www.sciencetranslationalmedicine.org/cgi/content/full/7/284/284ra58/DC1

Supplementary Materials for

A technology platform to assess multiple cancer agents simultaneously within a patient’s tumor

Richard A. Klinghoffer,* S. Bahram Bahrami, Beryl A. Hatton, Jason P. Frazier,

Alicia Moreno-Gonzalez, Andrew D. Strand, William S. Kerwin, Joseph R. Casalini, Derek J. Thirstrup, Sheng You, Shelli M. Morris, Korashon L. Watts, Mandana Veiseh,

Marc O. Grenley, Ilona Tretyak, Joyoti Dey, Michael Carleton, Emily Beirne, Kyle D. Pedro, Sally H. Ditzler, Emily J. Girard, Thomas L. Deckwerth,

Jessica A. Bertout, Karri A. Meleo, Ellen H. Filvaroff, Rajesh Chopra, Oliver W. Press, James M. Olson*

*Corresponding author. E-mail: rich.klinghoffer@presagebio.com (R.A.K.); jolson@fredhutch.org (J.M.O.)

Published 22 April 2015, Sci. Transl. Med. 7, 284ra58 (2015)

DOI: 10.1126/scitranslmed.aaa7489

The PDF file includes:

Methods Fig. S1. The inverse relationship of hypoxic and vascularized tumor regions quantified by CIVO Analyzer. Fig. S2. CIVO outcomes correlate with long-term response to vincristine in primary, patient-derived diffuse large B cell lymphoma. Fig. S3. Intratumoral doxorubicin autofluorescence is detectable with IVIS imaging after microinjection. Fig. S4. Co-microinjection of vincristine and prednisolone enhances the cell death response induced by vincristine alone. Fig. S5. A CIVO-enabled in vivo screen of oncology therapies identifies a dependence on mTOR signaling in Res-Ramos tumors. Table S1. Drug biodistribution after CIVO microinjection of radiolabeled compounds. Table S2. Drug efflux pump expression in Ramos and Res-Ramos cells. Table S3. The top five CIVO drug-screening hits were determined using CC3. Table S4. Patients’ pain experience. Table S5. Stakeholders’ input on the early CIVO prototype. References (27–36)

Supplementary Materials

METHODS

Cell culture

Ramos cells, a human Burkitt’s lymphoma cell line, were cultured in RPMI-1640 plus L-

glutamine medium (GibCo), supplemented with final concentrations of 10% fetal bovine serum

(HyClone), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco). Cultures were grown at

37⁰C, 5% CO2, in vented flasks. Cell density was maintained between 2 x105 and 2 ×106 viable

cells/ml.

Selection of doxorubicin resistant cell lines

Drug-naïve Ramos cells were seeded at 2 x 105 cells/ml in 25 ml of media containing 5 nM

doxorubicin (Pfizer Doxorubicin HCl injection 2 mg/ml). This culture was maintained, with cells

passaged by pelleting by low speed centrifugation (300 × g) and transferred to fresh drug-

positive media weekly, until a cell density of 1-2 ×106 cells/ml was reached. This process was

repeated while subjecting cells to incremental increases in doxorubicin following each recovery

period. Through several such cycles a cell line with a doubling time <36 hours in continuous

exposure to 500 nM doxorubicin (Res-Ramos) was selected.

Drug dose-response curves

Ten ml cultures of drug naïve and doxorubicin-resistant Ramos cell lines were seeded at 2 x105

cells/ml in drug-free media or media with various doxorubicin concentrations. After 48 or 72

hours of culture, the cell density and viability were determined by trypan blue dye exclusion

using a Vi-CELL cell viability analyzer (Beckman-Coulter). The cell densities of drug-positive

cultures were normalized to the drug-free culture’s cell density. For the CC-115 dose-response

analysis, drug naïve and doxorubicin-resistant Ramos cell lines were seeded in drug-free media

or media with various CC-115 concentrations in 96 well plates and cell viability determined

using the PrestoBlue assay (Molecular Probes) after 72 hours of drug exposure. Resulting

fluorescence was read using an EnVision Multilable Plate Reader (Perkin Elmer).

Establishing flank tumors

Drug-naïve or doxorubicin-resistant Ramos cell lines were cultured to a density of 1–2 × 106

cells/ml. Cells were pelleted then suspended in a 1:1 mixture of drug-free RPMI and BD

Matrigel Matrix (BD Biosciences) at 2.5–5 × 107 cells/ml (106 cells per graft). To establish

tumors, 200 µl of the cell suspension was injected subcutaneously into one flank of athymic

female Nude-Foxn1nu mice (Harlan Laboratories). Mice bearing primary, patient-derived tumors

(LY0055F diffuse large B cell lymphoma) were provided by the Jackson Laboratory. Tumor

fragments were directly engrafted into recipient NOD SCID gamma (NSG) mice immediately

following surgical resection and passaged as subcutaneous flank xenografts by the Jackson

Laboratory.

Systemic drug administration in mice

Cyclophosphamide was administered once per week (q1w) at 20 mg/kg I.V., doxorubicin

administered q1w at 3.3 mg/kg I.V., vincristine administered q1w at 0.5 mg/kg I.V., and

prednisone administered five times per week (q5d) at 0.2 mg/kg P.O as reported previously (27).

Dosing continued over a four-week treatment period. Mouse weights and tumor dimensions were

measured twice per week, and tumor volumes extrapolated using the equation volume (mm3) =

(A) × (B2/2), where A is the largest diameter (mm) and B is the smallest diameter (mm). Tumor

growth inhibition (TGI) was calculated using the following equation:

TGI = (ΔVmean[vehicle] - ΔV[treatment])/ΔVmean[vehicle])

where ΔV is the change in tumor volume from day 1 to 8.

CC-115 was administered daily (qd) at 5 mg/kg P.O. over a four-week treatment period.

Mouse weights and tumor dimensions were measured as above, and TGI calculated on day 25.

Autoradioluminographic analysis of intratumoral 3H-vincristine distribution

Tritiated (3H) vincristine sulfate from American Radiolabeled Chemicals (1.6 Ci/mmol) was

diluted with unlabeled vincristine sulfate (Sigma) and phosphate buffered saline to a

concentration of 0.25 mCi/ml and the activity verified by scintillation counting. Nude mice

bearing Ramos xenograft tumors were anesthetized with Avertin and their tumors microinjected

with a four-needle array. Mice were euthanized and tumors resected at 2, 8, 24, 48, and 72 h

after microinjection, with four replicate tumors injected per time point. Tumor tissue was frozen

on dry ice and shipped to Quest Pharmaceutical Services, where it was embedded along with 3H-

calibration standards in a carboxymethyl cellulose matrix, frozen, and sliced into 40-µm–thick

cross sections collected at 1-mm intervals. After dehydration, the cryosections were exposed to

phosphoimaging plates to obtain autoradioluminographic images of the distribution of the

radiolabeled vincristine at multiple depths at each microinjection position within each tumor.

The phosphoimaging plates were scanned at 25-µm resolution and the resulting intensity

distributions converted to vincristine concentrations. Radial distribution graphs were obtained by

averaging vincristine concentrations for annuli at various distances from the injection site over

multiple injections and depths.

Whole-slide scanning and image analysis

Images of every cell from each tissue section stained were captured by digital, automated, high-

resolution whole-tissue scanning (3D Histech Pannoramic 250 Flash). Tumor responses were

quantified from image files from each tissue section using Presage’s custom CIVO Analyzer

image analysis platform. Whole-tissue section images captured by the slide scanners were

automatically processed by CIVO Analyzer. Each cell from each tissue section was segmented

based on the nuclear (DAPI) signal and classified as biomarker-negative or -positive using Cell

Profiler (Broad Institute) (28). Following cellular segmentation and classification, regions of

interest (ROI) were localized around each microinjection site in each image by defining

concentric rings around the fluorescent injection tracking dye at each position. Images

representing parallel tissue sections stained for each biomarker were registered by CIVO

Analyzer to map each ROI to its corresponding location. Tumor responses were then plotted as

the fraction of biomarker-positive cells as a function of the radial distance from the center of the

microinjection site. Responses were quantified from tissue sections representing multiple depths

along the injection column that were each approximately 2 mm apart from each other.

RNA analysis and labeling

Total RNA was prepared from approximately 4 × 106 cells using Qiagen Qiashredder and

RNeasy kits per the manufacturer's instructions. Total RNA was quantified using a Trinean

DropSense96 spectrophotometer (Caliper Life Sciences). RNA integrity was tested using an

Agilent 2200 TapeStation (Agilent Technologies, Inc.). High-quality RNA samples were

converted to cDNA and biotin-labeled for microarray analysis using Ambion’s Illumina

TotalPrep RNA Amplification kit (Life Technologies). Labeled cRNAs were processed on a

HumanHT-12v4 Expression BeadChip (Illumina, Inc) and imaged using an Illumina iScan

system.

Microarray data analysis

Illumina HT-12V4 beadchip arrays were used to profile gene expression in Ramos cells, a

human Burkitt’s lymphoma cell line, and the Res-Ramos derivative that grows in 500 nM

doxorubicin. The complete set of microarray data was assessed for quality, followed by quantile

normalization using the Bioconductor package lumi (29). The dataset was filtered of probes that

were below a signal noise floor, which was established using the 75th percentile of the negative

control probe signals within each array. Additionally, probes with low variance were filtered

using the “shorth” function of the Bioconductor package genefilter. Differential gene expression

in the remaining set of probes was determined by pairwise comparison of each group of drug

resistant Dox samples to the Ramos samples using the Bioconductor package limma (30). A false

discovery rate (FDR) method was used to correct for multiple testing (31). Differential gene

expression was defined as |log2 (ratio)| ≥ 1 with the FDR set to 5%. The union of differentially

expressed probes across the five pairwise comparisons (n = 643) were mean-centered at the

probe-level and subjected to K-means clustering using the TM4 microarray software suite

MultiExperimental Viewer (32). Euclidian distance was used as a measure of similarity and the

number of clusters employed was selected using the figure of merit method (33). Gene ontology

enrichment analysis on differentially expressed genes and genes in different clusters was

performed using DAVID employing an FDR of 10% (34, 35). The data discussed in this

publication have been deposited in the NCBI’s Gene Expression Omnibus (36) and are

accessible through GEO Series accession number GSE61516 (www.ncbi.nlm.nih.gov/geo/).

Tissue processing, gross tissue imaging, and IHC

Mice were euthanized using CO2 inhalation and xenograft tumors were resected, cut into 2-mm

thick sections perpendicular to the injection columns, fixed in 10% buffered formalin for 48

hours, and scanned on a Xenogen IVIS in the near-infrared spectrum to assess injection quality:

(excitation 680 nm, emission 720 nm) to visualize the injection tracking dye; and (excitation

500nm, emission 600 nm) to visualize the doxorubicin auto-fluorescence.

Following IVIS scanning, 2 mm-thick tissue sections were processed for standard

paraffin embedding and histological analysis and tissue sections were sliced at a 4-µm thickness.

H&E staining was performed using standard methods. Rabbit polyclonal antibodies recognizing

γH2AX (Novus Biologicals, 1:500 dilution) or CC3 (Cell Signaling, 1:150 dilution), a rabbit

monoclonal antibody recognizing p4EBP1 (Cell Signaling, 1:100 dilution) or a mouse

monoclonal antibody recognizing pHH3 (Cell Signaling, 1:200 dilution) were used for IHC

analysis. For immunofluorescent detection, a secondary antibody conjugated to AlexaFluor555

(Invitrogen, 1:400 dilution) was applied according to manufacturer’s instructions and tissues

counterstained with DAPI. For co-stained tissues, secondary antibodies conjugated to

AlexaFluor488 or AlexaFluor555 (1:400 dilution) were applied together and tissues were

counterstained with DAPI.

Modifications of device and microinjection procedure in canine patients

Two major modifications were made following the pilot human study. (i) The placement of a

separate “guide needle” into the optimal injection site using ultrasound guidance. This type of

needle aligns the microinjection device to target all needles into the optimal location in the

lymph node. Ultrasound was used as the imaging modality for guidance. (ii) The use of a sterile

fluorescent tattoo dye (PS-2001 yellow color and/or PS-2002 magenta color) microinjected for

confirmation of drug delivery and to track tumor orientation during the surgical procedure and

sample sectioning. The fluorescent tattoo dye could be seen under the skin and throughout the

surgical procedure with the simple use of a blue light handheld flashlight and yellow filter

glasses (Fig. 7). The use of this fluorescent tattoo dye allowed the clinician to confirm successful

microinjection into the lymph node; it guided the surgeon during lymph node biopsy and the

pathologist during gross sectioning for retaining injection site orientation.

SUPPLEMENTARY FIGURES

Figure S1. The inverse relationship of hypoxic and vascularized tumor regions quantified by CIVO Analyzer . To understand how tumor responses to drugs are impacted by different tumor microenvironments, a battery of assays and imaging classifiers have been built into CIVO Analyzer. (A) Here, as an example, the relationship between hypoxic (GLUT1+) and vascularized (CD31+) regions of the tumor are shown. (B) An example of automated analysis with CIVO Analyzer shows the expected inverse relationship between hypoxia and vascularization of the microenvironment, with GLUT1 expression increasing as the distance from the nearest endothelial cell increases.

Figure S2. CIVO outcomes correlate with long-term response to vincristine in primary, patient-derived diffuse large B cell lymphoma. Mice bearing primary, patient-derived diffuse large B cell lymphoma tumors (LY0055F) were microinjected with vincristine and a vehicle control, and tumors resected 24 h later. (A) Tissue sections were stained for pHH3 or CC3 and the fraction of biomarker-positive cells was plotted as a function of radial distance from the injection site. Data are the average response ± S.E.M. (n = 9 tumors). *P < 0.05, **P < 0.001, Wald’s test. (B) Parallel LY0055F tumor-bearing mice were treated with saline (vehicle) or vincristine (0.5 mg/kg) intravenously once a week for 4 weeks. Efficacy was assessed by change in tumor volume. Data are means ± S.E.M. (n = 8 mice per cohort).

Figure S3. Intratumoral doxorubicin autofluorescence is detectable with IVIS imaging after microinjection. Ramos lymphoma tumors were microinjected with a vehicle control and five doxorubicin concentrations, each through a distinct needle within the array. The vehicle control was co-injected with an injection tracking dye (ITD). Tumors were resected 24 h after microinjection and gross tumor tissue imaged via a Xenogen IVIS. Excitation at 675 nm and emission at 720 nm reveals the ITD, whereas the doxorubicin fluorescence is visible with an ex/em of 500/600 nm. Staining of 4-µm tissue sections revealed the ITD at the vehicle position, and concentration-dependent γH2AX and CC3 tumor responses to doxorubicin.

Figure S4. Co-microinjection of vincristine and prednisolone enhances the cell death response induced by vincristine alone. Ramos tumors were microinjected with an array containing vincristine (1 µg) alone, or the combination or vincristine (1 µg) with prednisolone (50 µg) (together through the same needle in the array) and tumors resected 24 h after microinjection. Tissue sections were stained for CC3 and the fraction of biomarker-positive cells was plotted as a function of radial distance from the injection site. Data are the average response ± S.E.M. (n = 4 tumors). *P < 0.05, Wald’s test.

Figure S5. A CIVO-enabled in vivo screen of oncology therapies identifies a dependence on mTOR signaling in Res-Ramos tumors. (A) Ninety-seven approved oncology agents were tested for in vivo efficacy upon CIVO microinjection into replicate Res-Ramos tumors using an eight-needle array configuration. Tumors were resected 72 h following injection and tissue sections stained with H&E or an antibody recognizing CC3 (red). The ITD is in green. An example of a screen hit is indicated by the white arrow in the lower image. (B) Representative vehicle control or rapamycin injection sites from the screen. Four-micron sections were stained with H&E or CC3 and the fraction of CC3+ cells was plotted as a function of radial distance from the injection site. Data are the responses from each of two tumors. (C) Res-Ramos tumors were microinjected with rapamycin and resected at 2 h to examine in vivo pathway engagement prior to overt cell death. Parallel tissue sections were stained with H&E or antibodies recognizing phospho-4EBP1 (p4EBP1) or CC3. The p4EBP1– region designated by the red boxes is shown at high-magnification in the middle panels, and the p4EBP1+ region designated by the blue boxes is shown at high-magnification in the panels to the right. Data are average responses + S.E.M. (n ≥ 3). *P < 0.05, Wald’s test.

SUPPLMENTARY TABLES

Table S1. Drug biodistribution after CIVO microinje ction of radiolabeled compounds. Drugs were microinjected at one site and saline controls were injected at adjacent sites within the same tumor using the CIVO device. Core biopsies of all sites were measured for radioactivity by scintillation counting from a minimum of nine replicates per condition.

Isotope Drug Formula MW (g/mol)

Vd (l/kg, unless

otherwise indicated)

Protein-bound (%)

Half-life

Drug remaining in 2 mm radius vs. adjacent

saline control* (%) Ramos H2122 H292

C14 Chlorambucil C14H19Cl2NO2 304.22 0.14-0.24 99 1.5 h 94 97

H3 Cordycepin C10H13N5O3 251.20 97 98 97

H3 Didanosine C10H12N4O3 236.50 0.77 < 5 0.97-1.6 h

99 97 96

H3 2’,3’-

Dideoxyadenosine C10H13N5O2 235.20 99 98

H3 Flucortisone C24H30F2O6 452.50 1.3-1.7 h

98 97

C14 Mitoxantrone C22H28N4O4 444.49 1000 l/m2 78 5.8

days 97

H3 Paclitaxel C47H51NO14 853.91 89-98 5.8 h 95

C14 Mercaptopurine C5H6N4OS 170.19 0.59-0.9 5-37 20-47

min 97

*Represents combined data from 15 minute (n = 3-5), 4 hour (n = 3-5), and 24 hour (n = 3-5) time points.

Table S2. Drug efflux pump expression in Ramos and Res-Ramos cells.

Target ID Accession number

Mean centered intensity (log2) Fold change in Res-Ramos cells Ramos Res-Ramos

ABCB1 (MDR1) DR000893 -2.569681 1.6603004 18.76511726

ABCB1 (MDR1) DR000893 -1.5897073 1.1753292 6.797651971

ABCB9 (MDR9) DR003131 -2.4468904 0.14986765 4.914425417

ABCB9 (MDR9) DR003131 -2.1294227 0.18619162 3.845659633

ABCG1 DN996555 -1.0038419 -0.25202337 1.683914082

ABCG1 DN996555 -0.8154632 -0.17683513 1.556847972

ABCB4 (MDR4) NG_007118 -0.8673328 -0.3114056 1.470113151

Table S3. The top five CIVO drug-screening hits were determined using CC3. The total fraction of CC3-positive cells within 1200 µm from the injection site for each drug was quantified using CIVO Analyzer. The percentage of cells positive for CC3 are from the total number of cells within the same 1200 µm radius. A threshold of >15% total positive cells for CC3 was used to determine the top hits for CIVO-injected drugs in Res-Ramos tumors.

Drug name Mechanism of action CC3-positive cells (%)

Dactinomycin Noncovalent DNA binding 44.91

Bendamustine HCl Alkylating agent 35.98

Plicamycin Noncovalent DNA binding 33.25

Rapamycin mTOR inhibitor 24.46

Vorinostat Histone deacetylase inhibitor 17.41

Table S4. Patients’ pain experience. Pain was rated on a standard Mosby 0-10 scale, with 0 being no pain and 10 being the worst possible pain. Patient #4 did not respond to a request for follow-up after lymph node removal.

Patient ID #1 #2 #3 #4

Pain at microinjection

0.5 0 0.5 3

Pain after lymph node removal

2 2 0 N/A

Table S5. Stakeholders’ input on the early CIVO prototype.

CIVO device user/stakeholder

Input on early prototype

Oncologist microinjecting drugs into tumor

• Device was easy to insert through the skin and entire procedure was of short duration.

• Device was easy to use after minimal training and the accompanied manual was a good reference.

• Patients did not show signs of discomfort during injection. • Suggestion of improving the targeting of the ideal site of injection in the

tumor using ultrasonography, rather than simply measuring tumor size and depth prior to injection.

Pharmacist loading CIVO device

• The device was easy to load. • Device loading procedure was simple enough to perform within a reasonable

time period. • Suggestion of color coding the various needle positions to minimize drug

swapping errors. • Suggestion of improving loading procedure to only require aseptic technique.

Surgeon removing injected tumor

• It was sometimes difficult to identify the exact site and orientation of the injection site.

• Suggestion of improving internal and external marking methodology of injection site.

Pathologist handling resected tumor

• It was sometimes difficult to retain orientation of the injection sites without additional marks incorporated by surgeon.

• Suggestion of improving on external marking methodology to speed up the sample sectioning process.

Patient’s comments at microinjection

• Suggestion to improve patient experience by avoiding showing the multiple needles to the patient prior to injection