Cancer Stem Cells in Repopulation during Radiotherapy: Comparison
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Please note this is a comparison between Version 2 by Fanny Huang and Version 1 by Loredana G Marcu.

Tumour repopulation during treatment is a well acknowledged yet still challenging aspect of cancer management. The latest research results show clear evidence towards the existence of cancer stem cells (CSCs) that are responsible for tumour repopulation, dissemination, and distant metastases in most solid cancers. Cancer stem cell quiescence and the loss of asymmetrical division are two powerful mechanisms behind repopulation. Another important aspect in the context of cancer stem cells is cell plasticity, which was shown to be triggered during fractionated radiotherapy, leading to cell dedifferentiation and thus reactivation of stem-like properties. Repopulation during treatment is not limited to radiotherapy, as there is clinical proof for repopulation mechanisms to be activated through other conventional treatment techniques, such as chemotherapy. The dynamic nature of stem-like cancer cells often elicits resistance to treatment by escaping drug-induced cell death.

  • tumour regrowth
  • repopulation mechanisms
  • radiotherapy
  • cancer stem cells

1. Cancer Stem Cell Properties

Cancer stem cells are recognised as an important cellular category that is at the top of the hierarchical scale, presenting similar biological properties and expression profiles to normal stem cells [1]. While the origins of cancer stem cells in solid tumours are not fully elucidated, it is hypothesised that they arise from either differentiated tumour cells or from organ-specific adult stem cells [2].

1.1. Biological Characteristics of CSCs

Generally, cancer stem cells (CSCs) account for a very small percentage of the cells that constitute a malignant solid tumour [3]. The fraction of CSCs within a tumour was shown to be dependent on the neoplasm, but also on the in vivo assays and biomarkers employed for their identification [4]. An interesting observation derived from in vitro studies on CSCs is related to their heterogeneity [5]. Research undertaken on both HPV-positive and HPV-negative head and neck carcinoma cell lines revealed the existence of a diversity of CSCs that differed in their biological properties and response to therapy, explaining the more responsive behaviour of HPV-positive tumours [5]. This finding demonstrates than not only the absolute percentage of CSCs but also their phenotype dictate treatment outcome, showing the complexity that surrounds CSC identification and targeting. The link between oncogenic viruses and CSCs was also investigated in gastric cancers associated with the Epstein–Barr virus [6,7][6][7]. Epstein–Barr virus-encoded miRNAS were shown to have a strong impact on the maintenance of stemness through the regulation of epithelial–mesenchymal transition. Recent studies on gastric cancers identified a unique CSC marker (CD44v6/v9) for Epstein–Barr virus-associated carcinomas that might serve as a potential target for this subtype of gastric cancer [7].
Despite their low percentage, the biological properties specific to CSCs are powerful tools that influence the fate of the tumour. Among their biological features, pluripotency is perhaps one of the most notable ones, whereby CSCs are capable of unlimited self-renewal by maintaining an undifferentiated state, also being able to create the entire heterogeneous lineage of the original tumour through differentiation [8]. Stemness is maintained by cellular plasticity—a property that promotes tumour heterogeneity and CSC generation.

1.2. CSC and Hypoxia

A particular property of cancer stem cells is their preference for residing in specific microenvironmental niches, such as hypoxic ones, in order to preserve their functionality and stem-like properties [9]. Studies showed that the hypoxia inducible factor (HIF) plays a key role in the niche mechanism, being implicated in CSC survival and proliferation under hypoxic conditions as well as in the angiogenic switch activation [10]. Knowing the challenge of treatment resistance caused by the presence of hypoxia, tumour control is heavily impacted by the increased expression of HIF found in these hypoxic niches.

1.3. CSC Repopulation and Resistance to Therapy

CSCs are also characterised by quiescence, a state that fosters prolongation of their life span, preservation of cellular functions, and protection from therapeutic stress [11]. CSC quiescence was shown to induce treatment resistance and tumour recurrence while also promoting metastases. Another mechanism adopted by CSCs is cellular senescence, which was shown to be responsible for genetic reprogramming and the activation of stemness, contributing to tumour progression and distant spread [11].
All the above-mentioned CSC properties have important clinical implications, as stem-like cancer cells have been shown to be more resistant to treatment than their non-stem counterparts, often leading to treatment failure [12]. One of the reasons for poor treatment outcome is credited to tumour repopulation during therapy. Today it is known that repopulation during treatment is not limited to radiotherapy, as there is clinical proof of repopulation mechanisms being activated through other conventional treatment techniques, such as chemotherapy. The dynamic nature of stem-like cancer cells often elicits resistance to treatment by escaping drug-induced cell death.
The mechanisms behind repopulation are complex and most are attributed to the presence of CSCs [13,14,15][13][14][15]. According to previous research, CSCs adopt various repopulation mechanisms in response to the effect of chemo/radiotherapy. Among them, cell recruitment, shortening of cell cycle duration, and the loss of asymmetrical division are the most commonly investigated and agreed upon mechanisms [13,14,15,16][13][14][15][16]. Quiescence or dormancy, as previously mentioned, is a state outside the cell cycle where CSCs reside to prolong their lifespan and preserve functionality. However, a situation might arise, such as cellular eradication, which triggers the re-entry of CSCs into the cell cycle through the process of cell recruitment. Once in the mitotic cycle, stem cells can further activate repopulation mechanisms by shortening the length of their passage through the cycle, thus promoting faster cell turnover. Perhaps the most efficient repopulation mechanism is the symmetrical division of CSCs (i.e., the creation of two cancer stem cells) which is acquired through the loss of asymmetrical division (i.e., the creation of a stem and a differentiated cell) [15,17][15][17].
The goals of this scoping review are to gather evidence towards the role played by cancer stem cells in tumour repopulation during cancer treatment, and to identify techniques to either counteract these mechanisms or to overcome repopulation by direct targeting of cancer stem cells.

2. Repopulation during Radiotherapy

2.1. Cancer Stem Cells and Their Role in Repopulation during Radiotherapy

Tumour repopulation after radiotherapy remains a significant challenge in cancer therapy. Despite advancements in photon radiation therapy techniques, such as intensity modulated radiotherapy (IMRT) or volumetric modulated arc radiotherapy (VMAT), the recurrence of tumours post-treatment remains an issue for many cancers, including head and neck, breast, colorectal, prostate, and hepatocellular cancers [18,19,20,21][18][19][20][21]. Once again, clonogenic stem cells have emerged in the literature as primary drivers in the process of tumour repopulation that ultimately lead to treatment failure [22,23][22][23]. Because of CSCs’ propensity for renewal, differentiation into various cell types, as well as recruitment of differentiated cells and treatment resistance, they drive tumour repopulation during and post radiation therapy. In other words, the ongoing division of clonogenic cancer stem cells has been shown to be the cause of tumour regrowth in, for example, intestinal, breast, and brain tumours [24]. Similarly to chemotherapy, CSCs are often more resistant to radiation therapy compared to the bulk of tumour cells [25]. This CSC resistance is attributed to various mechanisms, including enhanced DNA repair capacity, quiescence, higher expression of drug efflux pumps, resting in tumour hypoxic regions, etc., which collectively contribute to their survival and capacity to initiate tumour repopulation and regrowth [26]. This phenomenon is a significant obstacle to achieving durable therapy responses in cancer patients. In addition to radiation resistance, another key characteristic of CSCs is their ability to be recruited from the quiescent state following the irradiation damage and re-enter a standard cell cycle, thus initiating cell proliferation and differentiation, leading to the regrowth of the tumour. Research has shown that about one third of CSCs in glioma and breast cancer cell lines remain quiescent but become active in the cell cycle following radiation treatment [27,28][27][28]. The work of Bao et al. [29], conducted in animal models using primary human gliomas, showed that CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, was not only enriched after irradiation but also capable of initiating xenografts from as few as 500 cells. The authors conclude that CD133-positive tumour cells represent the cellular population responsible for glioma radioresistance and potentially tumour recurrence after radiation therapy [29]. CSCs can also promote tumour heterogeneity by generating different types of CSCs through reversible transformations between stem and non-stem cells [30,31][30][31]. This heterogeneity further increases treatment resistance and contributes to cell survival and subsequent repopulation. Preclinical work by Lagadec et al. reports that upregulation of the embryonic transcription factors (such as Sox2, Oct4, Klf4, and Nanog) induced by radiation promotes nontumorigenic cancer cells to acquire CSC-like features [32]. Tumour repopulation itself after radiation therapy can be attributed to several processes. One key factor is the stimulation of surviving cancer cells through a phenomenon known as radiation-induced cytokine release. The irradiated tumour microenvironment can trigger the secretion of growth factors, such as TGF-β and VEGF, which promote the survival and proliferation of cancer cells [33]. Moreover, the radiation-induced DNA damage repair can lead to genetic mutations and clonal expansion of radioresistant cancer cells. This is further compounded by the fact that CSCs have pro-survival pathways that are upregulated and protect these cells from cell death, resulting in CSCs being resistant to radiation damage [34].

2.2. The Effect of Radiotherapy on CSCs

Fractionated radiation therapy, while necessary to mitigate the damage to healthy tissues, is selective in its cell kill. It eliminates, by and large, the differentiated tumour cells and enables the preferential survival of the most radiation therapy resistant and more tumorigenic CSCs, thus generating a so-called radiation-induced CSC phenotype [21,35,36][21][35][36]. This in turn enhances the malignancy of residual tumours. In breast cancer, the proportion of CSCs increases significantly after ionizing radiation, leading to enhanced proliferation, which accelerates tumour repopulation [32,37][32][37]. The cell line experiments by Lagadec et al. showed that these radiation-induced CSC phenotypes often exhibit enhanced sphere-forming and tumorigenic abilities. This means that the number of tumorigenic cells increases after treatment, potentially leading to a more rapid tumour recurrence and increased aggressiveness that can lead to metastatic disease [32]. Additionally, the selective killing that enables the radiation induced CSC phenotype is particularly relevant in cases where the overall treatment time is prolonged, which has been associated with a decrease in tumour control rates in certain types of cancers. A seminal study by Withers et al. identified rapid tumour regrowth during extensions of radiotherapy treatment from ~5–8 weeks in almost 500 patients with oropharyngeal cancer. They concluded that for rapidly proliferating cancers such as head and neck, radiotherapy should be completed within the shortest feasible time due to accelerated repopulation of CSCs that occurs while the tumour is still regressing. In view of this, the recommendation is that a delay in treatment initiation is preferable to delays during radiotherapy [18].

2.3. Strategies to Inhibit Tumour Regrowth after Radiotherapy and Future Perspectives

Fast tumour repopulation owing to CSCs gives justification for the implementation of shorter treatment courses and hypofractionation regimens, such as stereotactic ablative radiotherapy (SABR) or hyperfractionated regimens in head and neck cancers, to avoid quick repopulation between two subsequent doses. Where plausible, dose escalations also help to counteract the processes supporting tumour repopulation [38]. Additionally, combining radiation therapy with targeted therapies, such as EGFR inhibitors or immune checkpoint inhibitors, can enhance the radiosensitivity of cancer cells and suppress repopulation. Other options include the use of protons and heavy ions that generate more complex and direct DNA radiation-induced damage, which may be more independent of oxygen status, overcoming the resistance of hypoxic cells. Chiblak et al. showed that primary human glioma stem cells with resistance to photon therapy could be made sensitive to treatment with carbon ions due to their compromised ability to repair DNA double-strand breaks caused by carbon ion irradiation [39]. The interplay between CSCs and tumour repopulation post-radiotherapy is a complex and multifaceted phenomenon. The large heterogeneity in CSC radiosensitivity which leads to both inter- and intra-patient variability makes a one-size-fits-all treatment approach challenging. Understanding the biology and resistance mechanisms of these cells is critical for advancing cancer treatment strategies. Future research should focus on elucidating the molecular pathways involved and identifying innovative approaches to target clonogenic stem cells, ultimately improving the success rates of radiotherapy as well as combined therapies, thus reducing the risk of tumour recurrence. Efforts to mitigate tumour repopulation after radiotherapy have led to investigations into strategies that specifically target clonogenic stem cells. These strategies encompass the development of CSC-specific therapies, such as CSC-targeting drugs and combination treatments. While promising, challenges remain in effectively eradicating or sensitizing clonogenic stem cells to radiotherapy.

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