Dosimetric predictors of acute bowel toxicity after Stereotactic Body Radiotherapy (SBRT) in the definitive treatment of localized prostate cancer.

INTRODUCTION: SBRT is an increasingly popular treatment for localized prostate cancer, though considerable variation in technical approach is common and optimal dose constraints are uncertain. In this study, we sought to identify dosimetric and patient-related predictors of acute rectal toxicity. METHODS: Patients included in this study were treated with prostate SBRT on a prospective institutional protocol. Physician-graded toxicity and patient-reported outcomes were captured at one week, one month, and three months following SBRT. DVH data were extracted and converted into relative volume differential DVHs for NTCP modeling. Patient- and disease-related covariates along with NTCP model predictions were independently tested for significant association with physician-graded toxicity or a decline in bowel-related QoL. A multivariate model was constructed using forward selection, and significant parameter cutoff values were obtained with Fischer's exact test to group patients by risk of developing physician-graded toxicity or detriments in patient-reported QoL. RESULTS: One hundred and three patients treated for localized prostate cancer with SBRT were included in our analysis. 52% of patients experienced a clinically significant decline in bowel-related QOL within 1 week of completion of treatment, while only 27.5% of patients developed grade 2+ physician-graded rectal toxicity. Sequential feature selection multivariate logistic regression identified rectal V22.5 Gy (p = 0.001) and D19% (p = 0.001) as independent predictors of clinically significant toxicity, while rectal V20Gy (p = 0.004) and D25.3% (p = 0.007) were independently correlated with physician-graded toxicity. Global multivariate step-wise logistic regression identified only D19% (p = 0.001) and V20Gy (p = 0.004) as independent predictors of acute bowel bother or physician-graded rectal toxicity respectively. CONCLUSIONS: Moderate doses to large rectal volumes, D19% and V20Gy, were associated with an increased incidence of a clinically significant decrease in patient-reported bowel QOL and physician-scored grade 2+ rectal toxicity, respectively. These dosimetric parameters may help practitioners mitigate acute toxicity in patients treated with prostate SBRT.

Definitive hypofractionated radiation therapy for early stage breast cancer: Dosimetric feasibility of stereotactic ablative radiotherapy and proton beam therapy for intact breast tumors.

PURPOSE: Few definitive treatment options exist for elderly patients diagnosed with early stage breast cancer who are medically inoperable or refuse surgery. Historical data suggest very poor local control with hormone therapy alone. We examined the dosimetric feasibility of hypofractionated radiation therapy using stereotactic ablative radiotherapy (SABR) and proton beam therapy (PBT) as a means of definitive treatment for early stage breast cancer. METHODS AND MATERIALS: Fifteen patients with biopsy-proven early stage breast cancer with a clinically visible tumor on preoperative computed tomography scans were identified. Gross tumor volumes were contoured and correlated with known biopsy-proven malignancy on prior imaging. Treatment margins were created on the basis of set-up uncertainty and image guidance capabilities of the three radiation modalities analyzed (3-dimensional conformal radiation therapy [3D-CRT], SABR, and PBT) to deliver a total dose of 50 Gy in 5 fractions. Dose volume histograms were analyzed and compared between treatment techniques. RESULTS: The median planning target volume (PTV) for SABR, PBT, and 3-dimensional CRT was 11.91, 21.03, and 45.08 cm3, respectively, and were significantly different (P < .0001) between treatment modalities. Overall target coverage of gross tumor and clinical target volumes was excellent with all three modalities. Both SABR and PBT demonstrated significant dosimetric improvements, each in its own unique manner, relative to 3D-CRT. Dose constraints to normal structures including ipsilateral/contralateral breast, bilateral lungs, and heart were all consistently achieved using SABR and PBT. However, skin or chest wall dose constraints were exceeded in some cases for both SABR and PBT plans and was dictated by the anatomic location of the tumor. CONCLUSIONS: Definitive hypofractionated radiation therapy using SABR and PBT appears to be dosimetrically feasible for the treatment of early stage breast cancer. The anatomical location of the tumor relative to the skin and chest wall appears to be the primary limiting dosimetric factor.

Intensity-Modulated Radiation Therapy with Stereotactic Body Radiation Therapy Boost for Unfavorable Prostate Cancer: A Report on 3-Year Toxicity.

BACKGROUND: Recent data suggest that intensity-modulated radiation therapy (IMRT) plus brachytherapy boost for unfavorable prostate cancer provides improved biochemical relapse-free survival over IMRT alone. Stereotactic body radiation therapy (SBRT) may be a less invasive alternative to brachytherapy boost. Here, we report the 3-year gastrointestinal (GI) and genitourinary (GU) toxicities of IMRT plus SBRT boost. MATERIALS AND METHODS: Between March 2008 and September 2012, patients with prostate cancer were treated with robotic SBRT (19.5 Gy in three fractions) followed by fiducial-guided IMRT (45-50.4 Gy) on an institutional protocol. Toxicity was prospectively graded using the common terminology criteria for adverse events version 4.0 (CTCAEv.4) at the start of and at 1- to 6-month intervals after therapy. Rectal telangiectasias were graded using the Vienna Rectoscopy Score (VRS). RESULTS: At a median follow-up of 4.2 years (2.4-7.5), 108 patients (4 low-, 45 intermediate-, and 59 high-risk) with a median age of 74 years (55-92) were treated with SBRT plus IMRT, with 8% on anticoagulation and an additional 48% on antiplatelet therapy at the start of therapy. The cumulative incidence of late ≥grade 2 GI toxicity was 12%. Of these, 7% were due to late rectal bleeding, with six patients requiring up to two coagulation procedures. One patient with rectal telangiectasias was treated with hyperbaric oxygen (grade 3 toxicity). No rectal fistulas or stenoses were observed. Ten patients had multiple non-confluent telangiectasias (VRS grade 2), and three patients had multiple confluent telangiectasias (VRS grade 3). The cumulative incidence of late grade 3 GU toxicity was 6%. Most late toxicities were due to hematuria requiring bladder fulguration. There were no late ≥grade 4 GU toxicities. CONCLUSION: Rates of clinically significant GI and GU toxicities are modest following IMRT plus SBRT boost. Future studies should compare cancer control, quality of life, and toxicity with other treatment modalities for patients with high-risk prostate cancer.

Stereotactic Accelerated Partial Breast Irradiation for Early-Stage Breast Cancer: Rationale, Feasibility, and Early Experience Using the CyberKnife Radiosurgery Delivery Platform.

PURPOSE: The efficacy of accelerated partial breast irradiation (APBI) utilizing brachytherapy or conventional external beam radiation has been studied in early-stage breast cancer treated with breast-conserving surgery. Data regarding stereotactic treatment approaches are emerging. The CyberKnife linear accelerator enables excellent dose conformality to target structures while adjusting for target and patient motion. We report our institutional experience on the technical feasibility and rationale for stereotactic accelerated partial breast irradiation (SAPBI) delivery using the CyberKnife radiosurgery system. METHODS: Ten patients completed CyberKnife SAPBI (CK-SAPBI) in 2013 at Georgetown University Hospital. Four gold fiducials were implanted around the lumpectomy cavity prior to treatment under ultrasound guidance. The synchrony system tracked intrafraction motion of the fiducials. The clinical target volume was defined on contrast enhanced CT scans using surgical clips and post-operative changes. A 5 mm expansion was added to create the planning treatment volume (PTV). A total dose of 30 Gy was delivered to the PTV in five consecutive fractions. Target and critical structure doses were assessed as per the National Surgical Adjuvant Breast and Bowel Project B-39 study. RESULTS: At least three fiducials were tracked in 100% of cases. The Mean treated PTV was 70 cm(3) and the mean prescription isodose line was 80%. Mean dose to target volumes and constraints are as follows: 100% of the PTV received the prescription dose (PTV30). The volume of the ipsilateral breast receiving 30 Gy (V30) and above 15 Gy (V > 15) was 14 and 31%, respectively. The ipsilateral lung volume receiving 9 Gy (V9) was 3%, and the contralateral lung volume receiving 1.5 Gy (V1.5) was 8%. For left-sided breast cancers, the volume of heart receiving 1.5 Gy (V1.5) was 31%. Maximum skin dose was 36 Gy. At a median follow-up of 1.3 years, all patients have experienced excellent/good breast cosmesis outcomes, and no breast events have been recorded. CONCLUSION: CyberKnife stereotactic accelerated partial breast irradiation is an appealing technique for partial breast irradiation offering improvements over existing APBI techniques. Our early findings indicate that CK-SAPBI delivered in five daily fractions is feasible, well tolerated, and is a reliable platform for delivering APBI.

Four-dimensional computed tomography prediction of inter- and intrafractional upper gastrointestinal tumor motion during fractionated stereotactic body radiation therapy.

PURPOSE: Respiratory-induced tumor motion of upper gastrointestinal (GI) tumors during radiation therapy is often assessed using a single 4-dimensional computed tomography (4D-CT) and presumed to be representative during fractionated treatment regimens. The purpose of this study was to examine the intra- and interfraction correlations of tumor motion between pretreatment 4D-CT and real-time fiducial-based motion tracking in patients treated with fractionated stereotactic body radiation therapy (SBRT) for upper GI malignancies. METHODS AND MATERIALS: Fourteen patients with upper GI tumors underwent fractionated SBRT using the CyberKnife radiosurgical system with Synchrony respiratory motion management. Before treatment, each patient underwent a free-breathing 4D-CT scan and fiducial motion was tracked for each phase of the respiratory cycle. Real-time fiducial positions recorded during delivery of each SBRT fraction were extracted from the CyberKnife planning system. Displacements were compared between those predicted by 4D-CT and those recorded by Synchrony during treatment in the left-right (LR), anteroposterior (AP), and superoinferior (SI) directions. RESULTS: The 4D-CT scans demonstrated little correlation with real-time mean fiducial displacement as determined by Pearson correlation with coefficients of 0.45, 0.52, and 0.63 in the SI, AP, and LR directions, respectively. Cohort-averaged maximum fiducial displacements based on 4D-CT and real-time tracking were measured to be 3.86 ± 1.40 mm versus 10.73 ± 7.03 mm, 2.29 ± 1.02 mm versus 4.44 ± 3.33 mm, and 1.45 ± 0.49 mm versus 2.67 ± 2.49 mm in the SI, AP, and LR directions, respectively. Mean fiducial displacements were greater than that predicted by the maximum displacements on the corresponding 4D-CT scan in 39%, 22%, and 25% of SBRT fractions in the SI, AP, and LR directions, respectively. CONCLUSIONS: Comparison of 4D-CT with real-time fiducial tracking demonstrated significant inter- and intrafractional discrepancies, particularly in the SI direction, which could result in compromise of target coverage when planning with a single free-breathing 4D-CT.

Late urinary toxicity modeling after stereotactic body radiotherapy (SBRT) in the definitive treatment of localized prostate cancer.

BACKGROUND: Late urinary symptom flare has been shown to occur in a small subset of men treated with ultra- hypofractionated stereotactic body radiotherapy (SBRT) for prostate cancer. The purpose of this study was to use normal tissue complication probability modeling in an effort to derive SBRT specific dosimetric predictor's of late urinary flare. MATERIAL AND METHODS: Two hundred and sixteen men were treated for localized prostate cancer using ultra- hypofractionated SBRT. A dose of 35-36.25 Gy in 5 fractions was delivered to the prostate and proximal seminal vesicles. Functional surveys were conducted before and after treatment to assess late toxicity. Phenomenologic NTCP models were fit to bladder DVHs and late urinary flare outcomes using maximum likelihood estimation. RESULTS: Twenty-nine patients experienced late urinary flare within two years of completion of treatment. Fitting of bladder DVH data to a Lyman NTCP model resulted in parameter estimates of m, TD50, and n of 0.19 (0-0.47), 38.7 Gy (31.1-46.4), and 0.13 (-0.14-0.41), respectively. Subsequent fit to a hottest volume probit model revealed a significant association of late urinary flare with dose to the hottest 12.7% of bladder volume. Multivariate analysis resulted in a final model that included patient age and hottest volume probit model predictions. Kaplan-Meier analysis demonstrated a two-year urinary flare free survival of 95.7% in patients 65 years or older with a bladder D12.7% of 33.5 Gy or less, compared to 74.5% in patients meeting none of these criteria. CONCLUSION: NTCP modeling of late urinary flare after ultra-hypofractionated prostate SBRT demonstrates a relatively small volume effect for dose to the bladder, suggesting that reduction of volume receiving elevated dose will result in decreased incidence of late urinary toxicity. Future studies will be needed to examine the impact of dose to other potential sources of late genitourinary toxicity.

Noncontact conductivity and dielectric measurement for high throughput roll-to-roll nanomanufacturing.

Advances in roll-to-roll processing of graphene and carbon nanotubes have at last led to the continuous production of high-quality coatings and filaments, ushering in a wave of applications for flexible and wearable electronics, woven fabrics, and wires. These applications often require specific electrical properties, and hence precise control over material micro- and nanostructure. While such control can be achieved, in principle, by closed-loop processing methods, there are relatively few noncontact and nondestructive options for quantifying the electrical properties of materials on a moving web at the speed required in modern nanomanufacturing. Here, we demonstrate a noncontact microwave method for measuring the dielectric constant and conductivity (or geometry for samples of known dielectric properties) of materials in a millisecond. Such measurement times are compatible with current and future industrial needs, enabling real-time materials characterization and in-line control of processing variables without disrupting production.

Dysuria Following Stereotactic Body Radiation Therapy for Prostate Cancer.

BACKGROUND: Dysuria following prostate radiation therapy is a common toxicity that adversely affects patients' quality of life and may be difficult to manage. METHODS: Two hundred four patients treated with stereotactic body radiation therapy (SBRT) from 2007 to 2010 for localized prostate carcinoma with a minimum follow-up of 3 years were included in this retrospective review of prospectively collected data. All patients were treated to 35-36.25 Gy in five fractions delivered with robotic SBRT with real time fiducial tracking. Dysuria and other lower urinary tract symptoms were assessed via Question 4b (Pain or burning on urination) of the expanded prostate index composite-26 and the American Urological Association (AUA) Symptom Score at baseline and at routine follow-up. RESULTS: Two hundred four patients (82 low-, 105 intermediate-, and 17 high-risk according to the D'Amico classification) at a median age of 69 years (range 48-91) received SBRT for their localized prostate cancer with a median follow-up of 47 months. Bother associated with dysuria significantly increased from a baseline of 12% to a maximum of 43% at 1 month (p < 0.0001). There were two distinct peaks of moderate to severe dysuria bother at 1 month and at 6-12 months, with 9% of patients experiencing a late transient dysuria flare. While a low level of dysuria was seen through the first 2 years of follow-up, it returned to below baseline by 2 years (p = 0.91). The median baseline AUA score of 7.5 significantly increased to 11 at 1 month (p < 0.0001) and returned to 7 at 3 months (p = 0.54). Patients with dysuria had a statistically higher AUA score at baseline and at all follow-ups up to 30 months. Dysuria significantly correlated with dose and AUA score on multivariate analysis. Frequency and strain significantly correlated with dysuria on stepwise multivariate analysis. CONCLUSION: The rate and severity of dysuria following SBRT is comparable to patients treated with other radiation modalities.

Rapid Prototyping of Nanofluidic Slits in a Silicone Bilayer.

This article reports a process for rapidly prototyping nanofluidic devices, particularly those comprising slits with microscale widths and nanoscale depths, in silicone. This process consists of designing a nanofluidic device, fabricating a photomask, fabricating a device mold in epoxy photoresist, molding a device in silicone, cutting and punching a molded silicone device, bonding a silicone device to a glass substrate, and filling the device with aqueous solution. By using a bilayer of hard and soft silicone, we have formed and filled nanofluidic slits with depths of less than 400 nm and aspect ratios of width to depth exceeding 250 without collapse of the slits. An important attribute of this article is that the description of this rapid prototyping process is very comprehensive, presenting context and details which are highly relevant to the rational implementation and reliable repetition of the process. Moreover, this process makes use of equipment commonly found in nanofabrication facilities and research laboratories, facilitating the broad adaptation and application of the process. Therefore, while this article specifically informs users of the Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST), we anticipate that this information will be generally useful for the nanofabrication and nanofluidics research communities at large, and particularly useful for neophyte nanofabricators and nanofluidicists.

Prostate specific antigen kinetics following robotic stereotactic body radiotherapy for localized prostate cancer.

BACKGROUND: Stereotactic body radiotherapy (SBRT) has emerged as an effective treatment for localized prostate cancer. However, prostate specific antigen (PSA) kinetics after prostate SBRT have not been well characterized. The purpose of this study was to analyze the trend in PSA decline following robotic SBRT from a prospective cohort of patients. MATERIAL AND METHODS: In total 175 patients were treated definitively for localized prostate cancer to a dose of 35-36.25 Gy in 5 fractions using robotic SBRT in the absence of androgen deprivation therapy (ADT). PSA and testosterone were collected at regular intervals following treatment and patients were assessed for biochemical failure and benign PSA bounce. A PSA nadir threshold of 0.5 ng/ml was used as a predictor of long-term disease-free survival. Multivariate logistic regression was used to assess the effect of disease specific covariates on the likelihood of achieving a PSA nadir less than threshold. PSA kinetics were analyzed a multi-component exponential model accounting for benign and malignant sources of PSA. RESULTS AND CONCLUSION: At a median follow-up of 3 years, 70% of patients achieved a PSA nadir below 0.5 ng/ml with a median PSA nadir of 0.3 ng/ml at a median time to nadir of 30 months. In our cohort, 36.2% experienced a benign PSA bounce. Absence of PSA bounce, initial PSA, and testosterone at the time of nadir proved to be significant predictors of achieving a PSA nadir below threshold. PSA kinetics after prostate SBRT were well described with a functional volume model with fitted half-lives of 4.4 and 14.8 months for malignant and benign sources of PSA, respectively. Patients treated with prostate SBRT experience an initial period of rapid PSA decline followed by a slow decline which will likely result in lower PSA nadirs after longer follow-up. The long-term disease specific impacts of these results remain to be determined.

A dosimetric comparison of ultra-hypofractionated passively scattered proton radiotherapy and stereotactic body radiotherapy (SBRT) in the definitive treatment of localized prostate cancer.

BACKGROUND: We compared target and normal tissue dosimetric indices between ultra-hypofractionated passively scattered proton radiotherapy and stereotactic body radiotherapy (SBRT) in the definitive treatment of localized prostate cancer. MATERIAL AND METHODS: Ten patients were treated definitively for localized prostate cancer with SBRT to a dose of 36.25 Gy in 5 fractions prescribed to a volume encompassing the prostate only. Dose-volume constraints were applied to the rectum, bladder, penile bulb, femoral heads, and prostatic and membranous urethra. Three-field passively scattered proton plans were retrospectively generated using target volumes from the same patients. Dosimetric indices were compared between the SBRT and proton plans using the Wilcoxon signed rank test. RESULTS: All dose constraints were achieved using both ultra-hypofractionated passively scattered proton and SBRT planning. Proton plans demonstrated significant improvement over SBRT in mean dose delivered to the penile bulb (5.2 CGE vs. 11.4 Gy; p=0.002), rectum (6.7 CGE vs. 10.6 Gy; p=0.002), and membranous urethra (32.2 CGE vs. 34.4 Gy; p=0.006) with improved target homogeneity resulting in a significant reduction in hot spots and volumes of tissue exposed to low doses of radiation. Compared to proton planning, SBRT planning resulted in significant improvement in target conformality with a mean index of 1.17 versus 1.72 (p=0.002), resulting in a dose reduction to the volume of bladder receiving more than 90% of the PD (V32.6, 7.5% vs. 15.9%; p=0.01) and mean dose to the left (7.1 Gy vs. 10.4 CGE; p=0.004) and right (4.0 Gy vs. 10.9 CGE; p=0.01) femoral heads. CONCLUSION: Target and normal tissue dose constraints for ultra-hypofractionated definitive radiotherapy of localized prostate cancer are readily achieved using both CK SBRT and passively scattered proton-based therapy suggesting feasibility of either modality.

Comparison of heart and coronary artery doses associated with intensity-modulated radiotherapy versus three-dimensional conformal radiotherapy for distal esophageal cancer.

PURPOSE: To compare heart and coronary artery radiation exposure using intensity-modulated radiotherapy (IMRT) vs. four-field three-dimensional conformal radiotherapy (3D-CRT) treatment plans for patients with distal esophageal cancer undergoing chemoradiation. METHODS AND MATERIALS: Nineteen patients with distal esophageal cancers treated with IMRT from March 2007 to May 2008 were identified. All patients were treated to 50.4 Gy with five-field IMRT plans. Theoretical 3D-CRT plans with four-field beam arrangements were generated. Dose-volume histograms of the planning target volume, heart, right coronary artery, left coronary artery, and other critical normal tissues were compared between the IMRT and 3D-CRT plans, and selected parameters were statistically evaluated using the Wilcoxon rank-sum test. RESULTS: Intensity-modulated radiotherapy treatment planning showed significant reduction (p < 0.05) in heart dose over 3D-CRT as assessed by average mean dose (22.9 vs. 28.2 Gy) and V30 (24.8% vs. 61.0%). There was also significant sparing of the right coronary artery (average mean dose, 23.8 Gy vs. 35.5 Gy), whereas the left coronary artery showed no significant improvement (mean dose, 11.2 Gy vs. 9.2 Gy), p = 0.11. There was no significant difference in percentage of total lung volume receiving at least 10, 15, or 20 Gy or in the mean lung dose between the planning methods. There were also no significant differences observed for the kidneys, liver, stomach, or spinal cord. Intensity-modulated radiotherapy achieved a significant improvement in target conformity as measured by the conformality index (ratio of total volume receiving 95% of prescription dose to planning target volume receiving 95% of prescription dose), with the mean conformality index reduced from 1.56 to 1.30 using IMRT. CONCLUSIONS: Treatment of patients with distal esophageal cancer using IMRT significantly decreases the exposure of the heart and right coronary artery when compared with 3D-CRT. Long-term studies are necessary to determine how this will impact on development of coronary artery disease and other cardiac complications.

Enhanced interaction between Hsp90 and raptor regulates mTOR signaling upon T cell activation.

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase which plays a role in integrating environmental cues. mTOR signals via two complexes: TORC1, which contains the Regulatory Associated Protein of TOR (raptor), and TORC2, which contains the Rapamycin-insensitive Companion of TOR (rictor). The immunosuppressive/anti-cancer agent rapamycin inhibits TORC1 function by disrupting the mTOR-raptor interaction. In an effort to understand the downstream consequences of TORC1 activation in T cells we performed a proteomic analysis of raptor binding proteins. Using this approach we have identified Hsp90 as an activation-induced binding partner of raptor in T cells. Pharmacologic inhibition of Hsp90 leads to a decrease in raptor expression and TORC1 activity. Furthermore, full T cell activation during Hsp90 blockade leads to T cell tolerance in the form of anergy. Overall, our findings suggest that Hsp90 inhibitors might represent a novel means of promoting T cell tolerance.

The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment.

Effector T cell differentiation requires the simultaneous integration of multiple, and sometimes opposing, cytokine signals. We demonstrated mTOR's role in dictating the outcome of T cell fate. mTOR-deficient T cells displayed normal activation and IL-2 production upon initial stimulation. However, such cells failed to differentiate into T helper 1 (Th1), Th2, or Th17 effector cells. The inability to differentiate was associated with decreased STAT transcription factor activation and failure to upregulate lineage-specific transcription factors. Under normally activating conditions, T cells lacking mTOR differentiated into Foxp3(+) regulatory T cells. This was associated with hyperactive Smad3 activation in the absence of exogenous TGF-beta. Surprisingly, T cells selectively deficient in TORC1 do not divert to a regulatory T cell pathway, implicating both TORC1 and TORC2 in preventing the generation of regulatory T cells. Overall, our studies suggest that mTOR kinase signaling regulates decisions between effector and regulatory T cell lineage commitment.

Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites.

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.

Probing cellular mechanical responses to stimuli using ballistic intracellular nanorheology.

We describe a new method to measure the local and global micromechanical properties of the cytoplasm of single living cells in their physiological milieu and subjected to external stimuli. By tracking spontaneous, Brownian movements of individual nanoparticles of diameter>or=100 nm distributed within the cell with high spatial and temporal resolutions, the local viscoelastic properties of the intracellular milieu can be measured in different locations within the cell. The amplitude and the time-dependence of the mean-squared displacement of each nanoparticle directly reflect the elasticity and the viscosity of the cytoplasm in the vicinity of the nanoparticle. In our previous versions of particle tracking, we delivered nanoparticles via microinjection, which limited the number of cells amenable to measurement, rendering our technique incompatible with high-throughput experiments. Here we introduce ballistic injection to effectively deliver a large number of nanoparticles to a large number of cells simultaneously. When coupled with multiple particle tracking, this new method-ballistic intracellular nanorheology (BIN)-makes it now possible to probe the viscoelastic properties of cells in high-throughput experiments, which require large quantities of injected cells for seeding in various conditions. For instance, BIN allows us to probe an ensemble of cells embedded deeply inside a three-dimensional extracellular matrix or as a monolayer of cells subjected to shear flows.

A role for mammalian target of rapamycin in regulating T cell activation versus anergy.

Whether TCR engagement leads to activation or tolerance is determined by the concomitant delivery of multiple accessory signals, cytokines, and environmental cues. In this study, we demonstrate that the mammalian target of rapamycin (mTOR) integrates these signals and determines the outcome of TCR engagement with regard to activation or anergy. In vitro, Ag recognition in the setting of mTOR activation leads to full immune responses, whereas recognition in the setting of mTOR inhibition results in anergy. Full T cell activation is associated with an increase in the phosphorylation of the downstream mTOR target S6 kinase 1 at Thr(421)/Ser(424) and an increase in the mTOR-dependent cell surface expression of transferrin receptor (CD71). Alternatively, the induction of anergy results in markedly less S6 kinase 1 Thr(421)/Ser(424) phosphorylation and CD71 surface expression. Likewise, the reversal of anergy is associated not with proliferation, but rather the specific activation of mTOR. Importantly, T cells engineered to express a rapamycin-resistant mTOR construct are resistant to anergy induction caused by rapamycin. In vivo, mTOR inhibition promotes T cell anergy under conditions that would normally induce priming. Furthermore, by examining CD71 surface expression, we are able to distinguish and differentially isolate anergic and activated T cells in vivo. Overall, our data suggest that by integrating environmental cues, mTOR plays a central role in determining the outcome of Ag recognition.

Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

Ballistic intracellular nanorheology reveals ROCK-hard cytoplasmic stiffening response to fluid flow.

Cells in vivo are constantly subjected to mechanical shear stresses that play important regulatory roles in various physiological and pathological processes. Cytoskeletal reorganizations that occur in response to shear flow have been studied extensively, but whether the cytoplasm of an adherent cell adapts its mechanical properties to respond to shear is largely unknown. Here we develop a new method where fluorescent nanoparticles are ballistically injected into the cells to probe, with high resolution, possible local viscoelastic changes in the cytoplasm of individual cells subjected to fluid flow. This new assay, ballistic intracellular nanorheology (BIN), reveals that shear flow induces a dramatic sustained 25-fold increase in cytoplasmic viscosity in serum-starved Swiss 3T3 fibroblasts. By contrast, cells stimulated with the actin contractile agonist LPA show highly transient stiffening of much lower amplitude, despite the formation of similar cytoskeletal structures. Shear-induced cytoplasmic stiffening is attenuated by inhibiting actomyosin interactions and is entirely eliminated by specific Rho-kinase (ROCK) inhibition. Together, these results show that biochemical and biophysical stimuli may elicit the formation of qualitatively similar cytoskeleton structures (i.e. stress fibers and focal adhesions), but induces quantitatively different micromechanical responses. Our results suggest that when an adherent cell is subjected to shear stresses, its first order of action is to prevent detachment from its substratum by greatly stiffening its cytoplasm through enhanced actin assembly and Rho-kinase mediated contractility.