• Ludwig Institute For Cancer Research
• Anna Cvrljevdic

ELECTRONICS -Page 2 PAGE 2 INDEX: UPDATED!!! Search 101science.com pages: THEN: VERY IMPORTANT!! Use you browser's 'Find on this page' capability to search for words on a page.

Usually Ctrl + F will work. Knowing about good electronic shop practices begins with introduction to the basic tools and test instruments used in electronic repair, production and troubleshooting. It continues with 'hands-on' activity directed towards learning practical skills such as soldering and de-soldering and making connecting leads and cables. Also knowing how to make printed circuit boards and enclosures is important. As well as, component replacement techniques, testing and troubleshooting. We start below with learning how to solder, an everyday skill needed by anyone in electronics. Invest in a good bench-top or handheld multimeter.

Look for one with an auto-ranging feature. This feature allows the meter to automatically adjust to the proper range for testing. Measurements are a little slower than making the changes by hand but offer protection from incorrect switch settings and ease of use. This is particularly true of resistance reading where you may have no idea of where to start. Expect to pay $50-$150 for a new accurate auto-ranging meter. The second item of importance is an oscilloscope.

I feel this is the most important piece of test equipment in the shop. ( It should have an input bandwidth of at least 50 MHz or higher and a dual trace is recommended. A delayed horizontal trigger is also desired to allow viewing of short duration voltage changes. You should be able to find a good 100 MHz oscilloscope for around $100 at a local hamfest. A frequency counter of at least 100 MHz with an input sensitivity of around 25 Milivolts is the next item needed.

A variety of signal generators, transistor test sets, and capacitor test sets will round out your basic test equipment bench. Try to obtain name brand test equipment. Your initial investment will save you money in the long run as you will not be satisfied long with inferior test equipment.

See a listing of web links to test equipment sources. Check out the Build your own test equipment and much more. Commercial Test Equipment Dealer - URL: Title:- Electronic Test Equipment Sales, Rentals, Leasing and Service Description: Find Calright, Calright instruments, used test equipment, used electronic test equipment, refurbished test equipment and other pre-owned electronic test and measurement equipment to buy or rent. Step one is to have the proper soldering iron, solder and technique.

The soldering iron's tip size and wattage should match the size of the job. If you are working on printed circuit boards a 25 watt pencil sized iron is appropriate with tips 1/8th inch or smaller. Cordless irons work well here and may be less likely to induce unwanted voltages. Bench-top thermostatically controlled irons can be ideal for most work. The temperature of the iron should be adjusted just high enough to heat the part being solder to just above the melting point of the solder.

This is usually around 360 degrees F. The tip temperature will need to be somewhere around 460 to heat the part sufficiently and melt the solder.

You must also consider the problems of static discharges with any iron if you are working on circuits with installed sensitive integrated circuits or field effect transistors. You may also need to use a heat sink to protect installed parts from excessive heat.

Alligator clips or heat sink clamps will help. The main purpose of the iron is to heat the part being soldered to just high enough temperature to melt the solder. The solder (60/40 tin/lead of.025 to.040 inches in diameter) is place on the part being soldered not the iron. Although briefly touching the solder to the iron tip prior to the actual soldering can be helpful in transferring the irons heat to the part. Avoid touching the solder to the iron when soldering; touch the solder to the part.

This is insurance against a cold solder joint which is to be avoided. A cold solder joint is one where the part was not hot enough to melt the solder and results in a poor connection. Normally the use of a rosin core solder is sufficient for most soldering jobs.

Occasionally for larger work adding a separate soldering rosin paste may facilitate a better job. For larger work a handheld soldering gun is appropriate. Even larger work such as copper water pipes will require the use of a propane torch. For copper water pipes be sure the solder contains no lead. The larger the job the larger diameter solders are appropriate. Regardless of the size of the project the basic principles are the same.

The finished soldering job should be smooth and shiny, not dull. On new printed circuit boards it is often helpful to apply a tin coating to the copper runs to insure all the connections are of low resistance and avoid later problems due to corrosion. by Alan Winstanley - From Elecraft - Click on 'Builder Resources' - Kit Building Too! - From Mike WT9W - From EPE Online - From W8WWV - From K6MHE - From SolderIt - From IZ7ATH, Talino Tribuzio Soldering Guide Test anything with a scope by Homer L. Davidson Every shop needs a well regulated variable voltage power supply for bench top work. When building and testing new circuits a power supply is essential.

Here is a diagram for a LM317T Variable Voltage power supply - (Note: This is a link to one of Bill Bowden's wonderful pages.) Try this Electronic Calculator site made available by John Jenkens Also try Radio Calculators Test anything with a scope by Homer L. Davidson Every shop needs a well regulated variable voltage power supply for bench top work. When building and testing new circuits a power supply is essential. Here is a diagram for a LM317T Variable Voltage power supply - (Note: This is a link to one of Bill Bowden's wonderful pages.) Try this Electronic Calculator site made available by John Jenkens Also try Radio Calculators ELECTRONIC WORKBENCH While you can get by with a make-shift workbench made out of anything handy like a few boxes or boards but a real workbench is a delight.

Some use a door laid flat across some saw horses as a start. Building a real sturdy workbench out of two by four lumber is better.

A nice long electrical outlet strip is also most a must. You can see what I mean in the picture at the left. The outlet strip is just below the row of colored plastic bins. A shelf on your workbench to hold your test equipment is also nice.

Also, notice the light above the workbench - also a must. You can also add swinging lamps as well. If you can afford it you can purchase a nice workbench like in the picture already built. A good sturdy stool or chair is a must as well.

Lots of additional shelving and storage boxes will help you sort out parts you may have and keep the labeled for easy location when you need them. Try to keep your workbench free after you complete a job or project. Otherwise you will end up with no bench top space left to start new projects. That is why additional shelf and storage is a must.

If you follow these rules you will have a nice looking as well as an efficient place to work. Electrostatic protection of delicate high speed IC's should be considered also when constructing your work space. Carpet on the floor is a no-no because of static electricity it can generate. Also add a waste basket and a metal storage bin or cabinet of some sort to store chemicals.

You will collect control cleaner, glues and various other chemicals that need proper and safe storage A separate workbench could be added for drilling and other metal working. A nice vise, grinding wheels, and drill press are necessary at times for building metal enclosure boxes. Enjoy your new workbench and work space! HOW TO MAKE PRINTED CIRCUIT BOARDS - LINKS If you build your own electronic equipment, this is 'THE' book you need! Carr Also check out these great RF (Radio Frequency) books.; Chris Bowick, Christopher Bowick.; Irving M. Gottlieb.; Cotter W. Sayre.; Norm Dye, et al.

Television means 'seeing at a distance'. It may be described as a system for the conversion of light rays from still or moving scenes and pictures into electric signals for transmission or storage, and subsequent reconversion into visual images on a screen. Basically the image formed by the camera lens is focused on a light sensitive material that is scanned in horizontal lines with each line following closely beneath it.

The light intensity (and color) is converted into an electrical signal and transmitted over the air or through cables to a receiver. The 'TV' receiver converts the electrical signals back into scan lines traced on a cathode ray tube (CRT). The fluorescent material on the face of the CRT is activated by the CRT's scanning electron beam to re-form the picture.

NTSC signal information: Free television and video articles and guides Click to view a 'large list' of links related to television and video. FORMULAS: I = V/R V = I.R R = V/I P = V x I I = P/V V= P/I P = V^2/R R = V^2/P Series resistance formula: Rt = R1 + R2 + R3 + R., etc. Total power in a series circuit: Pt = P1 + P2 + P3 + P. Resistance's in parallel: 1/Rt = 1/R1 + 1/R2 + 1/R3 + 1/R., etc. Kirchhoff's Law - The algebraic sum of the currents into any point of the circuit must equal the algebraic sum of the currents out of that point.

Time vs frequency: Time T = 1/f or f = 1/T Wavelength = velocity/frequency Inductance in series or parallel: same formula as resistance's with L substituted for R. Series capacitance: use formula for parallel resistance and substitute C for R.

Parallel capacitance: use formula for series resistance and substitute C for R. ELECTRONIC SYMBOLS - Electronic schematics use a number of standard of symbols to display an electronic circuit on paper. These schematics are useful in troubleshooting unfamiliar circuits. To see some examples of electronic schematic symbols click ELECTRICAL ENGINEERING HAND HELD CALCULATOR - Electrical Engineering functionality in an electronic software upgrade for the Texas Instruments TI-89 and TI-92 Plus is available. Details click; Unit Conversions -Go to the 101science.com.

The Fourier Transform by Bartosz Milewski The Physics of Sound. Frequency Analyzer Whistle a melody and watch this program graph the pitch in real time. The. Definition DSP use of FFT Understanding Discrete Fourier Transforms MUST HAVE! TI-83 Plus BETTER! Voyage 200 The WWW links on this page will take you directly to the various web site pages.

Your browser URL address line will tell you the origin of the site. Tools - Links A must have book! CIRCUIT DESIGN ELECTRONICS SOFTWARE HOW TO USE SPICE AND WHY - PDF Note: SPICE is the basic program written may years ago and revised many times. Electronic Workbench a relatively expensive graphical program uses SPICE as the basic engine. Old SPICE is not all that hard to use and is very reliable.

(New Version) DUNCAN CIRCUIT DESIGN Free Down-loadable book list follows: Electronic Amplifier Circuits, Joseph Petit and Malcolm McWhorter, 1961, 325 pages This textbook is all about the theory and design of amplifiers. While mostly containing tube circuits it does include some information on transistor. Plenty of math in this one, it concentrates on wide band (e.g., video) amplifiers. 12 meg pdf format perfect copy for printing. Un-renewed copyright Theory and Applications OF Electron tubes. Army/air force 1952 From basic theory through all types of circuits. Audio, rf, osc etc.

229 pages, perfect for printing. 17 meg Handbook of Analog Computations. All about analog computers and how they work. Collage level book. 401 pages size 24 meg, unrenewed copyright Donated.

CP/M8000 by Digital Research. A set of 3 books programmers guide, system guide, users guide.

Total 510 pages. 23 meg, Donated United States Navy Electronic Training Course. Full course NOT scanned, made for cd by the navy. 24 modules 4100 pages. For full details and download Radio Laboratory Hand book.

Year 2004 in the public domain. All about cables, transmission lines and antennas.

133 pages not scanned direct pdf. Size 60 meg U. Navy Manual on Grounding.

Deep into grounding theory for your radio or test lab. Learn how to truly protect yourself and your equipment. This manual will teach you how. 814 pages searchable pdf format.

Size 10 meg Amateur Radio Handbook 1959 This was a bigger seller then the ARRL Handbook. Full 810 pages in pdf format size 60 meg unrenewed copyright Crystal Sets to Side Band by Frank W. Harris K01YE This is not scanned it is made for pdf. FULL color 372 pages. Copyright 2002 NOT to be used else where without written permission of K01YE size 4 meg Trouble Shooting Tektronix scopes. Written by Tektronix it is for their technicians to train repairing their scopes Donated 97 pages size 4 meg Navy Warfare and Radar Systems Handbook.

Contains a wealth of knowledge on electronics for radio, IEE buss's radar, microwaves, antennas etc for complete list and download Engineering Software (DOS) Links Links to Ward College of Technology of the University of Hartford - maintained by shows Fourier series graphically -Rotating phasors become V vs. Time graphs - Does calculations for RF systems - Simplied Nodal Analysis Program - simple, DC only - Transmission Line Analysis Program - shows phasors of V and I, with ANY load impedance - Does stub and L,C matching of loads on transmission lines - MORE Training software system. ELECTRONICS TRAINING SYSTEM UPDATED FILES. shows Fourier series graphically. collection of useful RF and ham radio software modules. Rotating phasors become V vs. Time graphs.

Smith Chart Analysis/Design. Does calculations for RF systems. Smith Chart Analysis/Design. Simplied Nodal Analysis Program - simple, DC only.

Transmission Line Analysis Program - shows phasors of V and I, with ANY load impedance. Simply wonderful, for Windows, for learning about transmission line circuits.

Does stub and L,C matching of loads on transmission lines. linear simulator from the UK. RFSim99 is a free linear S-parameter based circuit simulator offering schematic capture, simulation, 1 port and 2 port S-parameter display and file support, tolerance analysis, stability circles, and much more. Requires Windows 95, 98, NT or 2000. File size 2045KB. linear simulator from Denmark. This free linear circuit simulator is perfect for the rapid simulation of circuits containing resistors, capacitors, inductors, op amps, transformers and voltage and current sources.

Requires Windows 3.x, 95, 98, NT or 2000. File size 249KB. Test your electronics skills for the Federal Aviation Administration Basic Electronics Screening Test. RFSim99 Demo Become a better ham operator with these three programs. OS: Windows 95/98/NT Free Practice common electronics design with this specialized worksheet. OS: Windows 95/98/NT Shareware Download the Windows 95 drivers for the GoldStar monitor.

OS: Windows 95 Free Simulate digital circuits. OS: Windows 95/98/NT Shareware Use this complete electronics design system. OS: Windows 95/98/NT/2000 Shareware Convert dBM and dBV to RMS or Peak Voltages. OS: Windows 98/NT Free Learn or teach electronics.

OS: Windows 95/98/NT Shareware Use a VHDL-aware text editor. OS: Windows 95/NT Free Design and simulate digital electronic circuitry. OS: Windows 95/98/NT Demo Create professional circuit diagrams with this program. OS: Windows 95/98/NT Shareware Simulate a single-channel cathode ray oscilloscope. OS: Windows 3.x/95 Demo Keep track of users' bookmarks on a network. OS: Windows 95/98/NT Shareware Download the Delta CD-ROM Device Driver for Windows 95/98/NT.

OS: Windows 95/98/NT Free Decode the colored stripes on resistors in electronic components. OS: Windows 95/98/NT/2000 Free Use this tool to compare products while you are shopping online. OS: Windows 95/98/NT/2000 Free Obtain an operator's license for amateur radio. OS: Windows 95/98/NT/2000 Shareware Calculate values for electronics with this program. OS: Windows 95/98/Me (Note: NTE requires a registration form but the download is free.) (Tuned circuit analysis.) A simple ASCII schematic drawing program. Transceiving, single-turn, loop aerials of various regular shapes. Transceiving, single-turn, loop aerials of rectangular shape.

Receiving, multi-turn, square, loop aerials, ELF to HF. Design of single-layer solenoid Rx & Tx tuning & antenna loading coils. Centre-fed dipole analysis. Vary freq and feedler length for optimum match.

Helically-wound antenna performance, top-C loaded with rod or wire. Ferrite & iron-dust cored, toroidal coils. Size, Mu, Turns, uH, pF, Freq. Base-fed vertical antenna performance, coil-loaded at any height. DC & RF characteristics of a shallow-buried radial earth wire. VLF to HF Groundwave Propagation. Path loss over various terrains.

Earth Electrodes and Measurement of Soil Resistance. Performance on Long Waves of T Antennas above ground radials system. Performance of Inverted-L Antennas above system of ground radials.

Ludwig Institute For Cancer Research

Models Class-AB, Push-pull, Bipolar Linear RF amplifiers up to 30 MHz. Models HF transmission line transformers, impedance step-up ratio 4-to-1.

Analysis of openwire lines, 50Hz-1GHz, for any complex termination. Full analysis of balanced lines, 20Hz-1GHz, for any complex termination.

Full analysis of coaxial lines, 50Hz-1GHz, for any complex termination. Full analysis of balanced lines with facilities for line xfmr design. Design of 2-band trapped dipole antennas including trap details. Design of T & Pi phase-shifting networks for use with antenna arrays. Design of T & Pi impedance-matching and phase-shifting networks.

Simple & folded dipoles. Feedline VSWR versus freq. Enter soil characteristics. Display a table of skin depth vs frequency. Find safe input power for given cable dimensions and SWR. 0.3-3000 MHz. Compute load impedance from measured line input impedance, Zo & length.

Computer Assisted Circuit Design Analysis I nstruction Simulation simulation of digital circuits. Windows 3.1+. RF & Microwave circuit simulation package. Windows 3.1+ - Electronic circuit timing diagram generator. Windows 3.0+. This software can be used to create 3D circuit boards.

Windows 3.1+ Printed Circuit Board Design Misc. Computer Assisted color code interpreter.

Data for selecting the most popular 3 pin IC regulators. Power Factor Correction Program. Windows 3.1+ Resistor selection Links to Other Electronics Software Sites Learn how to identify various electronic components. OS: Windows (all) File Size: 2.47MB License: Free to try, $30 to buy 1,395 pop Learn about electronics, mechanics, and computing. OS: Windows (all) File Size: 6.48MB License: Free to try, $24 to buy 220 pop Implement complex electronic PCB designs on your PC. OS: Windows (all) File Size: 24.2MB License: Free, $45 to buy 17,662 pop Create professional circuit diagrams with this program.

OS: Windows 95/98/Me/2000/XP File Size: 2.61MB License: Free to try, $40 to buy 14,239 Convert dBM and dBV to RMS or Peak Voltages. OS: Windows 98/NT File Size: 192K License: Free 1,155 pop Enter the colors of a circuit's resistor and get the value of resistance. OS: Windows 95/98/NT/2000/XP File Size: 373K License: Free 408 pop Find out the value of your resistors. OS: Windows 95/98/Me/XP File Size: 419K License: Free 375 Build your own parallel port hardware. OS: Windows 95/98/Me/2000/XP File Size: 1.44MB License: Free to try, $27 to buy 295 pop Test your electronics resistor. OS: DOS File Size: 22.6K License: Free 282 pop Perform circuit and networks calculations, SWR and reactance conversions, and more. OS: Windows 98/Me/NT/2000/XP File Size: 1.17MB License: Free to try, $20 to buy 151 pop Find the nearest preferred resistor values within a given tolerance.

OS: Windows 95/98/NT/2000/XP File Size: 35K License: Free to try, $20 to buy 74.

Human sarcomas with a poor response to vascular endothelial growth factor-A (VEGF-A) inhibition and radiation therapy (RT) have upregulation of hypoxia-inducible factor 1α (HIF-1α) and HIF-1α target genes. This study examines the addition of the hypoxia-activated chemotherapy TH-302 to VEGF-A inhibition and RT (a.k.a. Trimodality therapy).Trimodality therapy was examined in two xenograft models and in vitro in tumour endothelial cells and sarcoma cell lines.In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy.

Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1α activity were reduced to 11-13% and 13-20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia.The combination of TH-302, VEGF-A inhibition, and RT is highly effective in preclinical models of sarcoma and is associated with increased DNA damage and apoptosis in endothelial cells and decreased HIF-1α activity. Results: In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy. Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1 α activity were reduced to 11–13% and 13–20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia.

Sarcomas account for over 20% of all pediatric solid malignancies and. Cell lines and reagents HT1080 human fibrosarcoma cells and SK-LMS-1 human leiomyosarcoma cells were obtained from the America Type Culture Collection (ATCC, Manassa, VA, USA).

MS4515 and MS5907 mouse undifferentiated pleomorphic sarcoma cell lines were derived from genetically engineered mouse models, which we have described previously. All sarcoma cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U ml −1 penicillin, 100 μg ml −1 streptomycin, and L-glutamine 2 m M. Human cancer cell lines were actively passaged for. Mouse studies All mouse protocols were approved by Institutional Animal Care and Use Committee. To generate subcutaneous flank tumour, 1 × 10 6 HT1080 or SK-LMS-1 cells were resuspended in 100 μl of Hank's balanced salt solution and injected subcutaneously into the right flank of athymic, nude, 6- to 8-week-old male BALB/c nu/nu mice following isoflurane anaesthesia.

Mice were assigned into treatment groups (5–6 mice per group) when tumours reached 50–100 mm 3 in volume, designated as day 0. DC101 (20 mg kg −1) or isotype control IgG 1s (20 mg kg −1) was injected intraperitoneally three times a week. TH-302 50 mg kg −1 was delivered by intraperitoneal injection 5 days per week. For tumours that were irradiated, radiation was delivered on day 0. Mice were anaesthetised using ketamine (125 mg kg −1) and xylazine (10 mg kg −1), placed in shielded device to expose only the flank tumour, and irradiated using a Gammacell 40 Exactor Irradiator (Best Theratronics, Ottawa, ON, Canada). When mice were treated with combination therapies, DC101 or control IgG was delivered first and TH-302 and/or radiation were delivered within 2 h of DC101 administration. For trimodality therapy, DC101 was administered followed by radiation, and then by TH-302.

Tumour volume (TV) was calculated by using the following formula: TV=length × (width) 2 × 0.52. Immunohistochemistry and immunofluorescence Mice were killed and tumours were harvested immediately and placed into 10% formalin for 2–3 days at 4 °C. Tumours were then rinsed and stored in saline and sent to our pathology core facility. Formalin-fixed, paraffin-embedded sections were deparaffinised by xylene and rehydrated. Immunohistochemistry was performed with Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) as per the manufacturer's protocol. For antigen retrieval, the sections were placed in citrate buffer (pH 6.0) and heated in a microwave oven for 10 min. For immunoperoxidase labelling, endogenous peroxidase was blocked by 0.3% H 2O 2 in absolute methanol for 15 min at room temperature.

The sections were then incubated overnight at 4 °C with primary antibody and washed with PBS containing 0.05% Triton X-100. Incubation with corresponding secondary antibody and the peroxidase–antiperoxidase complex were carried out for 30 min at room temperature. Immunoreactive sites were visualised by 3,3′-DAB. Later, the slices were counterstained by haematoxylin. Antibodies used were anti-TUNEL (ApoptoTag Peroxidase Kit; EMD Millipore, Temecula, CA, USA), anti-HIF-1 α (Ab-4; Novus Biologicals, Littleton, CO, USA), anti-CA9 (NB100-417; Novus), and anti-PCNA (sc-56; Santa Cruz Biotechnology, Dallas, TX, USA) CD31 immunohistochemical localisation and analysis of microvessel density were performed as described previously.

For detection of EC apoptosis, TUNEL and CD31 immunofluorescence was performed as described previously. Four tumours from each treatment group were analysed for total apoptosis, endothelial cell-specific apoptosis, microvessels density, HIF-1 α expression, and CA9 expression. Hypoxia in tumours was measured using the Hypoxyprobe-1 Kit (HPI, Burlington, MA, USA) as per the manufacturer's instructions. For examination of cells for γH2AX, CD31 and cleaved caspase-3, cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% Triton X-100 in PBS. Cells were incubated with the appropriate primary antibody in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4 °C overnight. Antibodies used were as follows: human anti- γH2AX (mouse polyclonal antibody, 1: 100, 05-636; EMD Millipore, Billerica, MA, USA), anti-cleaved caspase-3 (rabbit polyclonal antibody, 1: 100, no. 9661; Cell Signalling Technology, Beverly, MA, USA), and anti-CD31 (rat monoclonal antibody, 1: 00, DIA-310; Dianova, Hamburg, Germany).

Cells were then stained with anti-rat Alexa Flour 488, anti-rabbit Alexa Flour 594, and anti-mouse Alexa Flour 647 (Life Technologies, Norwalk, CT, USA). After 2 h, nuclei were counterstained using DAPI (Sigma-Aldrich). Stained cells were visualised with an inverted confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA). Image processing was performed using the Imaris 7.6 software (Bitplane, Zurich, Switzerland). Tumour endothelial cell isolation Tumour endothelial cell were harvested from HT1080 xenografts. When tumours reached 100–150 mm 3 in size, they were removed, minced, and digested in 25 ml of prewarmed collagenase I (2 mg ml −1; EMD Millipore) in PBS for 45 min at 37 °C. Cells were strained through a 100 μ M strainer, and collagenase activity was quenched with 10 ml of isolation medium (DMEM (high glucose 4500 mg l −1)+20% FCS+Pen/Strep).

Cells were resuspended in 5 ml of cold sterile PBS with 0.1% BSA, followed by centrifugation at 400 g for 8 min. The cloudy interface containing endothelial cells was removed and washed with sterile PBS containing 0.5% BSA.

Cells were then resuspended in 400 ml PBS containing 0.5% BSA. A measure of 2.5 μl of 1 μg ml anti-mouse CD31 (ab28364; Abcam, Cambridge, MA, USA) was added to the cell suspension and incubated for 20 min at room temperature. After using Dynal beads coated with CD31 antibody (110.35; Life Technologies), the cells were plated into a gelatin-coated T-75 flask. After 1–2 weeks (or if the attached cell clones are observed in dish), cells were used for experiments between passages 2 and 6 as described previously.

Proliferation and colony formation assays To assay for proliferation, 3000 cells were plated onto 24-well plates and maintained in media overnight. A colorimetric MTT assay was used to assess cell number by optical density after 3 days as described previously. Data reflect the mean of six samples. To assay for colony forming ability, 200–500 cells were plated onto 60 mm culture dishes and incubated for 7–14 days.

Colonies were stained with 0.5% crystal violet in 6% glutaraldehyde solution. The number of colonies consisting of 50 or more cells was scored. Data reflect the mean of nine samples. Hypoxia was created by placing cells into Heracell 150i Tri-Gas Incubator (Thermo Scientific, Waltham, MA, USA) with 1% oxygen, 94% nitrogen, and 5% CO 2. Each experiment was performed at least three times.

Western blot analysis For western blot analysis of HIF-1 α, cells were incubated in 21% oxygen or 1% oxygen for 24 h. Samples were collected in RIPA buffer (Sigma-Aldrich) containing Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN, USA), and protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed for HIF-1 α using the following antibodies: HIF-1 α (C-Term) Polyclonal Antibody (10006421; Cayman Chemical, Ann Arbor, MI, USA), anti-CD31 (rat monoclonal antibody, DIA-310; Dianova) and β-actin (A5441; Sigma-Aldrich).

Trimodality therapy with hypoxia-activated chemotherapy, VEGF-A inhibition, and radiation We hypothesised that directing cytotoxic chemotherapy to hypoxic regions within sarcomas would increase the efficacy of VEGF-A inhibition and radiation. We thus examined TH-302, DC101, and radiation (aka trimodality therapy) in an HT1080 fibrosarcoma xenograft model.

When tumours reached 50–100 mm 3, mice were randomised to eight treatment groups. After 12 days of treatment, trimodality therapy inhibited tumour growth by 91%, which was significantly better than single modality therapies (15–33%) and bimodality therapies (49–65%). Mice treated with trimodality therapy did not appear ill and did not lose weight compared with control mice.

Treatment of xenografts with trimodality therapy was stopped after 12 days, and tumours were observed. Even after 120 days, tumours treated with trimodality therapy were only an average of 125 mm 3±s.d. Trimodality therapy in HT1080 xenografts. ( A) Growth of HT1080 fibrosarcoma xenografts in athymic nude mice.

Groups were treated with control IgG 20 mg kg −1 three times per week, DC101 20 mg kg −1 three times. After the 12 days of treatment, tumours from each treatment group were harvested and analysed by immunohistochemistry. Representative H&E sections of tumours from the eight treatment groups are shown in. Tumours treated with trimodality therapy had significantly less cellularity and more necrosis compared with control tumours. When tumours were examined for overall apoptosis using TUNEL staining, TH-302 alone resulted in very little total apoptosis (4 cells per 5 fields).

TH-302 in combination with radiation or DC101 resulted in a modest induction of total apoptosis (15 cells per 5 fields). Trimodality therapy resulted in significantly more apoptosis (36 cells per 5 fields) compared with TH-302 in combination with radiation or DC101, but trimodality therapy did not cause more apoptosis than bimodality therapy with DC101 and radiation (32 cells per 5 fields). When tumour cells were examined for proliferation using PCNA staining, trimodality therapy led to a 30% reduction in the number of proliferating tumour cells, while bimodality therapies reduced proliferation by 12–18%. Thus, there did not appear to be synergistic effects with trimodality therapy on overall apoptosis or proliferation.

Given prior studies suggesting that VEGF-A inhibition and radiation have effects on tumour vasculature and hypoxia , we next examined microvessel density, endothelial cell-specific apoptosis, and HIF-1 α activity in treated HT1080 tumours. Trimodality therapy led to an 89% decrease in microvessel density compared with the control tumours and a 3.3-fold increase in endothelial cell-specific apoptosis compared with the next best bimodality therapy. Levels of nuclear HIF-1 α expression and cytoplasmic CA9 expression, as a measure of HIF-1 α target gene activation, were the lowest in tumours treated with trimodality therapy. Thus, trimodality therapy may block growth of HT1080 xenografts as least in part through induction of apoptosis in tumour endothelium and selective ablation of hypoxic cells. To determine if trimodality therapy would be effective against larger tumours, we again treated HT1080 xenografts with trimodality therapy, but this time waited to initiate therapy until tumours were about 400 mm 3 in size.

Mice were then randomised to treatment with vehicle alone or with trimodality therapy. After 2 weeks of treatment, tumours treated with trimodality therapy decreased to an average size of 273 mm 3, whereas control tumours grew to an average size of 1209 mm 3. The mean tumour weight of control mice was 545 mg and the mean tumour weight of treated mice was 83 mg. Mice were weighed every 2 days during the study, and there was no difference in body weight between control and treated mice.

At the end of the treatment period, mice were killed and tumours and blood samples were collected. There was no significant difference in the levels of hemoglobin, white blood cells or platelets between control mice and mice treated with trimodality therapy. Trimodality therapy has synergistic effects on tumour endothelial cells Given the significant effects of trimodality therapy on tumour vasculature and HIF-1 α activity, we next wanted to examine trimodality therapy on tumour endothelial cells in vitro.

We thus isolated endothelial cells from HT1080 xenografts using magnetic bead separation with CD31 antibody and confirmed that these cells upregulated HIF-1 α under hypoxia. In a proliferation assay, tumour endothelial cells tolerated hypoxia well with proliferation in hypoxia decreasing by only 15% compared with proliferation in normoxia.

Trimodality therapy reduced proliferation under normoxia and hypoxia by 64% and 87%, respectively. In a colony formation assay, the addition of TH-302 and withdrawal of VEGF-A had a minimal effect when added to radiation under normoxic conditions. In contrast under hypoxia, tumour endothelial cells decreased colony formation by 57% with 6 Gy of radiation alone and by 94% with trimodality therapy. We performed these same studies on HUVECs.

In contrast to tumour endothelial cells, HUVECs decrease proliferation by 58% when grown in hypoxic conditions. Despite this, we found similar effects of trimodality therapy on HUVEC proliferation and colony formation ( and ). Trimodality therapy in tumour endothelial cells. ( A) CD31 western blot of tumour cells following separation by CD31 magnetic beads. ( B) Western blot of isolated tumour endothelial cells in normoxia and hypoxia. Proliferation after 3 days ( C) and colony.

To determine if HT1080 xenografts possess areas of hypoxia adjacent to tumour blood vessels, we grew HT1080 xenografts to around 400 mm 3 and then treated with either vehicle alone or trimodality therapy for 15 days. At the end of the treatment period, mice were injected with hypoxyprobe and killed. Immunofluorescence on tumour sections for hypoxyprobe and for CD31 showed areas of hypoxia adjacent to CD31-positive tumour blood vessels in both control tumours and in tumours treated with trimodality therapy. The absolute amounts of hypoxia and CD31-positive vessels were significantly less in tumours treated with trimodality therapy.

Anna Cvrljevdic

DNA damage and apoptosis in tumour endothelial cells We next measured DNA damage in tumour endothelial cells by examining the levels of γH2AX expression and also by using Comet assay. ΓH2AX levels increase in response to DNA double-strand breaks.

ΓH2AX expression in tumour endothelial cells after trimodality therapy was 3.0- to 10.7-fold greater than that after any bimodality therapy when treatment was given under hypoxia. The effects under normoxia were not nearly as pronounced. In the Comet assay, mean tail moment for trimodality treatment under hypoxic conditions was 3.1- to 5.7-fold greater than any bimodality therapy. We then determined if DNA damage led to apoptosis by measuring expression of cleaved caspase-3. In hypoxia, cleaved caspase-3 expression in tumour endothelial cells was 2.3- to 4.8-fold greater following trimodality therapy compared with bimodality therapies.

The effects on apoptosis were much less pronounced under normoxic conditions. These same studies were performed using HUVECs, which also upregulate HIF-1 α under hypoxia , and we found similar results for γH2AX expression and cleaved caspase-3 expression. Cancer cell autonomous effects We next evaluated the cancer cell autonomous effects of trimodality therapy on four sarcoma cell lines in vitro. Vascular endothelial growth factor-A addition or withdrawal had no effect on proliferation or colony formation of sarcoma cell lines (data not shown), and thus experiments on these cell lines were conducted without VEGF-A.

All four sarcoma cell lines upregulated HIF-1 α under hypoxia. We found that combining TH-302 and radiation had a modest effect in blocking proliferation in both normoxia and hypoxia. We also examined colony formation in these four sarcoma cell lines in response to TH-302 and radiation under both normoxic and hypoxic conditions and again found only minimal increase with combination therapy ( and ). The combination of TH-302 and radiation did result in increases in DNA damage as measured by γH2AX expression ( and ) and apoptosis as measured by cleaved caspase-3 expression ( and ). However, these effects were not nearly as pronounced as the effects seen in tumour endothelial cells.

Trimodality therapy in SK-LMS-1 leiomyosarcoma xenografts To confirm the effects of trimodality therapy in a different type of sarcoma, we performed another mouse xenograft study using SK-LMS-1 leiomyosarcoma cells. Mice were again randomised to eight treatment groups after tumours reached 50–100 mm 3. Following 15 days of treatment, average tumour size and weight for the trimodality therapy group was 98 mm 3 and 79 mg, respectively, compared with control tumours that were 774 mm 3 and 567 mg, respectively. When tumours were harvested and analysed, trimodality therapy was found to have more than additive effects on microvessel density, endothelial cell-specific apoptosis, microvessel density, HIF-1 α nuclear expression, and VEGF-A expression.

Trimodality therapy in SK-LMS-1 xenografts. ( A) Growth of SK-LMS-1 leiomyosarcoma xenografts in athymic nude mice. Groups were treated with control immunoglobulin G (IgG) 20 mg kg −1 three times per week, DC101 20 mg kg. Multicolor immunofluorescence for γH2AX, cleaved caspase-3, and CD31 was performed on treated SK-LMS-1 tumours to quantify the number of endothelial cells and non-endothelial cells exhibiting DNA damage and apoptosis.

Trimodality therapy led to marked increases in both endothelial cells and non-endothelial tumour cells demonstrating both γH2AX and cleaved caspase-3 expression. The absolute number of CD31(+) cells per 5 visual fields demonstrating γH2AX and cleaved caspase-3 expression was 13.2 after trimodality therapy compared with 2.8–3.4 after bimodality therapies. Interestingly, CD31(−) cells also showed more than additive increases in γH2AX(−) and cleaved caspase-3+ cells (9.2 vs 3.0–4.0 cells per 5 fields). Given our in vitro studies in sarcoma cell lines showed at most additive effects of trimodality therapy on γH2AX and cleaved caspase-3 expression, the results of our SK-LMS-1 tumour analysis suggest that the efficacy of trimodality therapy may depend on in vivo interaction of the endothelial cell compartment and tumour cell compartment. We found similar results when we performed this multicolor immunofluorescence analysis on HT1080 tumours treated with trimodality therapy. The absolute number of CD31(+) cells per 5 visual fields demonstrating γH2AX and cleaved caspase-3 expression was 12.4 after trimodality therapy compared with 3.2–4.2 after bimodality therapies.

CD31(−) had 10.2 cells per 5 fields with both γH2AX(−) and cleaved caspase-3(+) after trimodality therapy and 4.4–5.0 cells per 5 fields after bimodality therapies. Discussion In this study, we examined the addition of TH-302, a hypoxia-activated chemotherapy, to VEGF-A inhibition and radiation. We found that this trimodality therapy blocked growth of HT1080 and SK-LMS-1 sarcoma xenografts significantly better than VEGF-A inhibition and radiation, and analysis of tumour samples and in vitro studies revealed that one primary mechanism of action was the induction of endothelial cell DNA damage and apoptosis resulting in loss of tumour vasculature. This induction of DNA damage and apoptosis was also seen in HT1080 and SK-LMS-1 cancer cells but to a lesser extent. As noted earlier, TH-302 is a 2-nitroimidazole-linked prodrug of Br-IPM. The 2-nitroimidazole moiety of TH-302 is a substrate for intracellular 1-electron reductases and, when reduced in deeply hypoxic conditions, releases Br-IPM.

In vitro cytotoxicity and clonogenic assays using human cancer cell lines show that TH-302 has little cytotoxic activity under normoxic conditions and greatly enhanced cytotoxic potency under hypoxic conditions. The activated prodrug works as a DNA crosslinking agent and is able to kill both nondividing and dividing cells. TH-302 enhances the activity of a wide range of chemotherapy drugs in numerous xenograft models including HT1080 xenografts (; ), and has been found to be a promising agent in combination with doxorubicin for patients with metastatic sarcoma. Found in two xenograft models that TH-302 combined with chemotherapy increases DNA damage and apoptosis throughout the tumour. TH-302 was found to be more effective in cell lines deficient in homologous recombination secondary to mutations in BRCA1 or BRCA2 (; ).

A phase III randomised trial of doxorubicin with or without TH-302 in patients with soft tissue sarcoma completed enrolment and results are pending. Mechanisms of resistance to antiangiogenic therapies include: upregulation of alternative proangiogenic signals, protection of the tumour vasculature either by recruiting proangiogenic inflammatory cells or by increasing protective pericyte coverage, accentuated invasiveness of tumour cells into local tissue to co-opt normal vasculature, and increased metastatic seeding and tumour cell growth in lymph nodes and distant organs. Upregulation of HIF-1 α and HIF-1 α target genes in response to hypoxia may contribute to all these resistance mechanisms. Found that VEGFR-2 inhibition of RIP1-Tag2 mouse pancreatic endocrine tumours led to an increase in intratumoral hypoxia along with increased tumour invasiveness and liver metastases , and found that sunitinib (which targets VEGF and other pathways) increased liver and lung metastases for both experimental and spontaneous metastases. Given the heterogeneity and genetic instability of tumours, combination of different therapies that target primary pathways as well as resistance pathways is a rational approach. This study demonstrates that destruction of hypoxic regions within tumours using TH-302 complements anti-VEGF-A therapy and radiation in sarcomas.

The mechanisms by which anti-VEGF agents may augment the efficacy of radiation include ‘vascular normalisation' of abnormally dilated and permeable tumour vessels leading to increased oxygenation and augmentation of endothelial cell cytotoxicity. Tumour response to radiation may be regulated by endothelial cell apoptosis.

Although VEGF-A inhibition has effects primarily on endothelial cells, TH-302 under hypoxic conditions will have cytotoxic effects via DNA alkylation on both cancer cells and tumour endothelial cells. In this study, we found the ability of trimodality therapy with TH-302, VEGF-A inhibition, and radiation to block tumour growth is primarily through induction of endothelial cell DNA damage and apoptosis rather than cancer cells autonomous effects. There are several limitations to this study. First, sarcomas are a heterogeneous group of tumours comprised of over 80 histologic subtypes, and thus the results of this study may not be applicable to all sarcoma subtypes.

However, we examined two xenograft models of sarcoma, two types of endothelial cells, and four sarcoma cell lines, and found generally consistent results. Moreover, the predominant effect of these trimodality therapies appears to be on the tumour vasculature, and thus cancer cell autonomous variations in sarcoma subtypes may be less important. Second, in this study we examined proliferation, colony formation, apoptosis, and DNA damage in cancer cells and endothelial cells but did not examine alternative mechanisms or effects on other cells in the tumour microenvironment. Such studies are currently ongoing but beyond the scope of this study. In conclusion, most solid tumours including sarcomas rely on several oncogenic pathways to drive tumour growth and to induce the tumour microenvironment to support this growth. Given the versatility of these tumours, blockade of a single pathway may have little effect or a transient effect as compensatory and resistance mechanisms become activated. Thus, combination therapies that target primary and secondary pathways make intuitive sense.

This study describes a three-pronged strategy to block the growth of sarcomas: radiation to induce endothelial cell DNA damage and apoptosis, VEGF-A inhibition to block the major survival factor for endothelial cells, and hypoxia-activated chemotherapy to eradicate hypoxic cells and block the hypoxic tumour response. In vivo and in vitro studies confirm that this trimodality approach leads.