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.

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.

Mechanistic insights into nanoparticle surface-bacterial membrane interactions in overcoming antibiotic resistance.

Antimicrobial Resistance (AMR) raises a serious concern as it contributes to the global mortality by 5 million deaths per year. The overall impact pertaining to significant membrane changes, through broad spectrum drugs have rendered the bacteria resistant over the years. The economic expenditure due to increasing drug resistance poses a global burden on healthcare community and must be dealt with immediate effect. Nanoparticles (NP) have demonstrated inherent therapeutic potential or can serve as nanocarriers of antibiotics against multidrug resistant (MDR) pathogens. These carriers can mask the antibiotics and help evade the resistance mechanism of the bacteria. The targeted delivery can be fine-tuned through surface functionalization of Nanocarriers using aptamers, antibodies etc. This review covers various molecular mechanisms acquired by resistant bacteria towards membrane modification. Mechanistic insight on 'NP surface-bacterial membrane' interactions are crucial in deciding the role of NP as therapeutic. Finally, we highlight the potential accessible membrane targets for designing smart surface-functionalized nanocarriers which can act as bacteria-targeted robots over the existing clinically available antibiotics. As the bacterial strains around us continue to evolve into resistant versions, nanomedicine can offer promising and alternative tools in overcoming AMR.

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.

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.

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.

P-glycoprotein inhibition of drug resistant cell lines by nanoparticles.

Several pharmaceutical excipients are known for their ability to interact with cell membrane lipids and reverse the phenomenon of multidrug resistance (MDR) in cancer. Interestingly, many excipients act as stabilizers and are key ingredients in a variety of nano-formulations. In this study, representatives of ionic and non-ionic excipients were used as surface active agents in nanoparticle (NP) formulations to utilize their MDR reversing potential. In-vitro assays were performed to elucidate particle-cell interaction and accumulation of P-glycoprotein (P-gp) substrates-rhodamine-123 and calcein AM, in highly drug resistant glioma cell lines. Chemosensitization achieved using NPs and their equivalent dose of free excipients was assessed with the co-administered anti-cancer drug doxorubicin. Among the excipients used, non-ionic surfactant, Cremophor® EL, and cationic surfactant, cetyltrimethylammonuium bromide (CTAB), demonstrated highest P-gp modulatory activity in both free solution form (up to 7-fold lower IC50) and as a formulation (up to 4.7-fold lower IC50) as compared to doxorubicin treatment alone. Solutol® HS15 and Tween® 80 exhibited considerable chemosensitization as free solution but not when incorporated into a formulation. Sodium dodecyl sulphate (SDS)-based nanocarriers resulted in slightly improved cytotoxicity. Overall, the results highlight and envisage the usage of excipient in nano-formulations in a bid to improve chemosensitization of drug resistant cancer cells towards anti-cancer drugs.

Evaluation of dual P-gp-BCRP inhibitors as nanoparticle formulation.

Overcoming multidrug resistance (MDR) in cancer is a major challenge and efforts are on-going to develop inhibitors against the most characterized and ubiquitous MDR transporters: P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and breast cancer resistance protein (BCRP). Recently reported, two 4-anilinoquinazolines (compounds 1 and 2), demonstrate potential MDR reversal activity against BCRP and to a lesser extent, P-gp. In this work, we formulated the compounds as polymeric nanoparticles (NPs) and assessed their MDR inhibitory activity in relevant BCRP and P-gp over-expressing cell line models. Particles in the size range 300-365nm with a loading efficiency of 69% (compound 1 NP) and 77% (compound 2 NP) respectively were obtained. BCRP inhibition was observed in Hoechst 33342 and pheophorbide A assays while P-gp inhibition was evaluated in calcein AM and rhodamine-123 assays. In cytotoxicity studies, while BCRP expressing cells showed complete reversal of drug resistance in nearly all treatment groups (both compounds and their respective NP); a higher reversal in NP treated group was obtained as compared with inhibitory compound treated group in P-gp expressing cells. These results demonstrate promising inhibitory activity of both formulations, especially against P-gp expressing cells; which is possibly due to a prolonged presence of encapsulated compounds in NPs and consequently a prolonged sensitization of transmembrane drug transporter. These formulations can therefore be considered as dual-transporter inhibitors and it is imperative to investigate both inhibitors in animal models of MDR owing to the presence of multiple efflux transporters in several cancer models.

Cargoing P-gp inhibitors via nanoparticle sensitizes tumor cells against doxorubicin.

Inhibitors against multidrug resistance (MDR) efflux transporters have failed in most clinical settings due to unfavorable pharmacokinetic interactions with co-administered anti-cancer drug and their inherent toxicities. Nanoparticles (NPs) have shown potential to overcome drug efflux by delivering and localizing therapeutic molecules within tumor mass. In this work, we investigated effect of nanocarrier surface charge and formulation parameters for a hydrophilic and lipophilic MDR inhibitor on their ability to reverse drug resistance. Active inhibition of efflux pumps was achieved by encapsulating first and third generation P-gp inhibitors- verapamil and elacridar respectively in non-ionic, anionic and cationic surfactant-based NPs. The ability of NPs to reverse P-glycoprotein (P-gp)-mediated MDR efflux was evaluated in sensitive (A2780) and resistant (A2780Adr) ovarian cancer cell lines by various in vitro accumulation and cytotoxicity assays. Uptake mechanism for NP appears to be caveolae-dependent with 20%-higher internalization in A2780Adr than A2780 cell lines which can be co-related to the biophysical membrane composition. Cationic- CTAB NPs showed highest reversal efficacy followed by PVA and SDS-NP (P+S NP) and PVA-NPs. As compared to doxorubicin treated drug resistant cells lines, blank-, verapamil- and elacridar-CTAB-NPs showed 2.6-, 20- and 193-fold lower IC50 values. This work highlights the importance of inhibitor-loaded charged particles to overcome cancer drug resistance.

Nanocarrier-based immunotherapy in cancer management and research.

Research in cancer immunotherapy has gained momentum in the last two decades, with many studies and clinical trials showing positive therapeutic outcomes. Immunotherapy can elicit not only a strong anticancer immune response which could even control metastases, but could also induce immunological memory, resulting in long-lasting protection in the prophylactic setting and protection against possible recurrence. Nanocarriers offer an attractive means for delivery of a multitude of therapeutic immunomodulators which are readily taken up by immune cells and can initiate a particular arm of an immunostimulatory cascade leading to tumor cell killing. This review focuses on recent advances in nanocarrier-mediated immunotherapy for the treatment of cancer. Both in vitro and in vivo studies as well as clinical progress are discussed in various sections. Description of the specific role of nanoparticle technology in immunotherapy highlights the way particles can be tailor-made in terms of size, structure, payload, and surface properties for active targeting to antigen-presenting cells and/or enhanced accumulation in the solid tumor.

Nanoparticle mediated co-delivery of paclitaxel and a TLR-4 agonist results in tumor regression and enhanced immune response in the tumor microenvironment of a mouse model.

Inefficiency of cancer chemotherapy to improve life expectancy in majority of patients raises serious concern and warrants development of novel therapeutic strategies. Immunotherapy in combination with chemotherapy has shown promising outcomes in recent years. Herein, we report better tumor regression and enhancement of antitumor immune response at the tumor microenvironment by co-delivery of paclitaxel and a TLR4 agonist through a PLGA based nanoparticle preparation (TLNP). Particle characterization showed high encapsulation of both components and retention of their biological activities. In vivo tumor regression studies demonstrated clear benefit of TLNP over the paclitaxel. The mean tumor volume of the TLNP treated animals was found to be 40% less than that of the Paclitaxel treated animals. Flow cytometric analysis of tumor infiltrating immune cells indicated activation of antigen presenting cells and T-cells providing evidence of Th1 immune response. In vivo results are promising and could pave way for novel chemo-immunotherapeutic treatment modality.

Combined chemo-immunotherapy as a prospective strategy to combat cancer: a nanoparticle based approach.

The prime objective of this study was to develop a combined chemo-immunotherapeutic formulation which could directly kill cancer cells as well as activate the immunosuppressed tumor microenvironment to mount a robust antitumor immune response. Paclitaxel (PTX) and SP-LPS (nontoxic derivative of lipopolysaccharide) were selected as anticancer drug and immunostimulant respectively. Poly(lactic-co-glycolic acid) (PLGA) based PTX and SP-LPS containing nanoparticles (TLNP) were prepared by the double-emulsion method (w/o/w) and characterized in terms of size, zeta potential and transmission electron microscopy (TEM). The release behavior of PTX and SP-LPS from the TLNP exhibited a biphasic pattern characterized by an initial burst followed by slow continuous release. In vitro anticancer activity of TLNP was found to be higher compared to PTX when studied in a tumor cell-splenocyte coculture system. TLNP activated murine monocytes induced the secretion of various proinflammatory cytokines. After iv administration of TLNP in tumor bearing C57BL/6 mice, the amount of PTX in the tumor mass was found to be higher in TLNP treated mice as compared to commercial Taxol group at all time points studied. In vitro studies suggest that nanoparticles containing PTX and SP-LPS have both direct cytotoxicity and immunostimulatory activity. Hence this might have potential as a chemo-immunotherapeutic formulation against cancer with advantage over present day chemotherapy with Taxol, in terms of tumor targeting, less toxicity and immunostimulation.