CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

A non-comparative, randomized, phase II trial of atezolizumab or atezolizumab plus tiragolumab for programmed death-ligand 1-positive recurrent cervical cancer (SKYSCRAPER-04).

OBJECTIVE: To evaluate tiragolumab (anti-TIGIT) and atezolizumab (anti-PD-L1) as second- or third-line therapy for PD-L1-positive persistent/recurrent cervical cancer. METHODS: In the open-label, non-comparative, randomized phase II SKYSCRAPER-04 trial (NCT04300647), patients with PD-L1-positive (SP263 tumor area positivity ≥5%) recurrent/persistent cervical cancer after 1-2 chemotherapy lines (≥1 platinum-based) were randomized 3:1 to atezolizumab 1200 mg with/without tiragolumab 600 mg every 3 weeks until disease progression or unacceptable toxicity. Stratification factors were performance status, prior (chemo)radiotherapy, and disease status. The primary endpoint was independent review committee-assessed confirmed objective response rate per RECIST v1.1 in patients receiving tiragolumab plus atezolizumab. An objective response rate ≥21% (one-sample z-test p≤0.0245) was required for statistical significance versus a historical reference. RESULTS: Protocol-defined independent review committee-assessed objective response rates were 19.0% (95% CI 12.6 to 27.0) in 126 patients receiving tiragolumab plus atezolizumab (p=0.0787 vs historical reference) and 15.6% (95% CI 6.5 to 29.5) in 45 atezolizumab-treated patients. Response rates were higher in PD-L1high (tumor area positivity ≥10%) than PD-L1low (tumor area positivity 5%-9%) subgroups with both regimens. At 8.5 months' median follow-up, independent review committee-assessed progression-free survival was 2.8 months (95% CI 1.7 to 4.1) with tiragolumab plus atezolizumab and 1.9 months (95% CI 1.5 to 3.0) with atezolizumab. In post hoc analyses (10.4 months' median follow-up), median overall survival was 11.1 months (95% CI 9.6 to 14.5) with the combination and 10.6 months (95% CI 6.9 to 13.8) with atezolizumab (crossover permitted). In the combination group, 3% of patients had adverse events requiring treatment discontinuation and 8% had grade ≥3 adverse events of special interest; corresponding values in the single-agent arm were 4% and 11%. There were no treatment-related deaths or new safety findings. CONCLUSION: The objective response rate with the tiragolumab-plus-atezolizumab combination was numerically higher than the historical reference but did not reach statistical significance.

An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins.

Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.

Macrophage-derived CCL23 upregulates expression of T-cell exhaustion markers in ovarian cancer.

BACKGROUND: Macrophages are an important component of the tumour immune microenvironment (TME) and can promote tumour growth and metastasis. Macrophage-secreted chemokine-ligand-23 (CCL23) induces ovarian cancer cell migration via chemokine-receptor 1 (CCR1). However, the effect of CCL23 on other immune cells in the TME is unknown. METHODS: CCL23 levels were measured by ELISA. The expression of surface markers in exhaustion assays was quantified by flow cytometry. Signalling pathways were identified by phosphokinase array and validated by western blot. RESULTS: Ascites from patients with high-grade serous ovarian cancer (HGSC) contain high levels of CCL23. Similarly, significantly higher CCL23 levels were found in plasma from HGSC patients compared to healthy individuals. RNA-seq analysis of ovarian cancer tissues from TCGA showed that expression of CCL23 correlated with the presence of macrophages. In tissues with high levels of CCL23 and macrophage content, the fraction of CD8 + T cells expressing exhaustion markers CTLA-4 and PD-1 were significantly higher compared to low-level CCL23 tissues. In vitro, CCL23 induced upregulation of immune checkpoint proteins on CD8 + T cells, including CTLA-4, TIGIT, TIM-3 and LAG-3 via phosphorylation of GSK3ß in CD8 + T cells. CONCLUSIONS: Our data suggest that CCL23 produced by macrophages contributes to the immune-suppressive TME in ovarian cancer by inducing an exhausted T-cell phenotype.

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.

Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.

Characterization of the cytokine storm reflects hyperinflammatory endothelial dysfunction in COVID-19.

BACKGROUND: Physicians treating patients with coronavirus disease 2019 (COVID-19) increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared with healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. OBJECTIVE: To identify and characterize the host inflammatory response to severe acute respiratory syndrome coronavirus 2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease in patients stratified as mild, moderate, and severe versus matched healthy controls. METHODS: Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. RESULTS: Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within patients with COVID-19, and strong biomarker association with patient response as measured by Ordinal Scale. As patients progress, we observe statistically significant dysregulation of IFN-γ, IL-1RA, IL-6, IL-10, IL-19, monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, CXCL9, CXCL10, CXCL5, ENRAGE, and poly (ADP-ribose) polymerase 1. Furthermore, in a limited series of patients who were sampled frequently, confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. CONCLUSIONS: These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of severe acute respiratory syndrome coronavirus 2 pathogenesis and the host response.

Utility of spinal angiography and arterial embolization in patients undergoing CT guided alcohol injection of aggressive vertebral hemangiomas.

PURPOSE: The purpose of this study is to evaluate the role of spinal angiography and arterial embolization in avoiding spinal cord ischemia in patients undergoing CT-guided alcohol injection of aggressive vertebral hemangiomas. METHODS: In this retrospective study, patients with vertebral hemangioma who underwent CT-guided direct alcohol injection between January 2007 and October 2018 were identified. Of 28 such patients, 26 had neurological deficits, and 2 had only back pain or radiculopathy. Direct alcohol injection without prior arterial embolization was done in 17 patients. Direct alcohol injection with prior arterial embolization was done in 11 patients. Clinical outcome was assessed immediately after the intervention and at follow-up. RESULTS: Three patients, who underwent alcohol injection without trans-arterial embolization, had worsening of neurological deficits in the post procedure period due to spinal cord ischemia. No complications related to spinal cord ischemia were noted in the embolization group. There was no significant difference in the outcomes between the two groups if the three patients with complications are excluded (p = 0.34). CONCLUSION: While CT-guided direct alcohol injection is effective in the management of symptomatic and aggressive vertebral hemangiomas, spinal angiography and trans-arterial embolization of the blood supply to the vertebral body hemangioma, prior to the direct transpedicular alcohol embolization of the lesion, improves the safety of the procedure.

Cell-based immunotherapy in gynecologic malignancies.

PURPOSE OF REVIEW: To provide an update on cell-based immunotherapies in solid tumors particularly in gynecological cancers. RECENT FINDINGS: Recent clinical trial results demonstrate safety and tolerability of different cell therapies in gynecological cancers. Novel approaches, such as harnessing the cells of the innate immune system are also under investigation in a phase I trial. SUMMARY: Cell-based therapies are gaining widespread attention as evidenced by the increasing number of clinical trials encompassing both, innate and adaptive cells to target gynecological cancers. A majority of these therapeutic approaches are well tolerated and show promising results in early trials.

Cell-based immunotherapy in gynecologic malignancies.

PURPOSE OF REVIEW: To provide an update on cell-based immunotherapies in solid tumors particularly in gynecological cancers. RECENT FINDINGS: Improvements have been made in engineering T cells to overcome the immunosuppressive environment in ovarian cancer. Significant efforts are underway to create 'off the shelf' cell therapies which leverage natural killer (NK) cells and would not rely on engineering a patient's T cells. SUMMARY: Efforts to target solid tumors using cell-based therapies are expanding into cell types other than T cells (NK cells and macrophages) which may have a lower risk of significant side effects and higher efficacy in solid tumors than chimeric antigen receptor T cells.

Tumor associated macrophages in gynecologic cancers.

The complex tumor microenvironment in gynecologic cancers plays a major role in modulating anti-tumor immune responses. The interaction of cancer cells with the diverse spectrum of immune effector cells has an important impact on the efficacy of standard chemotherapy and novel immunotherapy approaches. In this review, we specifically focus on the role of macrophages in ovarian, endometrial and cervical cancers. We discuss the origins of macrophages and their polarization state dictated by the microenvironment's cues. Within the tumor niche, tumor-associated macrophages (TAMs) promote tumor growth and mediate immune-suppression thereby effecting treatment responses. We outline clinical strategies that directly target TAMs, including inhibition of macrophage differentiation, prevention of the recruitment of monocytes to the tumor, enhancement of phagocytosis and immune checkpoint blockade.

Cell-based immunotherapy in gynecologic malignancies.

PURPOSE OF REVIEW: To provide an overview of the principles, safety and efficacy of adoptive cell therapy (ACT) in solid tumors particularly in gynecological cancers. RECENT FINDINGS: Efforts to target solid tumors using tumor-infiltrating lymphocytes and genetically modified T cells have shown promising efficacy in some patients. Two food and drug administration approvals for the treatment of leukemia are the first gene therapies available for cancer treatment in the United States. SUMMARY: Genetic engineering of antitumor immunity using T cells has the potential to target specific tumor-associated antigens and overcome obstacles to successful immunotherapy like immune-suppressive factors in the tumor microenvironment.

The role of Xpert MTB/RIF assay in the diagnosis of tubercular spondylodiscitis.

PURPOSE: This study aims to assess the accuracy of the Xpert MTB/RIF assay in the diagnosis of tubercular spondylodiscitis and to identify its role in detecting Rifampicin resistance in patients with infective spondylodiscitis. METHODS: A retrospective study including 348 patients suspected to have infective spondylodiscitis was done. Tissue/pus samples obtained were sent for culture, histopathology and Xpert MTB/RIF assay. All patients who were confirmed to have tubercular spondylodiscitis and those patients who were suspected on clinico-radiological basis were also treated with anti-tuberculous chemotherapy for a period of 9 months. The efficacy of the Xpert MTB/RIF assay was assessed in terms of sensitivity and specificity when compared to culture, histopathology, and Composite reference standard (CRS). RESULTS: During this study period of 24 months, a total of 348 patients were treated for infective spondylodiscitis. 254 patients were treated for tuberculosis following a smear positivity, culture positivity, and histopathology report or empirically based on clinico-radiological findings. The sensitivity and specificity of the Xpert MTB/RIF assay when compared to culture were 88.4 and 63.7%, respectively. When compared to both culture and histopathology reports it was 80.9 and 80.6%. The sensitivity and specificity of the Xpert MTB/RIF assay when compared to composite reference standard were 71.2 and 100%, respectively. The sensitivity of the assay to detect Rifampicin resistance was 100%. The prevalence of Rifampicin resistance was 5.1%. CONCLUSION: This study recommends Xpert MTB/RIF assay for early detection of Mycobacterium tubercular spondylodiscitis and Rifampicin resistance.

Vitamin D and/or calcium deficient diets may differentially affect muscle fiber neuromuscular junction innervation.

INTRODUCTION: There is evidence that supports a role for Vitamin D (Vit. D) in muscle. The exact mechanism by which Vit. D deficiency impairs muscle strength and function is not clear. METHODS: Three-week-old mice were fed diets with varied combinations of Vit. D and Ca2+ deficiency. Behavioral testing, genomic and protein analysis, and muscle histology were performed with a focus on neuromuscular junction (NMJ) -related genes. RESULTS: Vit. D and Ca2+ deficient mice performed more poorly on given behavioral tasks than animals with Vit. D deficiency alone. Genomic and protein analysis of the soleus and tibialis anterior muscles revealed changes in several Vit. D metabolic, NMJ-related, and protein chaperoning and refolding genes. CONCLUSIONS: These data suggest that detrimental effects of a Vit. D deficient or a Vit. D and Ca2+ deficient diet may be a result of differential alterations in the structure and function of the NMJ and a lack of a sustained stress response in muscles. Muscle Nerve 54: 1120-1132, 2016.

Targeting estrogen receptor ß in microglia and T cells to treat experimental autoimmune encephalomyelitis.

A therapeutic goal in the treatment of certain CNS diseases, including multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson disease, is to down-regulate inflammatory pathways. Inflammatory molecules produced by microglia are responsible for removal of damaged neurons, but can cause collateral damage to normal neurons located close to defective neurons. Although estrogen can inactivate microglia and inhibit the recruitment of T cells and macrophages into the CNS, there is controversy regarding which of the two estrogen receptors (ERs), ERα or ERß, mediates the beneficial effects in microglia. In this study, we found that ERß, but not ERα, is expressed in microglia. Using the experimental autoimmune encephalomyelitis (EAE) model in SJL/J mice, we evaluated the benefit of an ERß agonist as a modulator of neuroinflammation. Treatment of EAE mice with LY3201, a selective ERß agonist provided by Eli Lilly, resulted in marked reduction of activated microglia in the spinal cord. LY3201 down-regulated the nuclear transcription factor NF-κB, as well as the NF-κB-induced gene inducible nitric oxide synthase in microglia and CD3(+) T cells. In addition, LY3201 inhibited T-cell reactivity through regulation of indoleamine-2,3-dioxygenase. In the EAE model, treatment with LY3201 decreased mortality in the first 2 wk after disease onset, and also reduced the severity of symptoms in mice surviving for 4 wk. Our data show that ERß-selective agonists, by modulating the immune system in both microglia and T cells, offer promise as a useful class of drugs for treating degenerative diseases of the CNS.

Three-Dimensional in Vitro Model to Study Osteobiology and Osteopathology.

The bone is an amazing organ that grows and remodels itself over a lifetime. It is generally accepted that bone sculpting in response to stress and force is carried out by groups of cells contained within bone multicellular units that are coordinated to degrade existing bone and form new bone. Because of the nature of bone and the extensiveness of the skeleton, it is difficult to study bone remodeling in vivo. On the other hand, because the bone contains a complex environment of many cell types, is it possible to study bone remodeling in vitro? We propose that one can at minimum study the interaction between osteoblasts (bone formation) and osteoclasts (bone degradation) in a three dimensional (3D) "bioreactor". Furthermore, one can add bone degrading metastatic cancer cells, and study how they contribute to and take part in the bone degradation process. We have primarily cultured and differentiated MC3T3-E1 osteoblasts for long periods (2-10 months) before addition of bone marrow osteoclasts and/or metastatic (MDA-MB-231), metastasis suppressed (MDA-MB-231BRMS1) or non-metastatic (MCF-7) breast cancer cells. In the co-culture of osteoblasts and osteoclasts there was clear evidence of matrix degradation. Loss of matrix was also evident after co-culture with metastatic breast cancer cells. Tri-culture permitted an evaluation of the interaction of the three cell types. The 3D system holds promise for further studies of cancer dormancy, hormone, and cytokine effects and matrix manipulation.

Inhibition of activin A ameliorates skeletal muscle injury and rescues contractile properties by inducing efficient remodeling in female mice.

Activin A, a member of the transforming growth factor-ß superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

Reduction of dendritic spines and elevation of GABAergic signaling in the brains of mice treated with an estrogen receptor ß ligand.

An estrogen receptor (ER) ß ligand (LY3201) with a preference for ERß over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17ß is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD(+) terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP(+) astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERß was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERß agonist results in changes opposite to those reported in estradiol-17ß-treated mice and suggests that ERα and ERß play opposing roles in the brain.

In vitro mimics of bone remodeling and the vicious cycle of cancer in bone.

Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the "vicious cycle" of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue, it is difficult to study this system in vivo. In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases.