Antisense oligonucleotides and nucleic acids generate hypersensitive platelets.

INTRODUCTION: Despite the great promise for therapies using antisense oligonucleotides (ASOs), their adverse effects, which include pro-inflammatory effects and thrombocytopenia, have limited their use. Previously, these effects have been linked to the phosphorothioate (PS) backbone necessary to prevent rapid ASO degradation in plasma. The main aim of this study was to assess the impact of the nucleic acid portion of an ASO-type drug on platelets and determine if it may contribute to thrombosis or thrombocytopenia. METHODS: Platelets were isolated from healthy donors and men with advanced prostate cancer. Effects of antisense oligonucleotides (ASO), oligonucleotides, gDNA, and microRNA on platelet activation and aggregation were evaluated. A mouse model of lung thrombosis was used to confirm the effects of PS-modified oligonucleotides in vivo. RESULTS: Platelet exposure to gDNA, miRNA, and oligonucleotides longer than 16-mer at a concentration above 8 mM resulted in the formation of hypersensitive platelets, characterized by an increased sensitivity to low-dose thrombin (0.1 nM) and increase in p-Selectin expression (6-8 fold greater than control; p < 0.001). The observed nucleic acid (NA) effects on platelets were toll-like receptor (TLR) -7 subfamily dependent. Injection of a p-Selectin inhibitor significantly (p = 0.02) reduced the formation of oligonucleotide-associated pulmonary microthrombosis in vivo. CONCLUSION: Our results suggest that platelet exposure to nucleic acids independent of the presence of a PS modification leads to a generation of hypersensitive platelets and requires TLR-7 subfamily receptors. ASO studies conducted in cancer patients may benefit from testing the ASO effects on platelets ex vivo before initiation of patient treatment.

Multigene Profiling of Circulating Tumor Cells (CTCs) for Prognostic Assessment in Treatment-Naïve Metastatic Hormone-Sensitive Prostate Cancer (mHSPC).

The substantial biological heterogeneity of metastatic prostate cancer has hindered the development of personalized therapeutic approaches. Therefore, it is difficult to predict the course of metastatic hormone-sensitive prostate cancer (mHSPC), with some men remaining on first-line androgen deprivation therapy (ADT) for several years while others progress more rapidly. Improving our ability to risk-stratify patients would allow for the optimization of systemic therapies and support the development of stratified prospective clinical trials focused on patients likely to have the greatest potential benefit. Here, we applied a liquid biopsy approach to identify clinically relevant, blood-based prognostic biomarkers in patients with mHSPC. Gene expression indicating the presence of CTCs was greater in CHAARTED high-volume (HV) patients (52% CTChigh) than in low-volume (LV) patients (23% CTChigh; * p = 0.03). HV disease (p = 0.005, q = 0.033) and CTC presence at baseline prior to treatment initiation (p = 0.008, q = 0.033) were found to be independently associated with the risk of nonresponse at 7 months. The pooled gene expression from CTCs of pre-ADT samples found AR, DSG2, KLK3, MDK, and PCA3 as genes predictive of nonresponse. These observations support the utility of liquid biomarker approaches to identify patients with poor initial response. This approach could facilitate more precise treatment intensification in the highest risk patients.

Wnt Signaling Drives Prostate Cancer Bone Metastatic Tropism and Invasion.

Wnt signaling has been implicated as a driver of prostate cancer-related osteoblast differentiation, and previous studies have linked modifications in Wnt function with the induction of tumor metastasis. A unique aspect of prostate cancer bone metastases in mouse models is their relative predilection to the hindlimb (femur) compared to the forelimb (humerus). Comparative gene expression profiling was performed within the humerus and femur from non-tumor-bearing mice to evaluate differences in the microenvironments of these locations. This revealed the relative overexpression of the Wnt signaling inhibitors WIF1 and SOST in the humerus compared to the femur, with increased WNT5A expression in femur bone marrow, suggesting a coordinated upregulation of Wnt signals within the femur compared to the humerus. Conditioned medium (CM) from bone marrow stromal cells (HS-5 cells) was used to mimic the bone marrow microenvironment, which strongly promoted prostate cancer cell invasion (3.3-fold increase in PC3 cells, P < .05; 7-fold increase in LNCaP cells, P < .05). WNT5A shRNA knockdown within the CM-producing HS-5 cells significantly decreased PC3 (56%, P < .05) and LNCaP (60%, P < .05) cell invasion. Similarly, preincubation of CM with WIF1 significantly blocked LNCaP cell invasion (40%, P < .05). shRNA-mediated knockdown of the Wnt receptors FZD4 and FZD8 also strongly inhibited tumor cell invasion (60% inhibition shFZD4, P < .05; 63% shFZD8, P < .05). Furthermore, small molecule inhibition of JNK, which is an important component of the noncanonical Wnt signaling pathway, significantly inhibited CM-mediated tumor invasion. Overall, this study reveals a role for Wnt signaling as a driver of prostate cancer bone metastatic tropism and invasion.

Human lung organoids develop into adult airway-like structures directed by physico-chemical biomaterial properties.

Tissues derived from human pluripotent stem cells (hPSCs) often represent early stages of fetal development, but mature at the molecular and structural level when transplanted into immunocompromised mice. hPSC-derived lung organoids (HLOs) transplantation has been further enhanced with biomaterial scaffolds, where HLOs had improved tissue structure and cellular differentiation. Here, our goal was to define the physico-chemical biomaterial properties that maximally enhanced transplant efficiency, including features such as the polymer type, degradation, and pore interconnectivity of the scaffolds. We found that transplantation of HLOs on microporous scaffolds formed from poly (ethylene glycol) (PEG) hydrogel scaffolds inhibit growth and maturation, and the transplanted HLOs possessed mostly immature lung progenitors. On the other hand, HLOs transplanted on poly (lactide-co-glycolide) (PLG) scaffolds or polycaprolactone (PCL) led to tube-like structures that resembled both the structure and cellular diversity of an adult airway. Our data suggests that scaffold pore interconnectivity and polymer degradation contributed to the maturation, and we found that the size of the airway structures and the total size of the transplanted tissue was influenced by the material degradation rate. Collectively, these biomaterial platforms provide a set of tools to promote maturation of the tissues and to control the size and structure of the organoids.

Microporous Polymer Scaffolds for the Transplantation of Embryonic Stem Cell Derived Pancreatic Progenitors to a Clinically Translatable Site for the Treatment of Type I Diabetes.

Type I diabetes mellitus, which affects an estimated 1.5 million Americans, is caused by autoimmune destruction of the pancreatic beta cells that results in the need for life-long insulin therapy. Allogeneic islet transplantation for the treatment of type I diabetes is a therapy in which donor islets are infused intrahepatically, which has led to the transient reversal of diabetes. However, therapeutic limitations of allogeneic transplantation, which include a shortage of donor islets, long-term immunosuppression, and high risk of tissue rejection, have led to the investigation of embryonic or induced pluripotent stem cells as an unlimited source of functional beta-cells. Herein, we investigate the use of microporous scaffolds for their ability to promote the engraftment of stem cell derived pancreatic progenitors and their maturation toward mono-hormonal insulin producing ß-cells at a clinically translatable, extrahepatic site. Initial studies demonstrated that microporous scaffolds supported cell engraftment, and their maturation to become insulin positive; however, the number of insulin positive cells and the levels of C-peptide secretion were substantially lower than what was observed with progenitor cell transplantation into the kidney capsule. The scaffolds were subsequently modified to provide a sustained release of exendin-4, which has previously been employed to promote maturation of pancreatic progenitors in vitro and has been employed to promote engraftment of transplanted islets in the peritoneal fat. Transplantation of stem cell derived pancreatic progenitors on scaffolds releasing exendin-4 led to significantly increased C-peptide production compared to scaffolds without exendin-4, with C-peptide and blood glucose levels comparable to the kidney capsule transplantation cohort. Image analysis of insulin and glucagon producing cells indicated that monohormonal insulin producing cells were significantly greater compared to glucagon producing and polyhormonal cells in scaffolds releasing exendin-4, whereas a significantly decreased percentage of insulin-producing cells were present among hormone producing cells in scaffolds without exendin-4. Collectively, a microporous scaffold, capable of localized and sustained delivery of exendin-4, enhanced the maturation and function of pluripotent stem cell derived pancreatic progenitors that were transplanted to a clinically translatable site.