Background Pre-metastatic niches composed of mainly myeloid cells are recognized as critical for tumor metastasis. However, whether adaptive immune cells also play an important role in pre-metastatic niche formation remains to be explored.Methods CD4+ T cell accumulation in tumor-free lung tissues from mice bearing subcutaneous mouse tumors was detected by immunofluorescence/confocal microscopy. Tumor-conditioned media (TCM) from MB49-S1pr1high mouse bladder tumor cells or ID8 ovarian tumor cells were administered to tumor-free mice to induce pre-metastatic niche formation. We used mice lacking functional Signal Transducer and Activator of Transcription 3 (STAT3) in T cells and Il17a‒/‒ mice to investigate the roles of STAT3 and interleukin (IL)-17. In vivo time-course experiments were performed to assess whether CD4+ T cell clusters contribute to CD11b+ pre-metastatic clusters. CD4+ T cell migration and chemokine receptor expression assays were employed to identify tumor factors driving CD4+ T cell recruitment. A co-culture system with human MRC-5 lung fibroblasts, healthy donor-derived CD4+ T cells, myeloid cells, and TCM derived from human cancer cells was used to evaluate CD4+ T cell-driven fibroblast activation and IL-17A dependency for myeloid cell migration. Microscopic analyses were performed to confirm CD4+ T cell clusters in tumor-free lymph node tissues from patients with prostate cancer and postmortem lung and liver specimens from patients with ovarian cancer.Results We demonstrate that CD4+ T cells accumulate in tumor-free lungs and promote tumor metastasis in mouse models. CD4+ T-cell pre-metastatic niche formation requires STAT3, which regulates Th17 CD4+ T cells. TCM drives IL-6-dependent CCR4/CCR6 upregulation on naive CD4+ T cells. CD4+ T cell clusters contribute to myeloid cell accumulation, and ablating STAT3 in T cells abrogates both T cell and myeloid cluster formation. IL-17 inhibition reduces myeloid lung infiltration. In human co-cultures, CD4+ T cells amplify TCM-induced fibroblast pre-metastatic niche-like activation and myeloid recruitment in an IL-17A-dependent manner. CD4+ and IL-17+ or p-STAT3+ clusters were also detected in non-metastatic tissues from patients with several cancers.Conclusions CD4+ T cells form pre-metastatic niches through the STAT3-IL-17 axis, contributing to myeloid cell cluster formation, in part through amplifying fibroblast pre-metastatic niche-like activation. STAT3 and IL-17 in CD4+ T cells therefore are important for pre-metastatic niche formation and metastasis.

Mammalian Sterile 20-like protein kinase 3 (MST3) has been implicated in the progression of non-small cell lung cancer (NSCLC). However, the molecular mechanisms regarding MST3-mediated regulation of NSCLC metastasis remain to be elucidated. In this study, we demonstrate that MST3 enhances the invasion and migration of NSCLC cells by phosphorylating vimentin. Specifically, phosphorylation at residues S39 and S205 of vimentin contributes to MST3-induced metastatic behavior. The residues T190 and K65, which are essential for MST3 kinase activity, are required for MST3-mediated phosphorylation of vimentin, as demonstrated by in vitro phosphorylation assays showing phosphorylation at the S39 residue. Functional assays, including wound healing and transwell-based migration and invasion, further confirmed that the T190 and K65 residues are critical for MST3-driven cell motility and invasion. Immunofluorescence staining revealed co-localization of MST3 with vimentin, while co-immunoprecipitation and GST pull-down assays demonstrated a direct interaction between the two proteins. Collectively, these findings identify a novel mechanism by which MST3 drives NSCLC metastasis via vimentin phosphorylation, and suggest the MST3-vimentin axis as a potential therapeutic target for NSCLC intervention.

Starting at middle age, adults often suffer from visceral adiposity and associated adverse metabolic disorders. Lineage tracing in mice revealed that adipose progenitor cells (APCs) in visceral fat undergo extensive adipogenesis during middle age. Thus, despite the low turnover rate of adipocytes in young adults, adipogenesis is unlocked during middle age. Transplantations quantitatively showed that APCs in middle-aged mice exhibited high adipogenic capacity cell-autonomously. Single-cell RNA sequencing identified a distinct APC population, the committed preadipocyte, age-enriched (CP-A), emerging at this age. CP-As demonstrated elevated proliferation and adipogenesis activity. Pharmacological and genetic manipulations indicated that leukemia inhibitory factor receptor signaling was indispensable for CP-A adipogenesis and visceral fat expansion. These findings uncover a fundamental mechanism of age-dependent adipose remodeling, offering critical insights into age-related metabolic diseases.

Disrupting the interaction between tumor-cell surface PD-L1 and T cell membrane PD-1 can elicit durable clinical responses. However, only about 10% of ovarian cancer patients respond to PD-1/PD-L1 blockade. Here, we show that PD-L1 expression in ovarian cancer-patient tumors is predominantly intracellular. Notably, PARP inhibitor treatment highly increased intracellular PD-L1 accumulation in both ovarian cancer-patient tumor samples and cell lines. We investigated whether intracellular PD-L1 might play a critical role in ovarian cancer progression. Mutating the PD-L1 acetylation site in PEO1 and ID8Brca1-/- ovarian cancer cells significantly decreased PD-L1 levels and impaired colony formation, which was accompanied by cell cycle G2/M arrest and apoptosis induction. PEO1 and ID8Brca1-/- tumors with PD-L1 acetylation site mutation also exhibited significantly reduced growth in mice. Furthermore, targeting intracellular PD-L1 with a cell-penetrating antibody effectively decreased ovarian tumor-cell intracellular PD-L1 level and induced tumor-cell growth arrest and apoptosis, as well as enhanced DNA damage and STING activation, both in vitro and in vivo. In conclusion, we have shown the critical role of intracellular PD-L1 in ovarian cancer progression.

Adipose tissue inflammation contributes to obesity-induced insulin resistance. However, increasing evidence shows that high BMI (obesity) is not an accurate predictor of poor metabolic health in individuals. The molecular mechanisms regulating the metabolically activated M1 macrophage phenotype in the adipose tissues leading to insulin resistance remain largely unknown. Although the Janus Kinase (Jak)/signal transducer and activator of transcription 3 (Stat3) signaling in myeloid cells are known to promote the M2 phenotype in tumors, we demonstrate here that the Jak2/Stat3 pathway amplifies M1-mediated adipose tissue inflammation and insulin resistance under metabolic challenges. Ablating Jak2 in the myeloid compartment reduces insulin resistance in obese mice, which is associated with a decrease in infiltration of adipose tissue macrophages (ATMs). We show that the adoptive transfer of Jak2-deficient myeloid cells improves insulin sensitivity in obese mice. Furthermore, the protection of obese mice with myeloid-specific Stat3 deficiency against insulin resistance is also associated with reduced tissue infiltration by macrophages. Jak2/Stat3 in the macrophage is required for the production of pro-inflammatory cytokines that promote M1 macrophage polarization in the adipose tissues of obese mice. Moreover, free fatty acids (FFAs) activate Stat3 in macrophages, leading to the induction of M1 cytokines. Silencing the myeloid cell Stat3 with an in vivo siRNA targeted delivery approach reduces metabolically activated pro-inflammatory ATMs, thereby alleviating obesity-induced insulin resistance. These results demonstrate Jak2/Stat3 in myeloid cells is required for obesity-induced insulin resistance and inflammation. Moreover, targeting Stat3 in myeloid cells may be a novel approach to ameliorate obesity-induced insulin resistance.

Prenylated indole alkaloids, which are mainly produced by genera Aspergillus and Penicillium, are a class of structurally intriguing specialized metabolites with remarkable biomedical interests. In this study, chemically guided isolation of the Nicotiana tabacum-derived endophytic fungus Aspergillus japonicus TE-739D yielded eight structurally diverse prenylated indole alkaloids, including an undescribed compound, namely aspertaichamide B (ATB, 1), together with seven previously discovered derivatives (compounds 2 − 8). Their chemical structures as well as the stereochemical features were determined by integrated spectroscopic analyses, including HRESIMS, NMR, NMR calculations with DP4 + probability analysis, and a comparison of the experimental ECD data with computed DFT-based quantum chemical calculations. In vitro cytotoxic effects against the gastric cancer MFC cells revealed that the new compound ATB demonstrated considerable activity. Further studies found that ATB suppressed the viability, colony formation, and migration ability of MFC cells, and induced MFC cells apoptosis in a concentration-dependent way. Moreover, ATB stimulated ROS production in MFC cells and inhibited the tumor growth in the MFC-sourced subcutaneous tumor model while not significantly reducing the weight of mice. The pharmacological results suggested that the newly discovered ATB may be a promising anti-tumor lead compound. • Eight structurally diverse prenylated indole alkaloids including a new aspertaichamide B (ATB) were isolated from the fungus Aspergillus japonicus TE-739D. • The structure of ATB was elucidated by HRESIMS, NMR, NMR calculations with DP4 + probability analysis, and ECD calculations. • ATB inhibited cell proliferation, promoted apoptosis, and increased ROS production in gastric cancer cells, and exhibited inhibitory effects on tumor growth in vivo.

Background Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment.Methods By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG’s catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition.Results Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors.Conclusions We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

Immune checkpoint blockade (ICB) therapy has significantly benefited patients with several types of solid tumors and some lymphomas. However, many of the treated patients do not have a durable clinical response. It has been demonstrated that rescuing exhausted CD8+ T cells is required for ICB-mediated antitumor effects. We recently developed an immunostimulatory strategy based on silencing STAT3 while stimulating immune responses by CpG, a ligand for Toll-like receptor 9 (TLR9). The CpG-small interfering RNA (siRNA) conjugates efficiently enter immune cells, silencing STAT3 and activating innate immunity to enhance T cell-mediated antitumor immune responses. In the present study, we demonstrate that blocking STAT3 through locally delivered CpG-Stat3 siRNA enhances the efficacies of the systemic PD-1 and CTLA4 blockade against mouse A20 B cell lymphoma. In addition, locally delivered CpG-Stat3 siRNA combined with systemic administration of PD-1 antibody significantly augmented both local and systemic antitumor effects against mouse B16 melanoma tumors, with enhanced tumor-associated T cell activation. Furthermore, locally delivered CpG-Stat3 siRNA enhanced CD8+ T cell tumor infiltration and antitumor activity in a xenograft tumor model. Overall, our studies in both B cell lymphoma and melanoma mouse models demonstrate the potential of combinatory immunotherapy with CpG-Stat3 siRNA and checkpoint inhibitors as a therapeutic strategy for B cell lymphoma and melanoma.

Abstract Introduction Tumor‐associated macrophages, a major component of the tumor microenvironment, undergo polarization into M2 macrophages (M2), and thereby exert an immunosuppressive effect to induce cancer metastasis. This study strives to uncover a molecular mechanism underlying this event in hepatocellular carcinoma (HCC). Methods Proteasome subunit alpha 5 (PSMA5) expression in liver hepatocellular carcinoma (LIHC) tissues and its association with LIHC patients were predicted using StarBase. PSMA5 level in human HCC cells was manipulated via transfection. Exosomes were isolated from HCC cells, and internalized into macrophages which were cocultured with HCC cells. Exosome internalization was observed after fluorescence labeling. HCC cell migration and invasion were evaluated by wound healing and Transwell assays. Xenograft assay was performed to investigate the role of PSMA5 in in vitro tumorigenesis. M2 polarization was assessed by enzyme‐linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. PSMA5 expression in exosomes and Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) activation in macrophages and tumors were detected by Western blot analysis. Results High PSMA5 expression was observed in LIHC tissues and associated with compromised survival of LIHC patients. PSMA5 knockdown inhibited HCC cell migration and invasion. PSMA5 knockdown in HCC cells downregulated PSMA5 level in exosomes from these HCC cells. HCC cell‐isolated exosomes were successfully internalized into macrophages, and facilitated M2 polarization and JAK2/STAT3 pathway activation. HCC cell‐secreted exosomal PSMA5 knockdown inhibited the exosome‐induced effect on macrophages, and attenuated the promotion induced by exosome‐treated macrophages on HCC cell migration/invasion and tumorigenesis along with in vivo M2 polarization and JAK2/STAT3 pathway activation. Conclusion HCC cell‐secreted exosomal PSMA5 knockdown hinders M2 polarization to suppress cancer progression by restraining JAK2/STAT3 signaling.

Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

Targeting programmed cell death protein ligand 1 (PD-L1) remains one of the most essential immunotherapies in cancer1,2. PD-L1 has been detected in the nucleus in multiple malignancies, playing an oncogenic role independent of immune checkpoint regulation3-5. Howbeit, the regulatory function of nuclear PD-L1 (nPD-L1) remains to be fully understood. Here, we report that nPD-L1 is an endogenous accelerator for cancer angiogenesis. First, we found that an abundant proportion of PD-L1 was distributed within the nucleus of uveal melanoma samples, which is associated with an unfavorable outcome. Moreover, the capacity of promoting angiogenesis was largely attenuated in the nPD-L1-deficient cells both in vivo and in vitro. Mechanistically, nPD-L1 facilitates p-STAT3 binding to the promoter of early growth response-1 (EGR1), resulting in the activation of EGR1-mediated angiogenesis. Therapeutically, the inhibition of histone deacetylase 2 restores the normal acetylation level of PD-L1, blocking its nuclear translocation and thereby attenuating tumor angiogenesis. Conclusively, we reveal that nPD-L1 promotes angiogenesis in malignancies, and provide a novel anti-vascularization strategy through blocking aberrant PD-L1 nuclear translocation for tumor therapy.

Figure S1. Increased TLR9 expression in induced glioma spheres. Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs. Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth. Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs. Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site.

Supplementary Figure 1 - PDF file 231K, Inhibition of STAT3 activation in CHLA-255 cells by anti-IL-6R blocking antibody. A, Expression of pSTAT3 and STAT3 in CHLA-255 cells was examined by Western blot analysis as in Fig. 1A. When indicated, cells were treated with tocilizumab (2g/mL) for 12 hours before treatment with IL-6 with or without sIL-6R. The data are representative of 2 separate experiments showing similar results. B, Nuclear extracts from CHLA-255 treated with IL-6 and sIL-6R in the presence and absence of tocilizumab as above described were examined for STAT3 DNA-binding activity by EMSA as indicated in Materials and Methods. Lane 8 is same as lane 5, except for the addition of a supershifting anti-STAT3 antibody also added to lane 2 (DU145 control). Top band (labeled STAT3) represents STAT3/STAT3 homodimers bound to DNA. C, CHLA-255 transfected with a STAT3 Firefly luciferase vector and a Renilla luciferase control vector were treated with IL-6 (10ng/mL) alone or in combination with sIL-6R (25ng/mL) as shown in Fig. 1 in the absence or presence of tocilizumab (4g/mL). After 24 hours, the activity of the STAT3 reporter was examined by Dual Luciferase assay as indicated in Materials and Methods. The data represent the mean (SD) ratio Firefly/Renilla luciferase activity from five samples for each experimental condition

Supplementary Figure 6 from Toll-like Receptor 9 Activation of Signal Transducer and Activator of Transcription 3 Constrains Its Agonist-Based Immunotherapy

Suppl. Fig. S3 showing CTLA4 expression by tumor associated CD19 B cells

Suppl. Fig. S1 showing CTLA4 expression and CD86 cellular internalization by human BCL and human MM cells acquired by flow cytometry.

Supplementary Methods and Data- Changes are unmarked