenvironment remodeling mechanisms of cuproptosis-related genes in colorectal cancer.

BACKGROUND: Recently, there has been a great deal interest in cuproptosis, a form of programmed cell death that is mediated by copper. The specific mechanism through which cuproptosis-related genes impact the development of colorectal cancer (CRC) remains unknown. METHODS: Here, we combined bulk RNA-seq with scRNA-seq to investigate the CRGs functions within CRC. A number of 61 cuproptosis-related genes were chosen for further investigation. Nine prognostic CRGs were identified by Lasso-Cox. The RiskScore was created and the patients have been separated into two different groups, low- and high-RiskScore group. The CIBERSORT, ESTIMATE, MCP-counter, TIDE, and IPS have been employed to score the TME, and GSVA and GSEA were utilized to evaluate the pathway within the both groups. Further, we used cell communication analysis to explore the tumor microenvironment remodeling mechanisms of the COX17 and DLAT based on scRNA-seq. Finally, we used IHC and qPCR to validate the expression of COX17 and DLAT. RESULTS: AOC3, CCS, CDKN2A, COX11, COX17, COX19, DLD, DLAT, and PDHB have been recognized as prognostic CRGs in CRC. The high-risk group exhibited the worst prognosis, an immune-deficient phenotype, and were more resistant to ICB treatment. Further, scRNA-seq analysis revealed that elevated expression of COX17 in CD4-CXCL13Tfh could contribute to the immune evasion while DLAT had the opposite effect, reversing T cell exhaustion and inducing pyroptosis to boost CD8-GZMKT infiltration. CONCLUSIONS: The current investigation has developed a prognostic framework utilizing cuproptosis-related genes that is highly effective in predicting prognosis, TME type, and response to immunotherapy in CRC patients. Furthermore, our study reveals a novel finding that elevated levels of COX17 expression within CD4-CXCL13 T cells in CRC mediates T cell exhaustion and Treg infiltration, while DLAT has been found to facilitate the anti-tumor immunity activation through the T cell exhaustion reversal and the induction of pyroptosis.

Fluorophore-conjugated 4-1BB antibody enables early detection of T-cell responses in inflammatory arthritis via NIRF imaging.

PURPOSE: We first developed a 4-1BB-targeted optical probe, named IRDye-680RD-4-1BB mAb (monoclonal antibody), and evaluated its value for the detection of 4-1BB+ activated T cells in vivo as well as the diagnosis of rheumatoid arthritis (RA) in an adjuvant-induced arthritis (AIA) mouse model. METHODS: The 4-1BB expression pattern was analysed by flow cytometry and immunofluorescence (IF) staining. The 4-1BB mAb was conjugated with IRDye-680RD NHS ester, and characterized via fluorescence spectrum. A cell-binding assay was also performed to assess the interaction of this probe with activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed at 6, 24, 48, 72, and 96 h after probe administration. RESULTS: 4-1BB expression was highly upregulated during the pathogenesis of RA. Good colocalization was also observed between CD3 and 4-1BB by IF staining and t-SNE (T-distributed stochastic neighbour embedding) analysis, which indicates that 4-1BB was mainly expressed on T cells. Compared to the control group, a significantly higher signal was observed in the right hind paw (RP) of mice with AIA at all time points. The ex vivo biodistribution study results were consistent with the in vivo NIRF imaging results, which validated the accuracy of the region of interest (ROI) measurements. The sensitivity against 100% specificity observed in the receiver operator characteristic (ROC) curve analysis could distinguish the AIA group from the control group at all time points, indicating the value of IRDye-680RD-4-1BB mAb for RA diagnosis. CONCLUSION: We successfully developed a novel optical imaging probe, named IRDye-680RD-4-1BB mAb, for tracking 4-1BB+ activated T cells in vivo, and 4-1BB NIRF imaging is a promising strategy for noninvasively detecting the pathogenesis of RA.

PIK3CA mutation affects the proliferation of colorectal cancer cells through the PI3K-MEK/PDK1-GPT2 pathway.

The phosphatidylinositol­3­kinase catalytic subunit α (PIK3CA) gene is mutated in numerous human cancers. This mutation promotes the proliferation of tumor cells; however, the underlying mechanism is still not clear. In the present study, it was revealed that the PIK3CA mutation in colorectal cancer (CRC) HCT116 (MUT) rendered the cells more dependent on glutamine by regulating the glutamic­pyruvate transaminase 2 (GPT2). The dependence of glutamine increased the proliferation of cells in a normal environment and resistance to a suboptimal environment. Further study revealed that the mutated PIK3CA could regulate GPT2 expression not only through signal transduction molecule 3­phosphoinositide­dependent kinase (PDK1) but also through mitogen­activated protein kinase (MEK) molecules. In HCT116 cells, MEK inhibitor treatment could reduce the expression of GPT2 signaling molecules, thereby inhibiting the proliferation of CRC cells. A new signal transduction pathway, the PI3K/MEK/GPT2 pathway was identified. Based on these findings, MEK and PDK1 inhibitors were combined to inhibit the aforementioned pathway. It was revealed that the combined application of MEK and PDK1 inhibitors could promisingly inhibit the proliferation of MUT compared with the application of PI3K inhibitors, PDK1 inhibitors, or MEK inhibitors alone. In vivo, MEK inhibitors alone and combined inhibitors had stronger tumor­suppressing effects. There was no significant difference between the PDK1­inhibitor group and normal group in vivo. Thus, these results indicated that mutated PI3K affected GPT2 mediated by the MEK/PDK1 dual pathway, and that the PI3K/MEK/GPT2 pathway was more important in vivo. Inhibiting MEK and PDK1 concurrently could effectively inhibit the proliferation of CRC cells. Targeting the MEK and PDK1 signaling pathway may provide a novel strategy for the treatment of PIK3CA­mutated CRC.

Long noncoding RNA DLEU2 drives the malignant behaviors of thyroid cancer through mediating the miR-205-5p/TNFAIP8 axis.

OBJECTIVE: Considering the plight in thyroid cancer therapy, we aimed to find novel therapeutic targets from a molecular perspective. METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot assay were carried out to determine RNA and protein expression. Cell counting kit-8 (CCK8) assay, flow cytometry, transwell migration assay and aerobic glycolysis analysis were performed to analyze cell proliferation, apoptosis, migration and aerobic glycolysis of thyroid cancer cells. MiRcode and Starbase software were used to search the downstream genes of long noncoding RNA (lncRNA) deleted in lymphocytic leukemia 2 (DLEU2) and microRNA-205-5p (miR-205-5p), and the intermolecular combination was confirmed by dual-luciferase reporter assay. The in vivo role of DLEU2 in tumor growth was verified using the murine xenograft model. RESULTS: DLEU2 and tumor necrosis factor-α-induced protein 8 (TNFAIP8) were highly expressed in thyroid cancer tissues and cell lines. DLEU2 and TNRAIP8 promoted the proliferation, migration and aerobic glycolysis and restrained the apoptosis of thyroid cancer cells. DLEU2/miR-205-5p/TNFAIP8 signaling axis was identified in thyroid cancer cells. TNFAIP8 overexpression largely rescued the malignant phenotypes in DLEU2-silenced thyroid cancer cells. DLEU2 positively regulated TNFAIP8 expression by acting as miR-205-5p sponge in thyroid cancer cells. DLEU2 silencing blocked the growth of xenograft tumors in vivo. CONCLUSION: lncRNA DLEU2 exerted a pro-tumor role to promote proliferation, migration and aerobic glycolysis while repressing the apoptosis of thyroid cancer cells via miR-205-5p/TNFAIP8 axis.

Different Triple-Negative Breast Cancer Tumor Cell Lysates (TCLs) Induce Discrepant Anti-Tumor Immunity by PD1/PDL-1 Interaction.

BACKGROUND TCL-based immunotherapy has been applied in the field cancer therapy. However, it is un clear whether this therapy can be used to treat triple-negative breast cancer (TNBC), and different TNBC cells have distinct responses to this therapy. MATERIAL AND METHODS In the present work, we conducted 2 different TCL-based immunotherapies to treat TNBC and compared their anti-tumor effect on 4 TNBC cell lines: MDA-MB-231, MDA-MB-436, HCC1937, and HCC1187. RESULTS Peripheral blood mononuclear cells (PBMC) activated by TCL and peripheral blood lymphocytes (PBL) stimulated with TCL-loaded DC demonstrated the ability to kill TNBC cells in vitro, but the killing efficiency of PBL was much higher than that of PBMC. In vivo, PBL stimulated with TCL-loaded DC can also stop the growth of TNBC tumors in mice. HCC1187 and MDA-MD-231 best respond to TCL-based immunotherapy both in vitro and in vivo. The response of HCC1937 was weaker, and that of MDA-MB-436 was lowest among the 4 cell lines. Total mRNA microarray analysis of TNBC cells showed that PDL-1 mRNA expression in HCC1937 and MDA-MD-436 cells was higher than in the other 2 TNBC cell lines, and that of MDA-MB-436 was higher than that of HCC1937. PD1 blocking can decrease the apoptosis rate. These results show that different contents of PDL-1 in TCL, by interacting with PD expression on lymphocytes, can induce different ratios of lymphocyte apoptosis, and then result in distinct response of the 4 TNBC cell lines to TCL-based immunotherapy. CONCLUSIONS TCL-based immunotherapy has discrepant anti-tumor efficiency in different TNBC cell lines by PDL-1/PD interaction, providing the theoretical basis of TCL-based immunotherapy in TNBC.

Vaccination with FasL-/TCL plus MHSP65 induces improved anti-lung cancer immunity in mice.

In a previous study, we constructed a MHSP65-TCL anti-lung cancer vaccine with Lewis lung carcinoma TCL plus MHSP65, and illustrated its anti-lung cancer effect through specific and nonspecific anti-tumor immunity. However, TCL contains some immunoinhibit components such as FasL. If this component can be eliminated from TCL, the anti-tumor immunity of MHSP65-TCL constructed with TCL should be improved. In the present study, we knocked down FasL from Lewis lung carcinoma cells and prepared MHSP65-(FasL-/TCL) with this cell line's TCL. After further investigation, MHSP65-(FasL-/TCL) exhibited a better ability to reduce splenocytes apoptosis, promote its activation and secretion of secretingTNF-ß, IL-2 compared with MHSP65-(FasL+/TCL). Accordingly, specific and nonspecific antitumor immunity induced by MHSP65-(FasL-/TCL) is stronger than that of MHSP65-(FasL+/TCL). In vivo, MHSP65-(FasL-/TCL) immunization can prolong survival of Lewis lung carcinoma bearing mice. Thus, we report that the anti-lung cancer effect of MHSP65-TCL can be improved by removal of FasL from the TCL. It provides a new route to construct MHSP65-TCL and other antitumor vaccines based on TCL.

Detection of RACK1 and CTNNBL1­induced activation of mouse splenocytes using an immunoprecipitation­based technique.

Tumor cell lysates (TCLs) have been reported to induce antitumor immunity; however, it remains unclear which elements serve a role in this process. The present study identified 768 proteins that were upregulated in TCL prepared from Lewis lung cancer cells compared with the lysate from type II alveolar epithelial cells. Among the proteins that were upregulated in TCL, receptor for activated C kinase 1 (RACK1) and catenin ß­like 1 (CTNNBL1) are closely associated with cell proliferation and the inhibition of apoptosis. To determine the role of these proteins in TCL, a protein extraction method was designed, which was based on immunoprecipitation. Using this method, RACK1 and CTNNBL1 were extracted, whereas the other proteins within the TCL were not affected. The modified TCL exhibited a stronger ability to induce splenocyte apoptosis, whereas the ability to promote cell activation was reduced. These findings suggested that the TCL depends on RACK1 and CTNNBL1 to activate mouse immunocytes, including monocytes and B lymphocytes, and inhibit apoptosis. Therefore, the present study may provide information regarding the composition of TCLs and their positive regulatory effect on immunocytes.

Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer.

Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to α-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110α upregulates GPT2 gene expression through an AKT-independent, PDK1-RSK2-ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not with WT PIK3CA. Together, these data establish oncogenic PIK3CA mutations as a cause of glutamine dependency in CRCs and suggest that targeting glutamine metabolism may be an effective approach to treat CRC patients harbouring PIK3CA mutations.

A Protein Interaction between ß-Catenin and Dnmt1 Regulates Wnt Signaling and DNA Methylation in Colorectal Cancer Cells.

UNLABELLED: Aberrant activation of the Wnt signaling pathway is an important step in the initiation and progression of tumor development in diverse cancers. The central effector of canonical Wnt signaling, ß-catenin (CTNNB1), is a multifunctional protein, and has been extensively studied with respect to its roles in cell-cell adhesion and in regulation of Wnt-driven transcription. Here, a novel mass spectrometry-based proteomics technique in colorectal cancer cells expressing stabilized ß-catenin, was used to identify a protein-protein interaction between ß-catenin and DNA methyltransferase I (Dnmt1) protein, the primary regulator of DNA methylation patterns in mammalian cells. Dnmt1 and ß-catenin strongly colocalized in the nuclei of colorectal cancer cells, and the interaction is mediated by the central domain of the Dnmt1 protein. Dnmt1 protein abundance is dependent upon the levels of ß-catenin, and is increased in cells expressing stabilized mutant ß-catenin. Conversely, the Dnmt1 regulates the levels of nuclear ß-catenin and ß-catenin/TCF-driven transcription. In addition, lysine-specific demethylase 1 (LSD1/KDM1A), a regulator of DNMT1 stability, was identified as a component of the Dnmt1-ß-catenin protein complex and perturbation of the Dnmt1-ß-catenin interaction altered DNA methylation. In summary, a functional protein-protein interaction was identified between two critically important oncoproteins, in turn revealing a link between Wnt signaling and downstream nuclear functions mediated by Dnmt1. IMPLICATIONS: Two critical oncoproteins, Dnmt1 and ß-catenin, mutually regulate one each other's levels and activities in colorectal cancer cells.

Tumor cell lysate induces the immunosuppression and apoptosis of mouse immunocytes.

Although tumor cell lysate (TCL) is a type of immunocyte stimulator, its immunosuppressive function must not be ignored. The present study reported that TCL prepared from a Lewis lung cancer cell was able to induce the development of immunosuppressive macrophages (MΦ) and tolerogenic dendritic cells. In addition, TCL upregulated the expression of CD69 in mouse splenocytes, and cell apoptosis and the percentage of regulatory T cells in mouse splenocytes simultaneously increased. Furthermore, the present study found that the immunosuppressive factor, hyaluronan, and the apoptosis inducers, Fas ligand and transforming growth factor-ß, are present in TCL. These components may be associated with the emergence of immunosuppressive cells or splenocyte apoptosis. Thus, the present study has enriched our understanding of the composition of TCL and its negative regulatory effect on immunocytes.

[Effect of MHSP65-TCL anti-melanoma vaccine on the activity of immunocytes].

OBJECTIVE: To evaluate the therapeutic effect of MHSP65-TCL on melanoma and its effect on the activity of the immunocytes. METHODS: MHSP65-TCL was prepared by mixing MHSP65 with TCL derived from B16 melanoma cell lysate by repeated freezing and thawing. The MHSP65-TCL vaccine was administered in mice bearing B16 melanoma, and the changes in melanoma growth was observed. To investigate the influence of TCL in MHSP65-TCL on the activity of the immunocytes, we co-cultured TCL and mouse spleen cells in vitro, and analyzed CD69 expression on the cells, cell apoptosis, and levels of IL-10 and IFN-γ in the cell culture supernatant. RESULTS: The MHSP65-TCL vaccine showed an anti-melanoma effect in the tumor-bearing mice. In the in vitro experiment, TCL in MHSP65-TCL strongly stimulated the activation of mouse spleen cells while causing apoptosis in some of the immunocytes and promoting cellular IL-10 secretion, but not IFN-γ. CONCLUSIONS: MHSP65-TCL derived from B16 melanoma cells has an anti-melanoma effect mediated by the activation of immunocytes. TCL in MHSP65-TCL also has immunosuppressive effect on immunocytes possibly due to the presence of suppressive components in TCL, and identifying and eliminating these components may potentially improve the anti-tumor actovoty of MSHP65-TCL vaccine.

Vaccination with B16 tumor cell lysate plus recombinant Mycobacterium tuberculosis Hsp70 induces antimelanoma effect in mice.

Tumor cell lysate (TCL) has an advantage of containing an extensive repertoire of tumor antigens but requires proper adjuvants to enhance its immunogenicity when used as an efficient tumor vaccine. Mycobacterium tuberculosis-derived heat shock protein 70 (TBHsp70) has been shown to assist crosspresentation of exogenously applied tumor antigens and activate innate immunity against tumor cells. In this study, TBHsp70-B16TCL, a preparation generated by mixing recombinant TBHsp70 and TCL of B16 melanoma cells directly, was tested for its immunogenicity as a tumor vaccine. The TBHsp70-B16TCL induced a significant inhibition of the growth and metastasis of B16 melanoma in mice and prolonged the survival of B16 melanoma-bearing mice. The inhibition was correlated with the specific immune responses induced by TBHsp70-B16TCL. The data suggest that recombinant TBHsp70-adjuvanted TCL might be developed into effective tumor vaccines for melanomas and possibly for other tumors.

Vaccination with TCL plus MHSP65 induces anti-lung cancer immunity in mice.

To develop effective anti-lung cancer vaccines, we directly mixed mycobacterial heat shock protein 65 (MHSP65) and tumor cell lysate (TCL) from Lewis lung cancer cells in vitro and tested its efficacy on stimulating anti-tumor immunity. Our results showed that MHSP65-TCL immunization significantly inhibited the growth of lung cancer in mice and prolonged the survival of lung cancer bearing mice. In vivo and in vitro data suggest that MHSP65-TCL could induce specific CTL responses and non-specific immunity, both of which could contribute to the tumor inhibition. Thus, this report provides an easy approach to prepare an efficient TCL based tumor vaccine.