LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Ultrasound imaging guided targeted sonodynamic therapy enhanced by magnetophoretically controlled magnetic microbubbles.

Sonodynamic therapy (SDT) utilizes ultrasonic excitation of a sensitizer to generate reactive oxygen species (ROS) to destroy tumor. Two dimensional (2D) black phosphorus (BP) is an emerging sonosensitizer that can promote ROS production to be used in SDT but it alone lacks active targeting effect and showed low therapy efficiency. In this study, a stable dispersion of integrated micro-nanoplatform consisting of BP nanosheets loaded and Fe3O4 nanoparticles (NPs) connected microbubbles was introduced for ultrasound imaging guided and magnetic field directed precision SDT of breast cancer. The targeted ultrasound imaging at 18 MHz and efficient SDT effects at 1 MHz were demonstrated both in-vitro and in-vivo on the breast cancer. The magnetic microbubbles targeted deliver BP nanosheets to the tumor site under magnetic navigation and increased the uptake of BP nanosheets by inducing cavitation effect for increased cell membrane permeability via ultrasound targeted microbubble destruction (UTMD). The mechanism of SDT by magnetic black phosphorus microbubbles was proposed to be originated from the ROS triggered mitochondria mediated apoptosis by up-regulating the pro-apoptotic proteins while down-regulating the anti-apoptotic proteins. In conclusion, the ultrasound theranostic was realized via the magnetic black phosphorus microbubbles, which could realize targeting and catalytic sonodynamic therapy.

Targeting the adenosine signaling pathway in macrophages for cancer immunotherapy.

One of the ways in which macrophages support tumorigenic growth is by producing adenosine, which acts to dampen antitumor immune responses and is generated by both tumor and immune cells in the tumor microenvironment (TME). Two cell surface expressed molecules, CD73 and CD39, boost catalytic adenosine triphosphate, leading to further increased adenosine synthesis, under hypoxic circumstances in the TME. There are four receptors (A1, A2A, A2B, and A3) expressed on macrophages that allow adenosine to perform its immunomodulatory effect. Researchers have shown that adenosine signaling is a key factor in tumor progression and an attractive therapeutic target for treating cancer. Several antagonistic adenosine-targeting biological therapies that decrease the suppressive action of tumor-associated macrophages have been produced and explored to transform this result from basic research into a therapeutic advantage. Here, we'll review the newest findings from studies of pharmacological compounds that target adenosine receptors, and their potential therapeutic value based on blocking the suppressive action of macrophages in tumors.

Targeting TRIP13 for overcoming anticancer drug resistance (Review).

Cancer is one of the greatest dangers to human wellbeing and survival. A key barrier to effective cancer therapy is development of resistance to anti­cancer medications. In cancer cells, the AAA+ ATPase family member thyroid hormone receptor interactor 13 (TRIP13) is key in promoting treatment resistance. Nonetheless, knowledge of the molecular processes underlying TRIP13­based resistance to anticancer therapies is lacking. The present study evaluated the function of TRIP13 expression in anticancer drug resistance and potential methods to overcome this resistance. Additionally, the underlying mechanisms by which TRIP13 promotes resistance to anticancer drugs were explored, including induction of mitotic checkpoint complex surveillance system malfunction, promotion of DNA repair, the enhancement of autophagy and the prevention of immunological clearance. The effects of combination treatment, which include a TRIP13 inhibitor in addition to other inhibitors, were discussed. The present study evaluated the literature on TRIP13 as a possible target and its association with anticancer drug resistance, which may facilitate improvements in current anticancer therapeutic options.

TNFR2 antagonistic antibody induces the death of tumor infiltrating CD4+Foxp3+ regulatory T cells.

BACKGROUND: TNFR2 expression is a characteristic of highly potent immunosuppressive tumor infiltrating CD4+Foxp3+ regulatory T cells (Tregs). There is compelling evidence that TNF through TNFR2 preferentially stimulates the activation and expansion of Tregs. We and others, therefore, proposed that targeting TNFR2 may provide a novel strategy in cancer immunotherapy. Several studies have shown the effect of TNFR2 antagonistic antibodies in different tumor models. However, the exact action of the TNFR2 antibody on Tregs remained understood. METHOD: TY101, an anti-murine TNFR2 antibody, was used to examine the effect of TNFR2 blockade on Treg proliferation and viability in vitro. The role of TNFR2 on Treg viability was further validated by TNFR2 knockout mice and in the TY101 antagonistic antibody-treated mouse tumor model. RESULTS: In this study, we found that an anti-mouse TNFR2 antibody TY101 could inhibit TNF-induced proliferative expansion of Tregs, indicative of an antagonistic property. To examine the effect of TY101 antagonistic antibody on Treg viability, we treated unfractionated lymph node (L.N.) cells with Dexamethasone (Dex) which was known to induce T cell death. The result showed that TY101 antagonistic antibody treatment further promoted Treg death in the presence of Dex. This led us to find that TNFR2 expression was crucial for the survival of Tregs. In the mouse EG7 lymphoma model, treatment with TY101 antagonistic antibody potently inhibited tumor growth, resulting in complete regression of the tumor in 60% of mice. The treatment with TY101 antagonistic antibody elicited potent antitumor immune responses in this model, accompanied by enhanced death of Tregs. CONCLUSION: This study, therefore, provides clear experimental evidence that TNFR2 antagonistic antibody, TY101, can promote the death of Tregs, and this effect may be attributable to the antitumor effect of TNFR2 antagonistic antibody.

Collective analysis of the expression and prognosis for LEM-domain proteins in prostate cancer.

BACKGROUND: Mammalian LEM-domain proteins (LEMs) are encoded by seven genes, including LAP2, EMD, LEMD1, LEMD2, LEMD3, ANKLE1, and ANKLE2. Though some LEMs were involved in various tumor progression, the expression and prognostic values of LEMs in prostate adenocarcinoma (PRAD) have yet to be analyzed. METHODS: Herein, we investigated the expression, survival data, and immune infiltration levels of LEMs in PRAD patients from ATCG, TIMER, LinkedOmics, and TISIDB databases. We also further validated the mRNA and protein expression levels of ANKLE1, EMD, and LEMD2 in human prostate tumor specimens by qPCR, WB, and IHC. RESULTS: We found that all LEM expressions, except for that of LAP2, were markedly altered in PRAD compared to the normal samples. Among all LEMs, only the expressions of ANKLE1, EMD, and LEMD2 were correlated with advanced tumor stage and survival prognosis in PRAD. Consistent with the predicted computational results, the mRNA and protein expression levels of these genes were markedly increased in the PRAD group. We then found that ANKLE1, EMD, and LEMD2 expressions were markedly correlated with immune cell infiltration levels. High ANKLE1, EMD, and LEMD2 expressions predicted a worse prognosis in PRAD based on immune cells. DNA methylation or/and copy number variations may contribute to the abnormal upregulation of ANKLE1, EMD, and LEMD2 in PRAD. CONCLUSIONS: Taken together, this study implied that ANKLE1, EMD, and LEMD2 were promising prognosis predictors and potential immunotherapy targets for PRAD patients.

Signaling pathway(s) of TNFR2 required for the immunoregulatory effect of CD4+Foxp3+ regulatory T cells.

CD4+Foxp3+ regulatory T cells (Tregs), a subpopulation of CD4+ T cells, are engaged in maintaining the periphery tolerance and preventing autoimmunity. Recent studies showed that tumor necrosis factor receptor 2 (TNFR2) is preferentially expressed by Tregs and the expression of this receptor identifies the maximally suppressive Tregs. That is, TNFR2 is a liable phenotypic and functional surface marker of Tregs. Moreover, TNF activates and expands Tregs through TNFR2. However, it is very interesting which signaling pathway(s) of TNFR2 is required for the inhibitory effect of Tregs. Compelling evidence shows three TNFR2 signaling pathways in Tregs, including NF-κB, MAPK and PI3K-Akt pathways. Here, we summarize and discuss the latest progress in the studies on the downstream signaling pathways of TNF-TNFR2 for controlling Treg homeostasis, differentiation and proliferation.

Topical Application of Tetrandrine Nanoemulsion Promotes the Expansion of CD4+Foxp3+ Regulatory T Cells and Alleviates Imiquimod-Induced Psoriasis in Mice.

There is compelling evidence that CD4+Foxp3+ regulatory T cells (Tregs) are indispensable in the inhibition of autoimmune inflammatory responses, including psoriasis. Recently, we showed that systemically treatment with tetrandrine (TET), a two-pore channel inhibitor identified from the Chinese herb Stephania tetrandra S. Moor, could promote the proliferative expansion of Tregs in mice through stimulation of TNF-TNFR2 interaction. We thus hypothesized that topical administration of TET might also expand Tregs and consequently inhibit psoriasis. To this end, we developed a TET nanoemulsion and examined its effect on the expansion of Tregs after topical administration on mouse psoriasis induced by imiquimod. The result of our experiment showed that topical treatment with TET nanoemulsion markedly increased the proportion and number of Tregs in the spleen, as well as TNFR2 and Ki-67 expression by Tregs, in WT and TNFR1 KO mice, but not in TNFR2 KO mice. Consequently, TET nanoemulsion potently inhibited IL-17-expressing cells in the spleen and lymph nodes of imiquimod-treated WT mice, accompanied by decreased serum levels of IL-17A, INF-γ, and TNF and their mRNA levels in the flamed lesion. Importantly, TET nanoemulsion treatment markedly inhibited the development of psoriasis-like disease in WT and TNFR1 KO mice but not in TNFR2 KO mice. Therefore, our study indicates that the topical administration of TET could also stimulate the expansion of Tregs through the TNF-TNFR2 pathway. This effect of TET and its analogs may be useful in the treatment of inflammatory skin diseases such as psoriasis.

Circ_SAR1A regulates the malignant behavior of lung cancer cells via the miR-21-5p/TXNIP axis.

BACKGROUND: Lung cancer is one of the most common malignancies globally and a significant component of cancer-related deaths. The lack of early diagnosis accounts for detecting approximately 75% of cancer patients at an intermediate to an advanced stage, with a low 5-year survival rate. Therefore, a more comprehensive understanding of the molecular mechanisms of lung cancer development is necessary to find reliable and effective therapeutic and diagnostic biomarkers. METHODS: circ_SAR1A, miR-21-5p, and TXNIP in lung cancer tissues, animal xenografts, and cell lines were validated by qRT-PCR and western blotting analyses. RNase R digestion and nuclear/cytoplasm fractionation experiments were utilized to determine the stability and localization of circ_SAR1A in lung cancer cells. The binding between miR-21-5p and circ_SAR1A or TXNIP was confirmed by luciferase reporter, RNA pull-down, Spearman's correlation, and rescue assays. CCK-8, colony formation, flow cytometry, Transwell, and western blotting were utilized to illustrate the malignant behavior of lung cancer cells. RESULTS: circ_SAR1A and TXNIP were down-regulated while miR-21-5p was up-regulated in lung cancer samples and cells. circ_SAR1A was located predominantly in the cytoplasm; it inhibited lung cancer growth in vitro and in vivo by sponging to miR-21-5p. miR-21-5p silencing suppressed lung cancer malignancy by targeting TXNIP. CONCLUSIONS: circ_SAR1A is a critical negative regulator of lung carcinogenesis. circ_SAR1A/miR-21-5p/TXNIP attenuation inhibited lung cancer progression, presenting an ideal diagnostic and a potential therapeutic target.

Contrast-enhanced ultrasound imaging using long-circulating cationic magnetic microbubbles in vitro and in vivo validations.

Traditional encapsulated microbubbles are recently used as delivery carriers for drugs and genes, but they have low efficiency. If the local microbubble concentration could be increased, this might be able to improve the therapeutic efficacy of diseases. In this study, we developed novel cationic magnetic microbubbles (MBM), which could simultaneously realize targeted aggregation under a magnetic field as well as ultrasonographic real-time visualization. Their physicochemical properties, biocompatibility, ultrasonography, magnetic response characteristics, and biodistribution were systematically evaluated. Here, the MBM were 2.55 ± 0.14 µm in size with a positive zeta potential, and had a good biocompatibility. They were able to enhance ultrasonographic contrast both in vitro and in vivo. MBM could be attracted by an external magnet for directional movement and aggregation in vitro. We confirmed that MBM also had a great magnetic response in vivo, by means of fluorescence imaging and contrast-enhanced ultrasound imaging. Following intravenous injection into tumor-bearing mice, MBM showed excellent stability in the internal circulation, and could accumulate in the tumor vasculature through magnetic targeting. With the excellent combination of magnetic response and acoustic properties, cationic magnetic microbubbles (MBM) have promising potential for use as a new kind of drug/gene carrier for theranostics in the future.

Inhibition of two-pore channels in antigen-presenting cells promotes the expansion of TNFR2-expressing CD4+Foxp3+ regulatory T cells.

CD4+Foxp3+ regulatory T cells (Tregs) are pivotal for the inhibition of autoimmune inflammatory responses. One way to therapeutically harness the immunosuppressive actions of Tregs is to stimulate the proliferative expansion of TNFR2-expressing CD4+Foxp3+ Tregs via transmembrane TNF (tmTNF). Here, we report that two-pore channel (TPC) inhibitors markedly enhance tmTNF expression on antigen-presenting cells. Furthermore, injection of TPC inhibitors including tetrandrine, or TPC-specific siRNAs in mice, increases the number of Tregs in a tmTNF/TNFR2-dependent manner. In a mouse colitis model, inhibition of TPCs by tetrandrine markedly attenuates colon inflammation by expansion of Tregs Mechanistically, we show that TPC inhibitors enhance tmTNF levels by disrupting surface expression of TNF-α-converting enzyme by regulating vesicle trafficking. These results suggest that the therapeutic potential of TPC inhibitors is mediated by expansion of TNFR2-expressing Tregs and elucidate the basis of clinical use in the treatment of autoimmune and other inflammatory diseases.

The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells.

There is now compelling evidence that tumor necrosis factor (TNF) preferentially activates and expands CD4+Foxp3+ regulatory T cells (Tregs) through TNF receptor type II (TNFR2). However, it remains unclear which signaling transduction pathway(s) of TNFR2 is required for the stimulation of Tregs. Previously, it was shown that the interaction of TNF-TNFR2 resulted in the activation of a number of signaling pathways, including p38 MAPK, NF-κB, in T cells. We thus examined the role of p38 MAPK and NF-κB in TNF-mediated activation of Tregs, by using specific small molecule inhibitors. The results show that treatment with specific p38 MAPK inhibitor SB203580, rather than NF-κB inhibitors (Sulfasalazine and Bay 11-7082), abrogated TNF-induced expansion of Tregs in vitro. Furthermore, upregulation of TNFR2 and Foxp3 expression in Tregs by TNF was also markedly inhibited by SB203580. The proliferative expansion and the upregulation of TNFR2 expression on Tregs in LPS-treated mice were mediated by TNF-TNFR2 interaction, as shown by our previous study. The expansion of Tregs in LPS-treated mice were also markedly inhibited by in vivo treatment with SB203580. Taken together, our data clearly indicate that the activation of p38 MAPK is attributable to TNF/TNFR2-mediated activation and proliferative expansion of Tregs. Our results also suggest that targeting of p38 MAPK by pharmacological agent may represent a novel strategy to up- or downregulation of Treg activity for therapeutic purposes.

Deficiency in Fpr2 results in reduced numbers of Lin-cKit+Sca1+ myeloid progenitor cells.

The Lin-c-Kit+ Sca-1+ cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31+Ly6C+ (granulocytes and monocytes), CD31-/Ly6Cint (granuloid cells), and CD31-/Ly6Chigh (predominantly monocytes) cells. In particular, the number of Lin-c-Kit+Sca-1+ (LKS) cells was reduced in Fpr2-/- mouse BM. This was supported by observations of the reduced incorporation of intraperitoneally injected bromodeoxyuridine by cells in the c-Kit+ population from Fpr2-/- mouse BM. Purified c-Kit+ cells from Fpr2-/- mice showed reduced expansion when cultured in vitro with stem cell factor (SCF). SCF/c-Kit-mediated phosphorylation of P38, STAT1, Akt (Thr-308), and Akt (Ser-473) was also significantly reduced in c-Kit+ cells from Fpr2-/- mice. Furthermore, Fpr2 agonists enhanced SCF-induced proliferation of c-Kit+ cells. Colony-forming unit assays revealed that CFU-granulocyte-macrophage formation of BM cells from Fpr2-/- mice was significantly reduced. After heat-inactivated bacterial stimulation in the airway, the expansion of c-kit+ Sca-1+ cells in BM and recruitment of Ly6G+ cells to the lungs and CD11b+Ly6C+TNFα+ cells to the spleen of Fpr2-/- mice was significantly reduced. These results demonstrate an important role for Fpr2 in the development of myeloid lineage precursors in mouse BM.