Proliferation and apoptosis after whole-body irradiation: longitudinal PET study in a mouse model.

PURPOSE: A reliable method for regional in vivo imaging of radiation-induced cellular damage would be of great importance for the detection of therapy-induced injury to healthy tissue and the choice of adequate treatment of radiation emergency patients in both civilian and military events. This study aimed to investigate in a mouse model if positron emission tomography (PET) imaging with proliferation and apoptosis markers is potentially suitable for this purpose. METHODS: Four groups, including twenty mice (wild-type C57BL/6) each, were whole-body irradiated with 0 Gy, 0.5 Gy, 1 Gy, and 3 Gy and examined by PET over a six-month period at defined time points. 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) and 2-(5-[18F]fluoropentyl)-2-methyl malonic acid ([18F]ML-10) were used to visualise proliferation and apoptosis. Regional standard uptake values were compared with respect to irradiation dose over time. Histologic data and peripheral blood cell values were correlated with the PET results. RESULTS: The hematopoietic bone marrow showed a significantly increased [18F]FLT signal at early time points after radiation exposure (day 3 and day 7). This correlated with blood parameters, especially leukocytes, and histological data. A significantly increased [18F]FLT signal also occurred in the gastrointestinal tract and thymus at early time points. An increased [18F]ML-10 signal related to irradiation doses was observed in the bone marrow on day 8, but there was a high variability of standard uptake values and no correlation with histological data. CONCLUSION: [18F]FLT showed potential to visualise the extent, regional distribution and recovery from radiation-induced cellular damage in the bone marrow, gastrointestinal tract and thymus. The potential of [18F]FLT imaging to assess the extent of bone marrow affected by irradiation might be especially useful to predict the subsequent severity of hematopoietic impairment and to adapt the therapy of the bone marrow reserve. [18F]ML-10 PET proved to be not sensitive enough for the reliable detection of radiation induced apoptosis.

Systematic in vitro analysis of therapy resistance in glioblastoma cell lines by integration of clonogenic survival data with multi-level molecular data.

Despite intensive basic scientific, translational, and clinical efforts in the last decades, glioblastoma remains a devastating disease with a highly dismal prognosis. Apart from the implementation of temozolomide into the clinical routine, novel treatment approaches have largely failed, emphasizing the need for systematic examination of glioblastoma therapy resistance in order to identify major drivers and thus, potential vulnerabilities for therapeutic intervention. Recently, we provided proof-of-concept for the systematic identification of combined modality radiochemotherapy treatment vulnerabilities via integration of clonogenic survival data upon radio(chemo)therapy with low-density transcriptomic profiling data in a panel of established human glioblastoma cell lines. Here, we expand this approach to multiple molecular levels, including genomic copy number, spectral karyotyping, DNA methylation, and transcriptome data. Correlation of transcriptome data with inherent therapy resistance on the single gene level yielded several candidates that were so far underappreciated in this context and for which clinically approved drugs are readily available, such as the androgen receptor (AR). Gene set enrichment analyses confirmed these results, and identified additional gene sets, including reactive oxygen species detoxification, mammalian target of rapamycin complex 1 (MTORC1) signaling, and ferroptosis/autophagy-related regulatory circuits to be associated with inherent therapy resistance in glioblastoma cells. To identify pharmacologically accessible genes within those gene sets, leading edge analyses were performed yielding candidates with functions in thioredoxin/peroxiredoxin metabolism, glutathione synthesis, chaperoning of proteins, prolyl hydroxylation, proteasome function, and DNA synthesis/repair. Our study thus confirms previously nominated targets for mechanism-based multi-modal glioblastoma therapy, provides proof-of-concept for this workflow of multi-level data integration, and identifies novel candidates for which pharmacological inhibitors are readily available and whose targeting in combination with radio(chemo)therapy deserves further examination. In addition, our study also reveals that the presented workflow requires mRNA expression data, rather than genomic copy number or DNA methylation data, since no stringent correlation between these data levels could be observed. Finally, the data sets generated in the present study, including functional and multi-level molecular data of commonly used glioblastoma cell lines, represent a valuable toolbox for other researchers in the field of glioblastoma therapy resistance.

Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy.

BACKGROUND: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. METHODS: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. RESULTS: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. CONCLUSIONS: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Analysis of clonogenic growth in vitro.

The clonogenic assay measures the capacity of single cells to form colonies in vitro. It is widely used to identify and quantify self-renewing mammalian cells derived from in vitro cultures as well as from ex vivo tissue preparations of different origins. Varying research questions and the heterogeneous growth requirements of individual cell model systems led to the development of several assay principles and formats that differ with regard to their conceptual setup, 2D or 3D culture conditions, optional cytotoxic treatments and subsequent mathematical analysis. The protocol presented here is based on the initial clonogenic assay protocol as developed by Puck and Marcus more than 60 years ago. It updates and extends the 2006 Nature Protocols article by Franken et al. It discusses different strategies and principles to analyze clonogenic growth in vitro and presents the clonogenic assay in a modular protocol framework enabling a diversity of formats and measures to optimize determination of clonogenic growth parameters. We put particular focus on the phenomenon of cellular cooperation and consideration of how this can affect the mathematical analysis of survival data. This protocol is applicable to any mammalian cell model system from which single-cell suspensions can be prepared and which contains at least a small fraction of cells with self-renewing capacity in vitro. Depending on the cell system used, the entire procedure takes ~2-10 weeks, with a total hands-on time of <20 h per biological replicate.

Transcriptomic landscape of radiation-induced murine thyroid proliferative lesions.

Thyroid carcinoma incidence rates in western societies are among the fastest rising, compared to all malignant tumors over the past two decades. While risk factors such as age and exposure to ionizing radiation are known, early-state carcinogenic processes or pre-lesions are poorly understood or unknown. This study aims at the identification and characterization of early-state radiation-associated neoplastic processes by histologic and transcriptomic analyses of thyroid tissues derived from a mouse model. Comprehensive histological examination of 246 thyroids (164 exposed, 82 non-exposed) was carried out. Proliferative and normal tissues from exposed cases and normal tissue from non-exposed cases were collected by laser-capture microdissection, followed by RNAseq transcriptomic profiling using a low input 3'-library preparation protocol, differential gene expression analysis and functional association by gene set enrichment analysis. Nine exposed samples exhibited proliferative lesions, while none of the non-exposed samples showed histological abnormalities, indicating an association of ionizing radiation exposure with histological abnormalities. Activated immune response signaling and deregulated metabolic processes were observed in irradiated tissue with normal histology compared to normal tissue from non-exposed samples. Proliferative lesions compared to corresponding normal tissues showed enrichment for mainly proliferation-associated gene sets. Consistently, proliferative lesion samples from exposed mice showed elevated proliferation-associated signaling and deregulated metabolic processes compared to normal samples from non-exposed mice. Our findings suggest that a molecular deregulation may be detectable in histologically normal thyroid tissues and in early proliferative lesions in the frame of multi-step progression from irradiated normal tissue to tumorous lesions.

The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation.

BACKGROUND: The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. METHODS: A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. RESULTS: For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. CONCLUSIONS: Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms - a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition - apart from its radiosensitizing potential - obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells.

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer in vitro and in vivo. Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes in vivo. Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses - at least in models of triple-negative breast cancer.

Abscopal, immunological effects of radiotherapy: Narrowing the gap between clinical and preclinical experiences.

Radiotherapy-despite being a local therapy that meanwhile is characterized by an impressively high degree of spatial accuracy-can stimulate systemic phenomena which occasionally lead to regression and rejection of non-irradiated, distant tumor lesions. These abscopal effects of local irradiation have been observed in sporadic clinical case reports since the beginning of the 20th century, and extensive preclinical work has contributed to identify systemic anti-tumor immune responses as the underlying driving forces. Although abscopal tumor regression still remains a rare event in the radiotherapeutic routine, increasing numbers of cases are being reported, particularly since the clinical implementation of immune checkpoint inhibiting agents. Accordingly, interests to systematically exploit the therapeutic potential of radiotherapy-stimulated systemic responses are constantly growing. The present review briefly delineates the history of radiotherapy-induced abscopal effects and the activation of systemic anti-tumor immune responses by local irradiation. We discuss preclinical and clinical reports with specific focus on the corresponding controversies, and we propose issues that should be addressed in the future in order to narrow the gap between preclinical knowledge and clinical experiences.

A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer.

The chaperone heat shock protein 90 (HSP90) crucially supports the maturation, folding, and stability of a variety of client proteins which are of pivotal importance for the survival and proliferation of cancer cells. Consequently, targeting of HSP90 has emerged as an attractive strategy of anti-cancer therapy, and it appears to be particularly effective in the context of molecular sensitization towards radiotherapy as has been proven in preclinical models of different cancer entities. However, so far the clinical translation has largely been hampered by suboptimal pharmacological properties and serious hepatotoxicity of first- and second-generation HSP90 inhibitors. Here, we report on NW457, a novel radicicol-derived member of the pochoxime family with reduced hepatotoxicity, how it inhibits the DNA damage response and how it synergizes with ionizing irradiation to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer in vitro and in vivo.

Targeting the heat shock response in combination with radiotherapy: Sensitizing cancer cells to irradiation-induced cell death and heating up their immunogenicity.

Radiotherapy represents an essential treatment option for the majority of cancer patients in different stages of their disease. Physical achievements of the recent years led to the implementation of high precision treatment planning procedures, and image-guided dose delivery is current state of the art. Yet, radiotherapy still faces several limitations with cancer intrinsic radioresistance being a key driver of therapeutic failure. Accordingly, the mechanisms orchestrating radioresistance and their therapeutic targeting by combined modality approaches are in the center of attention of numerous radiation oncologists. In the present review, we summarize and discuss therapeutic approaches that exploit the heat shock response, either by hyperthermia or by pharmacological heat shock protein inhibition, in combination with radiotherapy. These strategies appear particularly promising, since they sensitize cancer cells to irradiation-induced cell death and at the same time have proven the potential to promote systemic anti-tumor immune mechanisms, which may target not only locally surviving tumor cells, but also distant out-of-field metastases.

Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated γ-irradiation.

BACKGROUND: Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells. METHODS: Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards γ-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry. RESULTS: Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to γ-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39. CONCLUSIONS: Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.