Transcription factor-nucleosome dynamics from plasma cfDNA identifies ER-driven states in breast cancer.

Genome-wide binding profiles of estrogen receptor (ER) and FOXA1 reflect cancer state in ER+ breast cancer. However, routine profiling of tumor transcription factor (TF) binding is impractical in the clinic. Here, we show that plasma cell-free DNA (cfDNA) contains high-resolution ER and FOXA1 tumor binding profiles for breast cancer. Enrichment of TF footprints in plasma reflects the binding strength of the TF in originating tissue. We defined pure in vivo tumor TF signatures in plasma using ER+ breast cancer xenografts, which can distinguish xenografts with distinct ER states. Furthermore, state-specific ER-binding signatures can partition human breast tumors into groups with significantly different ER expression and mortality. Last, TF footprints in human plasma samples can identify the presence of ER+ breast cancer. Thus, plasma TF footprints enable minimally invasive mapping of the regulatory landscape of breast cancer in humans and open vast possibilities for clinical applications across multiple tumor types.

Neoadjuvant endocrine therapy expands stromal populations that predict poor prognosis in estrogen receptor-positive breast cancer.

The tumor microenvironment (TME) is an important modulator of response and resistance to endocrine therapy in estrogen receptor alpha (ER) positive breast cancer. Endocrine therapy is highly effective at reducing tumor burden and preventing recurrence in most estrogen receptor alpha (ER) positive breast cancers. Existing drugs work either directly by targeting tumor-cell ER or indirectly by inhibiting estrogen production in stromal cells with aromatase inhibitors (AI). However, many stromal cells also express ER and the direct impact of endocrine therapies on ER + stromal cells remain unclear. In this study, we investigated how neoadjuvant endocrine therapy (NET) directly effects stromal cells by measuring changes in stomal components of the TME that favor tumor progression. We previously defined two major subsets of tumor-associated stromal cells (TASCs): CD146 positive/CDCP1 negative (TASCCD146 ), CD146 negative/CDCP1 positive (TASCCDCP1 ), and generated a differentially expressed genes list associated with each type. Here, we applied the TASC gene list for classification and an algorithm that estimates immune cell abundance (TIMEx) to METABRIC transcriptomic data for ER + breast cancer patients coupled with multiplex imaging and analysis of paired tissue samples pre- and post- NET with the AI exemestane. TASCCDCP1 composition predicted for decreased patient survival in the METABRIC cohort. Exemestane treatment significantly increased expression of TASCCDCP1 and decreased expression of TASCCD146 . The posttreatment shift toward TASCCDCP1 composition correlated with increased macrophage infiltration and increased CD8+ T-cell, B cell, and general stromal components. The effectiveness of NET is currently based solely on the reduction of ER+ breast cancer cells. Here, we show NET displays clear TME effects that promote the expansion of the less favorable TASCCDCP1 population which are correlated with TME remodeling and reshaping immune infiltration supportive of tumor progression. Our findings highlight the need to further understand the role of endocrine therapy on TME remodeling, tumor progression, and patient outcomes.

Analysis of circulating breast cancer cell heterogeneity and interactions with peripheral blood mononuclear cells.

For solid tumors, extravasation of cancer cells and their survival in circulation represents a critical stage of the metastatic process that lacks complete understanding. Gaining insight into interactions between circulating tumor cells (CTCs) and other peripheral blood mononuclear cells (PBMCs) may provide valuable prognostic information. The purpose of this study was to use single-cell RNA-sequencing (scRNA-seq) of liquid biopsies from breast cancer patients to begin defining intravascular interactions. We captured CTCs from the peripheral blood of breast cancer patients using size-exclusion membranes followed by scRNA-seq of enriched CTCs and carry-over PBMCs. Transcriptome analysis identified two populations of CTCs: one enriched for transcripts indicative of estrogen responsiveness and increased proliferation and another enriched for transcripts characteristic of reduced proliferation and epithelial-mesenchymal transition (EMT). We applied interactome and pathway analysis to determine interactions between CTCs and other captured cells. Our analysis predicted for enhanced immune evasion in the CTC population with EMT characteristics. In addition, PD-1/PD-L1 pathway activation and T cell exhaustion were predicted in T cells isolated from breast cancer patients compared with normal T cells. We conclude that scRNA-seq of breast cancer CTCs generally stratifies them into two types based on their proliferative and epithelial state and differential potential to interact with PBMCs. Better understanding of CTC subtypes and their intravascular interactions may help design treatments directed against CTCs with high metastatic and immune-evasive competence.

Fibroblast subtypes define a metastatic matrisome in breast cancer.

Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision-making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this analysis is insufficient - that the stromal microenvironment of the primary tumor plays a key role in tumor cell dissemination and implantation at distant sites. We previously described 2 cancer-associated fibroblasts (CAFs) that either express (CD146+) or lack (CD146-) CD146 (official symbol MCAM, alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer cells: CD146- fibroblasts promoted increased metastasis compared with CD146+ fibroblasts. Potentially novel quantitative and qualitative proteomic analyses showed that CD146+ CAFs produced an environment rich in basement membrane proteins, while CD146- CAFs exhibited increases in fibronectin 1, lysyl oxidase, and tenascin C, all overexpressed in aggressive disease. We also show clinically that CD146- CAFs predicted for likelihood of lymph node involvement even in small primary tumors (<5 cm). Clearly small tumors enriched for CD146- CAFs require aggressive treatments.

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo.

BACKGROUND AIMS: Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS: We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS: We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS: These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

Safety and pharmacokinetics of intravaginal rings delivering tenofovir in pig-tailed macaques.

Antiretroviral-based microbicides applied topically to the vagina may play an important role in protecting women from HIV infection. Incorporation of the nucleoside reverse transcriptase inhibitor tenofovir (TFV) into intravaginal rings (IVRs) for sustained mucosal delivery may lead to increased microbicide product adherence and efficacy compared with those of conventional vaginal formulations. Formulations of a novel "pod IVR" platform spanning a range of IVR drug loadings and daily release rates of TFV were evaluated in a pig-tailed macaque model. The rings were safe and exhibited sustained release at controlled rates over 28 days. Vaginal secretion TFV levels were independent of IVR drug loading and were able to be varied over 1.5 log units by changing the ring configuration. Mean TFV levels in vaginal secretions were 72.4 ± 109 µg ml(-1) (slow releasing) and 1.84 ± 1.97 mg ml(-1) (fast releasing). The mean TFV vaginal tissue concentration from the slow-releasing IVRs was 76.4 ± 54.8 µg g(-1) and remained at steady state 7 days after IVR removal, consistent with the long intracellular half-life of TFV. Intracellular tenofovir diphosphate (TFV-DP), the active moiety in defining efficacy, was measured in vaginal lymphocytes collected in the study using the fast-releasing IVR formulation. Mean intracellular TFV-DP levels of 446 ± 150 fmol/10(6) cells fall within a range that may be protective of simian-human immunodeficiency virus strain SF162p3 (SHIV(SF162p3)) infection in nonhuman primates. These data suggest that TFV-releasing IVRs based on the pod design have potential for the prevention of transmission of human immunodeficiency virus type 1 (HIV-1) and merit further clinical investigation.

An intravaginal ring for the simultaneous delivery of multiple drugs.

Intravaginal delivery of microbicide combinations is a promising approach for the prevention of sexually transmitted infections, but requires a method of providing simultaneous, independent release of multiple agents into the vaginal compartment. A novel intravaginal ring (IVR) platform has been developed for simultaneous delivery of the reverse-transcriptase inhibitor tenofovir (TFV) and the guanosine analogue antiviral acyclovir (ACV) with independent control of release rate for each drug. The IVR is based on a pod design, with up to 10 individual polymer-coated drug cores embedded in the ring releasing through preformed delivery channels. The release rate from each pod is controlled independently of the others by the drug properties, polymer coating, and size and number of delivery channels. Pseudo-zero-order in vitro release of TFV (144 ± 10 µg day) and ACV (120 ± 19 µg day⁻¹) from an IVR containing both drugs was sustained for 28 days. The mechanical properties of the pod IVR were evaluated and compared with the commercially available Estring® (Pfizer, NY, NY). The pod-IVR design enables the vaginal delivery of multiple microbicides with differing physicochemical properties, and is an attractive approach for the sustained intravaginal delivery of relatively hydrophilic drugs that are difficult to deliver using conventional matrix IVR technology.

Tenofovir and tenofovir disoproxil fumarate pharmacokinetics from intravaginal rings.

OBJECTIVES: To compare the distribution of tenofovir in sheep vaginal lumen, tissue, and plasma following topical delivery of the antiretroviral drug from intravaginal rings, either as tenofovir or the disoproxil fumarate prodrug. DESIGN: Comparative pharmacokinetic study in sheep. METHOD: Intravaginal rings formulated to achieve equivalent release rates of tenofovir and its disoproxil fumarate prodrug were evaluated for 28 days in sheep, with four animals in each group. Drug concentrations were measured by high-performance liquid chromatography-mass spectrometry. RESULTS: Tenofovir levels in cervicovaginal lavage were indistinguishable (P > 0.30) in both groups, but tissue levels in animals receiving the prodrug were 86-fold higher than those receiving tenofovir, and approximately 50 times higher than the level shown to be protective of HIV infection in the CAPRISA 004 trial. CONCLUSION: This is the first study to compare the pharmacokinetics of tenofovir and its disoproxil fumarate prodrug administered topically to the vaginal tract. These in-vivo data show that the prodrug leads to significantly higher drug tissue levels than tenofovir, a finding that may have important implications for the development of preexposure prophylaxis strategies based on topical delivery of antivirals to the female genital tract.

Simultaneous delivery of tenofovir and acyclovir via an intravaginal ring.

Vaginal microbicides may play an important role in protecting women from HIV infection. A strong synergy between HSV and HIV has been observed, and epidemiological studies demonstrate that HSV infection increases the risk of HIV acquisition. Incorporation of the antiretroviral tenofovir (TFV) along with the antiherpetic acyclovir (ACV) into combination intravaginal rings (IVRs) for sustained mucosal delivery of both compounds could lead to increased microbicide product adherence and efficacy compared with conventional vaginal formulations. A novel, dual-protection "pod IVR" platform developed in-house and delivering ACV and TFV was evaluated in rabbit and sheep models. The devices were safe and exhibited sustained release of both drugs independently and at controlled rates over the 28-day studies. Daily release rates were estimated based on residual drug content of the used devices: rabbits, 343 ± 335 µg day(-1) (ACV) and 321 ± 207 µg day(-1) (TFV); sheep, 174 ± 14 µg day(-1) (ACV) and 185 ± 34 µg day(-1) (TFV). Mean drug levels in sheep vaginal samples were as follows: secretions, 5.25 ± 7.31 µg ml(-1) (ACV) and 20.6 ± 16.2 µg ml(-1) (TFV); cervicovaginal lavage fluid, 118 ± 113 ng ml(-1) (ACV) and 191 ± 125 ng ml(-1) (TFV); tissue, 173 ng g(-1) (ACV) and 93 ng g(-1) (TFV). An in vitro-in vivo correlation was established for both drugs and will allow the development of future formulations delivering target levels for prophylaxis and therapy. These data suggest that the IVR based on the pod design has potential in the prevention of transmission of HIV-1 and other sexually transmitted pathogens.

Microbial biofilms on the surface of intravaginal rings worn in non-human primates.

Millions of intravaginal rings (IVRs) are used by women worldwide for contraception and for the treatment of vaginal atrophy. These devices also are suitable for local and systemic sustained release drug delivery, notably for antiviral agents in human immunodeficiency virus pre-exposure prophylaxis. Despite the widespread use of IVRs, no studies have examined whether surface-attached bacterial biofilms develop in vivo, an important consideration when determining the safety of these devices. The present study used scanning electron microscopy, fluorescence in situ hybridization and confocal laser scanning microscopy to study biofilms that formed on the surface of IVRs worn for 28 days by six female pig-tailed macaques, an excellent model organism for the human vaginal microbiome. Four of the IVRs released the nucleotide analogue reverse transcriptase inhibitor tenofovir at a controlled rate and the remaining two were unmedicated. Large areas of the ring surfaces were covered with monolayers of epithelial cells. Two bacterial biofilm phenotypes were found to develop on these monolayers and both had a broad diversity of bacterial cells closely associated with the extracellular material. Phenotype I, the more common of the two, consisted of tightly packed bacterial mats approximately 5 µm in thickness. Phenotype II was much thicker, typically 40 µm, and had an open architecture containing interwoven networks of uniform fibres. There was no significant difference in biofilm thickness and appearance between medicated and unmedicated IVRs. These preliminary results suggest that bacterial biofilms could be common on intravaginal devices worn for extended periods of time.