T2 Cells Research Articles

Anemoside A3 Inhibits Macrophage M2-Like Polarization to Prevent Triple-Negative Breast Cancer Metastasis

Triple negative breast cancer (TNBC) exhibits the characteristics of strong metastatic ability and a high recurrence rate, and M2-type macrophages play an important role in this process. Previous research data suggested that Anemoside A3 (A3), a monomeric component of Pulsatilla Chinensis, could prevent and treat TNBC by converting M0 macrophages into M1 immunogen phenotypes. This study showed that A3 significantly restrained the lung metastases of 4 T1-Luc cells with bioluminescence imaging in vivo and Hematoxylin and Eosin (H&E) staining. Meanwhile, the percentage of M2-type macrophages (CD206+ labeled cells) in the lung tissues was evidently decreased through immunohistochemical assay. We further proved that A3 markedly prevented M2-type polarization induced by IL-4 in vitro, as illustrated by the down-regulated expression of the cell surface marker CD206 protein by FACS and Arg-1, and of the Fizz1 and Ym1 genes by RT-PCR in M2-type macrophages. Furthermore, the invasion and migration of 4 T1 cells, which was promoted by the conditioned medium from M2-type macrophages, could be suppressed by A3. Luminex assay demonstrated that A3 treatment resulted in a reduction of the levels of CCL2, VEGF, CCL7, and MMP-9 in conditioned medium. Additionally, the expression of phosphorylated-STAT3 protein was inhibited by A3, which resulted in the macrophage M2-type polarization arrest, while no significant difference in JAK2 phosphorylation was detected. SiRNA transfection experiments suggested that STAT3 might be the target of A3 inhibiting M2-type polarization of macrophages. In conclusion, these results indicate that A3 could attenuate the metastasis of TNBC by inhibiting the M2-type polarization of macrophages, which may be related to the STAT3 pathway.

Polydopamine-guarded metal-organic frameworks as co-delivery systems for starvation-assisted chemo-photothermal therapy

Cutting off glucose provision by glucose oxidase (GOx) to famish tumors can be an assistance with chemotherapy to eliminate cancer cells. Co-encapsulation of GOx and chemotherapeutics (doxorubicin) within pH-sensitive metal-organic frameworks (MOFs) could disorder metabolic pathways of cancer cells and generate excessive intracellular reactive oxygen species (ROS), together. To prevent premature leach of GOx from the porous channels of MOFs, polydopamine (PDA) was deposited on the surface of MOFs, which endowed the delivery system with photothermal conversion ability. Our nanoscaled co-delivery system (denoted as DGZPNs) remains stable with low amount of drug leakage under simulated physiological conditions in vitro and internal environment, while they are triggered to release doxorubicin (DOX) and GOx in acid tumor microenvironment and at high temperature for reinforced chemotherapy. NIR laser irradiation also activates superior photothermal conversion efficiency of PDA (36.9 %) to initiate hyperthermia to ablate tumor tissue. After being phagocytized by 4 T1 cells (breast cancer cells), the DGZPNs delivery system showed a superior therapeutic efficacy with a tumor growth inhibition of 88.9 ± 6.6 % under NIR irradiation, which indicated that the starvation-assisted chemo-photothermal therapy prompts the significant advance of synergistic therapy in a parallelly controlled mode.

Stability and Survivability of Alginate Gum-Coated Lactobacillus rhamnosus GG in Simulated Gastrointestinal Conditions and Probiotic Juice Development

Survivability of probiotics is severely affected by harsh gastrointestinal conditions. In the present study, microbeads of Lactobacillus rhamnosus GG were formulated using alginate (1.5% w/v) and combination of alginate (1.5% w/v) with xanthan gum (0.5% w/v) through an emulsion technique to improve bacterial viability in low pH orange juice and in gastrointestinal conditions. The microbeads were tested for encapsulation efficiency, survivability in bile salt, SGF (simulated gastric juice), SIF (simulated intestinal fluid), and storage stability. Probiotic orange juice was formulated and tested for physicochemical parameters (pH, titratable acidity, and total sugars) and sensorial properties during storage. Gum-coated alginate microbeads (T3) showed higher encapsulation efficiency, i.e., 95.2% compared to alginate microbeads (T2), i.e., 86.85%. Similarly, T3 showed the highest resistance against bile salt (8.50 log CFU/g), SGF (7.95 log CFU/g), and SIF (8.0 log CFU/g) during 80 min exposure compared to T2 and free cells. The viability of gum-coated alginate beads (T3) remained above 107 CFU/g in gastrointestinal conditions and at the end of 21 days storage (8.3 log CFU/mL). All physicochemical parameters of probiotic juice were significantly ( p ≤ 0.05 ) decreased with respect to storage except acidity. In addition, minimal changes in physicochemical parameters were observed in T3 compared to other treatments. Treatment had no significant impact on the sensory characteristics of juice, but storage had a significant effect ( p ≤ 0.05 ) on the sensory characteristics of juice. The alginate gum microbeads improve the survivability of probiotics for targeted delivery. Hence, encapsulated probiotics can be used for functional beverage development to take advantage of their therapeutic benefits.

Inhibition of activin-signalling reduces the growth of LβT2 gonadotroph pituitary tumours in mouse.

Pituitary tumours are benign neoplasms that derive from hormone-producing cells of the pituitary gland. While medical treatments have emerged for most subtypes, Gonadotroph tumours that express FSH, and/or LH still lack therapeutic options apart from surgery and radiotherapy. Activin-ligands are physiological regulators of production and secretion of FSH by gonadotroph cells, but their role in gonadotroph tumorigenesis remains little explored. Using the LT2 mouse gonadotroph-cell line which produces FSH under Activin-stimulation, we first tested whether sub-cutaneous xenografts of LT2 cells resulted in tumour formation in Rag2KO mice. Histological analysis confirmed the presence of LT2-tumours with endothelial cells and macrophages in their microenvironment. FSH expression was found in a subset of clusters of LT2 cells in the tumours. We subsequently addressed the consequences of targeting activin-signalling via injection of a soluble activin decoy receptor (sActRIIB-Fc). sActRIIB-Fc treatment resulted in significantly decreased LT2 tumour volume. Reduced Smad2-phosphorylation as well as inhibition of tumour-induced FSH production confirmed the efficient targeting of activin-downstream signalling in treated tumours. More interestingly, treated tumours showed significantly fewer endothelial cells, associated with reduced Vegfa expression. In-vitro treatment of LT2 cells with sActRIIB-Fc had no effect on cell proliferation or apoptosis, but Vegfa expression was inhibited, pointing to a likely paracrine effect of LT2 cells on endothelial cells through activin-mediated Vegfa regulation. Further in-vitro and in-vivo studies are now needed to pinpoint the exact roles of activin-signalling in these processes prior to translating these observations to the clinic.

Activated T cell therapy targeting glioblastoma cancer stem cells

Naïve T cells become effector T cells following stimulation by antigen-loaded dendritic cells (DCs) and sequential cytokine activation. We aimed to develop procedures to efficiently activate T cells with tumor-associated antigens (TAAs) to glioblastoma (GBM) stem cells. To remove antigen presentation outside of the immunosuppressive tumor milieu, three different glioma stem cell (GSC) specific antigen sources to load DCs were compared in their ability to stimulate lymphocytes. An activated T cell (ATC) protocol including cytokine activation and expansion in culture to target GSCs was generated and optimized for a planned phase I clinical trial. We compared three different antigen-loading methods on DCs to effectively activate T cells, which were GBM patient-derived GSC-lysate, acid-eluate of GSCs and synthetic peptides derived from proteins expressed in GSCs. DCs derived from HLA-A2 positive blood sample were loaded with TAAs. Autologous T cells were activated by co-culturing with loaded DCs. Efficiency and cytotoxicity of ATCs were evaluated by targeting TAA-pulsed DCs or T2 cells, GSCs, or autologous PHA-blasts. Characteristics of ATCs were evaluated by Flow Cytometry and ELISpot assay, which showed increased number of ATCs secreting IFN-γ targeting GSCs as compared with non-activated T cells and unloaded target cells. Neither GSC-lysate nor acid-eluate loading showed enhancement in response of ATCs but the synthetic peptide pool showed significantly increased IFN-γ secretion and increased cytotoxicity towards target cells. These results demonstrate that ATCs activated using a TAA synthetic peptide pool efficiently enhance cytotoxicity specifically to target cells including GSC.

Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function.

Plant homeodomain fingers (PHD-fingers) are a family of reader domains that can recruit epigenetic proteins to specific histone modification sites. Many PHD-fingers recognise methylated lysines on histone tails and play crucial roles in transcriptional regulation, with their dysregulation linked to various human diseases. Despite their biological importance, chemical inhibitors for targeting PHD-fingers are very limited. Here we report a potent and selective de novo cyclic peptide inhibitor (OC9) targeting the Nε-trimethyllysine-binding PHD-fingers of the KDM7 histone demethylases, developed using mRNA display. OC9 disrupts PHD-finger interaction with histone H3K4me3 by engaging the Nε-methyllysine-binding aromatic cage through a valine, revealing a new non-lysine recognition motif for the PHD-fingers that does not require cation-π interaction. PHD-finger inhibition by OC9 impacted JmjC-domain mediated demethylase activity at H3K9me2, leading to inhibition of KDM7B (PHF8) but stimulation of KDM7A (KIAA1718), representing a new approach for selective allosteric modulation of demethylase activity. Chemoproteomic analysis showed selective engagement of OC9 with KDM7s in T cell lymphoblastic lymphoma SUP T1 cells. Our results highlight the utility of mRNA-display derived cyclic peptides for targeting challenging epigenetic reader proteins to probe their biology, and the broader potential of this approach for targeting protein-protein interactions.

Multiple breast cancer liver metastases response to macrophage-delivered nanotherapy evaluated via a 3D continuum model.

Breast cancer liver metastases (BCLM) are usually unresectable and difficult to treat with systemic chemotherapy. A major reason for chemotherapy failure is that BCLM are typically small, avascular nodules, with poor transport and fast washout of therapeutics from surrounding capillaries. We have previously shown that nanoalbumin-bound paclitaxel (nab-PTX) encapsulated in porous silicon multistage nanovectors (MSV) is preferentially taken up by tumour-associated macrophages (TAM) in the BCLM microenvironment. The TAM alter therapeutic transport characteristics and retain it in the tumour vicinity, increasing cytotoxicity. Computational modeling has shown that therapeutic regimens could be designed to eliminate single lesions. To evaluate clinically-relevant scenarios, this study develops a modeling framework to evaluate MSV-nab-PTX therapy targeting multiple BCLM. An experimental model of BCLM, splenic injection of breast cancer 4 T1 cells was established in BALB/C mice. Livers were analyzed histologically to determine size and density of BCLM. The data were used to calibrate a 3D continuum mixture model solved via distributed computing to enable simulation of multiple BCLM. Overall tumour burden was analyzed as a function of metastases number and potential therapeutic regimens. The computational model enables realistic 3D representation of metastatic tumour burden in the liver, with the capability to evaluate BCLM growth and therapy response for hundreds of lesions. With the given parameter set, the model projects that repeated MSV-nab-PTX treatment in intervals <7 days would control the tumour burden. We conclude that nanotherapy targeting TAM associated with BCLM may be evaluated and fine-tuned via 3D computational modeling that realistically simulates multiple metastases.

Synthesis, antiproliferative and anti-MDR activities of lathyrane diterpene derivatives based on configuration inversion strategy

A series of lathyrane-type Euphorbia diterpene derivatives featured 3R configuration (H-3β) were synthesized from natural rich Euphorbia factor L3via modified Mitsunobu reaction based on configuration inversion strategy. The antiproliferation activity and MDR reversal ability of the lathyrane derivatives were evaluated, and the most synthesized compounds showed moderate or strong potencies. Among them, diterpenes 21 (IC50 values of 2.6, 5.2 and 13.1 μM, respectively) and 25 (IC50 values of 5.5, 8.6 and 1.3 μM, respectively) presented the strong cytotoxicity against MCF-7, 4 T1 and HepG2 cells. Meanwhile, derivative 25 exhibited excellent MDR reversal ability with the reversal fold of 16.1 higher than that of verapamil. The cellular thermal shift assay and molecular docking proved direct engagement of diterpene 25 to ABCB1, suggesting 25 could be a promising MDR modulator. Furthermore, the preliminary SARs of these diterpenes were also discussed.

Kaurenoic acid nanocarriers regulates cytokine production and inhibit breast cancer cell migration.

Breast cancer is the type of cancer with the highest incidence in women around the world. Noteworthy, the triple-negative subtype affects 20% of the patients while presenting the highest death rate among subtypes. This is due to its aggressive phenotype and the capability of invading other tissues. In general, tumor-associated macrophages (TAM) and other immune cells, are responsible for maintaining a favorable tumor microenvironment for inflammation and metastasis by secreting several mediators such as pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, chemokines like CCL2, and other proteins, as metalloproteinases of matrix (MMP). On the other hand, immunomodulatory agents can interfere in the immune response of TAM and change the disease prognosis. In this work, we prepared nanostructured lipid carriers containing kaurenoic acid (NLC-KA) to evaluate the effect on cytokine production in vitro of bone marrow-derived macrophages (BMDM) and the migratory process of 4T1 breast cancer cells. NLC-KA prepared from a blend of natural lipids was shown to have approximately 90nm in diameter with low polydispersity index. To test the effect on cytokine production in vitro in NLC-KA treated BMDM, ELISA assay was performed and pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified. The formulation reduced the secretion of IL-1β and TNF-α cytokines while presenting no hemolytic activity. Noteworthy, an anti-migratory effect in 4T1 breast cancer cells treated with NLC-KA was observed in scratch assays. Further, MMP9 and CCL2 gene expressions in both BMDM and 4T1 treated cells confirmed that the mechanism of inhibition of migration is related to the blockade of this pathway by KA. Finally, cell invasion assays confirmed that NLC-KA treatment resulted in less invasiveness of 4T1 cells than control, and it is independent of CCL2 stimulus or BMDM direct stimulus. Ultimately, NLC-KA was able to regulate the cytokine production in vitro and reduce the migration of 4T1 breast cancer cells by decreasing MMP9 gene expression.

Increased Expression of POSTN Predicts Poor Prognosis: a Potential Therapeutic Target for Gastric Cancer

BackgroundPeriostin (POSTN) is involved in many biological processes and is associated with the occurrence and development of several cancers, while its role in gastric cancer is not clear. We aimed to demonstrate the relationship between POSTN and gastric cancer based on publicly available data from The Cancer Genome Atlas (TCGA) database. MethodsPOSTN expression data and corresponding clinical information were downloaded from TCGA database. We compared the expression of POSTN in gastric cancer samples and normal samples. POSTN-related differentially expressed genes (DEGs) were identified for further functional enrichment analysis. In addition, the relationships between POSTN expression and clinicopathological features and survival in patients with gastric cancer were also investigated through univariate and multivariate Cox regression analyses. Furthermore, a nomogram was constructed to predict the survival probability of gastric cancer patients. ResultsPOSTN expression in gastric cancer was significantly higher than that in normal gastric tissues (p < 0.001). High POSTN expression in gastric cancer was significantly related to poor prognostic features, including greater tumor extent (odds ratio [OR] = 1.638 for T3 and T4 vs. T1 and T2), worse histological type (OR = 0.329 for diffuse type vs. tubular type), and advanced histological grade (OR = 1.646 for grade 3 vs. grades 1 and 2) (all p < 0.05). The 118 identified DEGs were primarily enriched in pathways related to tumorigenesis and tumor progression, including the TGF-β signaling pathway, the WNT signaling pathway, and the signaling by VEGF. POSTN expression was positively correlated with the enrichment of the macrophages (r = 0.599, p < 0.001), helper T2 cells (r = 0.146, p = 0.005), and CD8 + T cells (r = 0.190, p < 0.001), but negatively correlated with the enrichment of Th17 cells (r = − 0.130, p = 0.012) and NK CD56bright cells. Kaplan–Meier survival analysis showed that high POSTN expression is associated with a short overall survival (hazard ratio [HR] = 1.54; p = 0.011). In the multivariate cox regression analysis, high POSTN expression was confirmed to be an independent predictor of poor overall survival (HR = 1.681; p = 0.017). The constructed nomogram can well predict the 1 and 3 years overall survival probability of patients with GC (0.696 [95% CI, 0.671–0.721]). ConclusionPOSTN plays an important role in the progression and prognosis of gastric cancer, and it may serve as a useful biomarker to predict survival in gastric cancer patients.

High performance carbon dots based prodrug Platform: Image-Guided photodynamic and chemotherapy with On-Demand drug release upon laser irradiation

The design of therapeutic nanoplatforms based on fluorescent carbon dots (CDs) has become a viable strategy because of their aqueous solubility, biocompatibility, and ease of further functionalization. By doping various heteroatoms into pristine CDs structures, we synthesized N-, Cl-, and S-doped CDs (NClS/CDs), as well as Se-, N-, and Cl-doped CDs (NClSe/CDs) with superior optoelectronic properties using rapid and straightforward microwave heating. The quantum efficiencies of these NClS/CDs and NClSe/CDs were enhanced to 30.7 % and 42.9 %, respectively, compared to those of undoped CDs (0.66 %). Owing to their better light absorption properties, NClS/CDs efficiently produced reactive oxygen species (ROS) under 532 nm laser irradiation for photodynamic therapy (PDT). Considering the ROS generation and surface carrier abilities of NClS/CDs, we designed the loading of camptothecin (CPT) drug via a thioketal linker (TL), resulting in h/CDs@CPT nanovesicles (NVs) with a drug-loading efficiency of 46.5 %. Under laser irradiation in an acidic environment, ROS-triggered CPT release was observed, with 50.2 % of CPT released following the breakdown of the ROS-sensitive TL. In vitro cellular studies revealed that h/CDs@CPT NVs possessed minimal cytotoxicity toward HeLa and 4 T1 cancer cells, despite the high clinical efficacy of PDT and ROS-induced chemotherapeutic response under laser treatment. Confocal microscopy of HeLa and 4 T1 cells revealed that h/CDs@CPT NVs produced red-emissive photographs for potential cancer cell detection. Therefore, our study presents an image-guided PDT and chemotherapeutic platform based on h/CDs@CPT NVs, which will be an attractive candidate for future cancer treatment.

Characteristics of lymphocyte subset alterations in COVID-19 patients with different levels of disease severity

BackgroundCoronavirus disease 2019 (COVID-19) is a respiratory disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which had rapidly spread all over the world and caused public health emergencies in the past two years. Although the diagnosis and treatment for COVID-19 have been well defined, the immune cell characteristics and the key lymphocytes subset alterations in COVID-19 patients have not been thoroughly investigated.MethodsThe levels of immune cells including T cells, B cells, and natural killer (NK) cells in 548 hospitalized COVID-19 patients, and 30 types of lymphocyte subsets in 125 hospitalized COVID-19 patients admitted to Wuhan Huoshenshan Hospital of China were measured using flow cytometry. The relationship between lymphocytes subsets with the cytokine interleukin-6 (IL-6) and the characteristics of lymphocyte subsets in single-cell RNA sequencing (scRNA-seq) data obtained from peripheral blood mononuclear cells (PBMCs) were also analysed in COVID-19 patients.ResultsIn this study, we found that patients with critical COVID-19 infection exhibited an overall decline in lymphocytes including CD4+ T cells, CD8+ T cells, total T cells, B cells, and NK cells compared to mild and severe patients. However, the number of lymphocyte subsets, such as CD21low CD38low B cells, effector T4 cells, and PD1+ depleted T8 cells, was moderately increased in critical COVID-19 patients compared to mild cases. Notably, except for effector memory T4 cells, plasma blasts and Tregs, the number of all lymphocyte subsets was markedly decreased in COVID-19 patients with IL-6 levels over 30-fold higher than those in healthy cases. Moreover, scRNA-seq data showed obvious differences in the distribution and numbers of lymphocyte subsets between COVID-19 patients and healthy persons, and subsets-specific marker genes of lymphocyte subsets including CD4, CD19, CCR7, and IL7R, were markedly decreased in COVID-19 patients compared with those in healthy cases.ConclusionA comprehensive decrease in immune cell and lymphocyte subsets in critical COVID-19 patients, and peripheral lymphocyte subset alterations showed a clear association with clinical characteristics.

Humanized T Cell Receptor-like Antibody Hu8F4 Reduces Leukemia By Fc-Mediated Mechanism

8F4, a T cell receptor (TCR) - like antibody which targets the leukemia-associated antigen PR1 (VLQELNVTV) when presented in the context of MHC class I (HLA-A2:01), is a mouse IgG2a monoclonal antibody (mAb) which mediates both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and eliminates AML in PDX (patient derived xenograft) mouse models. Recognizing the potential therapeutic value of 8F4, we produced a humanized IgG1 (Hu8F4) as a first-in-class mAb for preclinical development and clinical use. Characterization of Hu8F4 by ELISA and surface plasmon resonance showed high affinity (KD=6.5 nM) and specificity of Hu8F4 binding to PR1/HLA-A2 monomer. Staining of peptide-pulsed T2 cells confirmed Hu8F4 specificity to PR1, but not to control peptide MART1. To study anti-leukemia activity of Hu8F4 we used HLA-A2-transfected leukemia cell lines U937 (U937-A2) and MV411 (MV411-A2), as well as THP1 (endogenous HLA-A2+) as targets. In contrast to mouse 8F4, Hu8F4 did not induce CDC in vitro. Rather, Hu8F4 mediated both potent antibody-dependent cellular phagocytosis (ADCP) and ADCC of U937-A2 and THP1 cells in NFAT reporter assays, using FcgRI- (EC50 = 0.002 µg/ml), FcgRII- (EC50 = 1.49 µg/ml), and FcgRIII- (EC50 = 0.04 µg/ml) transfected Jurkat cells, as well as pooled human PBMC (EC50 = 0.95 µg/ml) and donor-derived macrophages (EC50 = 1.1 µg/ml), as effectors. Based on the ADCP activity of Hu8F4, we sought to examine the potential synergy of anti-CD47 antibody, a macrophage checkpoint inhibitor, with Hu8F4. Treatment of U937-A2 and THP1 target cells with anti-CD47 F(ab')2 neutralizing antibody made cells susceptible to Hu8F4-mediated phagocytosis by NSG mouse bone marrow derived macrophages (BMDM) in vitro. To confirm the critical role of CD47 in Hu8F4-mediated ADCP we created CD47 KO U937-A2 and THP1 AML lines. Within 96 hours of treatment with Hu8F4 both CD47 KO cell lines were completely phagocytosed by NSG BMDM, thus confirming the role of phagocytosis as a mechanism. To test Hu8F4 activity in vivo, we established xenografts using luciferase-transfected U937-A2 and MV411-A2 cell lines in NSG mice. Hu8F4 was then administered starting on either day 3 (U937-A2) or day 7 (MV411-A2). Treatment with Hu8F4 resulted in elimination of leukemia as measured by FACS and BLI and increased median survival (from 33 days to >85 days for U937-A2, p=0.0075; and from 125 days to >225 days for MV411-A2, p=0.0033). To further test Hu8F4 efficacy, we established PDX in NSG mice using cells from HLA-A2+ refractory/relapsed AML patients. Hu8F4 treatment reduced leukemia by 90.3% to 99.9% when assessed by FACS in four out of five PDX and resulted in increased median survival of the mice (from 41 days to 49 days, p=0.0002) in one PDX tested. To study the mechanism of action of Hu8F4, we produced a 3 aa mutant (Hu8F4 PG LALA), which completely abrogated binding to Fcg receptors. Hu8F4 PG LALA, as well as Hu8F4 F(ab')2, showed no in vivo activity against U937-A2 or MV411-A2 xenografts, confirming that the anti-leukemia activity of Hu8F4 is dependent on its ability to bind Fc receptors. Because FcgR may be expressed on AML we tested fratricide as a possible in vivo mechanism of Hu8F4. We utilized the CRISPR /Cas9 system to edit out both FcgRI and II in the U937-A2 cell line that is naturally deficient in FcgRIII (DKO U937-A2). We then used a retroviral system to stably express FcgRI or FcgRII in U937-A2 to create FcgRI-U937-A2 and FcgRII-U937-A2, respectively, and tested their susceptibility to Hu8F4 in the xenograft model. Compared to FcR-expressing wild-type U937-A2, Hu8F4 showed similar anti-leukemia activity in all three xenografts. There was no significant difference noted in the anti-leukemia potency of Hu8F4 between DKO U937-A2 or single FcR-expressing xenografts compared with FcgRI/FcgRII expressing wild-type U937-A2 xenografts, suggesting that fratricide is an unlikely mechanism in this model. Here we demonstrate high specificity, affinity, and remarkable anti-AML activity of Hu8F4, a first-in-class humanized TCR mimic monoclonal antibody that is currently being tested in a phase I clinical trial for HLA-A2+ refractory/relapsed AML and MDS. This study provides evidence for the importance of FcgR-mediated mechanisms (ADCC and ADCP) in the activity of Hu8F4. In addition, our results support future combination trials with treatments that promote phagocytosis.

Osteopontin: An important protein in the formation of kidney stones.

The incidence of kidney stones averages 10%, and the recurrence rate of kidney stones is approximately 10% at 1year, 35% at 5years, 50% at 10years, and 75% at 20years. However, there is currently a lack of good medicines for the prevention and treatment of kidney stones. Osteopontin (OPN) is an important protein in kidney stone formation, but its role is controversial, with some studies suggesting that it inhibits stone formation, while other studies suggest that it can promote stone formation. OPN is a highly phosphorylated protein, and with the deepening of research, there is growing evidence that it promotes stone formation, and the phosphorylated protein is believed to have adhesion effect, promote stone aggregation and nucleation. In addition, OPN is closely related to immune cell infiltration, such as OPN as a pro-inflammatory factor, which can activate mast cells (degranulate to release various inflammatory factors), macrophages (differentiated into M1 macrophages), and T cells (differentiated into T1 cells) etc., and these inflammatory cells play a role in kidney damage and stone formation. In short, OPN mainly exists in the phosphorylated form in kidney stones, plays an important role in the formation of stones, and may be an important target for drug therapy of kidney stones.

Low perforin expression in CD8+ T lymphocytes during the acute phase of severe SARS-CoV-2 infection predicts long COVID.

T cell cytotoxicity plays a major role in antiviral immunity. Anti-SARS-CoV-2 immunity may determine acute disease severity, but also the potential persistence of symptoms (long COVID). We therefore measured the expression of perforin, a cytotoxic mediator, in T cells of patients recently hospitalized for SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to Intensive Care Units (ICUs) or non-ICU, and 29 age- and sex-matched healthy controls (HCs). Amounts of intracellular perforin and granzyme-B, as well as cell surface expression of the degranulation marker CD107A were determined by flow cytometry. The levels of 15 cytokines in plasma were measured by Luminex. The frequency of perforin-positive T4 cells and T8 cells was higher in patients than in HCs (9.9 ± 10.1% versus 4.6 ± 6.4%, p = 0.006 and 46.7 ± 20.6% vs 33.3 ± 18.8%, p = 0.004, respectively). Perforin expression was neither correlated with clinical and biological markers of disease severity nor predictive of death. By contrast, the percentage of perforin-positive T8 cells in the acute phase of the disease predicted the onset of long COVID one year later. A low T8 cytotoxicity in the first days of SARS-CoV-2 infection might favor virus replication and persistence, autoimmunity, and/or reactivation of other viruses such as Epstein-Barr virus or cytomegalovirus, paving the way for long COVID. Under this hypothesis, boosting T cell cytotoxicity during the acute phase of the infection could prevent delayed sequelae.

Bcl-xL activity influences outcome of the mitotic arrest.

Microtubule-targeting (MT) drugs taxanes and vinca alkaloids are widely used as chemotherapeutic agents against different tumors for more than 30 years because of their ability to block mitotic progression by disrupting the mitotic spindle and activating the spindle assembly checkpoint (SAC) for a prolonged period of time. However, responses to mitotic arrest are different—some cells die during mitotic arrest, whereas others undergo mitotic slippage and survive becoming able for proliferation. Using normal fibroblasts and several cancer cell types we determined two critical doses, T1 and T2, of mitotic inhibitors (nocodazole, Taxol, and vinorelbine). T1 is the maximal dose cells can tolerate undergoing normal division, and T2 is the minimal mitostatic dose, wherein > 90% of mitotic cells are arrested in mitosis. In all studied cell lines after treatment with mitotic inhibitors in a dose above T2 cells had entered mitosis either die or undergo mitotic slippage. We show that for all three drugs used cell death during mitotic arrest and after slippage proceeded via mitochondria-dependent apoptosis. We determined two types of cancer cells: sensitive to mitotic arrest, that is, undergoing death in mitosis (DiM) frequently, and resistant to mitotic arrest, that is, undergoing mitotic slippage followed by prolonged survival. We then determined that inhibition of Bcl-xL, but not other anti-apoptotic proteins of the Bcl-2 group that regulate MOMP, make resistant cells susceptible to DiM induced by mitotic inhibitors. Combined treatment with MT drugs and highly specific Bcl-xL inhibitors A-1155643 or A-1331852 allows achieving 100% DiM in a time significantly shorter than maximal duration of mitotic arrest in all types of cultured cells tested. We further examined efficacy of sequential treatment of cultured cells using mitotic inhibitors followed by inhibitors of Bcl-xL anti-apoptotic protein and for the first time show that sensitivity to Bcl-xL inhibitors rapidly declines after mitotic slippage. Thus sequential use of mitotic inhibitors and inhibitors of Bcl-xL anti-apoptotic protein will be efficient only if the Bcl-xL inhibitor will be added before mitotic slippage occurs or soon afterward. The combined treatment proposed might be an efficient approach to anti-cancer therapy.

Abstract A017: Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor

Abstract Introduction: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit receptor. While highly effective, tyrosine kinase inhibitors like imatinib are not curative. The natural ligand for the Kit receptor is Kit Ligand (KitL), which exists in both soluble and membrane-bound forms. We sought to determine the role of KitL in the oncogenic Kit signaling of GIST. Methods: KitV558Δ/+ mice, which spontaneously develop an intestinal GIST, were treated with imatinib or vehicle for 1 week, and sorted non-immune (CD45-) cells from tumors were submitted for single-cell RNA sequencing (n=3/group). GIST T1 cells were treated with KitL with or without imatinib and oncogenic Kit signaling was evaluated. KitV558Δ/+ mice were crossed with various KitL mutant and inducible models and tumor weights were measured (n=3-4/group). Human GISTs were stained for KitL expression by IHC, and an existing bulk RNA sequencing human GIST dataset with matched human serum was analyzed. Results: Unsupervised clustering revealed endothelial, smooth muscle, and tumor cells had high expression of KitL RNA in untreated tumors from KitV558Δ/+ mice. Imatinib therapy increased KitL RNA in all 3 cell types, suggesting that extra-tumoral KitL expression is dependent on oncogenic signaling (p&amp;lt;0.05). In GIST T1 cells, exogenous KitL increased Kit activation and reduced imatinib’s efficacy. Tumors from mice with homozygous KitL deletion (KitV558Δ/+;Etv1Cre-ERT2/+;KitLflox/flox) were smaller than tumors from littermates with heterozygous KitL deletion (KitV558Δ /+;Etv1Cre-ERT2/+;KitLflox/+), providing evidence that KitL contributes to GIST development in vivo despite the presence of mutated Kit (p&amp;lt;0.05). Furthermore, tumors from KitV558Δ/+;KitLKitL1Δ/KitL1Δ mice, which lack the proteolytic cleavage site for soluble KitL, were smaller than matched KitV558Δ/+ mice, indicating that soluble KitL is important for maximal tumor growth in vivo (p&amp;lt;0.05). Heterogeneous KitL expression was confirmed in human GISTs by IHC. Human serum contained more KitL in patients with tumors resistant to imatinib and in those with tumors expressing more KitL RNA(p&amp;lt;0.05). Conclusions: KitL is produced by multiple cell types within GIST and contributes to oncogenesis. Targeting KitL may be important for optimal tumor therapy. Citation Format: Andrew D. Tieniber, Ferdinando Rossi, Andrew Hanna, Mengyuan Liu, Mark Etherington, Kevin Do, Laura Wang, Ronald P. DeMatteo. Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A017.

Effect of Thermal Ablation on Growth Plates: A Study to Explore the Thermal Threshold of Rabbit Growth Plates During Microwave Ablation.

To explore the temperature threshold of thermal damage to growth plates. Nine rabbits were divided into three groups for femoral ablation, exposing the growth plate to different temperatures (T1 = 43-45°C; T2 = 46-48°C; T3 = 49-51°C). After 5weeks, the changes in the femurs were assessed by macroscopic images, micro-CT, haematoxylin and eosin staining, and immunohistochemistry of Col2a1 (type II collagen). At the cellular level, rabbit epiphyseal chondrocytes were exposed to 37°C, 44°C, 47°C and 50°C for 5min. Then, proliferation and chondrogenic differentiation were detected. The rabbits in the T2 and T3 groups developed length discrepancies and axial deviations of femurs, abnormal newly formed bone in the marrow cavity, disorganized growth plates and decreased Col2a1 expression. At the cellular level, the cells exposed to 47°C and 50°C for 5min showed decreased viability, increased apoptosis, decreased extracellular matrix synthesis and decreased matrix mineralization. However, the changes in rabbits in the T1 group and cells at 44°C did not show a significant difference. The ablation of growth plates at temperatures above 45°C for 5min results in decreased chondrocyte viability and disorganized growth plates, leading to growth disturbances. Further studies are warranted to confirm these promising initial results.

The Understanding of ON-Edge Motion Detection Through the Simulation Based on the Connectome of Drosophila’s Optic Lobe

The optic lobe of the fly is one of the prominent model systems for the neural mechanism of the motion detection. How a fly who lives under various visual situations of the nature processes the information from at most a few thousands of ommatidia in their neural circuit for the detection of moving objects is not exactly clear though many computational models of the fly optic lobe as a moving objects detector were suggested. Here we attempted to elucidate the mechanisms of ON-edge motion detection by a simulation approach based on the TEM connectome of Drosophila. Our simulation model of the optic lobe with the NEURON simulator that covers the full scale of ommatidia, reproduced the characteristics of the receptor neurons, lamina monopolar neurons, and T4 cells in the lobula. The contribution of each neuron can be estimated by changing synaptic connection strengths in the simulation and measuring the response to the motion stimulus. Those show the paradelle pathway provide motion detection in the fly optic lobe has more robustness and is more sophisticated than a simple combination of HR and BL systems.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.