Preclinical Evaluation of the ATR Inhibitor BAY 1895344 as a Radiosensitizer for Head and Neck Squamous Cell Carcinoma.

PURPOSE: Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. METHODS AND MATERIALS: Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. RESULTS: ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. CONCLUSIONS: These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.

Evaluating Tumor Hypoxia Radiosensitization Via Electron Paramagnetic Resonance Oxygen Imaging (EPROI).

PURPOSE: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response. PROCEDURES: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival. RESULTS: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV. CONCLUSION: EPROI provides 3D high-resolution pO2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.

A Novel Preclinical Murine Model to Monitor Inflammatory Breast Cancer Tumor Growth and Lymphovascular Invasion.

Inflammatory breast cancer (IBC), an understudied and lethal breast cancer, is often misdiagnosed due to its unique presentation of diffuse tumor cell clusters in the skin and dermal lymphatics. Here, we describe a window chamber technique in combination with a novel transgenic mouse model that has red fluorescent lymphatics (ProxTom RFP Nu/Nu) to simulate IBC clinicopathological hallmarks. Various breast cancer cells stably transfected to express green or red fluorescent reporters were transplanted into mice bearing dorsal skinfold window chambers. Intravital fluorescence microscopy and the in vivo imaging system (IVIS) were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140 h. This short-term, longitudinal imaging time frame in studying transient or dynamic events of diffuse and collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility, and vessel characteristics can be expanded to investigate other cancer cell types exhibiting lymphovascular invasion, a key step in metastatic dissemination. It was found that these models were able to effectively track tumor cluster migration and dissemination, which is a hallmark of IBC clinically, and was recapitulated in these mouse models.

A Clinical Study to Assess Diffuse Reflectance Spectroscopy with an Auto-Calibrated, Pressure-Sensing Optical Probe in Head and Neck Cancer.

Diffuse reflectance spectroscopy (DRS) is a powerful tool for quantifying optical and physiological tissue properties such as hemoglobin oxygen saturation and vascularity. DRS is increasingly used clinically for distinguishing cancerous lesions from normal tissue. However, its widespread clinical acceptance is still limited due to uncontrolled probe-tissue interface pressure that influences reproducibility and introduces operator-dependent results. In this clinical study, we assessed and validated a pressure-sensing and automatic self-calibration DRS in patients with suspected head and neck squamous cell carcinoma (HNSCC). The clinical study enrolled nineteen patients undergoing HNSCC surgical biopsy procedures. Patients consented to evaluation of this improved DRS system during surgery. For each patient, we obtained 10 repeated measurements on one tumor site and one distant normal location. Using a Monte Carlo-based model, we extracted the hemoglobin saturation data along with total hemoglobin content and scattering properties. A total of twelve cancer tissue samples from HNSCC patients and fourteen normal tissues were analyzed. A linear mixed effects model tested for significance between repeated measurements and compared tumor versus normal tissue. These results demonstrate that cancerous tissues have a significantly lower hemoglobin saturation compared to normal controls (p < 0.001), which may be reflective of tumor hypoxia. In addition, there were minimal changes over time upon probe placement and repeated measurement, indicating that the pressure-induced changes were minimal and repeated measurements did not differ significantly from the initial value. This study demonstrates the feasibility of conducting optical spectroscopy measurements on intact lesions prior to removal during HNSCC procedures, and established that this probe provides diagnostically-relevant physiologic information that may impact further treatment.

Quantifying the effects of anesthesia on intracellular oxygen via low-cost portable microscopy using dual-emissive nanoparticles.

Intracellular oxygenation is an important parameter for numerous biological studies. While there are a variety of methods available for acquiring in vivo measurements of oxygenation in animal models, most are dependent on indirect oxygen measurements, restraints, or anesthetization. A portable microscope system using a Raspberry Pi computer and Pi Camera was developed for attaching to murine dorsal window chambers. Dual-emissive boron nanoparticles were used as an oxygen-sensing probe while mice were imaged in awake and anesthetized states. The portable microscope system avoids altered in vivo measurements due to anesthesia or restraints while enabling increased continual acquisition durations.

Dual-emissive, oxygen-sensing boron nanoparticles quantify oxygen consumption rate in breast cancer cells.

SIGNIFICANCE: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems. AIM: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay. APPROACH: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR. RESULTS: Using a Bland-Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation. CONCLUSIONS: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland-Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.

Cherenkov emissions for studying tumor changes during radiation therapy: An exploratory study in domesticated dogs with naturally-occurring cancer.

PURPOSE: Real-time monitoring of physiological changes of tumor tissue during radiation therapy (RT) could improve therapeutic efficacy and predict therapeutic outcomes. Cherenkov radiation is a normal byproduct of radiation deposited in tissue. Previous studies in rat tumors have confirmed a correlation between Cherenkov emission spectra and optical measurements of blood-oxygen saturation based on the tissue absorption coefficients. The purpose of this study is to determine if it is feasible to image Cherenkov emissions during radiation therapy in larger human-sized tumors of pet dogs with cancer. We also wished to validate the prior work in rats, to determine if Cherenkov emissions have the potential to act an indicator of blood-oxygen saturation or water-content changes in the tumor tissue-both of which have been correlated with patient prognosis. METHODS: A DoseOptics camera, built to image the low-intensity emission of Cherenkov radiation, was used to measure Cherenkov intensities in a cohort of cancer-bearing pet dogs during clinical irradiation. Tumor type and location varied, as did the radiation fractionation scheme and beam arrangement, each planned according to institutional standard-of-care. Unmodulated radiation was delivered using multiple 6 MV X-ray beams from a clinical linear accelerator. Each dog was treated with a minimum of 16 Gy total, in ≥3 fractions. Each fraction was split into at least three subfractions per gantry angle. During each subfraction, Cherenkov emissions were imaged. RESULTS: We documented significant intra-subfraction differences between the Cherenkov intensities for normal tissue, whole-tumor tissue, tissue at the edge of the tumor and tissue at the center of the tumor (p<0.05). Additionally, intra-subfraction changes suggest that Cherenkov emissions may have captured fluctuating absorption properties within the tumor. CONCLUSION: Here we demonstrate that it is possible to obtain Cherenkov emissions from canine cancers within a fraction of radiotherapy. The entire optical spectrum was obtained which includes the window for imaging changes in water and hemoglobin saturation. This lends credence to the goal of using this method during radiotherapy in human patients and client-owned pets.

Clinical and Pre-clinical Methods for Quantifying Tumor Hypoxia.

Hypoxia, a prevalent characteristic of most solid malignant tumors, contributes to diminished therapeutic responses and more aggressive phenotypes. The term hypoxia has two definitions. One definition would be a physiologic state where the oxygen partial pressure is below the normal physiologic range. For most normal tissues, the normal physiologic range is between 10 and 20 mmHg. Hypoxic regions develop when there is an imbalance between oxygen supply and demand. The impact of hypoxia on cancer therapeutics is significant: hypoxic tissue is 3× less radiosensitive than normoxic tissue, the impaired blood flow found in hypoxic tumor regions influences chemotherapy delivery, and the immune system is dependent on oxygen for functionality. Despite the clinical implications of hypoxia, there is not a universal, ideal method for quantifying hypoxia, particularly cycling hypoxia because of its complexity and heterogeneity across tumor types and individuals. Most standard imaging techniques can be modified and applied to measuring hypoxia and quantifying its effects; however, the benefits and challenges of each imaging modality makes imaging hypoxia case-dependent. In this chapter, a comprehensive overview of the preclinical and clinical methods for quantifying hypoxia is presented along with the advantages and disadvantages of each.

Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor.

The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.