| Summary: | <p>Ionizing radiation threatens cellular survival, a property strategically exploited in radiotherapy to eradicate cancer cells. However, the efficacy of radiotherapy is impeded by two co-existing challenges: the presence of radiotolerant cancer stem cells capable of withstanding treatment and subsequently driving cancer recurrence; coupled with collateral damage to normal stem cells, which are typically radiosensitive. Despite the pressing demand for radiotherapy, the underlying mechanisms governing radiosensitivity in normal stem cells and radiotolerance in cancer stem cells remain enigmatic, particularly within an in vivo setting. Here, we leverage the planarian <em>Schmidtea mediterranea</em>, which harbours abundant populations of experimentally tractable adult stem cells, as an in vivo model system to investigate stem cell radiation response and recovery. Employing a functional genomics approach, we aim to assess the transcriptional response of planarian stem cells to gamma radiation at both bulk and single-cell levels, as well as to uncover novel genes crucial for planarian radiotolerance.</p>
<p>We generated SPLiT-Seq libraries from irradiated planarians to explore their response to acute gamma radiation at the single-cell level. Our single-cell atlas delineates distinct stem cell clusters exhibiting varying sensitivity to gamma radiation. RNA velocity analyses further reveal dose and time-dependent changes in cell fate trajectories following irradiation. By integrating marker patterns and cell fate trajectories, we proposed a model linking radiosensitivity to cell cycle phase, with stem cells in S/G2/M phases exhibiting heightened sensitivity to gamma radiation.</p>
<p>Guided by insights from our single-cell and previous bulk RNA-sequencing data, we conducted an RNAi screen to identify novel regulators of planarian radiotolerance. We silenced the expression of 105 candidate genes and exposed animals to a sub-lethal dose of gamma radiation. Our screen unveiled six genes, namely Slmap, Kin-17, Dkc, Rab32, Rasl-12, and DMXL-1, as essential regulators of planarian radiotolerance in vivo. An additional 20 single-gene knockdowns were observed to significantly delay the post-irradiation stem cell recovery process, although these knockdowns alone were insufficient to cause animal mortality. Multiple members of the FHL gene family were amongst the single knockdowns causing this delay phenotype. To account for potential redundancies, we knocked down the FHL family members in all possible combinatorial pairs, revealing distinct RNAi pairs that rendered planarians sensitive to gamma radiation.</p>
<p>We then examined the in vivo functions of Slmap, Kin-17, Dkc, Rab32, Rasl-12, and DMXL-1 in the regulation of planarian radiotolerance. We determined that following sub-lethal irradiation, the residual surviving stem cells in Slmap, Kin-17, and Dkc RNAi-treated animals were proliferation incompetent. In contrast, RNAi of Rab32, Rasl-12, and DMXL-1 led to an abnormally prolonged state of cell-cycle arrest in the remaining stem cells. Whether it was complete abolition of stem cell recovery in the former set of gene knockdowns or partial recovery in the latter, the knockdown-induced impairments prevented animal survival following exposure to sub-lethal irradiation. Among these six genes, we identified Dkc as a key player for DNA strand break repair in planarians.</p>
<p>In the wider framework, the genes demonstrated in this study to be vital for planarian radiotolerance could be tested in other model organisms, potentially uncovering novel and conserved mechanisms employed by adult stem cells to counteract the deleterious effects of ionizing radiation. The potential conservation of these mechanisms in mammals may also hold biomedical relevance, as targeting these genes could be explored for imparting radioprotection to normal stem cells, or to sensitize cancer stem cells to radiotherapy.</p>
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