An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models

While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening

Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essen- tial component of the cellular homeostatic machinery in eukary- otes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co- chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilita- tor of Hsp90-mediated folding of kinase and non-kinase clients— including Tsc2—thereby preventing their ubiquitination and proteasomal degradation

Abstract 1122‐000218: Flow Reversal After Stent Diversion Causes In‐Stent Thrombosis and Anemia‐Driven Ischemia

Introduction: Large symptomatic ICA aneurysms are rare, but present a life threatening risk of rupture that increases with size, female sex, and age >50 at the time of diagnosis, among other risk factors. Historically, large carotid aneurysms have been treated with intentional carotid sacrifice, requiring recruitment of contralateral, posterior, and ECA‐supplied collaterals to provide flow to the anterior circulation previously supplied by the sacrificed ICA, lest the patient experience an iatrogenic stroke. While still a viable option in some cases, flow diverting stents provide an attractive alternative to vessel sacrifice. By providing a channel for blood to bypass the aneurysm, the stent can effectively exclude the aneurism from active circulation while preserving a path for blood to travel to the anterior cerebral circulation it currently provides. Methods: Here, we present a case of a 1.3 cm symptomatic left cavernous ICA aneurysm treated with such a flow diverting stent. Results: The patient presented to the emergency department with left sided ptosis. CTA head and neck revealed the 1.3 cm left sided cavernous ICA aneurysm. She was treated endovascularly under general anesthesia with continuous intra‐operative monitoring. The procedure was complicated by iatrogenic flow reversal through the Circle of Willis at the time of stent deployment and resultant in situ thrombosis of the stent without alteration in electrical signals recorded at the scalp – thus creating a de facto carotid sacrifice without intra‐operative complication. Follow up doppler study revealed a loss of flow through the left ICA and reversal of flow through the ophthalmic artery on the left side, thus confirming ECA collateral supply to the area. Post‐op course was complicated by extensive bleeding from the scalp electrode sites used for intraoperative monitoring due to hyper‐response to aspirin‐ticagrelor dual‐antiplatelet therapy. This gave rise to a symptomatic anemia that manifested as pressure‐dependent left‐sided circulatory failure on exam – specifically hemiparesis and aphasia. The symptoms ultimately resolved with pressure augmentation, blood transfusion, and supportive care in the Neuro ICU. The patient was successfully transitioned to a general neurology floor with subsequent resolution of the anemia and, correspondingly, the symptoms. Conclusions: The patient was discharged to rehab and at 4‐month follow‐up is again living independently with no residual deficits. This case has significance for pre‐operative anti‐platelet optimization for flow diverting stents, management of post‐operative complications of flow‐diverting stent placement including thrombosis and bleeding, and optimal critical care support for patients with pressure‐dependent ischemia. Specifically, the course of the patient’s symptoms and anemia raise the question of optimal hemoglobin targets in the subset of patients with pressure‐dependent ischemia, and how to best reach those targets

Abstract Number ‐ 224: Stroke Mimic—Patient with Symptomatic Amyloid‐Related Imaging Abnormalities Presenting with Stroke‐like Symptoms.

Introduction Amyloid‐related imaging abnormalities (ARIA) are rare phenomena in patients taking monoclonal antibodies directed against amyloid beta in Alzheimer’s disease clinical trials. The pathogenesis is unclear, but may be due to an induced inflammatory microangiopathy. While the majority of cases are asymptomatic and resolve without intervention, a small subset can experience symptoms of sudden or uncertain onset. Given the relative rarity of this phenomena and the striking imaging findings, ARIA can be misdiagnosed as ischemic stroke or primary microangiopathy when presenting with acute‐subacute symptoms. Following diagnosis there is no clinical consensus on the optimal management and approaches range from withdrawal of the study drug to pulse‐dose steroids. Methods We present a case study of a patient admitted to our inpatient stroke service after 4 days of rapidly progressive confusion and sudden vision loss with MRI workup reporting subacute stroke. The findings were ultimately determined to be secondary to ARIA. Results A 67‐year‐old man with early onset Alzheimer’s disease enrolled in a clinical trial for donamemab presented with four days of marked cognitive decline including increased forgetfulness, confusion, and episodic hallucinations following an episode of abrupt vision loss. An outpatient MRI brain demonstrated subcortical hyperintensity that was initially reported as a subacute left occipital stroke. Notably, an MRI performed 3 months prior showed no abnormality. Neurological exam was remarkable for right superior quadrantanopia, memory impairment, and mild confabulation. Repeat MRI demonstrated FLAIR white matter hyperintensities and extensive susceptibility artifact in the left parieto‐occipital lobe, consistent with an inflammatory cerebral amyloid angiopathy (or ARIA). Spot EEG demonstrated no associated epileptiform discharges. Patient was treated with 7‐day course of IV Solumedrol and had improvement in visual symptoms. He was discharged on a prednisone taper and a followup MRI brain 4 weeks later showed near‐ complete resolution of the previously noted subcortical white matter changes. Conclusions ARIA can be seen in patients receiving anti‐amyloid monoclonal antibody treatments. Surveillance MRIs have been implemented to screen for such cases as a minority of patients with ARIA present with symptoms. As noted with this case study, a relatively acute presentation and imaging characteristics can lead to an initial misdiagnosis as subacute ischemic stroke. Vascular neurologists should be aware of this clinical entity, noting the natural history and treatment are not well established. This patient was treated aggressively as an inflammatory cerebral amyloid angiopathy with IV steroids and noted positive outcome. More research is required to establish the optimal acute medical management of patients with ARIA

Abstract Number ‐ 89: Left M1 occlusion rescued by the Recurrent artery of Huebner: a spectrum of MCA collaterals

Introduction Proximal MCA occlusion has variable presentation and clinical course, ranging from MCA syndrome with completed infarct at presentation to asymptomatic, discovered incidentally on imaging. This variability is likely dictated by variable collateral circulation that ultimately fails on variable time courses. For discussion we can divide these into (1) rapid progressors, (2) standard, (3) slow progressors, (4) situationally symptomatic, and (5) asymptomatic. There is clear evidence for the role of endovascular therapy for those who are significantly symptomatic and progress along an acute timeline when combined with imaging criteria (1 and 2), but there is no clear guidance for those patients who progress slowly or are minimally symptomatic (3‐5). Methods We present selected cases of patients with left MCA occlusion from groups 3–5 along with their presentation and collateral flow as assessed by non‐invasive imaging and DSA. Results Case 1. Slow Progressor. A 63‐year‐old woman with past occipital lobe strokes presented with progressive aphasia of insidious onset that began at some point the previous day. NIHSS 4 for aphasia and LOC questions. Initial CTA revealed a left M1 occlusion with ASPECTS 9. Despite low NIHSS and extended time to LKW, she was brought to the angiography suite. Initial angiogram confirmed M1 occlusion with extensive collateralization via the left ACA and anterior temporal artery, but no reconstitution of the MCA proper. Thrombectomy was completed with TICI2B recanalization. Followup MRI revealed minimal infarct in the left insula. The patient was discharged with an NIHSS of 1 for a baseline hemianopia not appreciated on initial exam. Case 2. Situationally Symptomatic. A 55‐year‐old man with symptomatic epilepsy from bilateral subdural hygromas was admitted for video EEG monitoring of episodic dizziness and word‐finding difficulty. Initial exam revealed only orthostatic light‐headedness. Further history revealed these events often happed at night upon standing. No epileptiform events were captured. MRA revealed a complete occlusion of the proximal M1 with reconstitution at the M2 bifurcation in the sylvian fissure. Given lack of persistent clinical deficit and absence of infarct on MRI, DSA was deferred. The patient is followed outpatient without further symptom progression. Case 3. Asymptomatic. A 38 year‐old‐man with TBI presented to the emergency department with thunderclap headache. Initial exam was without deficits. CTA and MRA revealed complete occlusion of the left MCA. We proceeded to the angiography suite where DSA revealed complete left M1 occlusion with distal reconstitution at the M2 bifurcation primarily via the Recurrent Artery of Huebner in addition to ACA collaterals. Lumbar puncture was benign and his headache responded completely to typical migraine treatment. The patient was discharged to outpatient follow up without further sequalae. Conclusions We present a series of illustrative cases of patients with MCA occlusion of variable collateral supply and clinical course, none of whom met conventional criteria for endovascular intervention. More research is needed to establish clinical and imaging criteria to separate patients who would benefit from endovascular intervention from those who are best managed medically in these scenarios

Abstract Number ‐ 122: Minimally symptomatic left M1 occlusion from carotid web

Introduction Minimally symptomatic large vessel occlusion (LVO) is an area of prognostic and management uncertainty. Current guidelines recommend thrombectomy only for patients with NIHSS of 6 or greater when additional timeline and imaging criteria are met. However, patients with initial mild or rapidly improving symptoms with LVO can eventually deteriorate, raising the question of whether all patient with acute LVO should be offered thrombectomy. Methods We present a case of minimally symptomatic M1 occlusion due to ipsilateral carotid web managed without thrombectomy. Results A 42‐year‐old woman with Systemic Lupus Erythematosus (SLE) and hypertension presented to an outside hospital with sudden transient left monocular vision loss, aphasia, right‐side weakness, and dizziness. At the time of presentation (1h from LKW) exam revealed only mild aphasia (NIHSS 1) with normal blood pressure. CTA demonstrated left M1 occlusion with reconstitution of flow at the M2 bifurcation. She was given IV Alteplase and transferred to our center. On 7 h from LKW repeat imaging demonstrated persistence of the left M1 occlusion with an ipsilateral near‐occlusion suspicious for carotid web at the ICA origin, but exam was without deficit. She was monitored closely for neurological deterioration, but remained without significant symptoms. MRA taken the next day demonstrated complete recanalization of the left M1. She was taken to the angiography suite on an outpatient basis for stenting of the carotid web at the origin of the internal carotid artery. Initial diagnostic angiogram revealed near occlusion of the ICA at the origin with post‐stenotic dilation, which was treated with angioplasty and stenting with distal embolic protection. She has remained stable to 8‐month follow‐up with no recurrent stroke. Conclusions Minimally symptomatic LVO presents significant endovascular management uncertainty. While clinical trials support the use of mechanical thrombectomy for significantly symptomatic patients, there is no consensus to guide treatment decisions for patients with minimal symptoms. This uncertainty is driven at least in part by an inability to reliably distinguish between patients who are likely to progress and would benefit from intervention before their collateral circulation fails, and those who will recanalize or develop a compensated chronic occlusion. In this case, our patient received tPA and saw symptom improvement, but this did not remove her M1 occlusion. Angiographic investigation was limited as the patient was asymptomatic, however non‐invasive imaging revealed robust collateralization including reconstitution of MCA flow and left ICA injection failed to opacify the ipsilateral ACA despite patency in recent CT angiography, indicating a right to left flow dominance in circle of Willis collaterals. We opted for close monitoring with follow‐up imaging and outpatient correction of the carotid web, which resulted in a positive outcome. More work is needed to establish criteria to predict which patients with minimally symptomatic LVO are likely to progress

Reactivating mutant p53 using small molecules as zinc metallochaperones: awakening a sleeping giant in cancer

Tumor protein p53 (TP53) is the most commonly mutated gene in human cancer. The majority of mutations are missense, and generate a defective protein that is druggable. Yet, for decades, the small-molecule restoration of wild-type (WT) p53 function in mutant p53 tumors (so-called p53 mutant ‘reactivation’) has been elusive to researchers. The p53 protein requires the binding of a single zinc ion for proper folding, and impairing zinc binding is a major mechanism for loss of function in missense mutant p53. Here, we describe recent work defining a new class of drugs termed zinc metallochaperones that restore WT p53 structure and function by restoring Zn2+ to Zn2+-deficient mutant p53

Molecular evolution of V H 9 germline genes isolated from DBA, BALB, 129 and C57BL mouse strains and sublines

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