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How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.

While organ-confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumors with concomitant inhibition of two tumor suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having less than 50% 5-year secondary-therapy-free patient survival. Functionally, overexpression of PME-1, a methylesterase for the catalytic PP2A-C subunit, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumor xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1-deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME-1 supports anoikis resistance in PTEN-deficient PCa cells. Clinically, these results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.

Purpose: Only few predictive factors for the clinical activity of anti–epidermal growth factor receptor (EGFR) therapy are available. Mammary-derived growth inhibitor (MDGI) is a small cytosolic protein suggested to play a role in the differentiation of epithelial cells. Here, we have investigated the effect of MDGI expression on the EGFR signaling and cetuximab responsiveness of cancer cells.Experimental Design: MDGI mRNA expression was investigated in clinical breast and lung cancer samples and in nontransformed and malignant cell lines. The effect of ectopic expression of MDGI on EGFR, ErbB2, and integrin function and traffic was investigated in breast and lung cancer cell lines using multiple methods. The effect of anti-EGFR agents on these cells were tested by cell proliferation measurements and by assessing tumor growth of breast cancer cells in cetuximab treated and control athymic nude mice.Results: Here, we show that although MDGI is absent in cultured cell lines because of epigenetic silencing, MDGI mRNA is expressed in 40% of clinical breast carcinomas and 85% of lung cancers. Ectopic expression of MDGI rendered breast and lung cancer cells resistant to the anti-EGFR antibody cetuximab in vitro and in an orthotopic breast cancer xenograft model in vivo. When expressed in cancer cells, MDGI induces intracellular accumulation of EGFR, but not ErbB2, and the internalized receptor is phosphorylated and not degraded.Conclusions: MDGI-driven inherent desensitization of cancer cells is a novel molecular mechanism for resistance to the anti-EGFR antibody therapy, and MDGI may be a biomarker for responsiveness to anti-EGFR antibody therapy.(Clin Cancer Res 2009;15(21):6570–81)

Supplementary Data from CIP2A Is Associated with Human Breast Cancer Aggressivity

Supplementary Data from Mammary-Derived Growth Inhibitor Alters Traffic of EGFR and Induces a Novel Form of Cetuximab Resistance

Purpose: To investigate the clinical relevance of the recently characterized human oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) in human breast cancer.Experimental Design: CIP2A expression (mRNA and protein) was measured in three different sets of human mammary tumors and compared with clinicopathologic variables. The functional role of CIP2A in breast cancer cells was evaluated by small interfering RNA–mediated depletion of the protein followed by an analysis of cell proliferation, migration, anchorage-independent growth, and xenograft growth.Results: CIP2A mRNA is overexpressed (n = 159) and correlates with higher Scarff-Bloom-Richardson grades (n = 251) in samples from two independent human breast cancer patients. CIP2A protein was found to be overexpressed in 39% of 33 human breast cancer samples. Furthermore, CIP2A mRNA expression positively correlated with lymph node positivity of the patients and with the expression of proliferation markers and p53 mutations in the tumor samples. Moreover, CIP2A protein expression was induced in breast cancer mouse models presenting mammary gland–specific depletion of p53 and either BRCA1 or BRCA2. Functionally, CIP2A depletion was shown to inhibit the expression of its target protein c-Myc. Loss of CIP2A also inhibited anchorage-independent growth in breast cancer cells. Finally, CIP2A was shown to support MDA-MB-231 xenograft growth in nude mice.Conclusions: Our data show that CIP2A is associated with clinical aggressivity in human breast cancer and promotes the malignant growth of breast cancer cells. Thus, these results validate the role of CIP2A as a clinically relevant human oncoprotein and warrant further investigation of CIP2A as a therapeutic target in breast cancer treatment. (Clin Cancer Res 2009;15(16):5092–100)

Supplementary figures S1-5, materials and methods. 1) Supplementary figures and associated legends: - Figure S1. ERK silencing decreases breast cancer cell migration and invasion but does not influence cell proliferation. - Figure S2. ERK2 does not interact with actin or keratin 8 and ERK2 phosphorylation is supported by vimentin but not actin. - Figure S3. Fluorescence recovery of wild-type Slug-GFP after photobleaching (FRAP). - Figure S4. Mass spectrometric identification of ERK1/2-dependent phosphorylation sites on Slug and validation using a newly generated Slug phospho-serine-87 antibody. - Figure S5. The phosphorylation status of Slug does not influence repression of E-Cadherin, Slug nuclear localisation or stability. 2) Supplementary materials and methods 3) Supplementary references

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

In breast cancer, the currently approved anti‐receptor tyrosine‐protein kinase erbB‐2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2‐related therapeutic targets could offer a means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin‐related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2‐targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti‐SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cell density in a mouse xenograft model of HER2‐positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient‐derived explant three‐dimensional cultures. These results provide, for the first time, proof of principle that SorLA is a druggable target in breast cancer., SorLA interacts with HER2 and HER3 and positively regulates their oncogenic signaling. Here, we report that SorLA‐targeting antibody synergizes with trastuzumab to inhibit proliferation of HER2‐amplified, trastuzumab‐resistant breast cancer cells. In addition, SorLA ab monotherapy inhibits proliferation of breast cancer patient‐derived explant cultures. These results provide proof of principle that SorLA is a druggable target in breast cancer.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where numerous different activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds overactive mutant forms to limit oncogenic KRAS signalling and maintain RAS- activity at an optimal level. Depletion of SHANK3 results in hyperactivation of KRAS/mitogen-activated protein kinase (MAPK) signalling, which in turn selectively induces MAPK/ERK-dependent cell death in KRAS-mutant cancers. Furthermore, targeting of this therapeutic vulnerability through nanobody- or RNA interference- mediated disruption of the SHANK3-KRAS interaction reduces tumour growth in vivo. Thus, inhibition of the SHANK3-KRAS interaction represents a new pan-KRAS-mutant compatible strategy for selective killing of KRAS- mutant cancer cells through excessive signalling.Graphical abstractSchematic model of SHANK3-controlled cell fate in KRAS-mutant cancers. SHANK3 directly interacts with KRAS and competes with RAF for KRAS binding to sustain oncogenic RAS-MAPK/ERK signalling at an optimal level (i.e. below toxic oncogenic signalling) in KRAS-mutant cancers. SHANK3 silencing (1) and inhibition of SHANK3-KRAS interaction (2) drive KRAS-mutant cells into cell death.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

Cells sense and respond to the extracellular matrix (ECM) milieu through integrin proteins. Integrin availability on the plasma membrane, regulated by endosomal receptor uptake and recycling, has been extensively studied and regulates cell dynamics in various normal and pathological contexts1–5. In contrast, the role of integrin transport through the biosynthetic pathway has been considered primarily as a mechanism to replenish the receptor pool and too slow to influence cell dynamics6. Here, we adopted the RUSH (Retention Using Selective Hooks) assay to synchronize integrin anterograde transport from the endoplasmic reticulum (ER), allowing spatial and temporal analysis of newly synthesized receptor traffic. We observe that the delivery of new integrins to the plasma membrane is polarized in response to specific ECM ligands, facilitates integrin recruitment specifically to the membrane-proximal tip of focal adhesions (FA) and contributes to cell protrusion and FA growth. We explain the augmented adhesion growth using a computational molecular clutch model7, where increased integrin availability drives recruitment of additional integrins. Notably, a subset of newly synthesized integrins undergo rapid traffic from the ER to the cell surface to facilitate localized cell spreading, seemingly bypassing the Golgi. This unconventional secretion is dependent on cell adhesion and mediated by Golgi reassembling stacking proteins (GRASPs) association with the PDZ-binding motif in the integrin α5 cytoplasmic tail. This spatially targeted delivery of integrins through the biosynthetic pathway may propel cell dynamics by rapidly altering adhesion receptor availability, providing cells with an additional degree of plasticity to respond to their environment.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination. ispartof: DEVELOPMENTAL CELL vol:57 issue:20 pages:2350-+ ispartof: location:United States status: published

Coordinated Cell Motion In article number 2104808 by Swetha Raghuraman, Timo Betz, and co‐workers, a novel migration phenomenon is presented wherein cancer cells burst out from tumor aggregates as an act of pressure release into the surrounding in‐homogeneous soft extra‐cellular matrix (ECM) made up of collagen. The pressure within tumor aggregates manifests due to cellular swelling, leading to a super‐diffusive coordinated expulsion of cells within 12 hours, directed towards regions providing least mechanical resistance [Image: see text]

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions.

Epithelial-to-mesenchymal transition (EMT) contributes to tumor cell survival, immune evasion, migration, invasion, and therapy resistance. Across human cancer, tumors that are high grade, poorly differentiated, and have undergone EMT carry a worse prognosis with a higher likelihood of metastasis. AXL, a receptor tyrosine kinase, drives EMT and is implicated in tumor progression, metastasis, and therapy resistance in multiple cancer types including pancreatic cancer and breast cancer. TANK-binding kinase 1 (TBK1) is central to AXL-driven EMT yet, the mechanism of how TBK1 induces EMT remains unclear. Here, we report that AXL activation stimulates TBK1 binding and phosphorylation of AKT3. TBK1 activation of AKT3 drives binding and phosphorylation of slug/snail resulting in protection from proteasomal degradation and translocation of the complex into the nucleus. We show that nuclear translocation of AKT3 is required for AXL-driven EMT and metastasis. Congruently, nuclear AKT3 expression correlates with worse outcome in aggressive breast cancer. To advance AKT3 as a therapeutic target, an AKT3-isoform selective allosteric small molecule inhibitor, BGB214, was developed. BGB214 inhibits AKT3 nuclear translocation, EMT-TF stability, AKT3-mediated invasion of breast cancer cells and reduces tumor initiation in vivo. Our results suggest that AKT3 nuclear activity is an important feature of AXL-driven epithelial plasticity and that selective AKT3 inhibition represents a novel therapeutic avenue for treating aggressive cancer.SignificanceNuclear AKT3 activity is an important feature of AXL-TBK1 driven EMT and metastasis, thus selective AKT3 targeting represents a novel approach to treat aggressive cancer.

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell-cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell-cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer, where the tumor is encapsulated by a basement membrane (BM). At the invasive phase, the BM barrier is compromised enabling tumor cells to escape into the surrounding stroma. The molecular mechanisms that establish and maintain an epithelial BM barrier in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing motor protein implicated in metastasis and poor clinical outcome in patients with invasive breast cancer (IBC). We compared MYO10 expression in patient-matched normal breast tissue and DCIS lesions and found elevated MYO10 expression in DCIS samples, suggesting that MYO10 might facilitate the transition from DCIS to IBC. Indeed, MYO10 promoted the formation of filopodia and cell invasion in vitro and positively regulated the dissemination of individual cancer cells from IBC lesions in vivo. However, MYO10-depleted DCIS xenografts were, unexpectedly, more invasive. In these xenografts, MYO10 depletion compromised BM formation around the lesions resulting in poorly defined tumor borders and increased cancer cell dispersal into the surrounding stroma. Moreover, MYO10-depleted tumors showed increased EMT-marker-positive cells, specifically at the tumor periphery. We also observed cancer spheroids undergoing rotational motion and recruiting BM components in a filopodia-dependent manner to generate a near-continuous extracellular matrix boundary. Taken together, our data identify a protective role for MYO10 in early-stage breast cancer, where MYO10-dependent tumor cell protrusions support BM assembly at the tumor-stroma interface to limit cancer progression, and a pro-invasive role that facilitates cancer cell dissemination at later stages.Abstract FigureHighlights-Filopodia sculpt the tumor-proximal stroma in pre-invasive ductal carcinoma in situ (DCIS).-Filopodia-dependent basement membrane (BM) assembly limits invasive transition of DCIS-like tumors in vivo.-Loss of MYO10-dependent filopodia impairs BM assembly and induces an EMT-like phenotype at the tumor-stroma interface in vivo.-MYO10 filopodia are anti-invasive in DCIS but facilitate dissemination in invasive breast cancer.

MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with β3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-β receptor II (TGFBR2), a key modulator of TGF-β signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.

Summary Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10’s FERM domain, or mutation of its β1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10’s isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation., Graphical abstract, Highlights • Integrin activity is spatially regulated in filopodia • Integrin activation at filopodia tips is regulated by talin and MYO10 • MYO10 is required to activate (but not transport) integrins at filopodia tips • We propose a two-step integrin activation model in filopodia, Cells utilize cellular protrusions such as filopodia to explore their environment as they migrate. Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. Miihkinen et al. show that MYO10 and talin regulate integrin activity at filopodia tips and propose a two-step integrin activation model in filopodia.

Tissue architecture and function are orchestrated by an intricate repertoire of cellular adhesion and signalling receptors, and by the surrounding extracellular matrix (ECM). The essential role of cell-tissue interactions in guiding organogenesis was identified in experimental embryology studies over a century ago, and in 1954 Grobstein laid down the fundamental concept of ECM being the ultimate integrator of cellular systems. Long before the main cell adhesion receptors were identified, Abercrombie and colleagues proposed in 1971 that cell attachment to the ECM substratum was mediated through electron-dense plaques containing longitudinal cytoplasmic filaments that localise to areas of the ventral cell membrane that lie close to the substratum. In 1982, Bissell and co-workers proposed "the minimum required unit for expression of tissue specific functions", a model depicting a structure in which the nucleus links to the ECM via cytoskeletal filament bundles that connect to a hypothetical transmembrane ECM adhesion receptor.

Understanding the mechanisms of resistance to targeted therapies is a major challenge in epidermal growth factor receptor 2 (ERBB2)-amplified breast cancer and acquired resistance to ERBB2 inhibition is a common feature of recurrent tumors. In such a setting, the activation of intracellular oncogenic signaling pathways, through alternative growth factor receptors, adds a layer of complexity to therapeutic options. Our aim is to scrutinize the intracellular retrograde transport of ERBB-family receptors and to assess whether the key sorting proteins involved within this pathway represent a potential vulnerability in ERBB2-amplified breast cancer. Sortilin-related receptor (SorLA; SORL1) is a vacuolar protein sorting 10 (VPS10)-domain receptor well characterized for its role in Alzheimer’s and metabolic diseases. In a previous study, we demonstrated a previously unknown role of SorLA in cancer. SorLA increases ERBB2 stability by stimulating its recycling from endosomes back to the plasma membrane thus sustaining ERBB2 oncogenic signaling in vitro. In an orthotopic xenograft model, SorLA silencing inhibits tumor growth adding translational relevance for SorLA targeting in breast cancer. Here we investigate possible crosstalk inputs from other growth factor receptors, and we evaluate the potential of SorLA inhibition as a unique strategy to halt the progression of ERBB2-amplified tumors. Citation Format: Hussein Al Akhrass, James Conway, Mark Barok, Olav Andersen, Johanna Ivaska. Sortilin-related receptor regulates receptor tyrosine kinase traffic and signaling [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-08.

1380 Objectives: The purpose of this study was to investigate how gender, BMI, patient cross-sectional area and heart size affect the influence of time-of-flight (TOF) and point spread function (PSF) modeling in image reconstruction on regional absolute myocardial blood flow (MBF) measured with 82Rb PET in an obese population. Methods: Dynamic rest/stress 82Rb cardiac PET studies were performed for 30 obese subjects on a TOF PET scanner. Subject characteristics were (mean ± SD for continuous variables): gender 9 male, 21 female; weight 138.9 ± 32.8 kg; BMI 47.8 ± 9.5 kg/m2; heart size 92.4 ± 55.3 ml; cross-sectional area in a transaxial slice containing the heart 1133 ± 233 cm2. Images were reconstructed in four ways: (1) OSEM without TOF or PSF modeling (OSEM), (2) OSEM with TOF (TOF), (3) OSEM with PSF modeling (PSF) and (4) OSEM with TOF and PSF modeling (PSFTOF). All were with 3 iterations, 8 subsets and with a post-reconstruction 8mm 3D Gaussian filter. A one tissue compartmental model was used to compute MBF. Average rest and stress flow and myocardial flow reserve were computed in five regions: anterior, septal, inferior, lateral and apical. Results from TOF, PSF and PSFTOF were compared with those from OSEM, with subjects grouped by gender, and by tertiles (10 subjects each) for BMI (I: 32.5-40.5, II: 41.1-52.3, III: 53.3-75.6 (kg/m2)), cross-sectional area (I: 532 - 1086, II: 1089 - 1157, III: 1187 - 1662 (cm2)) and heart size (I: 19 -56, II: 66 - 93, III: 97 - 238 (ml)). The MBF or myocardial flow reserve (MFR) in a given region was compared with the corresponding OSEM value and the change was expressed as a percent. Results: The general trends for all subjects with TOF were similar MBF increases in the apical (+22.0%) and septal walls (+19.4%), and a slightly lower increase in the anterior wall (+11.3 %). Increases were small in the inferior (+4.6%) and lateral (+4.9%) walls. There was little effect of PSF modeling in any region. MBF trends with PSFTOF were similar to those for TOF alone. Rest and stress increases with TOF tended to be matched, so that MFR was little changed. Trends were examined for the groupings by patient characteristics. (1) Gender. MBF showed a greater increase with TOF for females (mean +20.9%) compared to males (mean +14.9%) in the apical, septal and anterior walls. Results with PSFTOF were similar. (2) BMI. The MBF increase with TOF in the apical, anterior and septal walls was greatest for tertile II (mean +26.9%) compared with tertile I (mean +13.6%) and tertile III (mean +16.7%). (3) Cross-sectional area. The MBF increase with TOF in the apical, anterior and septal walls was similar for tertiles II (mean +22.1%) and III (mean +22.6%), and was greater than that for tertile I (mean +12.6%). (4) Heart size. The MBF increase with TOF in the apical, anterior and septal walls was greater for smaller heart sizes: tertile I (mean +21.2%), tertile II (+20.7%) and tertile III (+15.3%). These results are for our patient population and particular hardware, software and processing implementation. Conclusions: Patient gender, BMI, cross-sectional area and heart size affect the degree to which MBF values change with TOF. The largest changes are observed for females, patients with larger BMI, larger cross-sectional area and smaller hearts. The greatest changes are found in the apical, septal and lateral walls. The studied patient characteristics have little effect on flow reserve. It is important to consider these patient-dependent effects when evaluating absolute MBF in a clinical environment, and to recognize that equipment or protocol changes may affect MBF results.

Key Points A negative feedback loop, integrin–PI3K–ARAP3–integrin, controls integrin inactivation. Integrin inactivation promotes neutrophil transendothelial migration and recruitment., Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.