Extracellular Vesicle- and Mitochondria-Based Targeting of Non-Small Cell Lung Cancer Response to Radiation: Challenges and Perspectives.

During the cell life cycle, extracellular vesicles (EVs) transport different cargos, including organelles, proteins, RNAs, DNAs, metabolites, etc., that influence cell proliferation and apoptosis in recipient cells. EVs from metastatic cancer cells remodel the extracellular matrix and cells of the tumor microenvironment (TME), promoting tumor invasion and metastatic niche preparation. Although the process is not fully understood, evidence suggests that EVs facilitate genetic material transfer between cells. In the context of NSCLC, EVs can mediate intercellular mitochondrial (Mt) transfer, delivering mitochondria organelle (MtO), mitochondrial DNA (mtDNA), and/or mtRNA/proteinaceous cargo signatures (MtS) through different mechanisms. On the other hand, certain populations of cancer cells can hijack the MtO from TME cells mainly by using tunneling nanotubes (TNTs). This transfer aids in restoring mitochondrial function, benefiting benign cells with impaired metabolism and enabling restoration of their metabolic activity. However, the impact of transferring mitochondria versus transplanting intact mitochondrial organelles in cancer remains uncertain and the subject of debate. Some studies suggest that EV-mediated mitochondria delivery to cancer cells can impact how cancer responds to radiation. It might make the cancer more resistant or more sensitive to radiation. In our review, we aimed to point out the current controversy surrounding experimental data and to highlight new paradigm-shifting modalities in radiation therapy that could potentially overcome cancer resistance mechanisms in NSCLC.

With a little help from my friends? Acculturation and mental health in Arabic-speaking refugee youth living with their families.

Introduction: Refugee youth are often faced with the compounding challenges of heightened exposure to traumatic events and acculturating to a new country during a developmental period when their sense of self is still forming. This study investigated whether refugee youth's acculturation orientation (separation, integration, marginalization, and assimilation) is associated with depressive and posttraumatic stress symptoms and aimed to identify additional indicators of acculturation that may contribute to mental health. Methods: A total of 101 Arabic-speaking refugee youths (aged 14-20 years), who were living with their families and attending school in Germany, took part in the study. They answered questions concerning traumatic exposure and posttraumatic stress symptoms, depressive symptoms, and several indicators of acculturation, including cultural orientation, positive and negative intra- and intergroup contact, language skills and friendship networks. All participants were categorized into one of four acculturation orientations using median splits. Results: Kruskal-Wallis rank sum tests revealed that acculturation orientation was not significantly associated with depressive symptoms [χ2 (3, 97) = 0.519, p = 0.915] or posttraumatic stress symptoms [χ2 (3, 97) = 0.263, p = 0.967]. Regression analysis revealed that German language skills were significantly associated with lower scores of depressive symptoms (p = 0.016) and number of friends in Germany was significantly associated with lower scores of depressive (p = 0.006) and posttraumatic stress symptoms (p = 0.002), respectively. Discussion: Policies that provide refugee youth with access to language classes and social activities with peers do not only enable them to actively participate in a new society but may also have a positive effect on their mental health.

Long-Term Cultured Human Glioblastoma Multiforme Cells Demonstrate Increased Radiosensitivity and Senescence-Associated Secretory Phenotype in Response to Irradiation.

The overall effect of senescence on cancer progression and cancer cell resistance to X-ray radiation (IR) is still not fully understood and remains controversial. How to induce tumor cell senescence and which senescent cell characteristics will ensure the safest therapeutic strategy for cancer treatment are under extensive investigation. While the evidence for passage number-related effects on malignant primary cells or cell lines is compelling, much less is known about how the changes affect safety and Senescence-Associated Secretory Phenotype (SASP), both of which are needed for the senescence cell-based vaccine to be effective against cancer. The present study aimed to investigate the effects of repeated passaging on the biological (self-renewal capacity and radioresistance) and functional (senescence) characteristics of the different populations of short- and long-term passaging glioblastoma multiforme (GBM) cells responding to senescence-inducing DNA-damaging IR stress. For this purpose, we compared radiobiological effects of X-ray exposure on two isogenic human U87 cell lines: U87L, minimally cultured cells (<15 passages after obtaining from the ATCC) and U87H, long-term cultured cells (>3 years of continuous culturing after obtaining from the ATCC). U87L cells displayed IR dose-related changes in the signs of IR stress-induced premature senescence. These included an increase in the proportion of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells, and concomitant decrease in the proportion of Ki67-positive cells and metabolically active cells. However, reproductive survival of irradiated short-term cultured U87L cells was higher compared to long-term cultured U87H cells, as the clonogenic activity results demonstrated. In contrast, the irradiated long-term cultured U87H cells possessed dose-related increases in the proportion of multinucleated giant cancer cells (MGCCs), while demonstrating higher radiosensitivity (lower self-renewal) and a significantly reduced fraction of DNA-replicating cells compared to short-term cultured U87L cells. Conditioned culture medium from U87H cells induced a significant rise of SA-ß-Gal staining in U87L cells in a paracrine manner suggesting inherent SASP. Our data suggested that low-dose irradiated long-term cultured GBM cells might be a safer candidate for a recently proposed senescence cell-based vaccine against cancer.

Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status.

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2-6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses.

The Molecular and Cellular Strategies of Glioblastoma and Non-Small-Cell Lung Cancer Cells Conferring Radioresistance.

Ionizing radiation (IR) has been shown to play a crucial role in the treatment of glioblastoma (GBM; grade IV) and non-small-cell lung cancer (NSCLC). Nevertheless, recent studies have indicated that radiotherapy can offer only palliation owing to the radioresistance of GBM and NSCLC. Therefore, delineating the major radioresistance mechanisms may provide novel therapeutic approaches to sensitize these diseases to IR and improve patient outcomes. This review provides insights into the molecular and cellular mechanisms underlying GBM and NSCLC radioresistance, where it sheds light on the role played by cancer stem cells (CSCs), as well as discusses comprehensively how the cellular dormancy/non-proliferating state and polyploidy impact on their survival and relapse post-IR exposure.

CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status.

Cancer stem cells (CSCs) play a critical role in the initiation, progression and therapy relapse of many cancers including non-small cell lung cancer (NSCLC). Here, we aimed to address the question of whether the FACS-sorted CSC-like (CD44 + &CD133 +) vs. non-CSC (CD44-/CD133- isogenic subpopulations of p53wt A549 and p53null H1299 cells differ in terms of DNA-damage signaling and the appearance of "dormant" features, including polyploidy, which are early markers (predictors) of their sensitivity to genotoxic stress. X-ray irradiation (IR) at 5 Gy provoked significantly higher levels of the ATR-Chk1/Chk2-pathway activity in CD44-/CD133- and CD133+ subpopulations of H1299 cells compared to the respective subpopulations of A549 cells, which only excited ATR-Chk2 activation as demonstrated by the Multiplex DNA-Damage/Genotoxicity profiling. The CD44+ subpopulations did not demonstrate IR-induced activation of ATR, while significantly augmenting only Chk2 and Chk1/2 in the A549- and H1299-derived cells, respectively. Compared to the A549 cells, all the subpopulations of H1299 cells established an increased IR-induced expression of the γH2AX DNA-repair protein. The CD44-/CD133- and CD133+ subpopulations of the A549 cells revealed IR-induced activation of ATR-p53-p21 cell dormancy signaling-mediated pathway, while none of the CD44+ subpopulations of either cell line possessed any signs of such activity. Our data indicated, for the first time, the transcription factor MITF-FAM3C axis operative in p53-deficient H1299 cells, specifically their CD44+ and CD133+ populations, in response to IR, which warrants further investigation. The p21-mediated quiescence is likely the predominant surviving pathway in CD44-/CD133- and CD133+ populations of A549 cells as indicated by single-cell high-content imaging and analysis of Ki67- and EdU-coupled fluorescence after IR stress. SA-beta-galhistology revealed that cellular-stress-induced premature senescence (SIPS) likely has a significant influence on the temporary dormant state of H1299 cells. For the first time, we demonstrated polyploid giant and/or multinucleated cancer-cell (PGCC/MGCC) fractions mainly featuring the progressively augmenting Ki67low phenotype in CD44+ and CD133+ A549 cells at 24-48 h after IR. In contrast, the Ki67high phenotype enrichment in the same fractions of all the sorted H1299 cells suggested an increase in their cycling/heterochromatin reorganization activity after IR stress. Our results proposed that entering the "quiescence" state rather than p21-mediated SIPS may play a significant role in the survival of p53wt CSC-like NSCLC cells after IR. The results obtained are important for the selection of therapeutic schemes for the treatment of patients with NSCLC, depending on the functioning of the p53 system in tumor cells.

IR-Surviving NSCLC Cells Exhibit Different Patterns of Molecular and Cellular Reactions Relating to the Multifraction Irradiation Regimen and p53-Family Proteins Expression.

Radiotherapy is a primary treatment modality for patients with unresectable non-small cell lung cancer (NSCLC). Tumor heterogeneity still poses the central question of cancer radioresistance, whether the presence of a particular cell population inside a tumor undergoing a selective outgrowth during radio- and chemotherapy give rise to metastasis and tumor recurrence. In this study, we examined the impact of two different multifraction X-ray radiation exposure (MFR) regimens, fraction dose escalation (FDE) in the split course and the conventional hypofractionation (HF), on the phenotypic and molecular signatures of four MFR-surviving NSCLC cell sublines derived from parental A549 (p53 wild-type) and H1299 (p53-null) cells, namely A549FR/A549HR, H1299FR/H1299HR cells. We demonstrate that sublines surviving different MFR regimens in a total dose of 60 Gy significantly diverge in their molecular traits related to irradiation regimen and p53 status. The observed changes regarding radiosensitivity, transformation, proliferation, metabolic activity, partial epithelial-to-mesenchymal transition (EMT) program activation and 1D confined migratory behavior (wound healing). For the first time, we demonstrated that MFR exposure led to the significant decrease in the expression of p63 and p73, the p53-family members, in p53null cells, which correlated with the increase in cell polyploidy. We could not find significant differences in FRA1 expression between parental cells and their sublines that survived after any MFR regimen regardless of p53 status. In our study, the FDE regimen probably causes partial EMT program activation in MFR-survived NSCLC cells through either Vimentin upregulation in p53null or an aberrant N-cadherin upregulation in p53wt cells. The HF regimen likely less influences the EMT activation irrespectively of the p53 status of MFR-survived NSCLC cells. Our data highlight that both MFR regimens caused overall higher cell transformation of p53null H1299FR and H1299HR cells than their parental H1299 cells. Moreover, our results indicate that the FDE regimen raised the radioresistance and transformation of MFR-surviving NSCLC cells irrespectively of their p53 status, though the HF regimen demonstrated a similar effect on p53null NSCLC cells only. Our data once again emphasize that NSCLC therapy approaches should become more personalized according to radiation therapy (RT) regimen, tumor histology, and molecular status of critical proteins.

The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status.

Ionizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) survived multifraction X-ray radiation exposure (MFR) at a total dose of 60 Gy were investigated three weeks after the MFR course. We compared radiosensitivity (colony formation), expression of epithelial-mesenchymal transition (EMT) markers, migration activity, autophagy, and HR-dependent DNA double-strand break (DSB) repair in the bulk and entire CD44high/CD166high CSC-like populations of both parental and MFR survived NSCLC cells. We demonstrated that the p53 status affected: the pattern of expression of N-cadherin, E-cadherin, Vimentin, witnessing the appearance of EMT-like phenotype of MFR-surviving sublines; 1D confined migratory behavior (wound healing); the capability of an irradiated cell to continue to divide and form a colony of NSCLC cells before and after MFR; influencing the CD44/CD166 expression level in MFR-surviving NSCLC cells after additional single irradiation. Our data further emphasize the impact of p53 status on the decay of γH2AX foci and the associated efficacy of the DSB repair in NSCLC cells survived after MFR. We revealed that Rad51 protein might play a principal role in MFR-surviving of p53 null NSCLC cells promoting DNA DSB repair by homologous recombination (HR) pathway. The proportion of Rad51 + cells elevated in CD44high/CD166high population in MFR-surviving p53wt and p53null sublines and their parental cells. The p53wt ensures DNA-PK-mediated DSB repair for both parental and MFR-surviving cells irrespectively of a subsequent additional single irradiation. Whereas in the absence of p53, a dose-dependent increase of DNA-PK-mediated non-homologous end joining (NHEJ) occurred as an early post-irradiation response is more intensive in the CSC-like population MFR-surviving H1299IR, compared to their parental H1299 cells. Our study strictly observed a significantly higher content of LC3 + cells in the CD44high/CD166high populations of p53wt MFR-surviving cells, which enriched the CSC-like cells in contrast to their p53null counterparts. The additional 2 Gy and 5 Gy X-ray exposure leads to the dose-dependent increase in the proportion of LC3 + cells in CD44high/CD166high population of both parental p53wt and p53null, but not MFR-surviving NSCLC sublines. Our data indicated that autophagy is not necessarily associated with CSC-like cells' radiosensitivity, emphasizing that careful assessment of other milestone processes (such as senescence and autophagy-p53-Zeb1 axis) of primary radiation responses may provide new potential targets modulated for therapeutic benefit through radiosensitizing cancer cells while rescuing normal tissue. Our findings also shed light on the intricate crosstalk between autophagy and the p53-related EMT, by which MFR-surviving cells might obtain an invasive phenotype and metastatic potential.

The p53-53BP1-Related Survival of A549 and H1299 Human Lung Cancer Cells after Multifractionated Radiotherapy Demonstrated Different Response to Additional Acute X-ray Exposure.

Radiation therapy is one of the main methods of treating patients with non-small cell lung cancer (NSCLC). However, the resistance of tumor cells to exposure remains the main factor that limits successful therapeutic outcome. To study the molecular/cellular mechanisms of increased resistance of NSCLC to ionizing radiation (IR) exposure, we compared A549 (p53 wild-type) and H1299 (p53-deficient) cells, the two NSCLC cell lines. Using fractionated X-ray irradiation of these cells at a total dose of 60 Gy, we obtained the survived populations and named them A549IR and H1299IR, respectively. Further characterization of these cells showed multiple alterations compared to parental NSCLC cells. The additional 2 Gy exposure led to significant changes in the kinetics of γH2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci numbers in A549IR and H1299IR compared to parental NSCLC cells. Whereas A549, A549IR, and H1299 cells demonstrated clear two-component kinetics of DNA double-strand break (DSB) repair, H1299IR showed slower kinetics of γH2AX foci disappearance with the presence of around 50% of the foci 8 h post-IR. The character of H2AX phosphorylation in these cells was pATM-independent. A decrease of residual γH2AX/53BP1 foci number was observed in both A549IR and H1299IR compared to parental cells post-IR at extra doses of 2, 4, and 6 Gy. This process was accompanied with the changes in the proliferation, cell cycle, apoptosis, and the expression of ATP-binding cassette sub-family G member 2 (ABCG2, also designated as CDw338 and the breast cancer resistance protein (BCRP)) protein. Our study provides strong evidence that different DNA repair mechanisms are activated by multifraction radiotherapy (MFR), as well as single-dose IR, and that the enhanced cellular survival after MFR is reliant on both p53 and 53BP1 signaling along with non-homologous end-joining (NHEJ). Our results are of clinical significance as they can guide the choice of the most effective IR regimen by analyzing the expression status of the p53-53BP1 pathway in tumors and thereby maximize therapeutic benefits for the patients while minimizing collateral damage to normal tissue.