ecm composition and rheology regulate growth, motility ......images were optimized for display in...

Cell Death and Survival

ECM Composition and Rheology RegulateGrowth, Motility, and Response toPhotodynamic Therapy in 3D Models ofPancreatic Ductal AdenocarcinomaGwendolyn M. Cramer1,2, Dustin P. Jones1,3, Hamid El-Hamidi1, and Jonathan P. Celli1

Abstract

Pancreatic ductal adenocarcinoma is characterized by promi-nent stromal involvement, which plays complex roles in regulat-ing tumor growth and therapeutic response. The extracellularmatrix (ECM)-rich stroma associated with this disease has beenimplicated as a barrier to drug penetration, although stromaldepletion strategies have had mixed clinical success. It remainsless clear how interactions with ECM, acting as a biophysicalregulator of phenotype, not only a barrier to drug perfusion,regulate susceptibilities and resistance to specific therapies. In thiscontext, an integrative approach is used to evaluate invasivebehavior and motility in rheologically characterized ECM asdeterminants of chemotherapy andphotodynamic therapy (PDT)responses. We show that in 3D cultures with ECM conditions thatpromote invasive progression, response to PDT is markedlyenhanced in the most motile ECM-infiltrating populations,whereas the same cells exhibit chemoresistance. Conversely,drug-resistant sublines with enhanced invasive potential weregenerated to compare differential treatment response in identical

ECM conditions, monitored by particle tracking microrheologymeasurements of matrix remodeling. In both scenarios, ECM-infiltrating cell populations exhibit increased sensitivity to PDT,whether invasion is consequent to selection of chemoresistance,or whether chemoresistance is correlated with acquisition ofinvasive behavior. However, while ECM-invading, chemoresis-tant cells exhibit mesenchymal phenotype, induction of EMT inmonolayers without ECM was not sufficient to enhance PDTsensitivity, yet does impart chemoresistance as expected. Inaddition to containing platformdevelopmentwith broader appli-cability to inform microenvironment-dependent therapeutics,these results reveal the efficacy of PDT for targeting the mostaggressive, chemoresistant, invasive pancreatic ductal adenocar-cinoma associated with dismal outcomes for this disease.

Implications: ECM-infiltrating and chemoresistant pancrea-tic tumor populations exhibit increased sensitivity to PDT.Mol Cancer Res; 15(1); 15–25. �2016 AACR.

IntroductionTumor growth and invasive progression are determined not

only by the biology of the tumor itself, but also by its inter-action with components of the microenvironment. The latterencompasses a broad set of factors including immune response,paracrine crosstalk with stromal cells, and the intertwinedbiochemical and biophysical properties of the extracellularmatrix (ECM; refs. 1–3). Extracellular rigidity associated withECM-rich stroma has been shown to promote malignantgrowth behavior (4–6), and type I collagen itself, a major ECMcomponent in solid tumors, promotes increased epithelial–

mesenchymal transition (EMT) and invasive behavior (7, 8).The role of these interactions is particularly provocative forpancreatic ductal adenocarcinoma, a lethal cancer with a medi-an survival of about 6 months (9, 10), which is also associatedwith exceptionally dense fibrotic stroma (11–13).

The abundant stroma associated with pancreatic tumors hasbeen explicitly linked with their poor response to chemotherapy,being implicated as a poorly vascularized barrier that physicallyinhibits drug perfusion (14). The paradigm of stromal depletionhas however had mixed results in the clinic, and further investi-gation revealed that stromal involvement plays complex rolesboth promoting and constraining tumor progression (15–17). Atthe same time, it is unclear how interactions with stroma, as abiophysical regulator of phenotype, not just a barrier to drugdelivery, also modulate response to specific therapies. Given thatthe 5-year survival rate for late-stage pancreatic cancer remains atonly about 2% (18), there is strong motivation not only toidentify new treatments, but also themanner in which prominentstromal involvement impacts upon their efficacy.

Investigation of mechanistically independent treatments forpancreatic ductal adenocarcinoma has motivated the develop-ment of photodynamic therapy (PDT), a photochemistry-basedmodality. In PDT, cancer cells are photosensitized by adminis-tration of an exogenous agent, which is activated by an appro-priate (usually red) light source to initiate photochemistry

1Department of Physics, University of Massachusetts Boston, Boston, Massachu-setts. 2Program in Molecular, Cellular and Organismal Biology, University of Massa-chusetts Boston, Boston, Massachusetts. 3Program in Biomedical Engineering andBiotechnology, University of Massachusetts Boston, Boston, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Jonathan P. Celli, University of Massachusetts Boston,100Morrissey Blvd, Boston, MA 02125. Phone: 617-287-5715; Fax: 617-287-6053;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0260

�2016 American Association for Cancer Research.

MolecularCancerResearch

www.aacrjournals.org 15

on January 16, 2021. © 2017 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst September 26, 2016; DOI: 10.1158/1541-7786.MCR-16-0260

http://mcr.aacrjournals.org/

resulting in tumor destruction by generating reactive oxygenspecies (19). Preclinical studies demonstrated PDT efficacy forpancreatic ductal adenocarcinoma and specifically in experi-ments using the photosensitizer verteporfin that PDT is effectiveagainst cells that are nonresponsive to gemcitabine (20).Although the delivery of light to the pancreas does present atechnical challenge to clinical implementation of PDT, a recentstudy (also using verteporfin) established the clinical efficacy ofthis approach using an innovative system of interstitial lightdelivery and dosimetry in patients with locally advanced unre-sectable disease (21). However, as noted above, there is compel-ling motivation to elucidate how PDT response in pancreaticductal adenocarcinoma cells is impacted by ECM propertieswhich in turn influence the characteristically aggressive growthand invasive behavior of this disease.

In this study, we combine an integrative set of cancer biologyand biophysical methods to identify how contrasting ECM-dependent growth and motility in pancreatic ductal adenocar-cinoma three-dimensional (3D) cell cultures leads also tocontrasting therapeutic susceptibilities. Motivated by the back-ground above, we specifically examine response to PDTin contrast to chemotherapy in pancreatic tumor spheroidstransplanted into laminin-rich and collagen I ECM environ-ments with differing physical and biological compositionshown in studies with breast cancer models to constrain andpromote invasion, respectively (22). As shown in the experi-mental schema (Fig. 1), the use of previously established highcontent imaging for 3D tumor models (23) allows us tocoregister treatment response with phenotypic parameters toexamine differential response in ECM invading and noninvad-ing populations. Surprisingly, this approach reveals that ECM-infiltrating populations, while displaying decreased response tochemotherapy, are markedly more sensitive to PDT, with themost motile leading cells exhibiting the most sensitivity. Wecorroborate this finding in similar investigation of phenotype-dependent response to PDT and chemotherapy but in matchedparent and drug-resistant cells with characterized contrastinginvasive potentials in identical ECM environments. In additionto the development of a more broadly applicable platformfor quantitatively interrogating microenvironment-dependenttherapeutic response, the data also reveals the therapeuticallyimportant insight that populations of pancreatic cancer cellswith increased chemoresistance and invasive/metastatic poten-tial, implicated in the otherwise dismal outcomes for thisdisease, are also the most responsive to PDT.

Materials and MethodsCell culture and reagents

PANC1, BxPC3, and MRC5 cell lines were obtained from theATCC, and grown in T75 cell culture flasks according to ATCCguidelines. RPMI,DMEM, andMEM(HyClone)were supplemen-ted with 10% FBS (HyClone), 100 IU/mL penicillin and 1%streptomycin (HyClone), and 0.5 mg/mL Amphotericin B (Corn-ing). Cell lines were passaged a maximum of 25 times beforediscarding and thawing cryogenically stored stocks.

Growth of adherent 3D cell culture on basement membraneoverlays

Growth factor reduced (GFR) Matrigel (240 mL; Corning) wasadded to each well of a chilled 24-well black-walled plate (Ibidi

USA Inc.) and incubated at 37�C for 30 minutes to allow forgelation. After polymerization, the Matrigel beds were overlaidwith single-cell suspensions of specified cell lines at a concentra-tion of 7,500 cells/mL in the appropriate media supplementedwith 2% Matrigel. Cultures were incubated at 37�C and main-tained with regular media changes and visual inspection bymicroscopy.

Preparation 3D spheroid transplants into contrasting ECMenvironments

For initial preparation of attachment-free multicellular spher-oids, single-cell suspensions of 1,000 cells/mL were added to thesurface of agarose menisci (1% w/v protein electrophoresis gradeagarose; Fisher BioReagents) formed previously in 96-well platesby dispensing heated agarose (50 mL/well) and allowing to set atroom temperature for 30 minutes. After 24 hours, multicellularaggregates were supplemented with complete medium contain-ing 2% Matrigel media. After 12 days (with regular media addi-tions), spheroids were transplanted into chilled 24-well platescontaining either Matrigel or 1 mg/mL bovine type I collagen(COL1; Corning) maintained in solution phase until spheroidwas incorporated. Matrigel layers were initially formed as aboveand COL1 was prepared in 10�MEM (Sigma-Aldrich) and sterilewater, adjusted to a neutral pH with NaOH (Fisher Chemical).COL1 or Matrigel gels were then set by incubation overnight at37�C, effectively embedding spheroids. Transplanted spheroidswere allowed to grow and invade into respective ECM micro-environments (with 500 mL DMEM/well) for 3 days prior to PDTor chemotherapy interventions.

PDT and chemotherapy treatmentsIn PDT-treated wells, media was replaced with complete medi-

um containing 250 nmol/L verteporfin (benzoporphyrin deriv-ative monoacid ring A, BPD (Sigma-Aldrich), replaced withregular media after 1 hour (or 2.5 hours for embedded spheroidsto offset delayed transport/uptake through ECM), prior to irra-diation using a 690-nm turn-key laser source (Intense). Total lightdose ranged from 0.5 to 25 J/cm2 at an irradiance of 100mW/cm2

at bottom surface of cell culture. For culture groups/spheroidsreceiving oxaliplatin chemotherapy treatments, oxaliplatin (Sell-eck Chemicals) was added to the media for each cell type at dosesranging from 0.1 to 500 mmol/L for 48 hours. In all therapeuticstudies, treatment conditions were prepared in at least triplicatewithin each batch including internal controls with sham manip-ulations. Therapeutic response was assessed following interven-tions by imaging, MTS colorimetric assay, or by replating forclonogenic survival as discussed below.

Generation of drug-resistant sublinesIncreasing concentrations of oxaliplatin and/or gemcitabine

(Tocris) were added to each cell type in regular media over thecourse of approximately 25 passages until a stable proliferativephenotype without chemotherapy was observed and maintainedfollowing cryopreservation and thawing. Drug resistance wasconfirmed by comparative dose response and measurement ofa statistically significant increase in IC50.

Induction of epithelial–mesenchymal transitionTo induce EMT under controlled conditions in monolayer cell

cultures, 10 ng/mL human recombinant TGFb (Gibco, ThermoFisher Scientific) in 1% FBS DMEM was added to designated

Cramer et al.

Mol Cancer Res; 15(1) January 2017 Molecular Cancer Research16




cultures for 48 hours. To induce EMTwith fibroblast-conditionedmedia (FCM), supernatant collected from MRC5 cells grown toconfluence in T75 flasks with 8-mLmedia filtered to 0.2 mm, thenadded to specified cell cultures for 48 hours.

Therapeutic assessmentFormonolayer treatments, viabilitywas assessed viaMTS, using

the CellTiter 96 AQueous One Solution Cell Proliferation Assay(Promega) at 490-nm absorbance in a BioTek Epoch MicroplateSpectrophotometer. For treatments of 3D cell cultures, adaptationof an imaging-basedmethodology previously described was used(23, 24). Briefly, this approach consists of image segmentationand quantitative analysis of fluorescence signal from vital dyescalcein AM (Thermo Fisher Scientific Molecular Probes) andethidium bromide (Fisher BioReagents). As in previous reports,cultures were terminally stained with vital dyes according prior toimaging.Multichannel fluorescence imageswere then obtained inacross all growth and treatment conditions in multiwell platesusing an automatedZeiss AxioObserver Z1microscope (Carl ZeissMicroscopy GmbH). In 3D spheroid ECM transplant experi-ments, the batch image analysis code was adapted to tabulateinvading cell positions for eachfieldwith respect topositionof theprimary spheroid (largest segmented object in the same field) andrelative live (cleaved calcein) and dead (intercalated ethidiumbromide) fluorescence signals to quantify number of live anddead invading cells with respect to invasion distance. In specifiedexperiments, treatment response was corroborated by clonogenicsurvival, as per established protocol (25).

ImmunofluorescenceFormaldehyde-fixed cells in optical-bottom multiwell plates

were incubated overnight at 4�C with primary antibodies againstE-cadherin and vimentin (Cell Signaling EMT Duplex) or withphalloidin (Thermo Fisher Scientific Molecular Probes) to stainfor F-actin. Afterwashingwith PBS, cellswere incubated for 1hourwith mouse or rabbit Alexa Fluor secondary antibodies (CellSignaling Technology). Cells were mounted with ProLong GoldAntifade reagent containing DAPI (Thermo Fisher ScientificMolecular Probes) and imaged after 24 hours using the sameexposure and fluorescence intensity settings for all treatmentgroups on an automated Zeiss AxioObserver Z1 or LSM 880confocal laser scanning microscope. Images were optimized fordisplay in figures using the ImageJ Hi-Lo lookup table or ZENsoftware for 3D reconstructions. Unedited images were analyzedusing custom MATLAB scripts, where fluorescent signal for eachprotein was normalized to the number of cells based on DAPI-stained nuclei.

Western blottingCells were lysed with cold RIPA (ThermoFisher Scientific)

containing 1�Halt protease andphosphatase inhibitors (ThermoFisher Scientific). Protein concentration was quantified using thePierce BCA protein assay (Thermo Fisher Scientific). After SDS-PAGE separation, transfer to nitrocellulose membranes, andblocking, membranes were incubated at 4�C overnight withantibodies against E-cadherin (BD Biosciences), vimentin (Sig-ma-Aldrich), and GAPDH (Cell Signaling Technology). Afterwashing, membranes were incubated with HRP-linked rabbitor mouse secondary antibodies (Cell Signaling Technology) for1 hour. ImmobilonWestern HRP Substrate (EMDMillipore) wasadded prior to imaging with the C-DiGit Blot Scanner (LI-COR).

Band density for each protein was normalized to the GAPDHloading control.

Bulk oscillatory shear rheologyFormechanical characterization, ECMpreparations identical to

those used in cell cultures (volumes of 300–400 mL) werepipetted, while ice cold, onto the lower peltier plate (held to 4�Cprior to contact with ECM) of a TA Instruments Discovery Hybridseries rheometer. While still in solution phase, a 40-mm parallelplate geometry was brought into contact while examining thesample spreading to achieve optimal filling of the gap. Peltierplate temperaturewas then regulated to37�C.After 45minutes forgelation and equilibration, rheology measurements were per-formed. An initial oscillatory strain was conducted at low strainvalues to ensure linear response (both components of G�(v)independent of applied strain). An appropriate strain value inthe linear regime was selected for subsequent dynamic oscillatoryshear measurements over a range of 1 < v < 100 rad/s.

Particle-tracking microrheologyTime-dependent changes in ECM compliance were obtained in

normal and drug resistant 3D pancreatic ductal adenocarcinomacell cultures using a methodology described previously (26). Inthis approach, themobility offluorescent tracer probes embeddedin ECM surrounding 3D tumor nodules is analyzed using theGeneralized Stokes Einstein Relation (GSER) to estimate localviscoelastic response, G�(v), of the material. Briefly, for thesemeasurements, 3D cultures were prepared as described above, butincorporating yellow-green fluorescent tracer probes (ThermoFisher Scientific Molecular Probes FluoSpheres). Video sequencesof 800 frames were obtained using a Zeiss AxioCamHRM cameramounted on a Zeiss AxioObserver Z1 microscope (Carl ZeissMicroscopy GmbH). The thermally driven motion of the probesin each video was analyzed using custom MATLAB routinesadapted from open source code of Maria Kilfoil to spatiallycoregister local rheological properties within 3D cell cultures.Changes in ECM stiffness are reported here as difference in theshear modulus (using the real component, G0(v) of the complexmodulus, atv¼ 10 rad/s) obtained from ensemble averages of allprobe trajectories in all replicates between the noted comparativeculture conditions after monitoring for 3 days.

Invasion assayInvasive potential of cells was also quantified by extent of

migration through basementmembrane in transwell inserts usingestablished methods (27). Briefly, 50 mL of diluted Matrigel wasadded to the topwells of aCorning 24-well transwell platewith an8-mmpore size membrane. Serum-starved cells were added to thetranswell over Matrigel beds, and FBS was used as a chemoat-tractant. After 24 hours, the Matrigel and noninvaded cells werecleaned off the membrane, and invaded cells were formalin-fixedthen stained with crystal violet (Thermo Fisher Scientific) andcounted.

Statistical analysisTwo-tailed Student t test was used to analyze normally distrib-

uted data. Results were considered significant if P < 0.05 (�), <0.01(��), <0.005 (��), or <0.001 (��), and ns is not significant. Errorbars indicate SEM in all figures. Figures show representative dataof at least three independent experiments, unless stated otherwisein figure legends.

ECM Regulates PDAC Invasive Motility and Response to Therapy

www.aacrjournals.org Mol Cancer Res; 15(1) January 2017 17




Results and DiscussionECM composition and rigidity regulate pancreatic ductaladenocarcinoma growth, invasion, and motility

Prior to treatment studies, we first characterized growth andinvasive behavior of pancreatic ductal adenocarcinoma 3Dcultures with respect to ECM conditions with contrasting bio-logical and biophysical properties (Figs. 1 and 2). We selectedreconstituted ECM materials modeling pancreatic ductal ade-nocarcinoma stroma rich in type I collagen (COL1), shown inprevious reports to promote invasive behavior (28, 29), con-trasted with laminin-rich basement membrane (GFR Matrigel).

PANC1 spheroids were initially grown in attachment-free con-ditions (agarose beds) for 12 days prior to transplantationinto either ECM condition. After transplanting, growth behav-ior in each condition was monitored nondestructively via dark-field microscopy prior to terminal immunofluorescence anal-ysis. For spheroids transplanted into COL1, within 24 hours theouter cells of the spheroid become more invasive and migrateinto the ECM, whereas spheroids embedded in laminin-richMatrigel do not exhibit significant invasive behavior (Fig. 2A),although a budding pattern is consistently observed on spher-oid surfaces. Darkfield image data were batch processed basedusing methods previously described (30) to obtain relative sizeand position of invading populations and quantify overallextent of invasion by ECM microenvironment. After 3 days,both the total number of invading cells and invasion distance issignificantly higher in COL1 (Fig. 2B). Spheroids fixed andstained after 3 days of growth in ECM show extensive F-actinstaining (Fig. 2C), increased vimentin and loss of E-cadherin ininvading cells (Fig. 2D) in COL1, consistent with a moremesenchymal phenotype in highly motile ECM-infiltratingcells. Conversely, cells in center (Fig. 2D, top right) exhibitmarkedly stronger honeycomb pattern E-cadherin staining,characteristic of adherens junctions and epithelial phenotype.The invasive phenotype of pancreatic ductal adenocarcinomacells observed here in COL1 ECM is consistent with previousreports showing increased EMT and invasion of both pancreaticductal adenocarcinoma and breast cancer cells in collagen-richmicroenvironments (7, 22, 28).

We further considered that the marked difference in pan-creatic ductal adenocarcinoma cell motility in Matrigel andCOL1 may be partly attributable to the contrasting mechanicalproperties of these materials. Identical preparations of both

Figure 1.

Workflow for imaging-based measurements of ECM-dependent growth,motility, and therapeutic response, showing (1) initial formation ofattachment-free spheroids on agarose beds for 12 days; (2) transplantationand embedding of spheroids in rheologically characterized Matrigel or COL1ECMs; (3) longitudinal and terminal (immunofluorescence) imaging ofgrowth and ECM invasion; (4) treatment with chemotherapy (oxaliplatin)or PDT; and (5) imaging-based assesment of therapeutic response,coregistered with phenotype.

Figure 2.

ECM composition and rigidity regulatepancreatic ductal adenocarcinomatumor growth and invasive behavior.A, Representantive darkfield snapshotsof PANC1 spheroids, 1 day followingtransplatation into COL1 or MatrigelECM, showing extensive invasion intoECM in the former. B, Analysis of ECMinvasion with respect to radial distancefrom spheroid edge, after 3 days in eachECM (COL1: n ¼ 5; Matrigel: n ¼ 10).C, Terminally fixed/stained COL1 andMatrigel PANC1 spheroids showingDAPI-stained nuclei and phalloidin-labeled F-actin. D, For COL1 ECM, arepresentative IF image showingincreased mesenchymal markers inECM infiltrating cells (lower right inset,increased vimentin and decreased E-cadherin) relative to inner spheroidpopulations with clear adherensjunctions (upper right inset). E, Bulkoscillatory shear rheology shows G0

(storage) and G00 (loss) moduli for bothECM materials used. Matrigel is asignificantly stronger gel than the softreconstituted COL1 used here, alsolikely contributing to increased motilityin the latter.

Cramer et al.





hydrogels as used for 3D cultures were characterized usingbulk oscillatory shear rheology (Fig. 2E). Both form viscoelas-tic gels with G0 (storage modulus) dominant over G00 (lossmodulus). Although both are soft gels, Matrigel is significantlystiffer with G0 � 90 Pa, within the range of previous reports(4, 31, 32) and roughly 20 times higher than that of the soft(1 mg/mL) COL1 hydrogel used here, which likely creates amore permissive environment for motility of invading cells.The observed differences in phenotype reported above arealmost certainly driven by both the biochemical and biophys-ical properties of the two ECM materials, although futureexperiments should be designed to isolate these influencesusing bioengineered hydrogels with independently tunablerigidity and biochemical properties (33–35). In the followingexperiments, we use the ECM conditions characterized aboveas a tool to interrogate how ECM-dependent regulation ofphenotype regulates response to PDT and chemotherapy.

ECM-infiltrating populations exhibit chemoresistance yetenhanced sensitivity to PDT

In the conditions established above, we examined differen-tial response to PDT and chemotherapy in invading popula-tions and the primary spheroid (Fig. 3). We used oxaliplatin, acomponent of the multidrug cocktail FOLFIRINOX, which hasshown increased effectiveness over the standard gemcitabinetreatment for pancreatic ductal adenocarcinoma (36), for che-motherapy treatment. After spheroid transplantation intoCOL1 or Matrigel, cultures were treated with oxaliplatin che-motherapy or verteporfin PDT using the equivalent monolayerLD90 dose for each modality as a basis for comparison acrosstherapies and growth conditions (Fig. 3B, arrow). As shownin Fig. 3A and C, oxaliplatin chemotherapy inhibits the growthof the primary spheroid to a greater extent than the equivalentPDT dose. However, populations of invading cells (zoomedregions in A and graph in C) exhibit the reverse trend, with no

Figure 3.

ECM invading populations exhibit chemoresistance but enhanced sensitivity to PDT. A, Representative PANC1 spheroids treated with either chemotherapy orPDT doses notes and stained with calcein (green) and ethidium bromide (red), showing viability of core spheroid cells and ECM infiltrating cells. B, Dose–response, evaluated via MTS, of PANC1 to oxaliplatin or PDT in monolayer, used to inform dose selection for each therapy. The LD90 dose (arrow) of eachis selected for subsequent comparison of therapies across contrasting ECM conditions. C, Dose–response (normalized residual volume from imagesegmentation) for primary spheroids shows modest growth inhibition of primary nodule by oxaliplatin, but n.s. for PDT. D, Dose–response for invading cells(analysis applicable for COL1 only) shows no response to oxaliplatin, approximately 50% killing from PDT at 25 J/cm2 (P < 0.01). E, Further analysis ofindividual invading cell viability with respect to radial distance from COL1 spheroid edge showing clear separation of PDT and chemotherapy response. F,Breakdown of response in leading (d > 200 mm) and lagging invaders for chemotherapy and PDT shows further enhancement in PDT response for leading cells.






significant decrease in viability in response to oxaliplatin evenat 500 mmol/L but significantly higher sensitivity to PDT. Thezoomed in regions of invading populations after oxaliplatinand PDT (Fig. 3A, right) show large numbers of dead cells (reddots) following PDT, but little evidence of cell death followingthe chemotherapy treatment. These images were processed toquantitatively report the fraction of surviving ECM-infiltratingcells, outside the segmented primary nodule volume, for eachtreatment condition (Fig. 3D). Although oxaliplatin treatmentappears to decrease proliferation to some extent even in inva-sive populations, these cells are significantly less chemosensi-tive than the cells of the primary spheroid. As expected, boththerapies are far less effective in 3D culture conditions than inmonolayer. Interestingly, when viability of ECM invaders in isplotted against invasion distance (Fig. 3E), PDT is found to bemost effective on the leading cells with highest invasive veloc-ity that have progressed more than 200 mm from the spheroidedge (Fig. 3F). This is an intriguing result, showing that not

only does PDT have enhanced efficacy in ECM-invading cells,but that the most invasive cells are the most sensitive to PDT.

Chemoresistant pancreatic ductal adenocarcinoma displaysincreased invasiveness in 3D culture

Having found phenotype-dependent treatment response relat-ed to ECM conditions, we further probed this in the inversescenario, using genetically matched pancreatic ductal adenocar-cinoma cells with contrasting invasive potential, but placed inidentical ECMmicroenvironments.Motivatedbyprevious reportsshowing acquisition of increased invasion and EMT in chemore-sistant cells (37), we generated and characterized drug-resistantpancreatic ductal adenocarcinoma sublines for further study.PANC1 and BxPC3 cells were exposed to oxaliplatin in increasingconcentration over consecutive passages to establish stable, resis-tant sublines, PANC1OR and BxPC3OR, respectively (Fig. 4). Inboth lines, increased IC50 and increased mesenchymal character-istics relative to their parent lines were confirmed (Fig. 4B and C).

Figure 4.

Generation chemoresistant sublines with increased mesenchymal characteristics. A, Workflow for experiments with chemoresistant sublines (Figs. 4–6),B, Confirmation of resistance to oxaliplatin from comparative dose–response in PANC1 and oxaliplatin resistant subline, PANC1OR (top), and for BxPC3and its oxaliplatin-resistant subline, BxPC3OR (bottom). C, Immunofluorescence and quantification show increased vimentin, decreased E-cadherin inresistant versus nonresistant cells, in a similar trend to invading versus noninvading in above experiments. D, Measurement of above markers viaWestern blot analysis quantified at right and normalized to GAPDH.

Cramer et al.





In addition, acquisition of chemoresistance in PANC1 led toincreased doubling time (2.1 days, vs. 1.7 days in the parent line;Supplementary Fig. S1), also likely protective from classic che-motherapy agents that target cell replication. As shown in Fig. 4Cand D, PANC1OR cells express significantly more vimentinand significantly less E-cadherin based on quantitative immuno-fluorescence verified by Western blot analysis, as well as thelow cell–cell contact and spindle-shaped morphology charac-teristic of a mesenchymal phenotype. In additional experi-ments, a PANC1 subline resistant to both oxaliplatin andgemcitabine (PANC1ORGR) has similar EMT marker expression(Supplementary Fig. S2A and S2B). The oxaliplatin-resistantBxPC3 cells also exhibit increased mesenchymal characteristicsto some extent with increased vimentin, but no observablechange in E-cadherin.

The 3D growth behavior of parent and resistant sublines wereevaluated using an establishedMatrigel overlay culture. Althoughboth lines initially formed compact 3D nodules on the ECM bed,at approximately day 12, PANC1OR cultures began exhibitingmorphologic changes and invasion deep into ECM (Fig. 5).

Confocal imaging of DAPI/phalloidin–stained PANC1 nodulesdisplays a compact 3D structure and minimal invasion intoMatrigel (dotted yellow line; Fig. 5A, top). In contrast, PANC1ORcultures form large invasive protrusions, spreading over the sur-face and invading into the ECM bed (Fig. 5A, bottom) with asimilar pattern of extensive invasion for the multidrug resistant3D cultures (Supplementary Fig. S2C). An established transwellinsert invasion assay quantitatively shows enhanced invasion ofPANC1OR through Matrigel ECM relative to PANC1 (Fig. 5B).

In parallel cultures with 1 mm tracer probes embedded toenable particle-tracking microrheology (PTMR) measurements,a decrease in ECM rigidity is also observed concomitant withinvasion. PTMR measurements show spatial variation in ECMcompliance with lower stiffness measurements obtained fromupper focal planes (UFP) close to 3D nodules on the surface,and stiffer ECM in lower focal planes (LFP; Fig. 5C). Asexpected, ECM degradation is more dramatic in drug-resistant3D cultures. Analysis of tracer probe trajectories close to theinterface of downward-progressing invaders shows an increasein thermally driven movement, reporting a drop in stiffness

Figure 5.

3D cultures of chemoresistant pancreatic ductal adenocarcinoma exhibit increased invasion. A, DAPI/phalloidin-stained PANC1 and PANC1OR overlaycultures on Matrigel. Both lines initially form compact multicellular aggregates consistent with previous characterization, but resistant lines in 3D culturesspontaneously develop highly invasive phenotypes after 12 days in 3D culture (shown). The dotted yellow line indicates approximate Matrigel surface,showing invasion through the ECM bed by PANC1OR cultures. B, Results of a transwell invasion characterizing increased invasive potential of PANC1ORthrough a Matrigel layer. C, PTMR measurements show spatial variation in ECM compliance with lower stiffness in measurements obtained from UFPclose to the 3D nodules on the surface, and stiffer ECM in LFP. D, Longitudinal analysis of PTMR measurements also shows increased rate of ECMdegradation (decrease in rigidity) in the drug-resistant cultures, concomitant with remodeling to enable motility (P < 0.01).






(G0(v)) over a 24-hour period after 12 days of 3D growth inPANC1OR cultures compared with PANC1 (Fig. 5D). Althoughthe laminin-rich basement membrane constrains invasion inthe parent cell line, both in overlays and in the transplantedspheroids (Fig. 1–3) the chemoresistant subline with increasedEMT is able to remodel ECM and enable 3D spreading andinvasive motility, similar to what is observed in spheroidsplaced in soft COL1 (Figs. 1–3). We then sought to determinewhether differential response to PDT and chemotherapy inresistant and nonresistant lines in identical ECM would parallelresults with the parent line in contrasting ECM environments.

Chemoresistant pancreatic ductal adenocarcinoma displaysenhanced PDT sensitivity in 3D culture

PANC1 and PANC1OR cultures were grown for 12 days (asabove) prior to intervention with oxaliplatin or PDT and terminalassessment via vital dye staining and quantitative treatmentassessment (qVISTA) previously described (23). As expected,PANC1OR retain their resistance to oxaliplatin when grown in3D cultures (Fig. 6A). However, PANC1OR cells exhibit signifi-cantly enhanced PDT response relative to PANC1 cells (Fig. 6Band C). Similarly, multidrug-resistant PANC1ORGR cells exhibitenhanced PDT response relative to PANC1 cells grown on Matri-gel beds (S2D). It is not unexpected that chemoresistant cancercells are responsive to PDT, particularly using verteporfin, whichis known to be a potent mitochondrial inducer of apoptosisthat bypasses mechanisms of drug resistance (38). This is, how-ever, to the best of our knowledge, the first time that conditionshave been identified in which drug-resistant cells acquireenhanced sensitivity to PDT relative to their parent cells. Yet, thisresult is also consistent with the spheroid transplantation experi-ments (Figs. 1–3), in which ECM conditions that drive enhancedinvasion, also are correlated with chemoresistance but enhancedresponse to PDT.

Collectively, both experimental designs, manipulating theECM environment of a given cell type and manipulating thebiology of a parent cell type in a given ECM, show that highlymotile ECM invading cells aremarkedlymore susceptible to PDT.Noting that in the conditions where enhanced response to PDT isobserved there is also acquisition of a mesenchymal phenotype,we further examinedwhether direct biochemical induction of thisphenotypic transition would have a similar effect on differentialsensitivity to chemotherapy and PDT.

EMT induction on plastic is not sufficient to enhancesensitivity to PDT

As shown in Fig. 7A and B, activation of TGFb in serum-starvedPANC1 results in increased vimentin and decreased E-cadherinconsistent with EMT and previous reports (39, 40). As expected,after oxaliplatin treatment, MTS evaluation of response showsthat TGFb-treated PANC1 cells remain significantlymore viable atmultiple oxaliplatin doses (Fig. 7C, top graph). However, PDTresponse for PANC1 induced with TGFb respond similarly to nonEMT-induced PANC1 (Fig. 7C, bottom graph). The same trend isevident in clonogenic survival assays (S3), which also show asignificant enhancement in viability for PANC1 cells treated withTGFb, but no difference in PDT response.

Similar experiments were carried out using BxPC3 cells, whichare SMAD4 deficient and do not have an activating KRAS muta-tion, both required for induction of EMT via TGFb signaling(39, 41). However, BxPC3 cells exposed to fibroblast conditioned

media (FCM) reveal similar increases in mesenchymal char-acteristics, although without measurable loss of E-cadherin(Fig. 7A and B). Similarly to PANC1 þ TGFb, BxPC3 þ FCMshow oxaliplatin resistance but no difference in PDT response(Fig. 7D). In both chemoresistant cell lines (BxPC3OR andPANC1OR), PDT treatment is not more effective than in parentcell lines (Fig. 7E). In addition, PANC1þ FCM increases EMTmarker expression and causes similar effects on oxaliplatin andPDT treatment response (Supplementary Fig. S4).

Treatment experiments using direct generation of chemore-sistant cell lines or EMT induction (using TGFb or FCM) areconsistent with the expectation that EMT populations areresistant to chemotherapy, but phenotype in monolayer appar-ently does not significantly affect PDT response (Fig. 7E com-pared with 6B). These results suggest that the observed increasein PDT efficacy in invasive populations requires interactionwith ECM in a 3D environment. This finding motivates further

Figure 6.

Chemoresistant pancreatic ductal adenocarcinoma displays enhanced PDTsensitivity in 3D culture. A, Comparison of response to oxaliplatin in PANC1 andPANC1OR 3D cultures shows that resistance to low doses of oxaliplatin ispreserved in 3D growth conditions, as expected. B, PDT response of PANC1and PANC1OR show dramatic increase in sensitivity in the latter. C,Representative images of PDT response in PANC1 and PANC1OR 3D culturesstained with vital dyes. Scale bars ¼ 100 mm.

Cramer et al.





mechanistic exploration to identify biological changes in thesecells (e.g., altered integrin signaling) that could be connectedwith susceptibility to PDT (e.g., oxidative stress response).Future directions for this research will also include developingculture methods to disentangle the observed biophysical andbiological effects of ECM composition on the motility andtherapeutic susceptibilities of pancreatic cancer cells.

Collectively, our results show that PDT targets invasive andchemoresistant pancreatic ductal adenocarcinoma populationsassociated with the aggressive metastatic spread and the notori-ously poor outcomes for this disease. Combined with recent

clinical studies establishing safety and feasibility of therapeuticlight delivery to the pancreas (21), these results suggest thepromise of PDT for targeting invasive and drug-resistant pancre-atic ductal adenocarcinoma. Within the context of optimizingclinical PDT for pancreatic cancer, these insights could help toinform treatment design, specifically targeting invading fieldscontaining early metastatic cells that would otherwise escapechemotherapy. This suggests that PDT could be particularly usefulfor relatively localized disease (if diagnosed in time), though iftreatmentfields are selected appropriately, the enhanced killing ofinvading and chemoresistant cells could havebenefit for disease at

Figure 7.

PDT efficacy is not enhanced if EMT is induced in the absence of ECM. A, Immunofluorescence images of PANC1, PANC1 þ TGFb, BxPC3, and BxPC3 þfibroblast conditioned media (FCM) show characteristic increased scattering and loss of adherens junctions in EMT-inducing conditions. B,Quantification of immunofluorescence at left. C, Dose–response comparison in PANC1 � TGFb for oxaliplatin (top) and PDT (bottom) showingexpected chemoresistance but not an enhancement in PDT response as seen in 3D ECM-invading cells. D, Dose–response comparison in BxPC3 andBxPC3 þ FCM for oxaliplatin (top) and PDT (bottom). E, PDT dose–response for oxaliplatin-resistant cell lines.






all stages. Building on the groundwork laid in the present study,this approachwill benefit from further investigation following thepath of other PDT-based strategies, such as targeting of tumorvasculature (42, 43) and stromal components (44). Furthermore,the insight that the primary proliferating spheroid mass in 3Dcultures is more susceptible to chemotherapy indicates that acombination would achieve more complete response, a themethat echoes numerous earlier studies on PDT-based combinations(45–49). To evaluate this specific insight into PDT for pancreaticductal adenocarcinoma, this study entailed development of a newtumor modeling and imaging framework for biophysicallyinformed therapeutic evaluation. This approach enables not onlydirect coregistration of cell motility and treatment response asdescribed above, but also lends itself to broader applicability toother cancer therapeutics going forward.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G.M. Cramer, J.P. CelliDevelopment of methodology: G.M. Cramer, D.P. Jones, H. El Hamidi,J.P. Celli

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G.M. Cramer, D.P. Jones, H. El HamidiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G.M. Cramer, D.P. Jones, H. El Hamidi, J.P. CelliWriting, review, and/or revision of the manuscript: G.M. Cramer,H. El Hamidi, J.P. CelliStudy supervision: J.P. Celli

AcknowledgmentsWe gratefully acknowledge funding from the National Cancer Institute,

productive discussions with Dr. Nabeel Bardeesy and Dr. Imran Rizvi ofMassachusetts General Hospital and assistance with cell culture and Westernblotting experiments provided by Rojin Jafari, Saipriya Sagiraju, andSathish Kasina.

Grant SupportThis work was supported by funding from the National Cancer Institute

(R00CA155045, principal investigator: J.P. Celli). G.M. Cramer also ackno-weldges support from a Sanofi Genzyme Doctoral Research Fellowship.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 27, 2016; revised September 5, 2016; accepted September 15,2016; published OnlineFirst September 26, 2016.

References1. Vonderheide RH. Tumor-promoting inflammatory networks in pancreatic

neoplasia: another reason to loathe Kras. Cancer Cell 2014;25:553–4.2. Cirri P, Chiarugi P. Cancer associated fibroblasts: the dark side of the coin.

Am J Cancer Res 2011;1:482–97.3. Pickup MW,Mouw JK, Weaver VM. The extracellular matrix modulates the

hallmarks of cancer. EMBO Rep 2014;15:1243–53.4. Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, et al.

Tensional homeostasis and the malignant phenotype. Cancer Cell 2005;8:241–54.

5. Lu P, Weaver VM, Werb Z. The extracellular matrix: a dynamic niche incancer progression. J Cell Biol 2012;196:395–406.

6. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, et al. Matrixcrosslinking forces tumor progression by enhancing integrin signaling. Cell2009;139:891–906.

7. Shintani Y, Hollingsworth MA, Wheelock MJ, Johnson KR. Collagen Ipromotes metastasis in pancreatic cancer by activating c-Jun NH(2)-ter-minal kinase 1 and up-regulating N-cadherin expression. Cancer Res2006;66:11745–53.

8. Shintani Y, Wheelock MJ, Johnson KR. Phosphoinositide-3 kinase-Rac1-c-Jun NH2-terminal kinase signaling mediates collagen I-induced cell scat-tering and up-regulation of N-cadherin expression in mouse mammaryepithelial cells. Mol Biol Cell 2006;17:2963–75.

9. Gong Z, Holly EA, Bracci PM. Survival in population-based pancreaticcancer patients: San Francisco Bay area, 1995–1999. Am J Epidemiol 2011;174:1373–81.

10. Goldstein D, El-Maraghi RH, Hammel P, Heinemann V, Kunzmann V,Sastre J, et al. nab-Paclitaxel plus gemcitabine for metastatic pancreaticcancer: long-term survival from a phase III trial. J Natl Cancer Inst2015;107:413 dju413.

11. Mollenhauer J, Roether I, Kern HF. Distribution of extracellular matrixproteins in pancreatic ductal adenocarcinoma and its influence on tumorcell proliferation in vitro. Pancreas 1987;2:14–24.

12. Mahadevan D, Von Hoff DD. Tumor-stroma interactions in pancreaticductal adenocarcinoma. Mol Cancer Ther 2007;6:1186–97.

13. Vonlaufen A, Phillips PA, Xu Z, Goldstein D, Pirola RC, Wilson JS, et al.Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance.Cancer Res 2008;68:7707–10.

14. Erkan M, Hausmann S, Michalski CW, Fingerle AA, Dobritz M,Kleeff J, et al. The role of stroma in pancreatic cancer: diagnostic

and therapeutic implications. Nat Rev Gastroenterol Hepatol 2012;9:454–67.

15. Gore J, Korc M. Pancreatic cancer stroma: friend or foe? Cancer Cell2014;25:711–2.

16. Ozdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu CC, SimpsonTR, et al. Depletion of carcinoma-associated fibroblasts and fibrosisinduces immunosuppression and accelerates pancreas cancer with reducedsurvival. Cancer Cell 2014;25:719–34.

17. Rhim AD, Oberstein PE, Thomas DH, Mirek ET, Palermo CF, Sastra SA,et al. Stromal elements act to restrain, rather than support, pancreatic ductaladenocarcinoma. Cancer Cell 2014;25:735–47.

18. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin2016;66:7–30.

19. Dougherty TJ, Gomer CJ,Henderson BW, JoriG, Kessel D, KorbelikM, et al.Photodynamic therapy. J Natl Cancer Inst 1998;90:889–905.

20. Celli JP, Solban N, Liang A, Pereira SP, Hasan T. Verteporfin-basedphotodynamic therapy overcomes gemcitabine insensitivity in a panel ofpancreatic cancer cell lines. Lasers Surg Med 2011;43:565–74.

21. Huggett MT, JermynM, Gillams A, Illing R, Mosse S, Novelli M, et al. PhaseI/II study of verteporfin photodynamic therapy in locally advanced pan-creatic cancer. Br J Cancer 2014;110:1698–704.

22. Nguyen-Ngoc KV, Cheung KJ, Brenot A, Shamir ER, Gray RS, Hines WC,et al. ECM microenvironment regulates collective migration and localdissemination in normal and malignant mammary epithelium. Proc NatlAcad Sci U S A 2012;109:E2595–604.

23. Celli JP, Rizvi I, Blanden AR, Massodi I, Glidden MD, Pogue BW, et al. Animaging-based platform for high-content, quantitative evaluation of ther-apeutic response in 3D tumour models. Sci Rep 2014;4:3751.

24. Anbil S, Rizvi I, Celli JP, Alagic N, Pogue BW, Hasan T. Impact of treatmentresponse metrics on photodynamic therapy planning and outcomes in athree-dimensional model of ovarian cancer. J Biomed Opt 2013;18:098004.

25. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenicassay of cells in vitro. Nat Protoc 2006;1:2315–9.

26. Jones DP, Hanna W, El-Hamidi H, Celli JP. Longitudinal measurement ofextracellular matrix rigidity in 3D tumor models using particle-trackingmicrorheology. J Vis Exp 2014;88:e51302.

27. Hall DM, Brooks SA. In vitro invasion assay using matrigel: a reconstitutedbasement membrane preparation. Methods Mol Biol 2014;1070:1–11.

Cramer et al.





28. Shields MA, Dangi-Garimella S, Krantz SB, Bentrem DJ, Munshi HG.Pancreatic cancer cells respond to type I collagen by inducing snailexpression to promotemembrane type 1matrix metalloproteinase-depen-dent collagen invasion. J Biol Chem 2011;286:10495–504.

29. Armstrong T, PackhamG,Murphy LB, BatemanAC,Conti JA, FineDR, et al.Type I collagen promotes the malignant phenotype of pancreatic ductaladenocarcinoma. Clin Cancer Res 2004;10:7427–37.

30. Celli JP, Rizvi I, Evans CL, Abu-Yousif AO, Hasan T. Quantitative imagingreveals heterogeneous growth dynamics and treatment-dependent residualtumor distributions in a three-dimensional ovarian cancer model.J Biomed Opt 2010;15:051603.

31. Semler EJ, Ranucci CS, Moghe PV. Mechanochemical manipulation ofhepatocyte aggregation can selectively induce or repress liver-specificfunction. Biotechnol Bioeng 2000;69:359–69.

32. Zaman MH, Trapani LM, Sieminski AL, Mackellar D, Gong H, Kamm RD,et al.Migration of tumor cells in 3Dmatrices is governed bymatrix stiffnessalong with cell-matrix adhesion and proteolysis. Proc Natl Acad Sci U S A2006;103:10889–94.

33. Wang L-S, Du C, Toh WS, Wan AC, Gao SJ, Kurisawa M. Modulation ofchondrocyte functions and stiffness-dependent cartilage repair using aninjectable enzymatically crosslinked hydrogel with tunable mechanicalproperties. Biomaterials 2014;35:2207–17.

34. Ulrich TA, Jain A, Tanner K, MacKay JL, Kumar S. Probing cellular mechan-obiology in three-dimensional culture with collagen–agarose matrices.Biomaterials 2010;31:1875–84.

35. BrancodaCunhaC, KlumpersDD, LiWA,Koshy ST,Weaver JC, ChaudhuriO, et al. Influence of the stiffness of three-dimensional alginate/collagen-Iinterpenetrating networks on fibroblast biology. Biomaterials 2014;35:8927–36.

36. ConroyT,Desseigne F, YchouM,BoucheO,GuimbaudR, Becouarn Y, et al.FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl JMed 2011;364:1817–25.

37. Shah AN, Summy JM, Zhang J, Park SI, Parikh NU, Gallick GE. Develop-ment and characterization of gemcitabine-resistant pancreatic tumor cells.Ann Surg Oncol 2007;14:3629–37.

38. Kessel D, Luo Y. Photodynamic therapy: a mitochondrial inducer ofapoptosis. Cell Death Differ 1999;6:28–35.

39. Ellenrieder V, Hendler SF, Boeck W, Seufferlein T, Menke A, Ruhland C,et al. Transforming growth factor beta1 treatment leads to an epithelial-

mesenchymal transdifferentiation of pancreatic cancer cells requiringextracellular signal-regulated kinase 2 activation. Cancer Res 2001;61:4222–8.

40. Fuxe J, Vincent T, Garcia de Herreros A. Transcriptional crosstalk betweenTGF-beta and stem cell pathways in tumor cell invasion: role of EMTpromoting Smad complexes. Cell Cycle 2010;9:2363–74.

41. Subramanian G, Schwarz RE, Higgins L, McEnroe G, Chakravarty S,Dugar S, et al. Targeting endogenous transforming growth factorbeta receptor signaling in SMAD4-deficient human pancreatic car-cinoma cells inhibits their invasive phenotype1. Cancer Res 2004;64:5200–11.

42. Chen B, Pogue BW, Luna JM, Hardman RL, Hoopes PJ, Hasan T. Tumorvascular permeabilization by vascular-targeting photosensitization:effects, mechanism, and therapeutic implications. Clin Cancer Res2006;12:917–23.

43. Scherz A, Salomon Y, Lindner U, Coleman J. Vascular-targeted photody-namic therapy in prostate cancer: frombench to clinic. PhotodynamicMed2016. p461–80.

44. Celli JP. Stromal interactions as regulators of tumor growth and therapeuticresponse: A potential target for photodynamic therapy? Isr J Chem 2012;52:757–66.

45. del Carmen MG, Rizvi I, Chang Y, Moor ACE, Oliva E, Sherwood M, et al.Synergism of epidermal growth factor receptor-targeted immunotherapywith photodynamic treatment of ovarian cancer in vivo. J Natl Cancer Inst2005;97:1516–24.

46. Kosharskyy B, Solban N, Chang SK, Rizvi I, Chang Y, Hasan T. A mech-anism-based combination therapy reduces local tumor growth andmetas-tasis in an orthotopic model of prostate cancer. Cancer Res 2006;66:10953–58.

47. Rizvi I, Celli JP, Evans CL, Abu-Yousif AO, Muzikansky A, Pogue BW, et al.Synergistic enhancement of carboplatin efficacy with photodynamic ther-apy in a three-dimensional model for micrometastatic ovarian cancer.Cancer Res 2010;70:9319–28.

48. Ferrario A, von Tiehl K, Wong S, Luna M, Gomer C. Cyclooxygenase-2inhibitor treatment enhances photodynamic therapy-mediated tumorresponse. Cancer Res 2002;62:3956–61.

49. Gomer CJ, Ferrario A, Luna M, Rucker N, Wong S. Photodynamic therapy:combined modality approaches targeting the tumor microenvironment.Lasers Surg Med 2006;38:516–21.






2017;15:15-25. Published OnlineFirst September 26, 2016.Mol Cancer Res Gwendolyn M. Cramer, Dustin P. Jones, Hamid El-Hamidi, et al. Ductal AdenocarcinomaResponse to Photodynamic Therapy in 3D Models of Pancreatic ECM Composition and Rheology Regulate Growth, Motility, and

Updated version

10.1158/1541-7786.MCR-16-0260doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2016/09/29/1541-7786.MCR-16-0260.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/15/1/15.full#ref-list-1

This article cites 48 articles, 16 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/15/1/15To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-16-0260

http://mcr.aacrjournals.org/content/suppl/2016/09/29/1541-7786.MCR-16-0260.DC1

http://mcr.aacrjournals.org/content/15/1/15.full#ref-list-1

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/15/1/15


ecm composition and rheology regulate growth, motility ......images were optimized for display in...

Documents