Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Fibrosis in the Spleens of Nonhuman Primates.

Although current antiretroviral therapy (ART) has increased life expectancy, a cure for human immunodeficiency virus (HIV) remains elusive due to the persistence of the virus in tissue reservoirs. In the present study, we sought to elucidate the relationship between antiretrovirals (ARVs) and viral expression in the spleen. We performed mass spectrometry imaging (MSI) of 6 different ARVs, RNAscope in situ hybridization of viral RNA, and immunohistochemistry of three different fibrosis markers in the spleens of 8 uninfected and 10 reverse transcriptase simian-human immunodeficiency virus (RT-SHIV)-infected rhesus macaques (infected for 6 weeks) that had been dosed for 10 days with combination ART. Using MATLAB, computational quantitative imaging analysis was performed to evaluate the spatial and pharmacological relationships between the 6 ARVs, viral RNA, and fibrotic deposition. In these spleens, >50% of the spleen tissue area was not covered by any detectable ARV response (any concentration above the limits of detection for individual ARVs). The median spatial ARV coverage across all tissues was driven by maraviroc followed by efavirenz. Yet >50% of RNA-positive cells were not exposed to any detectable ARV. Quantifiable maraviroc and efavirenz colocalization with RNA-positive cells was usually greater than the in vitro concentration inhibiting 50% replication (IC50). Fibrosis markers covered more than 50% of the spleen tissue area and had negative relationships with cumulative ARV coverages. Our findings suggest that a heterogeneous ARV spatial distribution must be considered when evaluating viral persistence in lymphoid tissue reservoirs.

Drug-Dependent Morphological Transitions in Spherical and Worm-Like Polymeric Micelles Define Stability and Pharmacological Performance of Micellar Drugs.

Significant advances in physicochemical properties of polymeric micelles enable optimization of therapeutic drug efficacy, supporting nanomedicine manufacturing and clinical translation. Yet, the effect of micelle morphology on pharmacological efficacy is not adequately addressed. This work addresses this gap by assessing pharmacological efficacy of polymeric micelles with spherical and worm-like morphologies. It is observed that poly(2-oxazoline)-based polymeric micelles can be elongated over time from a spherical structure to worm-like structure, with elongation influenced by several conditions, including the amount and type of drug loaded into the micelles. The role of different morphologies on pharmacological performance of drug loaded micelles against triple-negative breast cancer and pancreatic cancer tumor models is further evaluated. Spherical micelles accumulate rapidly in the tumor tissue while retaining large amounts of drug; worm-like micelles accumulate more slowly and only upon releasing significant amounts of drug. These findings suggest that the dynamic character of the drug-micelle structure and the micelle morphology play a critical role in pharmacological performance, and that spherical micelles are better suited for systemic delivery of anticancer drugs to tumors when drugs are loosely associated with the polymeric micelles.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Collagen in the Mesenteric Lymph Nodes of Nonhuman Primates.

Human immunodeficiency virus (HIV) persistence in tissue reservoirs is a major barrier to HIV cure. While antiretrovirals (ARVs) suppress viral replication, antiretroviral therapy (ART) interruption results in rapid rebound viremia that may originate from lymphoid tissues. To understand the relationship between anatomic distribution of ARV exposure and viral expression in lymph nodes, we performed mass spectrometry imaging (MSI) of 6 ARVs, RNAscope in situ hybridization for viral RNA (vRNA), and immunohistochemistry of collagen in mesenteric lymph nodes from 8 uninfected and 10 reverse transcriptase simian/human immunodeficiency virus (RT-SHIV)-infected rhesus macaques dosed to steady state with combination ART. MATLAB-based quantitative imaging analysis was used to evaluate spatial and pharmacological relationships between these ARVs, viral RNA (both vRNA+ cells and follicular dendritic cell [FDC]-bound virions), and collagen deposition. Using MSI, 31% of mesenteric lymph node tissue area was found to be not covered by any ARV. Additionally, 28% of FDC-trapped virions and 21% of infected cells were not exposed to any detected ARV. Of the 69% of tissue area that was covered by cumulative ART exposure, nearly 100% of concentrations were greater than in vitro 50% inhibitory concentration (IC50) values; however, 52% of total tissue coverage was from only one ARV, primarily maraviroc. Collagen covered ∼35% of tissue area but did not influence ARV distribution heterogeneity. Our findings are consistent with our hypothesis that ARV distribution, in addition to total-tissue drug concentration, must be considered when evaluating viral persistence in lymph nodes and other reservoir tissues.

Poly(2-oxazoline) nanoparticle delivery enhances the therapeutic potential of vismodegib for medulloblastoma by improving CNS pharmacokinetics and reducing systemic toxicity.

We report a nanoparticle formulation of the SHH-pathway inhibitor vismodegib that improves efficacy for medulloblastoma, while reducing toxicity. Limited blood-brain barrier (BBB) penetration and dose-limiting extitle/citraneural toxicities complicate systemic therapies for brain tumors. Vismodegib is FDA-approved for SHH-driven basal cell carcinoma, but implementation for medulloblastoma has been limited by inadequate efficacy and excessive bone toxicity. To address these issues through optimized drug delivery, we formulated vismodegib in polyoxazoline block copolymer micelles (POx-vismo). We then evaluated POx-vismo in transgenic mice that develop SHH-driven medulloblastomas with native vasculature and tumor microenvironment. POx-vismo improved CNS pharmacokinetics and reduced bone toxicity. Mechanistically, the nanoparticle carrier did not enter the CNS, and acted within the vascular compartment to improve drug delivery. Unlike conventional vismodegib, POx-vismo extended survival in medulloblastoma-bearing mice. Our results show the broad potential for non-targeted nanoparticle formulation to improve systemic brain tumor therapy, and specifically to improve vismodegib therapy for SHH-driven cancers.

scRNA-seq in medulloblastoma shows cellular heterogeneity and lineage expansion support resistance to SHH inhibitor therapy.

Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.

Heterogeneous antiretroviral drug distribution and HIV/SHIV detection in the gut of three species.

HIV replication within tissues may increase in response to a reduced exposure to antiretroviral drugs. Traditional approaches to measuring drug concentrations in tissues are unable to characterize a heterogeneous drug distribution. Here, we used mass spectrometry imaging (MSI) to visualize the distribution of six HIV antiretroviral drugs in gut tissue sections from three species (two strains of humanized mice, macaques, and humans). We measured drug concentrations in proximity to CD3+ T cells that are targeted by HIV, as well as expression of HIV or SHIV RNA and expression of the MDR1 drug efflux transporter in gut tissue from HIV-infected humanized mice, SHIV-infected macaques, and HIV-infected humans treated with combination antiretroviral drug therapy. Serial 10-µm sections of snap-frozen ileal and rectal tissue were analyzed by MSI for CD3+ T cells and MDR1 efflux transporter expression by immunofluorescence and immunohistochemistry, respectively. The tissue slices were analyzed for HIV/SHIV RNA expression by in situ hybridization and for antiretroviral drug concentrations by liquid chromatography-mass spectrometry. The gastrointestinal tissue distribution of the six drugs was heterogeneous. Fifty percent to 60% of CD3+ T cells did not colocalize with detectable drug concentrations in the gut tissue. In all three species, up to 90% of HIV/SHIV RNA was found to be expressed in gut tissue with no exposure to drug. These data suggest that there may be gut regions with little to no exposure to antiretroviral drugs, which may result in low-level HIV replication contributing to HIV persistence.

Virological and immunological responses to raltegravir and dolutegravir in the gut-associated lymphoid tissue of HIV-infected men and women.

BACKGROUND: Raltegravir (RTG) and dolutegravir (DTG) have different pharmacokinetic patterns in the gastrointestinal tract. To determine if this results in pharmacodynamic differences, we compared HIV RNA, HIV DNA and immunological markers in gut-associated lymphoid tissue (GALT) of HIV-infected participants receiving RTG or DTG with tenofovir+emtricitabine (TDF/FTC). METHODS: GALT specimens from the terminal ileum, splenic flexure and rectum were obtained by colonoscopy at a single time point in 20 adults treated with RTG (n=10) or DTG (n=10) with HIV RNA <50 copies/ml. Flow cytometry, drug concentrations, and HIV RNA and DNA were analysed in tissue. CD4/8+ T-cells were tested for γδ TCR, and markers of T-cell activation and exhaustion. Data are reported as median (Q1-Q3). RESULTS: A total of 15 men and 5 women were enrolled. There was no difference in time since HIV diagnosis for those on RTG (9.5 [4-22] years) and DTG (17 [1-24] years; P=0.6), although time on RTG (5.4 [2.3-6.7] years) was greater than DTG (1.0 [0.1-1.5] years; P<0.001). Concentrations of RTG and DTG in rectal tissue were similar to previous reports: median tissue:plasma ratio was 11.25 for RTG and 0.44 for DTG. RNA:DNA ratios were 1.14 (0.18-5.10) for the RTG group and 0.90 (0.30-18.87) for the DTG group (P=0.95). No differences (P≥0.1) between CD4+ and CD8+ T-cell markers were found. CONCLUSIONS: RTG produced higher tissue exposures than DTG, but no significant differences in GALT HIV RNA, DNA or most immunological markers were observed. ClinicalTrials.gov NCT02218320.