Enhanced radiation sensitivity, decreased DNA damage repair, and differentiation defects in airway stem cells derived from patients with chronic obstructive pulmonary disease.

Radiation therapy (RT) is a common treatment for lung cancer. Still, it can lead to irreversible loss of pulmonary function and a significant reduction in quality of life for one-third of patients. Preexisting comorbidities, such as chronic obstructive pulmonary disease (COPD), are frequent in patients with lung cancer and further increase the risk of complications. Because lung stem cells are crucial for the regeneration of lung tissue following injury, we hypothesized that airway stem cells from patients with COPD with lung cancer might contribute to increased radiation sensitivity. We used the air-liquid interface model, a three-dimensional (3D) culture system, to compare the radiation response of primary human airway stem cells from healthy and patients with COPD. We found that COPD-derived airway stem cells, compared to healthy airway stem cell cultures, exhibited disproportionate pathological mucociliary differentiation, aberrant cell cycle checkpoints, residual DNA damage, reduced survival of stem cells and self-renewal, and terminally differentiated cells post-irradiation, which could be reversed by blocking the Notch pathway using small-molecule γ-secretase inhibitors. Our findings shed light on the mechanisms underlying the increased radiation sensitivity of COPD and suggest that airway stem cells reflect part of the pathological remodeling seen in lung tissue from patients with lung cancer receiving thoracic RT.

The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways.

Radiotherapy is involved in the treatment of many cancers, but damage induced to the surrounding normal tissue is often inevitable. Evidence suggests that the maintenance of homeostasis and regeneration of the normal tissue is driven by specific adult tissue stem/progenitor cells. These tasks involve the input from several signaling pathways. Irradiation also targets these stem/progenitor cells, triggering a cellular response aimed at achieving tissue regeneration. Here we discuss the currently used in vitro and in vivo models and the involved specific tissue stem/progenitor cell signaling pathways to study the response to irradiation. The combination of the use of complex in vitro models that offer high in vivo resemblance and lineage tracing models, which address organ complexity constitute potential tools for the study of the stem/progenitor cellular response post-irradiation. The Notch, Wnt, Hippo, Hedgehog, and autophagy signaling pathways have been found as crucial for driving stem/progenitor radiation-induced tissue regeneration. We review how these signaling pathways drive the response of solid tissue-specific stem/progenitor cells to radiotherapy and the used models to address this.

NOTCH inhibition promotes bronchial stem cell renewal and epithelial barrier integrity after irradiation.

Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ-secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose-limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ-secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity. Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air-liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small-molecule NOTCH inhibitor/GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI-treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy.

FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence.

PURPOSE: One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity. EXPERIMENTAL DESIGN: Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc-/- mice exposed to bilateral thorax irradiation as well as human lung cells grown in vitro. RESULTS: In vitro studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc-/- mice harboring critically short telomeres and lack of telomerase activity. CONCLUSIONS: The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.

Radiation-Induced Lung Injury (RILI).

Radiation pneumonitis (RP) and radiation fibrosis (RF) are two dose-limiting toxicities of radiotherapy (RT), especially for lung, and esophageal cancer. It occurs in 5-20% of patients and limits the maximum dose that can be delivered, reducing tumor control probability (TCP) and may lead to dyspnea, lung fibrosis, and impaired quality of life. Both physical and biological factors determine the normal tissue complication probability (NTCP) by Radiotherapy. A better understanding of the pathophysiological sequence of radiation-induced lung injury (RILI) and the intrinsic, environmental and treatment-related factors may aid in the prevention, and better management of radiation-induced lung damage. In this review, we summarize our current understanding of the pathological and molecular consequences of lung exposure to ionizing radiation, and pharmaceutical interventions that may be beneficial in the prevention or curtailment of RILI, and therefore enable a more durable therapeutic tumor response.

NOTCH signaling promotes the survival of irradiated basal airway stem cells.

Radiation-induced lung injury to normal airway epithelium is a frequent side-effect and dose-limiting factor in radiotherapy of tumors in the thoracic cavity. NOTCH signaling plays key roles in self-renewal and differentiation of upper airway basal lung stem cells during development, and the NOTCH pathway is frequently deregulated in lung cancer. In preclinical lung cancer models, NOTCH inhibition was shown to improve the radiotherapy response by targeting tumor stem cells, but the effects in combination with irradiation on normal lung stem cells are unknown. NOTCH/γ-secretase inhibitors are potent clinical candidates to block NOTCH function in tumors, but their clinical implementation has been hampered by normal tissue side-effects. Here we show that NOTCH signaling is active in primary human- and murine-derived airway epithelial stem cell models and when combined with radiation NOTCH inhibition provokes a decrease in S-phase and increase in G1-phase arrest. We show that NOTCH inhibition in irradiated lung basal stem cells leads to a more potent activation of the DNA damage checkpoint kinases pATM and pCHK2 and results in an increased level of residual 53BP1 foci in irradiated lung basal stem cells reducing their capacity for self-renewal. The effects are recapitulated in ex vivo cultured lung basal stem cells after in vivo whole thorax irradiation and NOTCH inhibition. These results highlight the importance of studying normal tissue effects that may counteract the therapeutic benefit in the use of NOTCH/γ-secretase inhibitors in combination with radiation for antitumor treatment.

HIF-1α and HIF-2α Differently Regulate the Radiation Sensitivity of NSCLC Cells.

The hypoxia-inducible transcription factors (HIF)-1/2α are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-α isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.

Drug Resistance in Non-Small Cell Lung Cancer: A Potential for NOTCH Targeting?

Drug resistance is a major cause for therapeutic failure in non-small cell lung cancer (NSCLC) leading to tumor recurrence and disease progression. Cell intrinsic mechanisms of resistance include changes in the expression of drug transporters, activation of pro-survival, and anti-apoptotic pathways, as well as non-intrinsic influences of the tumor microenvironment. It has become evident that tumors are composed of a heterogeneous population of cells with different genetic, epigenetic, and phenotypic characteristics that result in diverse responses to therapy, and underlies the emergence of resistant clones. This tumor heterogeneity is driven by subpopulations of tumor cells termed cancer stem cells (CSCs) that have tumor-initiating capabilities, are highly self-renewing, and retain the ability for multi-lineage differentiation. CSCs have been identified in NSCLC and have been associated with chemo- and radiotherapy resistance. Stem cell pathways are frequently deregulated in cancer and are implicated in recurrence after treatment. Here, we focus on the NOTCH signaling pathway, which has a role in stem cell maintenance in non-squamous non-small lung cancer, and we critically assess the potential for targeting the NOTCH pathway to overcome resistance to chemotherapeutic and targeted agents using both preclinical and clinical evidence.