技术资料

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion,cell adhesion to bone,and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are,therefore,used in the treatment of patients with osteolytic metastases. However,an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al.,Cancer Res.,57: 3890-3894,1997). Here,we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate textgreater ibandronate textgreater NE-10244 (active pyridinium analogue of risedronate) textgreater clodronate. In addition,NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect,whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244,indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells,nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However,although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells,they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition,NE-10790 did not inhibit MMP activity,suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion,our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest,therefore,that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone. View Publication

Characterization and modeling of biological neural networks has emerged as a field driving significant advancements in our understanding of brain function and related pathologies. As of today,pharmacological treatments for neurological disorders remain limited,pushing the exploration of promising alternative approaches such as electroceutics. Recent research in bioelectronics and neuromorphic engineering have fostered the development of the new generation of neuroprostheses for brain repair. However,achieving their full potential necessitates a deeper understanding of biohybrid interaction. In this study,we present a novel real-time,biomimetic,cost-effective and user-friendly neural network capable of real-time emulation for biohybrid experiments. Our system facilitates the investigation and replication of biophysically detailed neural network dynamics while prioritizing cost-efficiency,flexibility and ease of use. We showcase the feasibility of conducting biohybrid experiments using standard biophysical interfaces and a variety of biological cells as well as real-time emulation of diverse network configurations. We envision our system as a crucial step towards the development of neuromorphic-based neuroprostheses for bioelectrical therapeutics,enabling seamless communication with biological networks on a comparable timescale. Its embedded real-time functionality enhances practicality and accessibility,amplifying its potential for real-world applications in biohybrid experiments. Beaubois et al. introduce a real-time biomimetic neural network for biohybrid experiments,providing a tool to study closed-loop applications for neuroscience and neuromorphic-based neuroprostheses. View Publication

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125),MUC4,and KRT24 . This cell state is enriched in metastases,predicted to be highly resistant to chemotherapy,and linked with poor survival. We also find enriched expression of TM4SF1,a transmembrane protein,in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner,highlighting its potential as a therapeutic target. Subject terms: Bladder cancer,Tumour biomarkers,Targeted therapies View Publication

Transforming growth factor beta (TGF-$$) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study,we have utilized a novel murine cell line NMuMG-ST,which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG,to investigate the role of autocrine TGF-$$ signaling in regulating their survival,metastatic ability,and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-$$ type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-$$ signaling. The expression of DNRII reduced TGF-$$ sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-$$ signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK,and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly,the abrogation of autocrine TGF-$$ signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition,mammosphere formation,and expression of stem cell markers. When xenografted in athymic nude mice,the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus,our results indicate that autocrine TGF-$$ signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells. View Publication

Cripto is a developmental oncoprotein that signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK),phosphatidylinositol 3-kinase (PI3K)/Akt and Smad2/3 pathways. However,the molecular basis for Cripto coupling to these pathways during embryogenesis and tumorigenesis is not fully understood. In this regard,we recently demonstrated that Cripto forms a cell surface complex with the HSP70 family member glucose-regulated protein-78 (GRP78). Here,we provide novel functional evidence demonstrating that cell surface GRP78 is a necessary mediator of Cripto signaling in human tumor,mammary epithelial and embryonic stem cells. We show that targeted disruption of the cell surface Cripto/GRP78 complex using shRNAs or GRP78 immunoneutralization precludes Cripto activation of MAPK/PI3K pathways and modulation of activin-A,activin-B,Nodal and transforming growth factor-beta1 signaling. We further demonstrate that blockade of Cripto binding to cell surface GRP78 prevents Cripto from increasing cellular proliferation,downregulating E-Cadherin,decreasing cell adhesion and promoting pro-proliferative responses to activin-A and Nodal. Thus,disrupting the Cripto/GRP78 binding interface blocks oncogenic Cripto signaling and may have important therapeutic value in the treatment of cancer. View Publication

In the context of pancreatic cancer,metastasis remains the most critical determinant of resectability,and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines,Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin,consistent with inhibition of epithelial-to-mesenchymal transition,and was mirrored by a striking reduction of in vitro invasive capacity (P textless 0.0001). Conversely,Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P textless 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model,cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of primary" tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced "ALDH-high" cells by approximately 3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites� View Publication

Strategies to induce p53 activation in wtp53-retaining tumors carry high potential in cancer therapy. Nutlin,a potent highly selective MDM2 inhibitor,induces non-genotoxic p53 activation. Although Nutlin shows promise in promoting cell death in hematopoietic malignancies,a major roadblock is that most solid cancers do not undergo apoptosis but merely reversible growth arrest. p53 inhibition by unopposed MDMX is one major cause for apoptosis resistance to Nutlin. The Hsp90 chaperone is ubiquitously activated in cancer cells and supports oncogenic survival pathways,many of which antagonize p53. The Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) is known to induce p53-dependent apoptosis. We show here that in multiple difficult-to-kill solid tumor cells 17AAG modulates several critical components that synergize with Nutlin-activated p53 signaling to convert Nutlin's transient cytostatic response into a cytotoxic killing response in vitro and in xenografts. Combined with Nutlin,17AAG destabilizes MDMX,reduces MDM2,induces PUMA and inhibits oncogenic survival pathways,such as PI3K/AKT,which counteract p53 signaling at multiple levels. Mechanistically,17AAG interferes with the repressive MDMX-p53 axis by inducing robust MDMX degradation,thereby markedly increasing p53 transcription compared with Nutlin alone. To our knowledge Nutlin+17AAG represents the first effective pharmacologic knockdown of MDMX. Our study identifies 17AAG as a promising synthetic lethal partner for a more efficient Nutlin-based therapy. View Publication

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines,but normal in vivo proliferation,increased accumulation of regulatory T cells,and preserved anticancer effects. Here,we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells,but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models,we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD,with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD,but led to treatment-limiting toxicity. In contrast,Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell-mediated human disorders. View Publication

Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study,we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a 20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures,glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions. View Publication

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells,which binds to CD47,a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells,therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer,several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However,the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited,suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end,DS-1103a,a newly developed anti-human SIRPα antibody (Ab),was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd),DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2,respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model,vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab,implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore,in syngeneic mouse models,both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together,this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs. View Publication

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients,we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence,we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition,we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays,CD69,CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155,one important TIGIT-ligand,is reliably expressed on AMLs,we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally,our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype,whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively,our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-024-03766-7. View Publication

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress,which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1? (IRE1?) is activated to splice X-box binding protein 1 (XBP1) mRNA,thereby increasing XBP1s protein,which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study,we examined whether IRE1?-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1? endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines,without toxicity in normal mononuclear cells. Importantly,it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG,even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress,evidenced by induction of XBP1s,which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946,associated with increased CHOP. Finally,MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo,associated with significant growth inhibition of MM cells. Taken together,our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1? endoribonuclease domain is a potential therapeutic opt View Publication