技术资料

High-grade serous ovarian cancer remains a poorly understood disease with a high mortality rate. Although most patients respond to cytotoxic therapies,a majority will experience recurrence. This may be due to a minority of drug-resistant cancer stem-like cells (CSC) that survive chemotherapy and are capable of repopulating heterogeneous tumors. It remains unclear how CSCs are supported in the tumor microenvironment (TME) particularly during chemotherapy exposure. Tumor-associated macrophages (TAM) make up half of the immune population of the ovarian TME and are known to support CSCs and contribute to cancer progression. TAMs are plastic cells that alter their phenotype in response to environmental stimuli and thus may influence CSC maintenance during chemotherapy. Given the plasticity of TAMs,we studied the effects of carboplatin on macrophage phenotypes using both THP1- and peripheral blood mononuclear cell (PBMC)–derived macrophages and whether this supports CSCs and ovarian cancer progression following treatment. We found that carboplatin exposure induces an M1-like proinflammatory phenotype that promotes SOX2 expression,spheroid formation,and CD117 + ovarian CSCs,and that macrophage-secreted CCL2/MCP-1 is at least partially responsible for this effect. Depletion of TAMs during carboplatin exposure results in fewer CSCs and prolonged survival in a xenograft model of ovarian cancer. This study supports a role for platinum-based chemotherapies in promoting a transient proinflammatory M1-like TAM that enriches for CSCs during treatment. Improving our understanding of TME responses to cytotoxic drugs and identifying novel mechanisms of CSC maintenance will enable the development of better therapeutic strategies for high-grade serous ovarian cancer. Significance: We show that chemotherapy enhances proinflammatory macrophage phenotypes that correlate with ovarian cancer progression. Given that macrophages are the most prominent immune cell within these tumors,this work provides the foundation for future translational studies targeting specific macrophage populations during chemotherapy,a promising approach to prevent relapse in ovarian cancer. View Publication

Autosomal recessive osteopetrosis (ARO) is a rare genetic disease,characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation,leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT),but its success is limited by several constraints. Conversely,gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. The Tcirg1 -defective oc/oc mouse model displays a short lifespan and high bone density,closely mirroring the human condition. In this work,we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. First,we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype,allowing long-term survival and reducing extramedullary haematopoiesis. Then,we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood,facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally,pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs,albeit at lower level than conventional total body irradiation,and led to long-term survival and correction of bone phenotype,in the absence of acute toxicity. These results will pave the way to the implementation of an effective GT protocol,reducing the transplant-related complication risks in the very young and severely affected ARO patients. View Publication

Epithelial-to-mesenchymal transition (EMT),cancer stem cells (CSCs),and colorectal cancer (CRC) therapy resistance are closely associated. Prior reports have demonstrated that sphingosine-1-phosphate (S1P) supports stem cells and maintains the CSC phenotype. We hypothesized that the EMT inducer SNAI1 drives S1P signaling to amplify CSC self-renewal capacity and chemoresistance. CRC cell lines with or without ectopic expression of SNAI1 were used to study the role of S1P signaling as mediators of cancer stemness and 5-fluorouracil (5FU) chemoresistance. The therapeutic ability of sphingosine kinase 2 (SPHK2) was assessed using siRNA and ABC294640,a SPHK2 inhibitor. CSCs were isolated from patient-derived xenografts (PDXs) and assessed for SPHK2 and SNAI1 expression. Ectopic SNAI1 expressing cell lines demonstrated elevated SPHK2 expression and increased SPHK2 promoter activity. SPHK2 inhibition with siRNA or ABC294640 ablated in vitro self-renewal and sensitized cells to 5FU. CSCs isolated from CRC PDXs express increased SPHK2 relative to the non-CSC population. Combination ABC294640/5FU therapy significantly inhibited tumor growth in mice and enhanced 5FU response in therapy-resistant CRC patient-derived tumor organoids (PDTOs). SNAI1/SPHK2 signaling mediates cancer stemness and 5FU resistance,implicating S1P as a therapeutic target for CRC. The S1P inhibitor ABC294640 holds potential as a therapeutic agent to target CSCs in therapy refractory CRC. View Publication

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance,they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors,which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT),a well-known checkpoint receptor involved in T-cell exhaustion,has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here,we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability,are rapidly and efficiently taken up by cells,and can be easily incorporated into cell product manufacturing. INTASYL PH-804,which targets TIGIT,suppresses TIGIT mRNA and protein expression in NK cells,resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition,thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy,particularly for hematological malignancies. The online version contains supplementary material available at 10.1007/s00262-024-03835-x. View Publication

Extracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising,natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture,human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However,hiPSC-EVs only have a moderate ability for brain delivery. Herein,we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor. An RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging. Here we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype,express pluripotency markers,and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways,compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency. Taken together,we have established stable,genetically engineered hiPSCs for producing EVs with RVG expression,offering the improved ability for brain-targeted drug delivery. The online version contains supplementary material available at 10.1186/s13287-024-03955-2. View Publication

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives,particularly extracellular vesicles (EVs),for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs,alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction,indicating a potential mechanism for their immunomodulatory effects. Furthermore,iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally,iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably,priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics,given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3. View Publication

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore,genetic engineering of the above-mentioned primary human cells has several safety needs,including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work,we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB,we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4 + T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34 + cells. A fraction of these cells is viable for up to 3 months. Next,we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4 + or CD34 + primary cells,and their growth was followed. By this approach,we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment,whereas tumor cells,after an adaptation phase,expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells. View Publication

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however,CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs,leading to disease relapse. To prevent this,new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34 + CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore,the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C 33 H 61 NO 5 S) is a structurally simplified analog of JCI-20679,the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here,we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34 + CML cells isolated from the bone marrow of untreated CML patients. Taken together,these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs,and may improve the treatment of CML. View Publication

Cancer cell plasticity contributes significantly to the failure of chemo- and targeted therapies in triple-negative breast cancer (TNBC). Molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance are largely unknown. Using a genome-wide CRISPR-Cas9 screen,we investigated escape mechanisms of NOTCH-driven TNBC treated with a gamma-secretase inhibitor (GSI) and identified SOX2 as a target of resistance to Notch inhibition. We describe a novel reciprocal inhibitory feedback mechanism between Notch signaling and SOX2. Specifically,Notch signaling inhibits SOX2 expression through its target genes of the HEY family,and SOX2 inhibits Notch signaling through direct interaction with RBPJ. This mechanism shapes divergent cell states with NOTCH positive TNBC being more epithelial-like,while SOX2 expression correlates with epithelial-mesenchymal transition,induces cancer stem cell features and GSI resistance. To counteract monotherapy-induced tumor relapse,we assessed GSI-paclitaxel and dasatinib-paclitaxel combination treatments in NOTCH inhibitor-sensitive and -resistant TNBC xenotransplants,respectively. These distinct preventive combinations and second-line treatment option dependent on NOTCH1 and SOX2 expression in TNBC are able to induce tumor growth control and reduce metastatic burden. View Publication

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Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here,we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks,enhancing the reconstitution capacity of edited HSPCs. However,this results in lower HDR efficiency,rendering ex vivo culture necessary yet detrimental. Mechanistically,ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation,which generates mitogenic reactive oxygen species (ROS),promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus,p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs,ultimately endowing edited cells with superior multi-lineage differentiation,persistence throughout serial transplantation,enhanced polyclonal repertoire,and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. View Publication

Pericytes play a crucial role in controlling inflammation and vascular functions in the central nervous system,which are disrupted in Parkinson’s disease (PD). Still,there is a lack of studies on the impact of pericytes on neurodegenerative diseases,and their involvement in the pathology of PD is unclear. Our objective was to investigate the molecular and functional differences between healthy pericytes and pericytes with the LRRK2 G2019S mutation,which is one of the most common mutations associated with PD. Our study employed pericyte-like cells obtained from induced pluripotent stem cells produced from PD patients with the LRRK2 G2019S mutation as well as from healthy individuals. We examined the gene expression profiles of the cells and analyzed how the alterations reflect on their functionality. We have shown differences in the expression of genes related to inflammation and angiogenesis. Furthermore,we observe modified migration speed in PD pericyte-like cells as well as enhanced secretion of inflammatory mediators,such as soluble VCAM-1 and MCP-1,in these pericyte-like cells following exposure to proinflammatory stimuli. In summary,our findings support the notion that pericytes play a role in the inflammatory and vascular changes observed in PD. Further investigation of pericytes could provide valuable insight into understanding the pathogenesis of PD. The online version contains supplementary material available at 10.1186/s12987-024-00592-y. View Publication