In conversation with

Ankita Das

on her recent study:

Das A, Nikhil A, Kumar A. Preparation of thermo-responsive polymer encapsulated exosomes and its role as a therapeutic agent for blood clot lysis. Colloids and Surfaces B: Biointerfaces. 2022 May 18:112580.

MFCEM: Congratulations Ankita to you and all authors of the study. Let me start by asking you, what gap does this study fill with respect to known and standard methods for thrombus degradation?
Ankita Das:
First of all, on behalf of all the authors, I would like to thank MFCEM for appreciating our work and thanks for the opportunity to discuss on the key aspects of the study.

To tackle the problem of arteries getting blocked by a clot, clinically the first line of defense is systemic injections of tissue plasminogen activators like streptokinase and urokinase. But this strategy has a downside in aspects of various side effects including bleeding and bruising at the injection site, blurred vision, fast heart rate, bleeding from the nose and gums, among others. So, such treatment has to be used with caution with other medications that alter platelet function and increase the risk of bleeding. Our group wanted to propose an effective solution for this problem with enhanced efficiency to fill the limitations of standard methods for thrombus degradation. So, in our study we selected a combinatorial injectable therapy where the treatment was provided locally to the clot, in comparison to the conventional systemic approach applied in the in vivo system. In this approach exosomes isolated from a cell line reported to have urokinase releasing activity have been utilized to check the thrombolytic effects and a thermo-responsive polymer was used as the delivery vehicle for exosomes. When exosome laden thermo-responsive polymer will be injected over the clot, polymer would undergo phase-change in situ and encapsulate the clot and there will be sustained release of the exosomes which will eventually lead to clot lysis. The sustained release approach has been adapted to avoid the risk of thrombo-embolism, which might occur if clot is detached from its site by direct treatment of exosome. Hence, the proposed treatment system will enable clot dissolution without associated limitations.

MFCEM: The study is very exhaustive, and I see that it spreads from harvesting exosomes from cell lines, to standardizing the polymer for encapsulation & release. What was the most challenging part of the study?
Ankita Das:
Yes, the study is quite extensive but has shown very interesting results. In this study, we have utilized different exosome concentrations and compared their activity with commercial tissue plasminogen activator (tPA) streptokinase. From the clot lysis activity assays which are routinely carried out for fibrinolytic drugs, we were able to deduce that these exosomes had the potential to serve as a replacement for streptokinase. Since, the activity of exosomes matched to that of streptokinase applied at a much higher concentration than the test tube equivalent (when a blood clot is placed in a static milieu), thereby showing the potential and application of these exosomes. The polymer solution was also optimized at a concentration where the released exosome will be sufficient enough in terms of concentration to carry out clot lysis. Challenge was to make a strategy that will be at par with the current clinical treatment strategies and study has to be designed such that its application will be clinically possible after its evaluation in pre-clinical trials. The ideology for this strategy was instigated by my mentor, Prof. Ashok Kumar, and our collective team effort yielded fruitful results.

MFCEM: Do you have any plans to test these polymer-encapsulated-exosomes for their efficacy in clot removal in animal models?
Ankita Das:
Yes, we are planning to test the efficacy of these polymer-encapsulated exosomes in animal model, where we will be generating a localized thrombus using chemical reagents and our combinatorial therapy will be utilized for clot degradation. The work will be carried out on a collaborative basis, and we are in the process of finalizing it.

MFCEM: What was the most satisfying aspect of this study?
Ankita Das:
Exosomes or extracellular vesicles (on a broader perspective) from stem cells have been utilized extensively for regenerative and therapeutic applications. In the present study, we wanted to explore the role of exosomes for thrombus degradation by taking into consideration previous literature reports about their plasminogen converting activity. We were able to satisfactorily lyse blood clots with these exosomes and also the use of a thermo-responsive polymer proved to be beneficial in encapsulating the blood clots so that the retained exosomes would be able to function better. Also, we were able to show that the exosome-based therapy is showing clot lysis effect similar to the clinically used drugs like streptokinase which has enhanced the scope and potential of the study many folds. This work was accompanied with high-risk, although the outcome came out as a high reward to us, and has also opened a new avenue in this field of research.

In conversation with first author, Sakhi Goel.

Transcriptional network involving ERG and AR orchestrates Distal-less homeobox-1 mediated prostate cancer progression.

Goel S, Bhatia V, Kundu S, Biswas T, Carskadon S, Gupta N, Asim M, Morrissey C, Palanisamy N, Ateeq B.

Nature communications. 2021 Sep 7;12(1):1-22.

Congratulations Sakshi to you and all authors for this wonderful study. What is the major gap in understanding of Prostate cancer that your study addresses?
Sakshi Goel: Distal-less homeobox-1 (DLX1) is widely recognized as a biomarker which is used to detect prostate cancer (PCa) owing to its higher expression in PCa patients. But the major gap existed that how the level of DLX1 is increased in PCa and what role does it play. Our study addressed these two important questions and established the role of DLX1 in promoting cancer growth and it’s spread to other organs. We also identified the role of well-known PCa promoting proteins AR and ERG in regulating DLX1. Further we demonstrate the treatment strategies which could help in reducing DLX1 expression, thereby regressing the tumor growth.

What was the most challenging part of the study?
Sakshi Goel: DLX1 is a homeobox gene which is known to play important role in development of jaws, neurons, and bone. It is not known to play any role during the development of prostate gland and hence is not expressed there. Additionally, not much was known about the role of DLX1 in PCa. Thus, the most challenging part was to identify the mechanism of DLX1 upregulation and its functional significance in the cancer type of an unrelated organ.

Where do you see the application of this study, particularly in the Indian context?
Sakshi Goel: Our study has identified that DLX1 and its tumor promoting ability can be targeted with the use of Bromodomain and extraterminal protein inhibitors either alone or coupled with the drugs against androgen receptor signaling. There are several commercially available urine-based diagnostics tests for detecting DLX1 levels in PCa patients. In western countries, these kits are used to detect PCa. If the DLX1 diagnostic test is utilized in India as well, the disease could be identified at the early stage, and it would be relatively easy to categorize PCa patients with higher DLX1 levels. This would further assist to predict the patients who could respond to suggested treatment strategy. Hence, the present study would be immensely helpful for better disease management of PCa patients with higher DLX1 levels.

How did the collaboration with other research group help?
Sakshi: The collaborations with other research groups provide a broader perspective to the study. Additionally, it helps in widening the biological tools to validate the findings thus providing a significant contribution in the research. In the present study our collaborations gave us access to the larger cohorts of both primary and advanced stage PCa patients. Using which we were able to identify that ~60% of PCa patients shows higher expression of DLX1. Further, we also validated the positive association between AR, ERG and DLX1 in the PCa patient samples.

Bioinspired Injectable Hydrogels Dynamically Stiffen and Contract to Promote Mechanosensing-Mediated Chondrogenic Commitment of Stem Cells.

Mahajan A, Singh A, Datta D, Katti DS.

ACS Applied Materials & Interfaces. 2022 Feb 4;14(6):7531-50.

The articular cartilage is a load-bearing tissue that acts as a shock absorber and ensures smooth movement of joints. Thus, not surprisingly, articular cartilages are highly prone to acute and chronic injuries, leading to joint impairment and disability. Further, due to limited number of tissues specific progenitor cells, compounded by lack of vasculature, cartilage has a limited capacity to repair or regenerate. Owing to which there is a dire need to develop strategies that can address the wear and tear of cartilage. Most of current tissue engineering strategies involving hydrogel-based scaffolding systems, that while promising, offer poor mechanical strength due to high-water content. In this study conducted by Prof. Katti and team, they display, a triplenetwork injectable hydrogel system, engineered using Bombyx mori silk fibroin, carboxymethyl cellulose (CMC), and gelatin that displayed a simultaneous increase in both stiffness and contraction over time, thereby imparting a four dimensional (4D) evolving niche to the cells. While resilience was provided by CMC, the dynamic alterations in the hydrogel matrix were attributed to the formation of β-sheets in silk. The engineered contraction facilitated condensation of cells that mimicked an important step during cartilage development. Subsequently, this led to downregulation of YAP signaling and enhanced chondrogenic commitment of stem cells. More importantly, the in vivo study showed that the ectopically regenerated cartilage was mature and closely resembled native articular cartilage. The study could prove to be a major step forward in area of cartilage biology and repair.