Supinoxin blocks Small Cell Lung Cancer Progression by Inhibiting Mitochondrial Respiration through the RNA Helicase DDX5.

DDX5 is a DEAD-box RNA helicase that is overexpressed and implicated in progression of several cancers 1-4. One of these is small cell lung cancer (SCLC). Our laboratory has demonstrated that the RNA helicase DDX5 is essential for the invasive growth of SCLC and mitochondrial respiration 5. SCLC is an extremely lethal, recalcitrant tumor 6,7, causing 250,000 deaths annually worldwide 8 and currently lacking effective treatments 9,10. Supinoxin (RX 5902), a compound having anti-cancer activity 11, is a known target of phosphor-DDX5 12,13; Supinoxin blocks the interaction between ß-catenin and phosphor-DDX5 13, thereby releasing ß-catenin and allowing its degradation. In an effort to repurpose Supinoxin for treatment of SCLC, we conducted a series of in vitro and in vivo experiments. Supinoxin has been observed to impede the proliferation of H69AR cell lines. Additionally, Supinoxin has the potential to mitigate both the growth of H69AR xenograft tumors and SCLC PDX tumors in vivo at a dosage of 70mg/kg in immunocompromised mice. The findings indicate that the administration of Supinoxin is effective in suppressing the growth of tumors and enhancing the survival rate of mice with SCLC tumors. Subsequently, an effort was made to explore the molecular pathways involved in the activity of Supinoxin in Small Cell Lung Cancer (SCLC) cells. Surprisingly, we did not see any decrease in ß-catenin levels or relocalization from the cytoplasm upon Supinoxin treatment. Moreover, we did not observe any decrease in the expression levels of ß-catenin target genes thereby contradicting the current model. Based on our current data we found that the current model of Supinoxin activity is inaccurate. Additional investigations were conducted to explore the mechanisms by which Supinoxin affects small cell lung cancer (SCLC). Treatment with Supinoxin induced mitochondrial dysfunction in the chemoresistant small cell lung cancer (SCLC) cell line H69AR. The latter was evidenced by down-regulation of genes associated with oxidative phosphorylation. Thus, Supinoxin is a new therapeutic agent for small cell lung cancer (SCLC).

synNotch-programmed iPSC-derived NK cells usurp TIGIT and CD73 activities for glioblastoma therapy.

Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT - CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such "decoy" receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

TIGIT contributes to the regulation of 4-1BB and does not define NK cell dysfunction in glioblastoma.

TIGIT is a receptor on human natural killer (NK) cells. Here, we report that TIGIT does not spontaneously induce inhibition of NK cells in glioblastoma (GBM), but rather acts as a decoy-like receptor, by usurping binding partners and regulating expression of NK activating ligands and receptors. Our data show that in GBM patients, one of the underpinnings of unresponsiveness to TIGIT blockade is that by targeting TIGIT, NK cells do not lose an inhibitory signal, but gains the potential for new interactions with other, shared, TIGIT ligands. Therefore, TIGIT does not define NK cell dysfunction in GBM. Further, in GBM, TIGIT+ NK cells are hyperfunctional. In addition, we discovered that 4-1BB correlates with TIGIT expression, the agonism of which contributes to TIGIT immunotherapy. Overall, our data suggest that in GBM, TIGIT acts as a regulator of a complex network, and provide new clues about its use as an immunotherapeutic target.

RNA helicase DDX5 modulates sorafenib sensitivity in hepatocellular carcinoma via the Wnt/ß-catenin-ferroptosis axis.

Reduced expression of the RNA helicase DDX5 associated with increased hepatocellular carcinoma (HCC) tumor grade and poor patient survival following treatment with sorafenib. While immunotherapy is the first-line treatment for HCC, sorafenib and other multi-tyrosine kinase inhibitors (mTKIs) are widely used when immunotherapy is contra-indicated or fails. Herein, we elucidate the role of DDX5 in sensitizing HCC to sorafenib, offering new therapeutic strategies. Treatment of various human HCC cell lines with sorafenib/mTKIs downregulated DDX5 in vitro and in preclinical HCC models. Conversely, DDX5 overexpression reduced the viability of sorafenib-treated cells via ferroptosis, suggesting a role for DDX5 in sorafenib sensitivity. RNAseq of wild-type vs. DDX5-knockdown cells treated with or without sorafenib identified a set of common genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps with Wnt signaling genes, including Disheveled-1 (DVL1), an indispensable Wnt activator and prognostic indicator of poor survival for sorafenib-treated patients. DDX5-knockout (DDX5KO) HCC cells exhibited DVL1 induction, Wnt/ß-catenin pathway activation, and ferroptosis upon inhibition of canonical Wnt signaling. Consistently, xenograft HCC tumors exhibited reduced growth by inhibition of Wnt/ß-catenin signaling via induction of ferroptosis. Significantly, overexpression of DDX5 in HCC xenografts repressed DVL1 expression and increased ferroptosis, resulting in reduced tumor growth by sorafenib. We conclude that DDX5 downregulation by sorafenib mediates adaptive resistance by activating Wnt/ß-catenin signaling, leading to ferroptosis escape. Conversely, overexpression of DDX5 in vivo enhances the anti-tumor efficacy of sorafenib by suppressing Wnt/ß-catenin activation and induction of ferroptosis. Thus, DDX5 overexpression in combination with mTKIs is a promising therapeutic strategy for HCC.

Inkjet-printed morphogenesis of tumor-stroma interface using bi-cellular bioinks of collagen-poly(N-isopropyl acrylamide-co-methyl methacrylate) mixture.

Recent advances in biomaterials and 3D printing/culture methods enable various tissue-engineered tumor models. However, it is still challenging to achieve native tumor-like characteristics due to lower cell density than native tissues and prolonged culture duration for maturation. Here, we report a new method to create tumoroids with a mechanically active tumor-stroma interface at extremely high cell density. This method, named "inkjet-printed morphogenesis" (iPM) of the tumor-stroma interface, is based on a hypothesis that cellular contractile force can significantly remodel the cell-laden polymer matrix to form densely-packed tissue-like constructs. Thus, differential cell-derived compaction of tumor cells and cancer-associated fibroblasts (CAFs) can be used to build a mechanically active tumor-stroma interface. In this methods, two kinds of bioinks are prepared, in which tumor cells and CAFs are suspended respectively in the mixture of collagen and poly (N-isopropyl acrylamide-co-methyl methacrylate) solution. These two cellular inks are inkjet-printed in multi-line or multi-layer patterns. As a result of cell-derived compaction, the resulting structure forms tumoroids with mechanically active tumor-stroma interface at extremely high cell density. We further test our working hypothesis that the morphogenesis can be controlled by manipulating the force balance between cellular contractile force and matrix stiffness. Furthermore, this new concept of "morphogenetic printing" is demonstrated to create more complex structures beyond current 3D bioprinting techniques.

Radiation-induced photodynamic therapy using calcium tungstate nanoparticles and 5-aminolevulinic acid prodrug.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) prodrug is a clinically tried and proven treatment modality for surface-level lesions. However, its use for deep-seated tumors has been limited due to the poor penetration depth of visible light needed to activate the photosensitizer protoporphyrin IX (PPIX), which is produced from ALA metabolism. Herein, we report the usage of poly(ethylene glycol-b-lactic acid) (PEG-PLA)-encapsulated calcium tungstate (CaWO4, CWO for short) nanoparticles (PEG-PLA/CWO NPs) as energy transducers for X-ray-activated PDT using ALA. Owing to the spectral overlap between radioluminescence afforded by the CWO core and the absorbance of PPIX, these NPs can serve as an in situ visible light activation source during radiotherapy (RT), thereby mitigating the limitation of penetration depth. We demonstrate that this effect is observed across different cell lines with varying radio-sensitivity. Importantly, both PPIX and PEG-PLA/CWO NPs exhibit no significant toxicities at therapeutic doses in the absence of radiation. To assess the efficacy of this approach, we conducted a study using a syngeneic mouse model subcutaneously implanted with inherently radio-resistant 4T1 tumors. The results show a significantly improved prognosis compared to conventional RT, even with as few as 2 fractions of 4 Gy X-rays. Taken together, these results suggest that PEG-PLA/CWO NPs are promising agents for application of ALA-PDT in deep-seated tumors, thereby significantly expanding the utility of the already established treatment strategy.

Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma.

Human dedifferentiated liposarcoma (DDLPS) is a rare but lethal cancer with no driver mutations being identified, hampering the development of targeted therapies. We and others recently reported that constitutive activation of Notch signaling through overexpression of the Notch1 intracellular domain (NICDOE) in murine adipocytes leads to tumors resembling human DDLPS. However, the mechanisms underlying the oncogenic functions of Notch activation in DDLPS remains unclear. Here, we show that Notch signaling is activated in a subset of human DDLPS and correlates with poor prognosis and expression of MDM2, a defining marker of DDLPS. Metabolic analyses reveal that murine NICDOE DDLPS cells exhibit markedly reduced mitochondrial respiration and increased glycolysis, mimicking the Warburg effect. This metabolic switch is associated with diminished expression of peroxisome proliferator-activated receptor gamma coactivator 1α (Ppargc1a, encoding PGC-1α protein), a master regulator of mitochondrial biogenesis. Genetic ablation of the NICDOE cassette rescues the expression of PGC-1α and mitochondrial respiration. Similarly, overexpression of PGC-1α is sufficient to rescue mitochondria biogenesis, inhibit the growth and promote adipogenic differentiation of DDLPS cells. Together, these data demonstrate that Notch activation inhibits PGC-1α to suppress mitochondrial biogenesis and drive a metabolic switch in DDLPS.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy.

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor ß-chain (ΔIL-2Rß) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

CAR-neutrophil mediated delivery of tumor-microenvironment responsive nanodrugs for glioblastoma chemo-immunotherapy.

Glioblastoma (GBM) is one of the most aggressive and lethal solid tumors in human. While efficacious therapeutics, such as emerging chimeric antigen receptor (CAR)-T cells and chemotherapeutics, have been developed to treat various cancers, their effectiveness in GBM treatment has been hindered largely by the blood-brain barrier and blood-brain-tumor barriers. Human neutrophils effectively cross physiological barriers and display effector immunity against pathogens but the short lifespan and resistance to genome editing of primary neutrophils have limited their broad application in immunotherapy. Here we genetically engineer human pluripotent stem cells with CRISPR/Cas9-mediated gene knock-in to express various anti-GBM CAR constructs with T-specific CD3ζ or neutrophil-specific γ-signaling domains. CAR-neutrophils with the best anti-tumor activity are produced to specifically and noninvasively deliver and release tumor microenvironment-responsive nanodrugs to target GBM without the need to induce additional inflammation at the tumor sites. This combinatory chemo-immunotherapy exhibits superior and specific anti-GBM activities, reduces off-target drug delivery and prolongs lifespan in female tumor-bearing mice. Together, this biomimetic CAR-neutrophil drug delivery system is a safe, potent and versatile platform for treating GBM and possibly other devastating diseases.

Monocytic Myeloid-Derived Suppressor Cells from Tumor Tissue Are a Differentiated Cell with Limited Fate Plasticity.

Owing to ease of access and high yield, most murine myeloid-derived suppressor cell (MDSC) knowledge comes from the study of spleen-derived MDSCs rather than those isolated from the tumor. Although several studies have identified subtle differences in suppressive function between these MDSCs, a recent report demonstrated that the whole peripheral myeloid compartment poorly reflects myeloid populations found at the tumor. We confirm and extend these observations by presenting data that indicate extensive differences exist between peripheral and tumor MDSCs, suggesting that it may be inappropriate to use spleen MDSCs as surrogates for studying tumor MDSCs. Using cytospins, we observed that tumor MDSCs have undergone a morphologic shift from immature myeloid cell forms commonly seen in bone marrow (BM) and spleen MDSCs and acquired mature myeloid cell characteristics. Spleen and BM monocyte-like MDSCs (M-MDSCs) readily responded to differentiation signals for multiple myeloid cell types whereas tumor M-MDSCs had remarkably reduced cellular plasticity. At the time of isolation, M-MDSCs from BM or spleen have little to no T cell suppressive activity whereas those from the tumor possess immediate and efficient T cell suppressive function. Finally, microarray analysis revealed that the transcriptomes of tumor and spleen M-MDSCs possessed >4500 differentially expressed transcripts. We conclude that tumor M-MDSCs are more differentiated and mature, and that they are morphologically, genetically, and functionally distinct from spleen and BM M-MDSCs. These observations have important implications for the design of anti-MDSC therapies and suggest that preclinical studies using nontumor MDSCs could lead to results not applicable to tumor MDSCs.

Engineered natural killer cells impede the immunometabolic CD73-adenosine axis in solid tumors.

Immunometabolic reprogramming due to adenosine produced by CD73 (encoded by the 5'-ectonucleotidase gene NT5E) is a recognized immunosuppressive mechanism contributing to immune evasion in solid tumors. Adenosine is not only known to contribute to tumor progression, but it has specific roles in driving dysfunction of immune cells, including natural killer (NK) cells. Here, we engineered human NK cells to directly target the CD73-adenosine axis by blocking the enzymatic activity of CD73. In doing so, the engineered NK cells not only impaired adenosinergic metabolism driven by the hypoxic uptake of ATP by cancer cells in a model of non-small-cell lung cancer, but also mediated killing of tumor cells due to the specific recognition of overexpressed CD73. This resulted in a 'single agent' immunotherapy that combines antibody specificity, blockade of purinergic signaling, and killing of targets mediated by NK cells. We also showed that CD73-targeted NK cells are potent in vivo and result in tumor arrest, while promoting NK cell infiltration into CD73+ tumors and enhancing intratumoral activation.

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy.

Neutrophils, the most abundant white blood cells in circulation, are closely related to cancer development and progression. Healthy primary neutrophils present potent cytotoxicity against various cancer cell lines through direct contact and via generation of reactive oxygen species. However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with chimeric antigen receptors (CARs) to enhance their antitumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells with synthetic CARs and differentiated them into functional neutrophils by implementing a chemically defined platform. The resulting CAR neutrophils present superior and specific cytotoxicity against tumor cells both in vitro and in vivo. Collectively, we established a robust platform for massive production of CAR neutrophils, paving the way to myeloid cell-based therapeutic strategies that would boost current cancer-treatment approaches.

Effect of Paclitaxel Stereochemistry on X-ray-Triggered Release of Paclitaxel from CaWO4/Paclitaxel-Coloaded PEG-PLA Nanoparticles.

For many locally advanced tumors, the chemotherapy-radiotherapy (CT-RT) combination ("chemoradiation") is currently the standard of care. Intratumoral (IT) CT-based chemoradiation has the potential to overcome the limitations of conventional systemic CT-RT (side effects). For maximizing the benefits of IT CT-RT, our laboratory has previously developed a radiation-controlled drug release formulation, in which anticancer drug paclitaxel (PTX) and radioluminescent CaWO4 (CWO) nanoparticles (NPs) are co-encapsulated with poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block copolymers ("PEG-PLA/CWO/PTX NPs"). These PEG-PLA/CWO/PTX NPs enable radiation-controlled release of PTX and are capable of producing sustained therapeutic effects lasting for at least one month following a single IT injection. The present article focuses on discussing our recent finding about the effect of the stereochemical structure of PTX on the efficacy of this PEG-PLA/CWO/PTX NP formulation. Stereochemical differences in two different PTX compounds ("PTX-S" from Samyang Biopharmaceuticals and "PTX-B" from Biotang) were characterized by 2D heteronuclear/homonuclear NMR, Raman spectroscopy, and circular dichroism measurements. The difference in PTX stereochemistry was found to significantly influence their water solubility (WS); PTX-S (WS ≈ 4.69 µg/mL) is about 19 times more water soluble than PTX-B (WS ≈ 0.25 µg/mL). The two PTX compounds showed similar cancer cell-killing performances in vitro when used as free drugs. However, the subtle stereochemical difference significantly influenced their X-ray-triggered release kinetics from the PEG-PLA/CWO/PTX NPs; the more water-soluble PTX-S was released faster than the less water-soluble PTX-B. This difference was manifested in the IT pharmacokinetics and eventually in the survival percentages of test animals (mice) treated with PEG-PLA/CWO/PTX NPs + X-rays in an in vivo human tumor xenograft study; at short times (<1 month), concurrent PEG-PLA/CWO/PTX-S NPs produced a greater tumor-suppression effect, whereas PEG-PLA/CWO/PTX-B NPs had a longer-lasting radio-sensitizing effect. This study demonstrates the importance of the stereochemistry of a drug in a therapy based on a controlled release formulation.

Piceatannol, a Dietary Polyphenol, Alleviates Adipose Tissue Loss in Pre-Clinical Model of Cancer-Associated Cachexia via Lipolysis Inhibition.

Cancer-associated cachexia (CAC) is the nutrition-independent loss of lean muscle and adipose tissues, and results in reduced chemotherapy effectiveness and increased mortality. Preventing adipose loss is considered a key target in the early stages of cachexia. Lipolysis is considered the central driver of adipose loss in CAC. We recently found that piceatannol, but not its analogue resveratrol, exhibits an inhibitory effect on lipolysis. The objective of this study was to investigate the role of piceatannol in cancer-associated lipolysis and cachexia-induced weight loss. Cancer cell-induced lipolysis in adipocytes was stimulated using cancer-conditioned media (CCM) or co-culture with human pancreatic cancer cells and the cachexia-associated cytokines TNF-α and interleukin-6 in 3T3-L1 adipocytes. C26 colon carcinoma-bearing mice were modeled using CAC in vivo. Piceatannol reduced cancer-associated lipolysis by at least 50% in both CCM and cytokine-induced lipolysis in vitro. Further gene and protein analysis confirmed that piceatannol modulated the stability of lipolytic proteins. Moreover, piceatannol protected tumor-bearing mice against weight-loss in early stages of CAC largely through preserving adipose tissue, with no effect on survival. This study demonstrates the use of a dietary compound to preserve adipose in models of early stage CAC and provides groundwork for further investigation of piceatannol or piceatannol-rich foods as alternative medicine in the preservation of body fat mass and future CAC therapy.

Pulmonary Pharmacokinetics of Polymer Lung Surfactants Following Pharyngeal Administration in Mice.

We have recently discovered that pulmonary administration of nanoparticles (micelles) formed by amphiphilic poly(styrene-block-ethylene glycol) (PS-PEG) block copolymers has the potential to treat a lung disorder involving lung surfactant (LS) dysfunction (called acute respiratory distress syndrome (ARDS)), as PS-PEG nanoparticles are capable of reducing the surface tension of alveolar fluid, while they are resistant to deactivation caused by plasma proteins/inflammation products unlike natural LS. Herein, we report studies of the clearance pathways and kinetics of PS-PEG nanoparticles from the lung, which are essential for designing further preclinical IND-enabling studies. Using fluorescently labeled PS-PEG nanoparticles, we found that, following pharyngeal aspiration in mice, the retention of these nanoparticles in the lungs extends over 2 weeks, while their transport into other (secondary) organs is relatively insignificant. An analysis based on a multicompartmental pharmacokinetic model suggests a biphasic mechanism involving a fast mucociliary escalator process through the conducting airways and much slower alveolar clearance processes by the action of macrophages and also via direct translocation into the circulation. An excessive dose of PS-PEG nanoparticles led to prolonged retention in the lungs due to saturation of the alveolar clearance capacity.

Chemically-defined generation of human hemogenic endothelium and definitive hematopoietic progenitor cells.

Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFß inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.

Bone Microenvironment-Suppressed T Cells Increase Osteoclast Formation and Osteolytic Bone Metastases in Mice.

Immunotherapies use components of the immune system, such as T cells, to fight cancer cells, and are changing cancer treatment, causing durable responses in some patients. Bone metastases are a debilitating complication in advanced breast and prostate cancer patients. Approved treatments fail to cure bone metastases or increase patient survival and it remains unclear whether immunotherapy could benefit patients. The bone microenvironment combines various immunosuppressive factors, and combined with T cell products could increase bone resorption fueling the vicious cycle of bone metastases. Using syngeneic mouse models, our study revealed that bone metastases from 4T1 breast cancer contain tumor-infiltrating lymphocyte (TILs) and their development is increased in normal mice compared to immunodeficient and T-cell depleted mice. This effect seemed caused by the TILs specifically in bone, because T-cell depletion increased 4T1 orthotopic tumors and did not affect bone metastases from RM-1 prostate cancer cells, which lack TILs. T cells increased osteoclast formation ex vivo and in vivo contributing to bone metastasis vicious cycle. This pro-osteoclastic effect is specific to unactivated T cells, because activated T cells, secreting interferon γ (IFNγ) and interleukin 4 (IL-4), actually suppressed osteoclastogenesis, which could benefit patients. However, non-activated T cells from bone metastases could not be activated in ex vivo cultures. 4T1 bone metastases were associated with an increase of functional polymorphonuclear and monocytic myeloid-derived suppressor cells (MDSCs), potent T-cell suppressors. Although effective in other models, sildenafil and zoledronic acid did not affect MDSCs in bone metastases. Seeking other therapeutic targets, we found that monocytic MDSCs are more potent suppressors than polymorphonuclear MDSCs, expressing programmed cell death receptor-1 ligand (PD-L1)+ in bone, which could trigger T-cell suppression because 70% express its receptor, programmed cell death receptor-1 (PD-1). Collectively, our findings identified a new mechanism by which suppressed T cells increase osteoclastogenesis and bone metastases. Our results also provide a rationale for using immunotherapy because T-cell activation would increase their anti-cancer and their anti-osteoclastic properties. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Changes in lipid profile and SOX-2 expression in RM-1 cells after co-culture with preimplantation embryos or with deproteinated blastocyst extracts.

The embryonic environment can modify cancer cell metabolism, and it is reported to induce the loss of tumorigenic properties and even affect the differentiation of cancer cells into normal tissues. The cellular mechanisms related to this remarkable phenomenon, which is likely mediated by cell-to-cell communication, have been previously investigated with particular focus on the proteins and genes involved. In this study we report the optimization and results of a straightforward in vitro system where mouse prostate carcinoma (RM-1) cells were co-cultured for three days with preimplantation mouse embryos or spiked with deproteinated extracts from mouse blastocysts. Compared to controls, both treatments induced RM-1 cells to increase the expression of the SOX-2 gene, which is related to cellular stemness, as well as altered their lipid composition. Specific acyl-carnitines, diacylglycerols, phosphatidylglycerols, phosphatidylinositols, phosphatidylserines and cardiolipins selected using an elastic net model discriminated the treated RM-1 cells from controls. Note that the tumorigenic properties of the treated RM-1 cells were not evaluated in this research. Due to the nature of the lipids impacted in the treated RM-1 cells, we hypothesize that mitochondrial metabolism has been altered, and that small molecules both secreted from and present within the embryos might be involved in the induction of metabolic changes observed in the RM-1 cells. These molecules, which could influence cancer cell metabolism, may still be unknown (i.e. structure, role).