Investigations of hypoxia-induced deoxyhemoglobin as a contrast agent for cerebral perfusion imaging.

The assessment of resting perfusion measures (mean transit time, cerebral blood flow, and cerebral blood volume) with magnetic resonance imaging currently requires the presence of a susceptibility contrast agent such as gadolinium. Here, we present an initial comparison between perfusion measures obtained using hypoxia-induced deoxyhemoglobin and gadolinium in healthy study participants. We hypothesize that resting cerebral perfusion measures obtained using precise changes of deoxyhemoglobin concentration will generate images comparable to those obtained using a clinical standard, gadolinium. Eight healthy study participants were recruited (6F; age 23-60). The study was performed using a 3-Tesla scanner with an eight-channel head coil. The experimental protocol consisted of a high-resolution T1-weighted scan followed by two BOLD sequence scans in which each participant underwent a controlled bolus of transient pulmonary hypoxia, and subsequently received an intravenous bolus of gadolinium. The resting perfusion measures calculated using hypoxia-induced deoxyhemoglobin and gadolinium yielded maps that looked spatially comparable. There was no statistical difference between methods in the average voxel-wise measures of mean transit time, relative cerebral blood flow and relative cerebral blood volume, in the gray matter or white matter within each participant. We conclude that perfusion measures generated with hypoxia-induced deoxyhemoglobin are spatially and quantitatively comparable to those generated from a gadolinium injection in the same healthy participant.

Perfusion MRI using endogenous deoxyhemoglobin as a contrast agent: Preliminary data.

PURPOSE: To demonstrate the feasibility of mapping cerebral perfusion metrics with BOLD MRI during modulation of pulmonary venous oxygen saturation. METHODS: A gas blender with a sequential gas delivery breathing circuit was used to implement rapid isocapnic changes in the partial pressure of oxygen of the arterial blood. Partial pressure of oxygen was initially lowered to a baseline of 40 mmHg. It was then rapidly raised to 95 mmHg for 20 s before rapidly returning to baseline. The induced cerebral changes in deoxyhemoglobin concentration were tracked over time using BOLD MRI in 6 healthy subjects and 1 patient with cerebral steno-occlusive disease. BOLD signal change, contrast-to-noise ratio, and time delay metrics were calculated. Perfusion metrics such as mean transit time, relative cerebral blood volume, and relative cerebral blood flow were calculated using a parametrized method with a mono-exponential residue function. An arterial input function from within the middle cerebral artery was used to scale relative cerebral blood volume and calculate absolute cerebral blood volume and cerebral blood flow. RESULTS: In normal subjects, average gray and white matter were: BOLD change = 6.3 ± 1.2% and 2.5 ± 0.6%, contrast-to-noise ratio = 4.3 ± 1.3 and 2.6 ± 0.7, time delay = 2.3 ± 0.6 s and 3.6 ± 0.7 s, mean transit time = 3.9 ± 0.6 s and 5.5 ± 0.6 s, relative cerebral blood volume = 3.7 ± 0.9 and 1.6 ± 0.4, relative cerebral blood flow = 70.1 ± 8.3 and 20.6 ± 4.0, cerebral blood flow volume = 4.1 ± 0.9 mL/100 g and 1.8 ± 0.5 mL/100 g, and cerebral blood flow = 97.2 ± 18.7 mL/100 g/min and 28.7 ± 5.9 mL/100 g/min. CONCLUSION: This study demonstrates that induced abrupt changes in deoxyhemoglobin can function as a noninvasive vascular contrast agent that may be used for cerebral perfusion imaging.

STING-Activating Polymer-Drug Conjugates for Cancer Immunotherapy.

The stimulator of interferon genes (STING) pathway links innate and adaptive antitumor immunity and therefore plays an important role in cancer immune surveillance. This has prompted widespread development of STING agonists for cancer immunotherapy, but pharmacological barriers continue to limit the clinical impact of STING agonists and motivate the development of drug delivery systems to improve their efficacy and/or safety. To address this challenge, we developed SAPCon, a STING-activating polymer-drug conjugate platform based on strain-promoted azide-alkyne cycloaddition of dimeric-amidobenzimidazole (diABZI) STING agonists to hydrophilic polymer chains through an enzyme-responsive chemical linker. To synthesize a first-generation SAPCon, we designed a diABZI prodrug modified with a DBCO reactive handle a cathepsin B-cleavable spacer for intracellular drug release and conjugated this to pendant azide groups on a 100 kDa poly(dimethyla acrylamide-co-azide methacrylate) copolymer backbone to increase circulation time and enable passive tumor accumulation. We found that intravenously administered SAPCon accumulated at tumor sites where they it was endocytosed by tumor-associated myeloid cells, resulting in increased STING activation in tumor tissue compared to a free diABZI STING agonist. Consequently, SAPCon promoted an immunogenic tumor microenvironment, characterized by increased frequency of activated macrophages and dendritic cells and improved infiltration of CD8+ T cells, resulting in inhibition of tumor growth, prolonged survival, and increased response to anti-PD-1 immune checkpoint blockade in orthotopic models of breast cancer. Collectively, these studies position SAPCon as a modular and programmable platform for improving the efficacy of systemically administered STING agonists for cancer immunotherapy.

A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

DSC MRI in the human brain using deoxyhemoglobin and gadolinium-Simulations and validations at 3T.

Introduction: Dynamic susceptibility contrast (DSC) MRI allows clinicians to determine perfusion parameters in the brain, such as cerebral blood flow, cerebral blood volume, and mean transit time. To enable quantification, susceptibility changes can be induced using gadolinium (Gd) or deoxyhemoglobin (dOHb), the latter just recently introduced as a contrast agent in DSC. Previous investigations found that experimental parameters and analysis choices, such as the susceptibility amplitude and partial volume, affect perfusion quantification. However, the accuracy and precision of DSC MRI has not been systematically investigated, particularly in the lower susceptibility range. Methods: In this study, we compared perfusion values determined using Gd with values determined using a contrast agent with a lower susceptibility-dOHb-under different physiological conditions, such as varying the baseline blood oxygenation and/or magnitude of hypoxic bolus, by utilizing numerical simulations and conducting experiments on healthy subjects at 3T. The simulation framework we developed for DSC incorporates MRI signal contributions from intravascular and extravascular proton spins in arterial, venous, and cerebral tissue voxels. This framework allowed us to model the MRI signal in response to both Gd and dOHb. Results and discussion: We found, both in the experimental results and simulations, that a reduced intravascular volume of the selected arterial voxel, reduced baseline oxygen saturation, greater susceptibility of applied contrast agent (Gd vs. dOHb), and/or larger magnitude of applied hypoxic bolus reduces the overestimation and increases precision of cerebral blood volume and flow. As well, we found that normalizing tissue to venous rather than arterial signal increases the accuracy of perfusion quantification across experimental paradigms. Furthermore, we found that shortening the bolus duration increases the accuracy and reduces the calculated values of mean transit time. In summary, we experimentally uncovered an array of perfusion quantification dependencies, which agreed with the simulation framework predictions, using a wider range of susceptibility values than previously investigated. We argue for caution when comparing absolute and relative perfusion values within and across subjects obtained from a standard DSC MRI analysis, particularly when employing different experimental paradigms and contrast agents.

Focal Cryo-Immunotherapy with Intratumoral IL-12 Prevents Recurrence of Large Murine Tumors.

Focal ablation technologies are routinely used in the clinical management of inoperable solid tumors but they often result in incomplete ablations leading to high recurrence rates. Adjuvant therapies, capable of safely eliminating residual tumor cells, are therefore of great clinical interest. Interleukin-12 (IL-12) is a potent antitumor cytokine that can be localized intratumorally through coformulation with viscous biopolymers, including chitosan (CS) solutions. The objective of this research was to determine if localized immunotherapy with a CS/IL-12 formulation could prevent tumor recurrence after cryoablation (CA). Tumor recurrence and overall survival rates were assessed. Systemic immunity was evaluated in spontaneously metastatic and bilateral tumor models. Temporal bulk RNA sequencing was performed on tumor and draining lymph node (dLN) samples. In multiple murine tumor models, the addition of CS/IL-12 to CA reduced recurrence rates by 30-55%. Altogether, this cryo-immunotherapy induced complete durable regression of large tumors in 80-100% of treated animals. Additionally, CS/IL-12 prevented lung metastases when delivered as a neoadjuvant to CA. However, CA plus CS/IL-12 had minimal antitumor activity against established, untreated abscopal tumors. Adjuvant anti-PD-1 therapy delayed the growth of abscopal tumors. Transcriptome analyses revealed early immunological changes in the dLN, followed by a significant increase in gene expression associated with immune suppression and regulation. Cryo-immunotherapy with localized CS/IL-12 reduces recurrences and enhances the elimination of large primary tumors. This focal combination therapy also induces significant but limited systemic antitumor immunity.

STING-activating nanoparticles normalize the vascular-immune interface to potentiate cancer immunotherapy.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.