Overcoming MET-mediated resistance in oncogene-driven NSCLC.

This study evaluates the efficacy of combining targeted therapies with MET or SHP2 inhibitors to overcome MET-mediated resistance in different NSCLC subtypes. A prevalence study was conducted for MET amplification and overexpression in samples from patients with NSCLC who relapsed on ALK, ROS1, or RET tyrosine kinase inhibitors. MET-mediated resistance was detected in 37.5% of tissue biopsies, which allow the detection of MET overexpression, compared to 7.4% of liquid biopsies. The development of drug resistance by MET overexpression was confirmed in EGFRex19del-, KRASG12C-, HER2ex20ins-, and TPM3-NTRK1-mutant cell lines. The combination of targeted therapy with MET or SHP2 inhibitors was found to overcome MET-mediated resistance in both in vitro and in vivo assays. This study highlights the importance of considering MET overexpression as a resistance driver to NSCLC targeted therapies to better identify patients who could potentially benefit from combination approaches with MET or SHP2 inhibitors.

Improving Delineation of True Tumor Volume With Multimodal MRI in a Rat Model of Brain Metastasis.

PURPOSE: Brain metastases are almost universally lethal with short median survival times. Despite this, they are often potentially curable, with therapy failing only because of local relapse. One key reason relapse occurs is because treatment planning did not delineate metastasis margins sufficiently or accurately, allowing residual tumor to regrow. The aim of this study was to determine the extent to which multimodal magnetic resonance imaging (MRI), with a simple and automated analysis pipeline, could improve upon current clinical practice of single-modality, independent-observer tumor delineation. METHODS AND MATERIALS: We used a single rat model of brain metastasis (ENU1564 breast carcinoma cells in BD-IX rats), with and without radiation therapy. Multimodal MRI data were acquired using sequences either in current clinical use or in clinical trial and included postgadolinium T1-weighted images and maps of blood flow, blood volume, T1 and T2 relaxation times, and apparent diffusion coefficient. RESULTS: In all cases, independent observers underestimated the true size of metastases from single-modality gadolinium-enhanced MRI (85 ± 36 µL vs 131 ± 40 µL histologic measurement), although multimodal MRI more accurately delineated tumor volume (132 ± 41 µL). Multimodal MRI offered increased sensitivity compared with independent observer for detecting metastasis (0.82 vs 0.61, respectively), with only a slight decrease in specificity (0.86 vs 0.98). Blood flow maps conferred the greatest improvements in margin detection for late-stage metastases after radiation therapy. Gadolinium-enhanced T1-weighted images conferred the greatest increase in accuracy of detection for smaller metastases. CONCLUSIONS: These findings suggest that multimodal MRI of brain metastases could significantly improve the visualization of brain metastasis margins, beyond current clinical practice, with the potential to decrease relapse rates and increase patient survival. This finding now needs validation in additional tumor models or clinical cohorts.

Quantitative blood flow measurement in rat brain with multiphase arterial spin labelling magnetic resonance imaging.

Cerebral blood flow is an important parameter in many diseases and functional studies that can be accurately measured in humans using arterial spin labelling (ASL) MRI. However, although rat models are frequently used for preclinical studies of both human disease and brain function, rat CBF measurements show poor consistency between studies. This lack of reproducibility is due, partly, to the smaller size and differing head geometry of rats compared to humans, as well as the differing analysis methodologies employed and higher field strengths used for preclinical MRI. To address these issues, we have implemented, optimised and validated a multiphase pseudo-continuous ASL technique, which overcomes many of the limitations of rat CBF measurement. Three rat strains (Wistar, Sprague Dawley and Berlin Druckrey IX) were used, and CBF values validated against gold-standard autoradiography measurements. Label positioning was found to be optimal at 45°, while post-label delay was optimised to 0.55 s. Whole brain CBF measures were 109 ± 22, 111 ± 18 and 100 ± 15 mL/100 g/min by multiphase pCASL, and 108 ± 12, 116 ± 14 and 122 ± 16 mL/100 g/min by autoradiography in Wistar, SD and BDIX cohorts, respectively. Tumour model analysis shows that the developed methods also apply in disease states. Thus, optimised multiphase pCASL provides robust, reproducible and non-invasive measurement of CBF in rats.

Tumor pH and Protein Concentration Contribute to the Signal of Amide Proton Transfer Magnetic Resonance Imaging.

Abnormal pH is a common feature of malignant tumors and has been associated clinically with suboptimal outcomes. Amide proton transfer magnetic resonance imaging (APT MRI) holds promise as a means to noninvasively measure tumor pH, yet multiple factors collectively make quantification of tumor pH from APT MRI data challenging. The purpose of this study was to improve our understanding of the biophysical sources of altered APT MRI signals in tumors. Combining in vivo APT MRI measurements with ex vivo histological measurements of protein concentration in a rat model of brain metastasis, we determined that the proportion of APT MRI signal originating from changes in protein concentration was approximately 66%, with the remaining 34% originating from changes in tumor pH. In a mouse model of hypopharyngeal squamous cell carcinoma (FaDu), APT MRI showed that a reduction in tumor hypoxia was associated with a shift in tumor pH. The results of this study extend our understanding of APT MRI data and may enable the use of APT MRI to infer the pH of individual patients' tumors as either a biomarker for therapy stratification or as a measure of therapeutic response in clinical settings. SIGNIFICANCE: These findings advance our understanding of amide proton transfer magnetic resonance imaging (APT MRI) of tumors and may improve the interpretation of APT MRI in clinical settings.

Sensitivity of Multiphase Pseudocontinuous Arterial Spin Labelling (MP pCASL) Magnetic Resonance Imaging for Measuring Brain and Tumour Blood Flow in Mice.

Brain and tumour blood flow can be measured noninvasively using arterial spin labelling (ASL) magnetic resonance imaging (MRI), but reliable quantification in mouse models remains difficult. Pseudocontinuous ASL (pCASL) is recommended as the clinical standard for ASL and can be improved using multiphase labelling (MP pCASL). The aim of this study was to optimise and validate MP pCASL MRI for cerebral blood flow (CBF) measurement in mice and to assess its sensitivity to tumour perfusion. Following optimization of the MP pCASL sequence, CBF data were compared with gold-standard autoradiography, showing close agreement. Subsequently, MP pCASL data were acquired at weekly intervals in models of primary and secondary brain tumours, and tumour microvessel density was determined histologically. MP pCASL measurements in a secondary brain tumour model revealed a significant reduction in blood flow at day 35 after induction, despite a higher density of blood vessels. Tumour core regions also showed reduced blood flow compared with the tumour rim. Similarly, significant reductions in CBF were found in a model of glioma 28 days after tumour induction, together with an increased density of blood vessels. These findings indicate that MP pCASL MRI provides accurate and robust measurements of cerebral blood flow in naïve mice and is sensitive to changes in tumour perfusion.

PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma.

Cellular senescence is a tumor suppressor mechanism where cells enter a permanent growth arrest following cellular stress. Oncogene-induced senescence (OIS) is induced in non-malignant cells following the expression of an oncogene or inactivation of a tumor suppressor. Previously, we have shown that protein kinase C iota (PKCι) depletion induces cellular senescence in glioblastoma cells in the absence of a detectable DNA damage response. Here we demonstrate that senescent glioblastoma cells exhibit an aberrant centrosome morphology. This was observed in basal levels of senescence, in p21-induced senescence, and in PKCι depletion-induced senescence. In addition, senescent glioblastoma cells are polyploid, Ki-67 negative and arrest at the G1/S checkpoint, as determined by expression of cell cycle regulatory proteins. These markers are all consistent with cells that have undergone mitotic slippage. Failure of the spindle assembly checkpoint to function properly can lead to mitotic slippage, resulting in the premature exit of mitotic cells into the G1 phase of the cell cycle. Although in G1, these cells have the replicated DNA and centrosomal phenotype of a cell that has entered mitosis and failed to divide. Overall, we demonstrate that PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma cells. To our knowledge, this is the first evidence of markers of mitotic slippage directly in senescent cells by co-staining for senescence-associated ß-galactosidase and immunofluorescence markers in the same cell population. We suggest that markers of mitotic slippage be assessed in future studies of senescence to determine the extent of mitotic slippage in the induction of cellular senescence.