Target Cells Research Articles

Map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.

Phthalate monoesters affect membrane fluidity and cell-cell contacts in endometrial stromal adherent cell lines and spheroids

Phthalate monoesters have been identified as endocrine disruptors in a variety of models, yet understanding of their exact mechanisms of action and molecular targets in cells remains incomplete. Here, we set to determine whether epidemiologically relevant mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) can affect biological processes by altering cell plasma membrane fluidity or formation of cell-cell contacts. As a model system, we chose endometrial stromal cell lines, one of which was previously used in a transcriptomic study with MEHHP or MEHHP-containing mixtures. A short-term exposure (1 h) of membrane preparations to endocrine disruptors was sufficient to induce changes in membrane fluidity/rigidity, whereas different mixtures showed different effects at various depths of the bilayer. A longer exposure (96 h) affected the ability of cells to form spheroids and highlighted issues with membrane integrity in loosely assembled spheroids. Finally, in spheroids assembled from T-HESC cells, MEHHP interfered with the formation of cell-cell contacts as indicated by the immunostaining of zonula occludens 1 protein. Overall, this study emphasized the need to consider plasma membrane, membrane-bound organelles, and secretory vesicles as possible biological targets of endocrine disruptors and offered an explanation for a multitude of endocrine disruptor roles documented earlier.

Insights into the binding recognition and computational design of IL-2 muteins with enhanced predicted binding affinity to the IL-2 receptor α

Interleukin-2 (IL-2) is an immune system regulator that has received approval for cancer treatment. However, high-dose IL-2 therapy has seen restricted use due to its low efficacy and on-target toxicity. To enhance the effectiveness of IL-2 therapy, it is essential to engineer IL-2 molecules to enhance their specificity toward target cell populations. In this study, molecular dynamics (MD) simulations and Rosetta software were utilized to design novel high-affinity IL-2Rα-binding IL-2 muteins. MD simulations were used to identify the target residues of IL-2 for design, and Rosetta software were then employed to predict potential IL-2 muteins with higher binding affinity toward IL-2Rα. Rosetta generated two potential designed IL-2 muteins. The results of the MD validation and MM/GBSA analysis indicated that both designed IL-2 muteins exhibited greater predicted binding affinities toward IL-2Rα than that of the native proteins. RMSF analysis demonstrated that the structural fluctuations of free IL-2 and designed muteins were similar, indicating that the mutations did not alter the intramolecular force responsible for IL-2's stability and folding. These designed IL-2 muteins may have potential benefits for cancer immunotherapy.

Stage-specific modulation of multinucleation, fusion, and resorption by the long non-coding RNA DLEU1 and miR-16 in human primary osteoclasts

Osteoclasts are the only cells able to resorb all the constituents of the bone matrix. While the modulation of osteoclast activity is well established for preventing bone-related diseases, there is an increasing demand for novel classes of anti-resorption agents. Herein, we investigated non-coding RNA molecules and proposed DLEU1 and miR-16 as potential candidates for modulating osteoclast functions. DLEU1 and miR-16 target cell fusion at both the early and late stages of osteoclastogenesis but operate through independent pathways. DLEU1 silencing hinders the fusion process, leading to abrogation of the phagocytic cup fusion modality and a reduction in the fusion events between mononucleated precursors and multinucleated osteoclasts, while miR-16 influences monocyte-to-osteoclast differentiation, impairing osteoclasts formation but not the number of nuclei at early stages. On the other hand, using these non-coding RNAs to engineer mature osteoclasts has implications for bone resorption. Both DLEU1 and miR-16 influence the speed of resorption in pit-forming osteoclasts, without affecting the resorbed area. However, the impact of increasing miR-16 levels extends more broadly, affecting trench-forming osteoclasts as well, leading to a reduction in their percentage, speed, and resorbed area. These findings offer potential new therapeutic targets to ameliorate bone destruction in skeletal diseases.

Disulfide stoppered polyrotaxanes with enhanced cellular uptake and intracellular cyclodextrin release

Background and AimPolyrotaxanes are molecular necklaces composed of polymers, threaded macrocycles, and bulky stopper molecules to inhibit the decomposition of the first two. These supramolecular assemblies are promising active ingredients for treating lysosomal storage disorders. This study aimed to synthesize such polyrotaxanes with a novel, simple method, resulting in high threading efficacy, enhanced cellular uptake, and intracellular cyclodextrin (CD) release. MethodsIn this study, we developed two novel poly(ethylene glycol) (PEG) based polyrotaxanes, with threaded α-cyclodextrin (α-CD) or its mixture with 2-hydroxypropyl-α-CD (HP-α-CD) and glutathione sensitive disulfide connected stopper molecules. The structure and composition of these polyrotaxanes were determined by 1H NMR spectroscopy and gel permeation chromatography, while the cellular uptake was investigated by flow cytometry and confocal microscopy. ResultsHigh threading efficacy, as well as molar mass of 17.9 and 13.1 kDa, was found for the polymeric supramolecules with threaded α-CD and α-CD/HP-α-CD, respectively. Glutathion-triggered reductive removal of the stopper molecules showed potential decomposition of these polyrotaxanes in target cells. Flow cytometry revealed an up to 52-fold enhancement in cellular uptake of α- and HP-α-CD by the polyrotaxanes compared to free CD, which was also visualized by confocal microscopy. Conclusion and scope of applicationPolyrotaxanes based on α-CD and its derivative were tested in vitro for application in the treatment of lysosomal storage disease for the first time. Based on these results, polyrotaxanes with disulfide stopper molecules might be promising supramolecular excipients for cellular delivery of α-CDs.

Preventative Cancer Vaccine-Elicited Human Anti-MUC1 Antibodies Have Multiple Effector Functions.

Mucin-1 (MUC1) is a transmembrane glycoprotein that is overexpressed and hypoglycosylated in premalignant and malignant epithelial cells compared to normal cells, creating a target antigen for humoral and cellular immunity. Healthy individuals with a history of advanced colonic adenomas and at high risk for colon cancer were enrolled in a clinical trial to evaluate the feasibility of using a MUC1 peptide vaccine to prevent colon cancer. Anti-MUC1 antibodies elicited by this vaccine were cloned using peripheral blood B cells and sera collected two weeks after a one-year booster. Twelve of these fully human monoclonal antibodies (mAb) were tested for binding to MUC1+ target cells, and three with the highest binding were further evaluated for various effector functions important for tumor rejection. Immune cells were incubated together with target cells expressing variations in the number, distance, and membrane anchoring properties of the MUC1 epitope in the presence of each mAb. All three mAbs mediated antibody-dependent cytokine release (ADCR), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Two also mediated antibody-dependent trogocytosis/trogoptosis (ADCT). None were capable of complement-dependent cytotoxicity (CDC). ADCP and ADCT functions were more efficient when antibodies bound epitopes proximal to and anchored to the membrane, providing insight for future therapeutic antibody validation strategies.

Intrinsic Synergy and Selectivity for the Inhibition of Cancer Cell Growth Generated by a Polymer Ligand of Proximal Enzymes.

A fundamental understanding of the design of polymer ligands of proximal enzymes is essential for the precise targeting of cancer cells, but it is still in its infancy. In this study, we systematically investigated the contribution of the chain length, ligand density, and ligand ratio of proximal enzyme-targeted polymers to the efficacy, synergy, and selectivity for the inhibition of cancer cell proliferation. The results revealed that employing a moderate chain length as a scaffold allowed for an intrinsically high efficacy and synergy of proximal enzyme-targeted polymers, in contrast to single enzyme-targeted polymers that prefer longer chain length for efficacy. The synergy obtained in proximal enzyme targeting was not provided by the combination of the corresponding small molecules. Moreover, the maturation of the synergistic efficacy of the proximal enzyme-targeted polymers also improved selectivity. This study proposes a rational design for polymer inhibitors and/or ligands for cancer cells with a high efficacy and selectivity.

High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells

RNAs and their encoded proteins intricately regulate diverse cell types and states within the human body. Dysregulated RNA expressions or mutations can lead to various diseased cell states, including tumorigenesis. Detecting and manipulating these endogenous RNAs offers significant promise for restoring healthy cell states and targeting tumors both in research and clinical contexts. This study presents an RNA-IN and RNA-OUT genetic circuit capable dynamically sensing and manipulating any RNA target in a programmable manner. The RNA-IN module employes a programmable CRISPR-associated protease (CASP) complex for RNA detection, while the RNA-OUT module utilizes an engineered protease-responsive dCas9-VPR activator. Additionally, the CASP module can detect point mutations by harnessing an uncovered dual-nucleotide synergistic switching effect within the CASP complex, resulting in the amplification of point-mutation signals from initially undetectable levels (1.5-fold) to a remarkable 94-fold. We successfully showcase the circuit’s ability to rewire endogenous RNA-IN signals to activate endogenous progesterone biosynthesis pathway, dynamically monitor adipogenic differentiation of mesenchymal stem cells (MSCs) and the epithelial-to-mesenchmal trans-differentiation, as well as selective killing of tumor cells. The programmable RNA-IN and RNA-OUT circuit exhibits tremendous potential for applications in gene therapy, biosensing and design of synthetic regulatory networks.

Microfluidic post-insertion of polyethylene glycol lipids and KK or RGD high functionality and quality lipids in milk-derived extracellular vesicles

To achieve the desired delivery effect, extracellular vesicles (EVs) must bypass rapid clearance from circulation and exhibit affinity for target cells; however, it is difficult to simultaneously incorporate two materials into EVs. Post-insertion is a general modification method that can be performed by simply mixing different solutions. Previously, we have developed a microfluidic post-insertion method that supported fast and upscaled modification of EVs using KK-modified high-functionality and -quality (HFQ) lipids. Here, we used microfluidic post-insertion to achieve simultaneous incorporation of polyethylene glycol (PEG) lipids and KK or RGD-modified HFQ lipids into milk-derived EVs to avoid uptake from the reticuloendothelial system and increase the uptake into target cells. PEG lipid and HFQ lipids were formulated to produce micelles and subsequently mixed with EV solution using a microfluidic device. Compared to bulk mixing, microfluidic post-insertion showed higher cellular association. Altered cellular association capacities and endocytic pathways indicated simultaneous incorporation. The cellular association of modified EVs can be adjusted by altering the ratio of (EK)4-KK in micelles with slight changes in physicochemical properties. Furthermore, microfluidic post-insertion is also suitable for (SG)5-RGD, which is insoluble in phosphate-buffered saline (PBS). Our results may be valuable for the development and manufacture of functional EVs as drug delivery systems for clinical applications.

Mind the GAPS: Glia associated with psychological stress.

Glial cells are an integral component of the nervous system, performing crucial functions that extend beyond structural support, including modulation of the immune system, tissue repair, and maintaining tissue homeostasis. Recent studies have highlighted the importance of glial cells as key mediators of stress responses across different organs. This review focuses on the roles of glial cells in peripheral tissues in health and their involvement in diseases linked to psychological stress. Populations of glia associated with psychological stress ("GAPS") emerge as a promising target cell population in our basic understanding of stress-associated pathologies, highlighting their role as mediators of the deleterious effects of psychological stress on various health conditions. Ultimately, new insights into the impact of stress on glial cell populations in the periphery may support clinical efforts aimed at improving the psychological state of patients for improved health outcomes.

Application of novel CAR technologies to improve treatment of autoimmune disease.

Chimeric antigen receptor (CAR) T cell therapy has become an important treatment for hematological cancers, and its success has spurred research into CAR T cell therapies for other diseases, including solid tumor cancers and autoimmune diseases. Notably, the development of CAR-based treatments for autoimmune diseases has shown great progress recently. Clinical trials for anti-CD19 and anti-BCMA CAR T cells in treating severe B cell-mediated autoimmune diseases, like systemic lupus erythematosus (SLE), have shown lasting remission thus far. CAR T cells targeting autoreactive T cells are beginning clinical trials for treating T cell mediated autoimmune diseases. Chimeric autoantigen receptor (CAAR) T cells specifically target and eliminate only autoreactive B cells, and they have shown promise in treating mucosal pemphigus vulgaris and MuSK myasthenia gravis. Regulatory CAR T cells have also been developed, which show potential in altering autoimmune affected areas by creating a protective barrier as well as helping decrease inflammation. These new treatments are only the beginning of potential CAR T cell applications in treating autoimmune disease. Novel CAR technologies have been developed that increase the safety, potency, specificity, and efficacy of CAR T cell therapy. Applying these novel modifications to autoimmune CARs has the potential to enhance the efficacy and applicability of CAR therapies to autoimmune disease. This review will detail several recently developed CAR technologies and discuss how their application to autoimmune disease will improve this emerging field. These include logic-gated CARs, soluble protein-secreting CARs, and modular CARs that enable CAR T cell therapies to be more specific, reach a wider span of target cells, be safer for patients, and give a more potent cytotoxic response. Applying these novel CAR technologies to the treatment of autoimmune diseases has the potential to revolutionize this growing application of CAR T cell therapies.

Cutting-edge approaches to B-cell depletion in autoimmune diseases.

B-cell depletion therapy (BCDT) has been employed to treat autoimmune disease for ~20 years. Immunoglobulin G1 (IgG1) monoclonal antibodies targeting CD20 and utilizing effector function (eg, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis) to eliminate B cells have historically been the predominant therapeutic approaches. More recently, diverse BCDT approaches targeting a variety of B-cell surface antigens have been developed for use in hematologic malignancies, including effector-function-enhanced monoclonal antibodies, chimeric antigen receptor T-cell (CAR-T) treatment, and bispecific T-cell engagers (TCEs). The latter category of antibodies employs CD3 engagement to augment the killing of target cells. Given the improvement in B-cell depletion observed with CAR-T and TCEs compared with conventional monospecific antibodies for treatment of hematologic malignancies and the recent case reports demonstrating therapeutic benefit of CAR-T in autoimmune disease, there is potential for these mechanisms to be effective for B-cell-mediated autoimmune disease. In this review, we discuss the various BCDTs that are being developed in autoimmune diseases, describing the molecule designs, depletion mechanisms, and potential advantages and disadvantages of each approach as they pertain to safety, efficacy, and patient experience. Additionally, recent advances and strategies with TCEs are presented to help broaden understanding of the potential for bispecific antibodies to safely and effectively engage T cells for deep B-cell depletion in autoimmune diseases.

Exploring miRNA therapies and gut microbiome-enhanced CAR-T cells: advancing frontiers in glioblastoma stem cell targeting.

Glioblastoma multiforme (GBM) presents a formidable challenge in oncology due to its aggressive nature and resistance to conventional treatments. Recent advancements propose a novel therapeutic strategy combining microRNA-based therapies, chimeric antigen receptor-T (CAR-T) cells, and gut microbiome modulation to target GBM stem cells and transform cancer treatment. MicroRNA therapies show promise in regulating key signalling pathways implicated in GBM progression, offering the potential to disrupt GBM stem cell renewal. CAR-T cell therapy, initially successful in blood cancers, is being adapted to target GBM by genetically engineering T cells to recognise and eliminate GBM stem cell-specific antigens. Despite early successes, challenges like the immunosuppressive tumour microenvironment persist. Additionally, recent research has uncovered a link between the gut microbiome and GBM, suggesting that gut dysbiosis can influence systemic inflammation and immune responses. Novel strategies to modulate the gut microbiome are emerging, enhancing the efficacy of microRNA therapies and CAR-T cell treatments. This combined approach highlights the synergistic potential of these innovative therapies in GBM treatment, aiming to eradicate primary tumours and prevent recurrence, thereby improving patient prognosis and quality of life. Ongoing research and clinical trials are crucial to fully exploit this promising frontier in GBM therapy, offering hope to patients grappling with this devastating disease.

T-cell engagers: model interrogation as a tool to quantify the interplay of relative affinity and target expression on trimer formation.

T-cell engagers (TCEs) represent a promising therapeutic strategy for various cancers and autoimmune disorders. These bispecific antibodies act as bridges, connecting T-cell receptors (TCRs) to target cells (either malignant or autoreactive) via interactions with specific tumour-associated antigens (TAAs) or autoantigens to form trimeric synapses, or trimers, that co-localise T-cells with target cells and stimulate their cytotoxic function. Bispecific TCEs are expected to exhibit a bell-shaped dose-response curve, with a defined optimal TCE exposure for maximizing trimer formation. The shape of the dose-response is determined by a non-trivial interplay of binding affinities, exposure and antigens expression levels. Furthermore, excessively low binding to the TCR may reduce efficacy, but mitigate risk of over-stimulating cytokine secretion or induce effector cell exhaustion. These inevitable trade-off highlights the importance of quantitatively understanding the relationship between TCE concentration, target expression, binding affinities, and trimer formation. We utilized a mechanistic target engagement model to show that, if the TCE design parameters are close to the recommended ranges found in the literature, relative affinities for TCR, TAA and target expression levels have qualitatively different, but predictable, effects on the resulting dose-response curve: higher expression levels shift the curve upwards, higher antigen affinity shifts the curve to the left, and higher TCR affinity shifts the curve upwards and to the left.

Development of folate receptor targeting chimeras for cancer selective degradation of extracellular proteins.

Targeted protein degradation has emerged as a novel therapeutic modality to treat human diseases by utilizing the cell's own disposal systems to remove protein target. Significant clinical benefits have been observed for degrading many intracellular proteins. Recently, the degradation of extracellular proteins in the lysosome has been developed. However, there have been limited successes in selectively degrading protein targets in disease-relevant cells or tissues, which would greatly enhance the development of precision medicine. Additionally, most degraders are not readily available due to their complexity. We report a class of easily accessible Folate Receptor TArgeting Chimeras (FRTACs) to recruit the folate receptor, primarily expressed on malignant cells, to degrade extracellular soluble and membrane cancer-related proteins in vitro and in vivo. Our results indicate that FRTAC is a general platform for developing more precise and effective chemical probes and therapeutics for the study and treatment of cancers.

Inhibitory Effects on RNA Binding and RNase H Induction Activity of Prodrug-Type Oligodeoxynucleotides Modified with a Galactosylated Self-Immolative Linker Cleavable by β-Galactosidase.

Prodrug-type oligonucleotides (prodrug-ONs) are a class of oligonucleotide designed for activation under specific intracellular conditions or external stimuli. Prodrug-ONs can be activated in the target tissues or cells, thereby reducing the risk of adverse effects. In this study, we synthesized prodrug-type oligodeoxynucleotides activated by β-galactosidase, an enzyme that is overexpressed in cancer and senescent cells. These oligodeoxynucleotides (ODNs) contain a modified thymidine conjugated with galactose via a self-immolative linker at the O4-position. UV-melting analysis revealed that the modifications decreased the melting temperature (Tm) compared with that of the unmodified ODN when hybridized with complementary RNA. Furthermore, cleavage of the glycosidic bond by β-galactosidase resulted in the spontaneous removal of the linker from the nucleobase moiety, generating unmodified ODNs. Additionally, the introduction of multiple modified thymidines into ODNs completely inhibited the RNase H-mediated cleavage of complementary RNA. These findings suggest the possibility of developing prodrug-ONs, which are specifically activated in cancer cells or senescent cells with high β-galactosidase expression.

Gene Therapy for Hearing Loss: A Review

Background: Hearing impairment is the most prevalent sensory deficiency among humans. Scientists have been actively studying the genetic factors behind human hearing loss in recent years. A considerable proportion of hearing impairment cases can be due to genetic aetiologies. Numerous specific genes have been linked to inherited hearing impairments. At present, scientists globally are engaged in genomics-driven research and development related to hearing. Methods: The search for recent research articles on gene therapy for hearing loss was done with databases Scopus, PubMed, Medline and Google Scholar. This review article discusses the gene therapy for hearing loss. Results: Screening approaches, coupled with next-generation sequencing technologies, are being employed to analyse groups of individuals affected by hearing loss who are suspected to share common genetic mutations. This approach aims to accurately diagnose patients with hearing impairments. Conclusion: This review article discusses gene therapy techniques for the restoration or preservation of hearing, focusing on methods for delivering genes to target cells using viral or non-viral vectors. The review also covers technology targeted at creating gene therapy as a potential therapeutic option in the near future and emphasises recent developments in gene therapy for hearing loss.

Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection.

Under some conditions, dengue virus (DENV) can hijack IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR)-a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this unusual IgG-mediated infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout (KO) screens in an in vitro system poorly permissive to infection in the absence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates the binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired the binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that promote efficient ADE of DENV infection. Our findings represent a first step toward advancing fundamental knowledge behind the biology of a non-canonical infection route implicated in disease.IMPORTANCEAntibodies can paradoxically enhance rather than inhibit dengue virus (DENV) infection in some cases. To advance knowledge of the functional requirements of antibody-dependent enhancement (ADE) of infection beyond existing descriptive studies, we performed a genome-scale CRISPR knockout (KO) screen in an optimized in vitro system permissive to efficient DENV infection only in the presence of IgG. In addition to FcgRIIa, a known receptor that facilitates IgG-mediated uptake of IgG-bound, but not naked DENV particles, our screens identified TBC1D24 and SV2B, cellular factors with no known role in DENV infection. We validated a functional role for TBC1D24 and SV2B in mediating ADE of all four DENV serotypes in different cell lines and using various antibodies. Thus, we identify cellular factors beyond Fc gamma receptors that promote ADE mechanisms. This study represents a first step toward advancing fundamental knowledge beyond a poorly understood non-canonical viral entry mechanism.

Disulfiram ameliorates bone loss in ovariectomized mice by suppressing osteoclastogenesis.

Disulfiram (DSF), known as an anti-alcoholism drug, has been reported to suppress osteoclast differentiation in vitro; however, it remains uncertain whether DSF is effective in preventing osteoclastogenesis in vivo. This study aimed to investigate the effect of DSF administration in osteoporotic mice and its contribution to osteoclastogenesis in vivo. The bone phenotype of ovariectomized mice, both treated and untreated with DSF, was examined using microcomputed tomography analysis. Osteoclastic and osteoblastic parameters were assessed through bone morphometric analysis. The direct effect of DSF on osteoblastogenesis in vitro was evaluated via a primary osteoblast culture experiment. The expression of genes related to DSF targets (Nup85, Ccr2, and Ccr5) in osteoclast-lineage cells was examined using scRNA-seq analysis and flow cytometry analysis using the bone marrow cells from ovariectomized mice. The impact of DSF on osteoclast-lineage cells was assessed using primary cultures of osteoclasts. DSF administration ameliorated ovariectomy-induced bone loss and mitigated the increase of osteoclasts without affecting osteoblastogenesis. The scRNA-seq data revealed that osteoclast precursor cells expressed Nup85, Ccr2, and Ccr5. CCR2 and CCR5-positive cells in osteoclast precursor cells within bone marrow increased following ovariectomy, and this increase was canceled by DSF administration. Finally, we found that DSF had a significant inhibitory effect on osteoclastogenesis in the early stage by suppressing Tnfrsf11a expression. This study demonstrates that DSF could be a candidate for osteoporosis therapies because it suppresses osteoclastogenesis from an early stage in vivo.

Immune-reactive tumor organoids system to determine the effects of microbial metabolites on cancer immunity and immunotherapies

Immunotherapies are a revolutionary approach to treating cancer by utilizing the body’s immune system to target and combat cancer cells. This approach offers promising alternatives to traditional chemotherapies. Its potential to induce long-lasting remissions and specificity for cancer cells, which minimizes side effects, makes it a cutting-edge treatment with tremendous potential. With the increase of the clinical usage of immunotherapy, evidence emerges of the microbiome’s impact on both tumor growth and response to immunotherapy. The proposed involvement of the microbiome can change treatment efficacy by altering drug metabolism and reshaping the immune system response. Understanding the specific interactions between tumor cells, immune cells, and the microbiome is a critical step in the advancement of immunotherapy. To study the complex interaction between cancer immunity and the microbiome, various preclinical in vivo and in vitro models have been developed. We have recently described the use of an ex vivo preclinical model for anti-cancer treatment outcome prediction –tumor tissue equivalents (organoids). Specifically, immune-reactive tumor organoids are proposed as a novel tool for understanding how the microbiome influences cancer immunity and immunotherapy. More importantly, this platform can utilize patient samples to dissect patient-specific elements regulating cancer immune response and microbiome influence. This review presents the rationale for using the immune-reactive tumor organoids model to study the interactions between the microbiome and cancer immunotherapy. It will discuss available components of the model and analyze their interplay, summarize relevant experimental data, and assess their validity. Additionally, it explores the potential of immune-reactive organoids for personalized treatment approaches. Understanding the microbiome’s role in immunotherapy outcomes will lead to transformative cancer treatment via a simple change of diet or other microbiome manipulations. Ongoing research on microbiome-cancer interactions utilizing the described model systems will lead to innovative treatment strategies and improved patient outcomes.