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Cytostatic hypothermia and its impact on glioblastoma and survival

Syed Faaiz Enam, Cem Y. Kilic, Jianxi Huang, Brian J. Kang, Reed Chen, Connor S. Tribble, Martha I. Betancur, Ekaterina Ilich, Stephanie J. Blocker, Steven Owen, Johnathan G. Lyon, Ravi V. Bellamkonda

Preprint posted on March 26, 2021 https://www.biorxiv.org/content/10.1101/2021.03.25.436870v1.full

Cytostatic hypothermia: a very “cool” way to stop brain tumor progression and prevent tumor recurrence

Selected by Kristina Kuhbandner

Background

The effects of hypothermia, defined as body temperature below 35°C, and its application as a therapeutic strategy in various diseases has been studied for a long time (1). Today, mild therapeutic hypothermia is widely used in emergency medicine to improve the rates of long-term survival without neurological damage in patients with sudden cardiac arrest (2). First experiments using hypothermia in the field of neurosurgery date back to the mid-twentieth century, when the neurosurgeon Temple Fay utilized quite unconventional tools (including a beer cooler-machine pump) to locally lower the temperature in a brain tumor patient (3).

Glioblastoma is the most common type of malignant brain tumor, and affected individuals have a very poor survival rate. This might be partly due to residual cancer cells in the resection margin after surgical removal of the malignant tissue. Subsequent postoperative treatments such as radio- and chemotherapy can activate these dormant glioblastoma cells and ultimately lead to a recurrence of the tumor (4). This emphasizes the need for alternative treatment strategies able to inhibit cancer recurrence without damaging the healthy brain tissue. Recently, it has been suggested that moderate hypothermia might be a promising adjuvant glioblastoma therapy through inhibition of cancer cell proliferation (5). This preprint by Enam and colleagues investigates the effect of cytostatic hypothermia on different glioblastoma cell lines in vitro and in vivo using two different rat glioblastoma models.

“Cool” results

First, the authors aimed to define an optimal temperature range sufficient to stop tumor growth but with minimal cytotoxicity for healthy cells, which they termed “cytostatic hypothermia”. For the human glioblastoma cell lines used in this assay this ranged from 20-25°C.  Notably, cycles of hypothermia with intervals of normal temperature showed comparable cytostatic results and were even less cytotoxic. Further, they determined the effect of hypothermia on cell cycle, metabolism and cytokine synthesis. Any kind of hypothermia treatment resulted in cell cycle arrest in the G2 phase, with a concurrent decrease in metabolite consumption and production and reduced secretion of inflammatory cytokines.

Next, Elan et al. applied different simulation tools and considered parameters from previous studies to model local intracranial hypothermia in the rat brain. Based on these data, they built a hypothermia device consisting of 1) an implantable interface which attaches to the skull with a gold needle reaching into the tumor and 2) a removable cooling element. For a preliminary imaging study, rats were inoculated with tumor cells followed by implantation of the hypothermia device. MRI analysis showed that tumor size was significantly reduced in rats receiving a seven-day hypothermia treatment compared to control animals with cooler switched off.

To assess the effect of hypothermia treatment on the survival rate, two different rat models (Fisher rats inoculated with F98 and RNU rats inoculated with human U-87 MG cells, respectively) were used. In both models, cytostatic hypothermia significantly prolonged animal survival and reduced tumor burden. Finally, the authors investigated whether hypothermia can be used in combination with traditional therapeutic strategies, namely chemotherapy or CAR (chimeric antigen receptor) T immunotherapy, in vitro. While hypothermia boosted the growth-inhibiting effects of the chemotherapeutic agent temozolomide, reduced temperatures seemed to slightly impact the potency of CAR T cells to kill tumor cells.

Why I chose this preprint

Although I am not very familiar with neurosurgery, this preprint immediately caught my attention. Firstly, glioblastomas are very aggressive, and I personally knew a patient who died following tumor recurrence a few years after tumor removal. Therefore, the development of additional treatment options able to prevent this scenario is key to improve survival chances. Enam et al. show that using hypothermia might be a promising strategy to inhibit tumor cell reactivation and consequently tumor progression and recurrence. Furthermore, their results might also be useful for the treatment of other temperature-sensitive cancer types. I also like their approach going from cell culture experiments to computer-based simulations to in vivo models. Although the beneficial effect of hypothermia in vitro has been known before, this is the first study to translate these findings to the in vivo situation. Last but not least, the history and evolution of hypothermia research is absolutely astonishing and I enjoyed delving deeper into this topic reading many fascinating stories.

Questions to the authors

  • Your in vitro studies show that F98 cells are much more resistant to hypothermia. Do you have an explanation for this?
  • Developing such a hypothermia device is an enormous technical challenge and an awesome achievement. However, refinements are necessary as some of the devices failed during the trial especially when rats were more active. Furthermore, it would be very helpful if the thermistor shows compatibility with MRI. Do you already have ideas how these improvements could be realized?
  • The last experiment on possible combination therapies indicates that temperature reduction could affect the functionality of CAR T cells in vitro. Are you also planning to further investigate this and the co-therapy with chemotherapeutics or radiotherapy in vivo?
  • Considering the differences between rats and humans, what are the biggest challenges in constructing a device for application in humans?

References

  1. Bohl MA, Martirosyan NL, Killeen ZW, Belykh E, Zabramski JM, Spetzler RF, et al. The history of therapeutic hypothermia and its use in neurosurgery. J Neurosurg. 2018 May 25;130(3):1006–20.
  2. Hunter BR, Ellender TJ. Targeted temperature management in emergency medicine: current perspectives. Open Access Emerg Med OAEM. 2015 Sep 28;7:69–77.
  3. Fay T. Early Experiences with Local and Generalized Refrigeration of the Human Brain. J Neurosurg. 1959 May 1;16(3):239–60.
  4. Wion D. Therapeutic dormancy to delay postsurgical glioma recurrence: the past, present and promise of focal hypothermia. J Neurooncol. 2017 Jul 1;133(3):447–54.
  5. Fulbert C, Chabardès S, Ratel D. Adjuvant therapeutic potential of moderate hypothermia for glioblastoma. J Neurooncol [Internet]. 2021 Mar 19 [cited 2021 Mar 27]; Available from: https://doi.org/10.1007/s11060-021-03704-y

Tags: brain tumor, cancer recurrence, hypothermia

Posted on: 10th April 2021 , updated on: 21st April 2021

doi: https://doi.org/10.1242/prelights.28191

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Author's response

Syed Faaiz Enam shared

1) Your in vitro studies show that F98 cells are much more resistant to hypothermia. Do you have an explanation for this?
We do not currently have a clear explanation for this but it is fascinating. We hypothesize that it may be due to temperature-resistant metabolic or transcriptomic pathways which need to be elucidated with further studies. Currently, our data show that cell morphology of F98 is significantly different between 25°C and 20°C. Apart from that, we still see an accumulation of cells in the G2-phase even at 25°C and a reduction in metabolite production/consumption at 25°C and 20°C. Regardless, this raises questions of whether there are cells that are even more resilient (re: cell division and temperature), what the mechanisms driving this are, and whether it is hypothetically possible to evolve around cytostatic hypothermia!
 
2) Developing such a hypothermia device is an enormous technical challenge and an awesome achievement. However, refinements are necessary as some of the devices failed during the trial especially when rats were more active. Furthermore, it would be very helpful if the thermistor shows compatibility with MRI. Do you already have ideas how these improvements could be realized?
Working with a small animal model for this application has been technically challenging but there is a lot of room for improvement. As we mentioned, MRI-compatible thermistors do exist but we experienced unreliability when using them. Currently we do not know why those thermistors failed but were unable to identify any major fault in the way they were wired to the Interface. That said, it may be worth giving them another shot because it would make longitudinal tumor volume assessment possible. The problem of active rats eventually dislodging their Interface would likely best be solved with either further miniaturization or switching to a liquid-based cooler that is hidden under the scalp. These are strategies we are considering for future studies.
 
3) The last experiment on possible combination therapies indicates that temperature reduction could affect the functionality of CAR T cells in vitro. Are you also planning to further investigate this and the co-therapy with chemotherapeutics in vivo?
Absolutely! Both findings were happy surprises. While we want to test these in vivo (with different hypothermia dosing paradigms) we also want to understand the mechanisms of why there is synergism with Temozolomide and why CAR T cells retain and lose some functionality. A mechanistic study could elucidate the scope of chemotherapy-hypothermia synergism and provide ways to overcome reduced CAR T cell functioning.
 
4) Considering the differences between rats and humans, for example much bigger tumor volume in humans, what are the biggest challenges in constructing a device for application in humans?
Currently the biggest challenge is designing a fully-implantable device that provides homogenous cytostatic hypothermia to a large region of tissue that is also practicable, potentially indefinitely, for a patient. We are working on this!

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