Protein detection in blood with single-molecule imaging.

The ability to characterize individual biomarker protein molecules in patient blood samples could enable diagnosis of diseases at an earlier stage, when treatment is typically more effective. Single-molecule imaging offers a promising approach to accomplish this goal. However, thus far, single-molecule imaging methods have not been translated into the clinical setting. The detection limit of these methods has been confined to the picomolar (10-12 M) range, several orders of magnitude higher than the circulating concentrations of biomarker proteins present in many diseases. Here, we describe single-molecule augmented capture (SMAC), a single-molecule imaging technique to quantify and characterize individual protein molecules of interest down to the subfemtomolar (<10-15 M) range. We demonstrate SMAC in a variety of applications with human blood samples, including the analysis of disease-associated secreted proteins, membrane proteins, and rare intracellular proteins. SMAC opens the door to the application of single-molecule imaging in noninvasive disease profiling.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Ultrasensitive high-resolution profiling of early seroconversion in patients with COVID-19.

Sensitive assays are essential for the accurate identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report a multiplexed assay for the fluorescence-based detection of seroconversion in infected individuals from less than 1 µl of blood, and as early as the day of the first positive nucleic acid test after symptom onset. The assay uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens. A logistic regression model trained using samples collected during the pandemic and samples collected from healthy individuals and patients with respiratory infections before the first outbreak of coronavirus disease 2019 (COVID-19) was 99% accurate in the detection of seroconversion in a blinded validation cohort of samples collected before the pandemic and from patients with COVID-19 five or more days after a positive nasopharyngeal test by PCR with reverse transcription. The high-throughput serological profiling of patients with COVID-19 allows for the interrogation of interactions between antibody isotypes and viral proteins, and should help us to understand the heterogeneity of clinical presentations.

TLR9 acts as a sensor for tumor-released DNA to modulate anti-tumor immunity after chemotherapy.

The tumor microenvironment exists in a state of dynamic equilibrium, in which a balance of agonist and antagonist signals govern the anti-tumor immune responses. Previous studies have shown that chemotherapy could shift this balance in favor of agonistic signals for the anti-tumor immune responses mounted by CD8+ cytotoxic T lymphocytes (CTL), providing sufficiently high antigen density within the tumor. We undertook the current study to characterize the anti-tumor immune response following chemotherapy and its underlying mechanisms. We show that this 'adjuvant effect' of chemotherapy is, at least partially, mediated by the release of tumor DNA and acts through the Toll-like receptor 9 (TLR9) pathway. We found that tumor-released DNA causes accumulation, antigen uptake, and maturation of dendritic cells (DCs) in the tumor in a TLR9-dependent manner. These DCs subsequently migrate into the draining lymph nodes and prime tumor-specific CTLs. Our study provides novel insights to the molecular and cellular mechanisms by which chemotherapy converts the tumor microenvironment into a site permissive for the activation of a potent tumor-specific adaptive immune response.

Mitochondrial reprogramming via ATP5H loss promotes multimodal cancer therapy resistance.

The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1α stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer.

Programmed self-assembly of peptide-major histocompatibility complex for antigen-specific immune modulation.

A technology to prime desired populations of T cells in the body-particularly those that possess low avidity against target antigen-would pave the way for the design of new types of vaccination for intractable infectious diseases or cancer. Here, we report such a technology based on positive feedback-driven, programmed self-assembly of peptide-major histocompatibility complex (pMHC) directly on the membrane of cognate T cells. Our design capitalizes on the unique features of the protein annexin V (ANXA5), which-in a concerted and synergistic manner-couples the early onset of TCR signaling by cognate pMHC with a surge in pMHC-TCR affinity, with repeated pMHC encounters, and with widespread TCR cross-linking. In our system, ANXA5 is linked to pMHC and firmly engages the plasma membrane of cognate T cells upon (and only upon) the early onset of TCR signaling. ANXA5, in turn, exerts a mechanical force that stabilizes interactions at the TCR-pMHC interface and facilitates repeated, serial pMHC encounters. Furthermore, ANXA5 quickly arranges into uniform 2D matrices, thereby prompting TCR cross-linking. Fusion of ANXA5 to pMHC augments lymphocyte activation by several orders of magnitude (>1,000-fold), bypasses the need for costimulation, and breaks tolerance against a model self-antigen in vivo. Our study opens the door to the application of synthetic, feedback-driven self-assembly platforms in immune modulation.

HDAC1 Upregulation by NANOG Promotes Multidrug Resistance and a Stem-like Phenotype in Immune Edited Tumor Cells.

Cancer immunoediting drives the adaptation of tumor cells to host immune surveillance. Immunoediting driven by antigen (Ag)-specific T cells enriches NANOG expression in tumor cells, resulting in a stem-like phenotype and immune resistance. Here, we identify HDAC1 as a key mediator of the NANOG-associated phenotype. NANOG upregulated HDAC1 through promoter occupancy, thereby decreasing histone H3 acetylation on K14 and K27. NANOG-dependent, HDAC1-driven epigenetic silencing of cell-cycle inhibitors CDKN2D and CDKN1B induced stem-like features. Silencing of TRIM17 and NOXA induced immune and drug resistance in tumor cells by increasing antiapoptotic MCL1. Importantly, HDAC inhibition synergized with Ag-specific adoptive T-cell therapy to control immune refractory cancers. Our results reveal that NANOG influences the epigenetic state of tumor cells via HDAC1, and they encourage a rational application of epigenetic modulators and immunotherapy in treatment of NANOG+ refractory cancer types. Cancer Res; 77(18); 5039-53. ©2017 AACR.

Local administration of granulocyte macrophage colony-stimulating factor induces local accumulation of dendritic cells and antigen-specific CD8+ T cells and enhances dendritic cell cross-presentation.

Immunotherapy has emerged as a promising treatment strategy for the control of HPV-associated malignancies. Various therapeutic HPV vaccines have elicited potent antigen-specific CD8+ T cell mediated antitumor immune responses in preclinical models and are currently being tested in several clinical trials. Recent evidence indicates the importance of local immune activation, and higher number of immune cells in the site of lesion correlates with positive prognosis. Granulocyte macrophage colony-stimulating factor (GMCSF) has been reported to posses the ability to induce migration of antigen presentation cells and CD8+ T cells. Therefore, in the current study, we employ a combination of systemic therapeutic HPV DNA vaccination with local GMCSF application in the TC-1 tumor model. We show that intramuscular vaccination with CRT/E7 DNA followed by GMCSF intravaginal administration effectively controls cervicovaginal TC-1 tumors in mice. Furthermore, we observe an increase in the accumulation of E7-specific CD8+ T cells and dendritic cells in vaginal tumors following the combination treatment. In addition, we show that GMCSF induces activation and maturation in dendritic cells and promotes antigen cross-presentation. Our results support the clinical translation of the combination treatment of systemic therapeutic vaccination followed by local GMCSF administration as an effective strategy for tumor treatment.

Gain of HIF-1α under normoxia in cancer mediates immune adaptation through the AKT/ERK and VEGFA axes.

PURPOSE: Adaptation to host immune surveillance is now recognized as a hallmark of cancer onset and progression, and represents an early, indispensable event in cancer evolution. This process of evolution is first instigated by an immune selection pressure imposed by natural host surveillance mechanisms and may then be propagated by vaccination or other types of immunotherapy. EXPERIMENTAL DESIGN: We developed a system to simulate cancer evolution in a live host and to dissect the mechanisms responsible for adaptation to immune selection. Here, we show that the oxygen-sensitive α subunit of hypoxia-inducible factor 1 (HIF-1α) plays a central role in cancer immune adaptation under conditions of normal oxygen tension. RESULTS: We found that tumor cells gain HIF-1α in the course of immune selection under normoxia and that HIF-1α renders tumor cells resistant to lysis by tumor-specific cytotoxic T lymphocytes (CTL) in culture and in mice. The effects of HIF-1α on immune adaptation were mediated through VEGFA-dependent activation of the AKT and ERK signaling pathways, which induced an antiapoptotic gene expression network in tumor cells. CONCLUSIONS: Our study therefore establishes a link between immune selection, overexpression of HIF-1α, and cancer immune adaptation under normoxia, providing new opportunities for molecular intervention in patients with cancer.

Immune-mediated tumor evolution: Nanog links the emergence of a stem like cancer cell state and immune evasion.

Tumor cells undergo molecular evolution under immune pressure. Using a murine metastatic lung cancer model, we recently reported that evolutionary pressure enforced through vaccination incites gain of Nanog, a master transcription factor that mediates both emergence of a stem-like cancer cell state and immune evasion. Thus, therapeutic strategies aiming to blunt NANOG's expression in patient tumors may improve the clinical management of cancer.

Chemotherapy acts as an adjuvant to convert the tumor microenvironment into a highly permissive state for vaccination-induced antitumor immunity.

Multiple classes of pharmacologic agents have the potential to induce the expression and release of proinflammatory factors from dying tumor cells. As a result, these cells can in theory elicit an immune response through various defined mechanisms to permanently eradicate disseminated cancer. However, the impact of chemotherapy on the tumor-specific immune response in the context of the tumor microenvironment is largely unknown. Within the tumor microenvironment, the immune response promoted by chemotherapy is antagonized by an immune-suppressive milieu, and the balance of these opposing forces dictates the clinical course of disease. Here, we report that high antigen exposure within the tumor microenvironment following chemotherapy is sufficient to skew this balance in favor of a productive immune response. In elevating antigen exposure, chemotherapy can achieve long-term control of tumor progression without the need of an additional adjuvant. We found that chemotherapy initiated this phenomenon in the tumor microenvironment through an accumulation of dendritic cells, which stimulated CD8(+) T cells and the type I IFN pathway. From this conceptual base, we developed a simple approach to cancer therapy combining chemotherapy and vaccination that may be widely applicable.

Innovative DNA vaccine to break immune tolerance against tumor self-antigen.

Vaccination is, in theory, a safe and effective approach for controlling disseminated or metastatic cancer due to the specificity of the mammalian immune system, yet its success in the clinic has been hampered thus far by the problem of immune tolerance to tumor self-antigen. Here we describe a DNA vaccination strategy that is able to control cancer by overcoming immune tolerance to tumor self-antigen. We engineered a DNA construct encoding a dimeric form of a secreted single-chain trimer of major histocompatibility complex class I heavy chain, ß2-microglobulin, and peptide antigen linked to immunoglobulin G (SCT-Ag/IgG). The chimeric protein was able to bind to antigen-specific CD8(+) T cells with nearly 100% efficiency and strongly induce their activation and proliferation. In addition, the chimeric protein was able to coat professional antigen-presenting cells through the F(c) receptor to activate antigen-specific CD8(+) T cells. Furthermore, intradermal vaccination with DNA-encoding SCT-Ag/IgG could generate significant numbers of cytotoxic effector T cells against tumor self-antigen and leads to successful therapeutic outcomes in a preclinical model of metastatic melanoma. Our data suggest that the DNA vaccine strategy described in the current study is able to break immune tolerance against endogenous antigen from melanoma and result in potent therapeutic antitumor effects. Such strategy may be used in other antigenic systems for the control of infections and/or cancers.

Nanog signaling in cancer promotes stem-like phenotype and immune evasion.

Adaptation of tumor cells to the host is a major cause of cancer progression, failure of therapy, and ultimately death. Immune selection drives this adaptation in human cancer by enriching tumor cells with a cancer stem cell-like (CSC-like) phenotype that makes them resistant to CTL-mediated apoptosis; however, the mechanisms that mediate CSC maintenance and proliferation are largely unknown. Here, we report that CTL-mediated immune selection drives the evolution of tumor cells toward a CSC-like phenotype and that the CSC-like phenotype arises through the Akt signaling pathway via transcriptional induction of Tcl1a by Nanog. Furthermore, we found that hyperactivation of the Nanog/Tcl1a/Akt signaling axis was conserved across multiple types of human cancer. Inhibition of Nanog in a murine model of colon cancer rendered tumor cells susceptible to immune-mediated clearance and led to successful, long-term control of the disease. Our findings establish a firm link among immune selection, disease progression, and the development of a stem-like tumor phenotype in human cancer and implicate the Nanog/Tcl1a/Akt pathway as a central molecular target in this process.

Tumor-targeted delivery of IL-2 by NKG2D leads to accumulation of antigen-specific CD8+ T cells in the tumor loci and enhanced anti-tumor effects.

Interleukin-2 (IL-2) has been shown to promote tumor-specific T-cell proliferation and differentiation but systemic administration of IL-2 results in significant toxicity. Therefore, a strategy that can specifically deliver IL-2 to the tumor location may alleviate concerns of toxicity. Because NKG2D ligands have been shown to be highly expressed in many cancer cells but not in healthy cells, we reason that a chimeric protein consisting of NKG2D linked to IL-2 will lead to the specific targeting of IL-2 to the tumor location. Therefore, we created chimeric proteins consisting of NKG2D linked to Gaussia luciferase (GLuc; a marker protein) or IL-2 to form NKG2D-Fc-GLuc and NKG2D-Fc-IL2, respectively. We demonstrated that NKG2D linked to GLuc was able to deliver GLuc to the tumor location in vivo. Furthermore, we showed that TC-1 tumor-bearing mice intramuscularly injected with DNA encoding NKG2D-Fc-IL2, followed by electroporation, exhibited an increased number of luciferase-expressing E7-specific CD8+ T cells at the tumor location. More importantly, treatment with the DNA construct encoding NKG2D-Fc-IL2 significantly enhanced the therapeutic anti-tumor effects generated by intradermal vaccination with therapeutic HPV DNA in tumor-bearing mice. Therefore, by linking NKG2D to IL2, we are able to specifically deliver IL-2 to the tumor location, enhancing antigen-specific T-cell immune response and controlling tumor growth. Our approach represents a platform technology to specifically deliver proteins of interest to tumor loci.

Cancer vaccination drives Nanog-dependent evolution of tumor cells toward an immune-resistant and stem-like phenotype.

Due to the exquisite specificity and potency of the immune system, vaccination is in theory the most precise and powerful approach for controlling cancer. However, current data from clinical trials indicate that vaccination rarely yields significant benefits for cancer patients in terms of tumor progression and long-term survival. The poor clinical outcomes of vaccination are primarily caused by mechanisms of immune tolerance, especially within the tumor microenvironment. Here, we report that vaccination drives the evolution of tumor cells toward an immune-resistant and stem-like phenotype that promotes tumor growth and nullifies the CTL response. The emergence of this phenotype required the transcription factor Nanog, which is induced as a consequence of immune selection. Nanog expression enhanced the stem-like features of tumor cells and protected them from killing by tumor-reactive CTLs. Delivery of siNanog into tumor-bearing mice rendered the tumor vulnerable to immune surveillance and strongly suppressed its growth. Together, our findings show tumor adaptation to vaccination through gain of an immune-resistant, stem-like phenotype and identify Nanog as a central molecular target in this process. Future vaccination technology should consider Nanog an important target to enhance the immunotherapeutic response.

In vivo microRNA-155 expression influences antigen-specific T cell-mediated immune responses generated by DNA vaccination.

BACKGROUND: MicroRNA (miRNA) molecules are potent mediators of post-transcriptional gene silencing that are emerging to be critical in the regulation of innate and adaptive immunity. RESULTS: Here we report that miR-155--an oncogenic miRNA with important function in the mammalian immune system--is induced in dendritic cells (DCs) upon maturation and potentially attenuates their ability to activate T cells. Biolistic epidermal transfection with DNA encoding miR-155 suppressed the induction of antigen-specific T cell-mediated immunity, whereas reduction of endogenous miR-155 by a partially complementary antisense sequence reversed this effect. Because DCs represent a significant component of epidermal tissue and are among the most potent of antigen-presenting cells, the inhibitory actions of miR-155 could be mediated through this subset of cells. CONCLUSIONS: These results suggest that miR-155 may repress the expression of key molecules involved in lymph node migration, antigen presentation, or T cell activation in DCs, and thus forms part of a negative regulatory pathway that dampens the generation of T cell-mediated immune responses. Modulation of miR-155 expression in epidermis therefore represents a potentially promising form of gene therapy for the control of diseases ranging from autoimmunity to cancer and viral infection.

Inhibitory RNA molecules in immunotherapy for cancer.

Over the past few decades, our expanding knowledge of the mammalian immune system - how it is developed, activated, and regulated - has fostered hope that it may be harnessed in the future to successfully treat human cancer. The immune system activated by cancer vaccines may have the unique ability to selectively eradicate tumor cells at multiple sites in the body without inflicting damage on normal tissue. However, progress in the development of cancer vaccines that effectively capitalize on this ability has been limited and slow. The immune system is restrained by complex, negative feedback mechanisms that evolved to protect the host against autoimmunity and may also prevent antitumor immunity. In addition, tumor cells exploit a plethora of strategies to evade detection and elimination by the immune system. For these reasons, the field of cancer immunotherapy has suffered considerable setbacks in the past and faces great challenges at the present time. Some of these challenges may be overcome through the use of RNA interference, a process by which gene expression can be efficiently and specifically "knocked down" in cells. This chapter focuses on the current status and future prospects in the application of small interfering RNA and microRNA, two main forms of RNA interference, to treat cancer by curtailing mechanisms that attenuate the host immune response.

Combined administration with DNA encoding vesicular stomatitis virus G protein enhances DNA vaccine potency.

DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8(+) T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)-together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen-elicited robust E7-specific CD8(+) T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.

Treatment with LL-37 peptide enhances antitumor effects induced by CpG oligodeoxynucleotides against ovarian cancer.

There is an urgent need for innovative therapies against ovarian cancer, one of the leading causes of death from gynecological cancers in the United States. Immunotherapy employing Toll-like receptor (TLR) ligands, such as CpG oligodeoxynucleotides (CpG-ODN), may serve as a potentially promising approach in the control of ovarian tumors. The CpG-ODN requires intracellular delivery into the endosomal compartment, where it can bind to TLR9 in order to activate the immune system. In the current study, we aim to investigate whether the antimicrobial polypeptide from the cathelicidin family, LL-37, could enhance the immunostimulatory effects of CpG-ODN by increasing the uptake of CpG-ODN into the immune cells, thus enhancing the antitumor effects against ovarian cancer. We found that treatment with the combination of CpG-ODN and LL-37 generated significantly better therapeutic antitumor effects and enhanced survival in murine ovarian tumor-bearing mice compared with treatment with CpG-ODN or LL-37 alone. We also observed that treatment with the combination of CpG-ODN and LL-37 enhanced proliferation and activation of natural killer (NK) cells, but not CD4(+) or CD8(+) T cells, in the peritoneal cavity. Furthermore, in vivo antibody depletion experiments indicated that peritoneal NK cells played a critical role in the observed antitumor effects. Thus, our data suggest that the combination of CpG-ODN with LL-37 peptide may lead to the control of ovarian tumors through the activation of innate immunity.

RNA interference-mediated in vivo silencing of fas ligand as a strategy for the enhancement of DNA vaccine potency.

Intradermal administration of DNA vaccines encoding luciferase represents a convenient method to assess gene expression in vivo. Gene silencing by intradermal gene gun administration of DNA encoding short hairpin RNA (shRNA) may represent an effective technique for the specific knockdown of gene expression in vivo. In the current study, we characterized luciferase gene expression over time in vivo by noninvasive bioluminescence imaging. Furthermore, we characterized in vivo luciferase gene silencing with DNA encoding shRNA targeting luciferase. We also characterized human papillomavirus type 16 (HPV-16) E7-specific CD8(+) T cell immune responses in mice immunized with E7 DNA and DNA encoding shRNA targeting Fas ligand (FasL), a key proapoptotic signaling protein. Our results indicated that coadministration of DNA encoding shRNA targeting luciferase significantly reduced luciferase expression in mice intradermally administered luciferase DNA. Furthermore, we observed that mice vaccinated with E7-expressing DNA coadministered with DNA encoding shRNA targeting FasL generated significantly enhanced E7-specific CD8(+) cytotoxic T cell responses as well as potent therapeutic antitumor effects against E7-expressing tumors. Thus, intradermal administration of DNA encoding shRNA represents a plausible approach to silence genes in vivo and a potentially useful tool to enhance DNA vaccine potency.