Deciphering the functional role of clinical mutations in ABCB1, ABCC1, and ABCG2 ABC transporters in endometrial cancer.

ATP-binding cassette transporters represent a superfamily of dynamic membrane-based proteins with diverse yet common functions such as use of ATP hydrolysis to efflux substrates across cellular membranes. Three major transporters-P-glycoprotein (P-gp or ABCB1), multidrug resistance protein 1 (MRP1 or ABCC1), and breast cancer resistance protein (BCRP or ABCG2) are notoriously involved in therapy resistance in cancer patients. Despite exhaustive individual characterizations of each of these transporters, there is a lack of understanding in terms of the functional role of mutations in substrate binding and efflux, leading to drug resistance. We analyzed clinical variations reported in endometrial cancers for these transporters. For ABCB1, the majority of key mutations were present in the membrane-facing region, followed by the drug transport channel and ATP-binding regions. Similarly, for ABCG2, the majority of key mutations were located in the membrane-facing region, followed by the ATP-binding region and drug transport channel, thus highlighting the importance of membrane-mediated drug recruitment and efflux in ABCB1 and ABCG2. On the other hand, for ABCC1, the majority of key mutations were present in the inactive nucleotide-binding domain, followed by the drug transport channel and membrane-facing regions, highlighting the importance of the inactive nucleotide-binding domain in facilitating indirect drug efflux in ABCC1. The identified key mutations in endometrial cancer and mapped common mutations present across different types of cancers in ABCB1, ABCC1, and ABCG2 will facilitate the design and discovery of inhibitors targeting unexplored structural regions of these transporters and re-engineering of these transporters to tackle chemoresistance.

Oncolytic virotherapy stimulates anti­tumor immune response and demonstrates activity in advanced sarcoma: Report of two cases.

Sarcoma is derived from mesenchymal neoplasms and has numerous subtypes, accounting for 1% of all adult malignancies and 15% of childhood malignancies. The prognosis of metastatic or recurrent sarcoma remains poor. The current study presents two cases of sarcoma enrolled in a phase I dose escalation trial for solid tumor, who had previously failed all standard therapies. These patients were treated with VG161, an immune-stimulating herpes simplex virus type 1 oncolytic virus with payloads of IL-12, IL-15 and IL-15 receptor α unit, and a programmed cell death 1 (PD-1)/PD-1 ligand 1 blocking peptide. Both cases demonstrated stable disease as the best response, accompanied by a noteworthy prolongation of progression-free survival (11.8 months for chondrosarcoma and 11.9 months for soft tissue sarcoma, respectively) at a dose of 2.5×108 PFU/cycle. In addition, the treatment led to the activation of anti-cancer immunity, as evident from cytokine, lymphocyte subset and related pathway analyses of peripheral blood and/or tumor biopsy samples. These promising results suggest that VG161 monotherapy holds promise as an effective treatment for sarcoma and warrants further investigation through clinical trials. The two reported patients were part of a phase I clinical trial conducted and registered on the Australian New Zealand Clinical Trials Registry in Australia (registration no. ACTRN12620000244909; registration date, 26 February, 2020).

Prophylactic Vaccination and Intratumoral Boost with HER2-Expressing Oncolytic Herpes Simplex Virus Induces Robust and Persistent Immune Response against HER2-Positive Tumor Cells.

The development of effective cancer vaccines remains a significant challenge due to immune tolerance and limited clinical benefits. Oncolytic herpes simplex virus type 1 (oHSV-1) has shown promise as a cancer therapy, but efficacy is often limited in advanced cancers. In this study, we constructed and characterized a novel oHSV-1 virus (VG22401) expressing the human epidermal growth factor receptor 2 (HER2), a transmembrane glycoprotein overexpressed in many carcinomas. VG22401 exhibited efficient replication and HER2 payload expression in both human and mouse colorectal cancer cells. Mice immunized with VG22401 showed significant binding of serum anti-HER2 antibodies to HER2-expressing tumor cells, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, mice primed with VG22401 and intratumorally boosted with the same virus showed enhanced antitumor efficacy in a bilateral syngeneic HER2(+) tumor model, compared to HER2-null backbone virus. This effect was accompanied by the induction of anti-HER2 T cell responses. Our findings suggest that peripheral priming with HER2-expressing oHSV-1 followed by an intratumoral boost with the same virus can significantly enhance antitumor immunity and efficacy, presenting a promising strategy for cancer immunotherapy.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Breaking the niche: multidimensional nanotherapeutics for tumor microenvironment modulation.

Most of the current antitumor therapeutics were developed targeting the cancer cells only. Unfortunately, in the majority of tumors, this single-dimensional therapy is found to be ineffective. Advanced research has shown that cancer is a multicellular disorder. The tumor microenvironment (TME), which is made by a complex network of the bulk tumor cells and other supporting cells, plays a crucial role in tumor progression. Understanding the importance of the TME in tumor growth, different treatment modalities have been developed targeting these supporting cells. Recent clinical results suggest that simultaneously targeting multiple components of the tumor ecosystem with drug combinations can be highly effective. This type of "multidimensional" therapy has a high potential for cancer treatment. However, tumor-specific delivery of such multi-drug combinations remains a challenge. Nanomedicine could be utilized for the tumor-targeted delivery of such multidimensional therapeutics. In this review, we first give a brief overview of the major components of TME. We then highlight the latest developments in nanoparticle-based combination therapies, where one drug targets cancer cells and other drug targets tumor-supporting components in the TME for a synergistic effect. We include the latest preclinical and clinical studies and discuss innovative nanoparticle-mediated targeting strategies.

Extracellular Vesicle-Derived DNA vs. CfDNA as a Biomarker for the Detection of Colon Cancer.

Liquid biopsy has emerged as a promising non-invasive way to diagnose tumor and monitor its progression. Different types of liquid biopsies have different advantages and limitations. In the present research, we compared the use of two types of liquid biopsy, extracellular vesicle-derived DNA (EV-DNA) and cell-free DNA (cfDNA) for identifying tumor mutations in patients with colon carcinoma. METHOD: DNA was extracted from the tumor tissue of 33 patients diagnosed with colon carcinoma. Targeted NGS panel, based on the hotspots panel, was used to identify tumor mutations. Pre-surgery serum and plasma were taken from the patients in which mutation was found in the tumor tissue. Extracellular vesicles were isolated from the serum followed by the extraction of EV-DNA. CfDNA was extracted from the plasma. The mutations found in the tumor were used to detect the circulating tumor DNA using ultra-deep sequencing. We compared the sensitivity of mutation detection and allele frequency obtained in EV-DNA and cfDNA. RESULTS: The sensitivity of mutation detection in EV-DNA and cfDNA was 61.90% and 66.67%, respectively. We obtained almost identical sensitivity of mutation detection in EV-DNA and cfDNA in each of the four stages of colon carcinoma. The total DNA concentration and number mutant copies were higher in cfDNA vs. EV-DNA (p value = 0.002 and 0.003, respectively). CONCLUSION: Both cfDNA and EV-DNA can serve as tumor biomarkers. The use of EV-DNA did not lead to improved sensitivity or better detection of tumor DNA in the circulation.

Therapeutic Gene Silencing Using Targeted Lipid Nanoparticles in Metastatic Ovarian Cancer.

Ovarian cancer is an aggressive tumor owing to its ability to metastasize from stage II onward. Herein, lipid nanoparticles (LNPs) that encapsulate combination of small interfering RNAs (siRNAs), polo-like kinase-1 (PLK1), and eukaryotic translation-initiation factor 3c (eIF3c), to target different cellular pathways essential for ovarian cancer progression are generated. The LNPs are further modified with hyaluronan (tNPs) to target cluster of differentiation 44 (CD44) expressing cells. Interestingly, hyaluronan-coated LNPs (tNPs) prolong functional activity and reduce growth kinetics of spheroids in in vitro assay as compared to uncoated LNPs (uNPs) due to ≈1500-fold higher expression of CD44. Treatment of 2D and 3D cultured ovarian cancer cells with LNPs encapsulating both siRNAs result in 85% cell death and robust target gene silencing. In advanced orthotopic ovarian cancer model, intraperitoneal administration of LNPs demonstrates CD44 specific tumor targeting of tNPs compared to uNPs and robust gene silencing in tissues involved in ovarian cancer pathophysiology. At very low siRNA dose, enhanced overall survival of 60% for tNPs treated mice is observed compared to 10% and 20% for single siRNA-, eIF3c-tNP, and PLK1-tNP treatment groups, respectively. Overall, LNPs represent promising platform in the treatment of advanced ovarian cancer by improving median- and overall-survival.

MexXY RND pump of Pseudomonas aeruginosa PA7 effluxes bi-anionic ß-lactams carbenicillin and sulbenicillin when it partners with the outer membrane factor OprA but not with OprM.

Antibiotic resistance in Pseudomonas aeruginosa is a serious concern in healthcare systems. Among the determinants of antibiotic resistance in P. aeruginosa, efflux pumps belonging to the resistance-nodulation-division (RND) family confer resistance to a broad range of antibacterial compounds. The MexXY efflux system is widely overexpressed in P. aeruginosa isolates from cystic fibrosis (CF) patients. MexXY can form functional complexes with two different outer membrane factors (OMFs), OprA and OprM. In this study, using state-of-the-art genetic tools, the substrate specificities of MexXY-OprA and MexXY-OprM complexes were determined. Our results show, for the first time, that the substrate profile of the MexXY system from P. aeruginosa PA7 can vary depending on which OM factor (OprM or OprA) it complexes with. While both MexXY-OprA and MexXY-OprM complexes are capable of effluxing aminoglycosides, the bi-anionic ß-lactam molecules carbenicillin and sulbenicillin were found to only be the substrate of MexXY-OprA. Our study therefore shows that by partnering with different OMF proteins MexY can expand its substrate profile.

An ovarian spheroid based tumor model that represents vascularized tumors and enables the investigation of nanomedicine therapeutics.

The failure of cancer therapies in clinical settings is often attributed to the lack of a relevant tumor model and pathological heterogeneity across tumor types in the clinic. The objective of this study was to develop a robust in vivo tumor model that better represents clinical tumors for the evaluation of anti-cancer therapies. We successfully developed a simple mouse tumor model based on 3D cell culture by injecting a single spheroid and compared it to a tumor model routinely used by injecting cell suspension from 2D monolayer cell culture. We further characterized both tumors with cellular markers for the presence of myofibroblasts, pericytes, endothelial cells and extracellular matrix to understand the role of the tumor microenvironment. We further investigated the effect of chemotherapy (doxorubicin), nanomedicine (Doxil®), biological therapy (Avastin®) and their combination. Our results showed that the substantial blood vasculature in the 3D spheroid model enhances the delivery of Doxil® by 2.5-fold as compared to the 2D model. Taken together, our data suggest that the 3D tumors created by simple subcutaneous spheroid injection represents a robust and more vascular murine tumor model which is a clinically relevant platform to test anti-cancer therapy in solid tumors.

Endovascular Therapy for Vasculogenic Erectile Dysfunction: A Systematic Review and Meta-Analysis of Arterial and Venous Therapies.

PURPOSE: To systematically review and perform a meta-analysis on the safety and efficacy of endovascular therapy in the treatment of the two most common etiologies of vasculogenic erectile dysfunction (ED): veno-occlusive dysfunction (VOD) and arterial insufficiency (AI). MATERIALS AND METHODS: PubMed, Web of Science, ScienceDirect, and Scopus databases were searched for published English literature regarding endovascular ED treatments. Case series (n ≥ 3) were included. Multiple data points were obtained, including demographic data, etiology, diagnosis method, imaging studies, treatment approach, technical success, clinical success, complications, and follow-up. RESULTS: Sixteen relevant articles were obtained and a total of 212 patients with VOD and 162 with AI were identified. The VOD cohort were treated either percutaneously (60.4%; n = 128) or after surgical exposure of the deep dorsal vein (33.5%, n = 71), or it was unspecified (6.1%; n = 13). The most common embolic used was n-butyl cyanoacrylate (51.9%; n = 109). Meta-analysis found an overall clinical success rate of 59.8% in VOD patients. Complications occurred in 5.2% of patients (n = 11), with 9 considered to be mild and 2 considered to be severe. The AI cohort contained 162 patients most commonly treated via stenting of the internal pudendal artery (40.1%; n = 65). Meta-analysis found an overall clinical success rate of 63.2% in AI patients. Complications occurred in 4.9% of patients (n = 8), with 4 considered to be mild and 4 considered to be severe. CONCLUSIONS: Endovascular therapy for medically refractory ED is safe and may provide a treatment alternative to more invasive surgical management; however, conclusions are limited by the heterogeneity of clinical success definitions among the included studies.

Balloon-Occluded Transarterial Chemoembolization for the Treatment of Hepatocellular Carcinoma: A Single-Center US Preliminary Experience.

This report describes a single-center experience with balloon-occluded transarterial chemoembolization for liver-directed therapy. A total of 26 patients (11 male, 4 female; mean age, 65 y ± 7) with 28 tumors (mean diameter, 2.7 cm; range, 1.1-5.9 cm) were treated. Technical success rate was 100% (28 of 28 cases), with 1 minor complication of left portal vein thrombosis and small liver infarct. Of the 15 tumors analyzed for response, 60% (9 of 15) exhibited complete response, 33.3% (5 of 15) exhibited partial response, and 6.6% (1 of 15) had stable disease on follow-up. Eight patients exhibited overall progression with a new hepatic lesion and a median time to progression of 7.9 months (range, 5-11 mo).

Erythromelanosis follicularis faciei et colli: A clinicoepidemiologic study.

We describe 25 cases of erythromelanosis follicularis faciei et colli from India. The male:female ratio was 5.25:1 and the average age of onset was 12.3 years. The cheeks, preauricular area, and submandibular region were the sites most commonly affected. Keratosis pilaris was seen in 22 (88%) of the patients.

MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections.

In this study, we analyzed 15 multidrug-resistant cystic fibrosis isolates of Pseudomonas aeruginosa from chronic lung infections for expression of 4 different multidrug efflux systems (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY), using quantitative reverse transcriptase PCR. Overexpression of MexXY pump was observed in all of the isolates tested. Analysis of regulatory genes that control the expression of these 4 efflux pumps revealed a number of previously uncharacterized mutations. Our work shows that MexXY pump overexpression is common in cystic fibrosis isolates and could be contributing to their reduced aminoglycoside susceptibility. Further, we also identified novel mutations in the regulatory genes of the 4 abovementioned Resistance-Nodulation-Division superfamily pumps that may be involved in the overexpression of these pumps.

MDR in cancer: Addressing the underlying cellular alterations with the use of nanocarriers.

Multidrug resistance (MDR) is associated with a wide range of pathological changes at different cellular and intracellular levels. Nanoparticles (NPs) have been extensively exploited as the carriers of MDR reversing payloads to resistant tumor cells. However, when properly formulated in terms of chemical composition and physicochemical properties, NPs can serve as beyond delivery systems and help overcome MDR even without carrying a load of chemosensitizers or MDR reversing molecular cargos. Whether serving as drug carriers or beyond, a wise design of the nanoparticulate systems to overcome the cellular and intracellular alterations underlying the resistance is imperative. Within the current review, we will initially discuss the cellular changes occurring in resistant cells and how such changes lead to chemotherapy failure and cancer cell survival. We will then focus on different mechanisms through which nanosystems with appropriate chemical composition and physicochemical properties can serve as MDR reversing units at different cellular and intracellular levels according to the changes that underlie the resistance. Finally, we will conclude by discussing logical grounds for a wise and rational design of MDR reversing nanoparticulate systems to improve the cancer therapeutic approaches.

Budd-Chiari Syndrome Demonstrated on PET/CT.

Budd-Chiari syndrome is a rare clinical condition that involves diminution or interruption of hepatic venous blood flow from the liver. We describe a case of Budd-Chiari syndrome visualized via F-FDG PET/CT in a 60-year-old woman with medical history of B-cell non-Hodgkin lymphoma who presented with abdominal pain. FDG PET/CT fusion images demonstrate increased FDG uptake with SUVmax of 2.2 along the course of the middle hepatic vein with decreased attenuation within both the middle and right hepatic veins, consistent with thrombus.

Sustaining Screening, Brief Intervention and Referral to Treatment (SBIRT) services in health-care settings.

AIMS: To assess the sustainability of Screening, Brief Intervention and Referral to Treatment (SBIRT) services after cessation of initial start-up funding. DESIGN: Descriptive study with quantitative and qualitative data collected from 34 staff participants from six grantees (comprising 103 sites) funded previously through a large, federally supported SBIRT program. SETTING: Primary care out-patient clinics and hospitals in the United States. PARTICIPANTS: Thirty-four grantee-related staff members, including administrators, evaluators, key stakeholders and SBIRT service providers from six grantees. MEASUREMENTS: Changes to levels and types of service delivery activities after federal funding stopped, alternative sources of funding and obstacles to delivery of services. FINDINGS: Of the 103 original sites in the six SBIRT grantee programs, 69 sites continued providing services in some capacity (same as before, reduced, modified or expanded). Most of the 69 sites (67%) adapted and redesigned the delivery of SBIRT services post-initial grant funding. In addition, new sites were added after grant funding ended, bringing the total number of sites to 88. Analysis of participant responses identified four primary factors that influenced SBIRT sustainability: presence of champions, funding availability, systemic change and SBIRT practitioner characteristics. CONCLUSIONS: Almost 70% of the Screening, Brief Intervention and Referral to Treatment (SBIRT) services in the United States funded initially through a federal program were able to sustain operations after federal funding ceased and some expanded SBIRT services beyond the original sites. The key factors related to sustainability were securing new funding, having champions, adapting and making system changes and managing program staffing challenges.

Screening, brief intervention and referral to treatment (SBIRT): implementation barriers, facilitators and model migration.

AIMS: To identify barriers and facilitators associated with initial implementation of a US alcohol and other substance use Screening, Brief Intervention, and Referral to Treatment (SBIRT) grant program, and to identify modifications in program design that addressed implementation challenges. DESIGN: A mixed-method approach used quantitative and qualitative data, including SBIRT provider ratings of implementation barriers and facilitators, staff interview responses and program documentation. SETTING: Multiple sites within the first seven programs funded in a national demonstration program in the United States. PARTICIPANTS: One hundred and two SBIRT providers were surveyed; 221 SBIRT stakeholders and staff were interviewed. MEASUREMENTS: Mean ratings of barriers and facilitators were calculated using provider survey responses. An inductive content analysis of interview responses identified factors perceived to support and challenge implementation; program modifications that occurred over time were recorded. FINDINGS: Providers rated pre-selected implementation facilitators higher than barriers. Content analysis of interview responses revealed six themes: committed leaders; intra- and inter-organizational communication/collaboration; provider buy-in and model acceptance; contextual factors; quality assurance; and grant requirements. Over time, programs tended to: adopt more efficient 'pre-screen' item sets; screen for risk factors in addition to alcohol/substance use; use contracted specialists to deliver SBIRT services; conduct services in high-volume emergency department and trauma center settings; and implement on-site and telephonic treatment delivery. CONCLUSIONS: Screening, Brief Intervention and Referral to Treatment program implementation in the United States is facilitated by committed leadership and the use of substance use specialists, rather than medical generalists, to deliver services. Many implementation challenges can be addressed by an adequate start-up phase focused on comprehensive education and training, and on the development of intra- and inter-organizational communication and collaboration; opinion leader support; and practitioner and host site buy-in.

RNA nanomedicines: the next generation drugs?

RNA therapeutics could represent the next generation personalized medicine. The variety of RNA molecules that can inhibit the expression of any mRNA using, for example, RNA interference (RNAi) strategies, or increase the expression of a given protein using modified mRNA together with new gene editing strategies open new avenues for manipulating the fate of diseased cells while leaving healthy cells untouched. In addition, these therapeutic RNA molecules can maximize the treatment of diseases and minimize its adverse effects. Yet, the promise of RNA therapeutics is hindered by the lack of efficient delivery strategies to selectively target these molecules into specific cells. Herein, we will focus on the challenges and opportunities of the delivery of therapeutic RNAi molecules into cancer cells with special emphasis on solid tumors. Solid tumors represent more than 80 percent of cancers and some are very challenging to treat, not merely due to physiological barriers but also since the tumor microenvironment (TME) is a complex milieu of accessory cells besides the cancerous cells. In this review, we will highlight various limiting factors to successful delivery, current clinical achievements and future outlook focusing on RNAi therapeutics to the TME.