First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Paul Ann Riya and Budhaditya Basu are co-first authors on ‘ HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells’, published in JCS. Paul Ann Riya is a PhD student in the lab of Dr Jackson James at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India, investigating the key mechanisms that maintain stem cells. Budhaditya Basu is a PhD student in same lab, where he works on understanding the role of circular RNA in axon path finding.

Paul Ann Riya

How would you explain the main findings of your paper in lay terms?

P.A.R. and B.B.: Cancer stem cells (CSCs) are a miniscule population among numerous other tumor cell types. These cells are like seeds that can give rise to the entire tumor if required. Major cancer therapies that are in the market target these stem cells in order to stop tumor regrowth. We showed that CSCs are not a homogenous mixture of cells that can be targeted by one single mechanism, and even if CSCs are eliminated, there is still a possibility for them to return back from other tumor cell types, which might lead to tumor relapse. Notch signaling and its downstream regulator HES1 is one of the major signaling pathways required for the maintenance and proliferation of CSCs. In our paper, we have shown the existence of heterogenic HES1 expression in neuroblastoma CSCs through which these stem cells switch from one subtype to another subtype.

Were there any specific challenges associated with this project? If so, how did you overcome them?

P.A.R.: The initial step for all experiments in the manuscript was sorting of NDHes-1 and NIHes-1 cell populations by FACS. The NIHes-1 population was only 1% of the total population, and we could not get enough cells for the experiments. This was the first challenge that we faced. To overcome that we performed multiple FACS sorting experiments and generated an enriched culture of NIHes-1-expressing cells.

Budhaditya Basu

B.B.: To explore the biology behind the CSCs, we had to perform transcriptomic analysis and look at existing high-throughput data. Learning bulk and single-cell transcriptomic analysis took a lot of effort and time. We are really thankful to Dr Achuthsankar Sukumaran Nair and Dr Shijulal Nelson-Sati for providing access to their high-end computing facility and appropriate software to play around with our data. COVID was a challenging time, especially when we had to stop all running in vitro experiments, as well as planned in vivo experiments. This significantly delayed our research progress, but at the same time I feel that has helped us to develop new interests in coding skills, which actually helped how we analyzed and visualized our data.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

P.A.R.: Actually, the whole work was initiated from a fortunate stroke of serendipity. We started to generate an IMR32 cell line with a reporter construct to screen Notch target drugs as part of other work in the lab. While generating the cell line, we sorted red fluorescent NDHes-1-expressing cells and green fluorescent NIHes-1-expressing cells by FACS and cultured each separately. We could observe red cells in green-sorted culture plates after 6 days in culture. This was the first ‘eureka’ moment, which led to the initiation of the project. We found that NIHes-1-expressing cells can give rise to NDHes-1-expressing cells. The next ‘eureka’ moment was when we saw NIHes-1 expression at the margins of NDHes-1 spheroids and during budding of secondary spheres. We were very excited to find a reverse transition of NDHes1 cells to NIHes-1 state in 3D culture.

B.B.: Initially, we were focused on the transition between two different modes of HES1 expression among the CSCs. When we revisited our time-lapse images and found that bulk cancer cells are capable of reverting to the CSC state, that significantly changed our perception. We then looked at all our previous data again and found many insights into their behavior. Sometimes we are biased by our own hypothesis, but some days, we actually break our own hypothesis.

Why did you choose Journal of Cell Science for your paper?

P.A.R.: We explain the cell state transitions in IMR32 cells, which is well suited for publication in JCS. JCS has a wide audience, which makes it highly appropriate for publishing our novel data.

B.B.: It is a reputed cell biology journal and we wanted to publish our findings there so they get the attention of a relevant readership.

Notch-dependent Hes-1-expressing CSCs at the margin transitioning to Notch-independent Hes-1-expressing CSCs in a 3D spheroid model, as indicated with arrows.

Notch-dependent Hes-1-expressing CSCs at the margin transitioning to Notch-independent Hes-1-expressing CSCs in a 3D spheroid model, as indicated with arrows.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

P.A.R.: Dr Jackson James was the most supportive mentor someone could ever have. Even though the lab focus was on embryonic brain development, he allowed me to venture into a new field. His enthusiasm made me work even if I was tired at times.

B.B.: Yes. I would like to mention my supervisor, Dr Jackson James, who has always encouraged us to plan our own thesis projects and experiments, and discuss the results even if they did not work. He always looked at our potential and inclination and based on that he suggested taking up our projects. I feel that he created an atmosphere of thinking freely. Above all, I learnt a lot from him about how to run a lab with a constrained budget and how to get things done when administration becomes a bottleneck for pursuing research.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

P.A.R.: I was interested in science from childhood onwards. During undergraduate training, we isolated DNA and I could see a white thread-like structure floating in the test tube, which was one of the most exciting moments that I have experienced. In fact, I was able to do karyotyping of my own chromosome, which was a booster dose for my excitement. I will always get excited about moving cells, even if I see that a million times. I am sure that I can never stop experimenting in science.

B.B.: I think the news of the human genome project and a curious reading of its potential motivated me to pursue a career in science. The most interesting moment to me was the day when I first saw a hippocampal neuron culture. The amount of fascinating neuroscience that is happening around the globe always encourages me to ask questions.

Who are your role models in science? Why?

P.A.R.: Dr Lubert Stryer was the one who taught me to ask questions. He was the best teacher who taught me to study science with enthusiasm through his books. He helped me to decide on my interest and to pursue my career in science.

B.B.: Srinivasa Ramanujan and Rosalind Franklin. These are the names that come first to my mind. The sheer dedication towards their work motivates me always. I do enjoy reading the work of Professor Erin Schuman.

What's next for you?

P.A.R.: Currently I am about to submit my PhD thesis. Understanding signaling pathways always excited me, and I would like to do post-doctoral training in neuro-oncology. I would like to continue in academia as a scientist who will motivate many, especially women, to science.

B.B.: Currently I am in the last stage of my PhD. I want to continue research in neuroscience in the future. At the same time, I want to take my coding skills to the next level.

Tell us something interesting about yourself that wouldn't be on your CV

P.A.R.: I can handle the kitchen very nicely, which helped me a lot in managing disciplined cell culture experiments.

B.B.: I always enjoy listening to Shastriya Sangeet (Indian classical music) and love playing badminton.

Paul Ann Riya's and Budhaditya Basu's contact details: Rajiv Gandhi Centre for Biotechnology, Jagathy, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India.

E-mail: riyaann@rgcb.res.in; budhaditya@rgcb.res.in

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et al. 
(
2022
).
HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells
.
J. Cell Sci.
135
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jcs260157
.