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Therapeutics, Targets, and Chemical Biology

Preclinical Efficacy of Bevacizumab withCRLX101, an Investigational Nanoparticle–DrugConjugate, in Treatment of MetastaticTriple-Negative Breast CancerElizabeth Pham1, Melissa Yin2, Christian G. Peters3, Christina R. Lee1, Donna Brown3,Ping Xu1, Shan Man1, Lata Jayaraman3, Ellen Rohde3, Annabelle Chow1, Douglas Lazarus3,Scott Eliasof3, F. Stuart Foster2,4, and Robert S. Kerbel1,4

Abstract

VEGF pathway–targeting antiangiogenic drugs, such as beva-cizumab, when combined with chemotherapy have changedclinical practice for the treatment of a broad spectrum of humancancers. However, adaptive resistance often develops, and onemajor mechanism is elevated tumor hypoxia and upregulatedhypoxia-inducible factor-1a (HIF1a) caused by antiangiogenictreatment. Reduced tumor vessel numbers and function followingantiangiogenic therapy may also affect intratumoral delivery ofconcurrently administered chemotherapy. Nonetheless, combin-ing chemotherapy and bevacizumab can lead to improvedresponse rates, progression-free survival, and sometimes, overallsurvival, the extent of which can partly depend on the chemo-therapy backbone. A rational, complementing chemotherapypartner for combination with bevacizumab would not onlyreduce HIF1a to overcome hypoxia-induced resistance, but alsoimprove tumor perfusion tomaintain intratumoral drug delivery.Here, we evaluated bevacizumab and CRLX101, an investigation-

al nanoparticle–drug conjugate containing camptothecin, in pre-clinical mouse models of orthotopic primary triple-negativebreast tumor xenografts, including a patient-derived xenograft.We also evaluated long-term efficacy of CRLX101 and bevacizu-mab to treat postsurgical, advanced metastatic breast cancer inmice. CRLX101 alone and combined with bevacizumab washighly efficacious, leading to complete tumor regressions, reducedmetastasis, and greatly extended survival of mice with metastaticdisease. Moreover, CRLX101 led to improved tumor perfusionand reduced hypoxia, as measured by contrast-enhanced ultra-sound and photoacoustic imaging. CRLX101 durably suppressedHIF1a, thus potentially counteracting undesirable effects of ele-vated tumor hypoxia caused by bevacizumab. Our preclinicalresults show pairing a potent cytotoxic nanoparticle chemother-apeutic that complements and improves concurrent antiangio-genic therapy may be a promising treatment strategy for meta-static breast cancer. Cancer Res; 76(15); 4493–503. �2016 AACR.

IntroductionPatients with triple-negative breast cancer (TNBC) have the

highest risk of recurrence and metastasis (1). Various targetedtherapies have been investigated, but as yet, none are currentlyapproved. One notable example is bevacizumab, a VEGF-tar-geting antibody, which was granted accelerated FDA approvalwith weekly paclitaxel for first-line treatment of metastaticbreast cancer, but this approval was later revoked after fol-low-up phase III clinical trials with different chemotherapy

backbones showed less impressive benefits in progression-freesurvival (PFS; ref. 2). More recently, however, there has beenrenewed interest in reconsidering bevacizumab for the treat-ment of breast cancer based on several phase III clinical trialresults, including one in the neoadjuvant and adjuvant setting(NSABP-B40) and two in the maintenance metastatic setting(IMELDA and TANIA; refs. 3–5). Importantly, a number of trialresults and meta-analyses suggest the extent of beneficial effect(and associated toxicities) of adding bevacizumab to chemo-therapy may depend on the concurrent chemotherapy regimenused (2, 4, 6, 7). Thus, an appropriate chemotherapy partner,one with better efficacy, manageable toxicity, and complemen-tary modes of action, may be critical to gaining optimal benefitout of adding bevacizumab (and vice versa). One promisinginvestigational chemotherapy drug is a nanoparticle–drug con-jugate (NDC) known as CRLX101, which contains the payloadcamptothecin, a highly potent cytotoxic agent that inhibitstopoisomerase-I (8, 9).

Camptothecin showed impressive preclinical antitumoractivity but had low solubility,metabolic instability of the activelactone form, rapid renal clearance, and severe toxicities, whichresulted in disappointing results in early-phase clinical trials(10, 11). A number of analogues were thus developedto improve the solubility of camptothecin, including the

1Biological Sciences Platform, Sunnybrook Research Institute, Tor-onto, Ontario, Canada. 2Physical Sciences Platform, SunnybrookResearch Institute, Toronto, Ontario, Canada. 3Cerulean Pharma Inc.,Waltham,Massachusetts. 4Department ofMedical Biophysics, Univer-sity of Toronto, Toronto, Ontario, Canada.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Robert S. Kerbel, Biological Sciences Platform, Sunny-brook Research Institute, 2075 Bayview Avenue S217, Toronto, Ontario M4N3M5, Canada. Phone: 416-480-5711; Fax: 416-480-5884; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-3435

�2016 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst June 20, 2016; DOI: 10.1158/0008-5472.CAN-15-3435

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subsequently approved drugs, topotecan (Hycamtin, Glaxo-SmithKline) and irinotecan (Camptosar, Pfizer; refs. 9, 12).Currently, there is no topoisomerase-I inhibitor approved forbreast cancer treatment. Although both irinotecan and topote-can have been evaluated in phase II trials for metastatic breastcancer, they have not been approved as treatments for thatindication given the associated high-grade toxicities experiencedby patients for a modest benefit gain (13–17). Nonetheless,lessons learned from early trials using camptothecin (18), topo-tecan, and irinotecan suggest camptothecin analogues areindeed active in the treatment of breast cancer, but furtherclinical development requires better drug solubility, improvedlactone ring stabilization, and less systemic toxicity. Notably, arecent phase III trial (BEACON) evaluated etirinotecan pegol, anew formulation of irinotecan, in patients with recurrent ormetastatic breast cancer (19). It showed single-agent PFS ben-efits similar to treatment of physician's choice. Moreover,subgroup analyses showed etirinotecan pegol significantly pro-longed overall survival in patients with a history of brain or livermetastases, and with two or more sites of disease (19). As anNDC of camptothecin, CRLX101 was designed to have superiorsolubility and stabilization of the lactone ring, as well as favor-able safety and pharmacokinetics in patients (20, 21).

CRLX101 is composed of a cyclodextrin-containing polymerconjugated to camptothecin. In mice, the cyclodextrin polymeritself has no observable side effects or antitumor efficacy whentested up to 240 mg/kg (22). CRLX101 was designed to haveimproved accumulation within tumors by the enhanced per-meability and retention (EPR) effect and thus reduced systemicexposure and toxicity (8, 20). The drug has been administeredto more than 300 patients to date and appears to be generallywell tolerated, achieving an overall response rate of 16% in 19patients in a phase II clinical trial of platinum-resistant ovariancancer as a monotherapy (23) and 23% in 22 patients in aphase I/II study of metastatic renal cell carcinoma in combi-nation with bevacizumab (21).

We recently reported that CRLX101 plus bevacizumab resultedin synergistic antitumor efficacy in a preclinical model ofadvanced, metastatic ovarian cancer (23). CRLX101 was alsoshown to effectively and durably suppress elevated hypoxia-induced upregulation of hypoxia-inducible factor-1a (HIF1a)following therapy with bevacizumab, thus downregulatingexpression of downstream HIF1a-regulated markers, such ascarbonic anhydrase IX (CAIX; ref. 23), and blocking the inductionof cancer stem cells (24).

Here, we evaluated the combination of CRLX101 and bev-acizumab in long-term therapy experiments of orthotopic pri-mary TNBC xenografts either derived from tumor tissue frag-ment implantation of a patient-derived xenograft (PDX) calledHCI-002 (25, 26) or cell injection of LM2-4, a luciferase-taggedvariant of the established cell line MDA-MB-231 serially select-ed in vivo for aggressive spontaneous metastatic properties (27).We also evaluated long-term efficacy of CRLX101 and bevaci-zumab in a preclinical model of postsurgical, advanced met-astatic TNBC (28), a model that recapitulates the morechallenging clinical treatment of systemic metastatic disease(28, 29). We report that CRLX101 alone and in combinationwith bevacizumab is an effective treatment for advanced met-astatic TNBC in these mouse models and provide new mech-anistic results to help explain this encouraging antitumoractivity.

Materials and MethodsCell line and patient-derived tumor fragments

MDA-MB-231/LM2-4luc16þ (LM2-4) is a highly aggressivevariant of MDA-MB-231 [parental line obtained from Jeff Lem-ontt (Genzyme Corp., Boston, MA) in 2000] and maintained inRPMI1640 supplemented with 5% FBS (27). LM2-4 was lastauthenticated in 2013 by Genetica DNA Laboratories (a LabCorpSpecialty Testing Group) using analytical procedures for DNAextraction, PCR, and capillary electrophoresis on a 3130xlGeneticAnalyzer (Applied Biosystems). The 13 core CODIS short tandemrepeat (STR) loci plus PENTA E and PENTA D, and the gender-determining locus, amelogenin, were analyzed using the com-mercially available PowerPlex 16 HS Amplification Kit (PromegaCorporation) and GeneMapper ID v3.2.1 software (Applied Bio-systems)with appropriate positive andnegative controls. Authen-tication of cell lines is confirmed by entering the STR DNA profileof each tested cell line into known repository cell line databases;authentication is defined as having a percent match with thereference STR profile �80% when using the ANSI/ATCC guide-lines (ASN-0002-2011) orhaving a "unique" STRDNAprofile (nomatches found) for "in-house" cell lines not distributed by anycell line repository.

PDX HCI-002 tumor fragments were generously provided byDr. Alana Welm (University of Utah, Salt Lake City, UT). Tumorfragments were originally obtained from a patient with TNBC andverified by histology to have retained morphology and character-istics as matched patient samples (25, 26). HCI-002 tumor frag-ments were serially passaged in SCID mice, and only earlypassages were used for all experiments.

Primary tumor implantation into the mammary fat padEight-week-old female YFP-SCID mice were bred in-house.

Procedures involving animals and their care were conducted instrict conformity with the guidelines of Sunnybrook HealthScience Centre (Toronto, Ontario, Canada) and the CanadianCouncil of Animal Care. Mammary fat pad injections of LM2-4(5� 105 cells) and tumor fragment implantations (pieces of 2–5mm3) of HCI-002 were carried out as described previously (26,27). Mice were randomized by tumor volume prior to treatmentinitiation. All doses of CRLX101 are reported as camptothecin-equivalent doses.

Contrast-enhanced ultrasound photoacoustic imagingIn vivo imaging of primary tumors was conducted using a

commercially available high-frequency laser-integrated ultra-sound system (VevoLAZR, VisualSonics), which allowed for bothphotoacoustic and contrast-enhanced imaging (30). Contrastdata were quantified with VevoCQ using time intensity curves(TIC) generated from the wash-in of microbubbles. Two para-meters are taken from the TIC: peak enhancement and wash-inrate. Dual-wavelength PA imaging was used for real-time mon-itoring and calculation of oxygen saturation with the VevoLABsoftware.

Postsurgical, advanced metastatic breast cancer therapy modelLM2-4 cells were orthotopically implanted into the right mam-

mary fat pad and resected when tumors reached 400 to 500mm3,as described in ref. 27. Following primary tumor resection, distantvisceralmetastases can be detected by total bodybioluminescenceimaging using an IVIS200 Xenogen. Mice with metastatic disease

Pham et al.

Cancer Res; 76(15) August 1, 2016 Cancer Research4494




were randomized on the basis of metastatic load and locationbefore therapies were initiated (27, 29).

Statistical analysisResults were reported as mean � SD or SEM, as indicated.

Tumor growth curveswere reported asmean� SD. Survival curveswere plotted by the method of Kaplan and Meier and tested forsurvival differences with the log-rank test. Statistical significancewas assessed by one-way ANOVA (Kruskal–Wallis test with Dunnpost hoc) or t test (Mann–Whitney, two-tailed) using GraphPadPrism 4 (P < 0.05 was used as the threshold of statisticalsignificance).

ResultsCRLX101 treatment can lead to complete primary tumorregressions

The MTD (8 mg/kg i.p. once weekly) of CRLX101 was previ-ously established for SCID mice and is well tolerated even whencombined with bevacizumab for long-term studies (23). In aprimary tumor model derived from cell injection of LM2-4,CRLX101 caused rapid and durable tumor regressions (Fig. 1).Lower doses of CRLX101monotherapy resulted in slower rates oftumor shrinkage compared with 8 mg/kg, resulting in smalltumors that still eventually caused hind leg mobility impairment(smaller tumor volumes compared with vehicle but not statisti-cally significant). Although the addition of bevacizumab to 2mg/kg CRLX101 showed a trend for tumor growth delay, this was notstatistically significant compared with 2 mg/kg CRLX101 mono-therapy. Nonetheless, the addition of bevacizumab to 4 mg/kgCRLX101 significantly improved antitumor efficacy (P < 0.05compared with 4 mg/kg CRLX101 alone), showing improvedtumor growth suppression and shrinkage despite bevacizumabalone having no antitumor efficacy. However, despite early tumorsuppression and continued therapy, bioluminescence imaging ofmice treated with CRLX101 4 mg/kg plus bevacizumab showedthese tumors eventually continued to grow. Notably, while micewere on-therapy continuously for 6 months, 8 mg/kg CRLX101quickly and dramatically shrank established primary tumors,resulting in complete regressions (P < 0.001), with 3 of 5 miceessentially cured by 6 months of therapy (no observable biolu-minescence signals). Similarly, 8 mg/kg CRLX101 plus bevacizu-mab also led to complete primary tumor regressions (P < 0.001compared with vehicle, no statistically significant difference com-pared with 8 mg/kg CRLX101 monotherapy). Four of 5 micetreatedwith 8mg/kgCRLX101 andbevacizumabdidnot have anyresidual disease or regrowth even after being off-therapy for anadditional 4 months (Supplementary Fig. S1).

Compared with primary tumors derived from LM2-4, PDXHCI-002 tumors were highly vascular. In contrast to bevacizu-mab's absence of efficacy when treating LM2-4 primary tumors,bevacizumab led to significantly delayed HCI-002 tumor growthwhile mice were on-therapy continuously for 5 months (P < 0.05compared with vehicle; Fig. 2). Nonetheless, both doses ofCRLX101 durably suppressed tumor growth (P ¼ 0.002 for 4mg/kg and P < 0.001 for 8 mg/kg CRLX101). Notably, thecombination of either dose with bevacizumab significantlyimproved the antitumor efficacy compared with either drug alone(P < 0.001 compared with monotherapy). In this PDX model,despite tumor growth suppression or regression while mice wereon-therapy, when all therapies were stopped after 5 months,

tumor regrowth did occur but was delayed longer if mice wereoriginally treated with a higher dose of CRLX101 or with con-current bevacizumab.

In separate experiments, tumors from mice treated for twoweeks were evaluated for changes in microvessel density (MVD)and any corresponding changes in HIF1a (Fig. 3 and Supple-mentary Figs. S2 and S3). Bevacizumab reduced the number oftumor vessels by approximately 50% in LM2-4 tumors (Fig. 3A)and approximately 65% in HCI-002 tumors (SupplementaryFig. S2). This reduced MVD by bevacizumab monotherapycorresponded to an increase in HIF1a protein levels (Fig.3B), which was confirmed by similar trends observed usingimmunohistochemical staining of HIF1a and CAIX, a down-streammarker of HIF1a activity (Fig. 3C and D; ref. 23). In vitro,higher doses of CRLX101 showed a trend (although not sta-tistically significant) of decreasing VEGF levels (SupplementaryFig. S3). However, in vivo, CRLX101 monotherapies did notsignificantly decrease MVD even though CRLX101 maintainedlow levels of HIF1a protein levels. Although CRLX101 mono-therapies did not change the extent of tumor cell proliferation(Fig. 3E), there was a significant increase in apoptosis at alldoses administered (Fig. 3F). Tumors from mice treated withconcurrent bevacizumab and CRLX101 showed decreased MVDto levels similar to bevacizumab monotherapy. Nonetheless,concurrent CRLX101 was able to suppress any bevacizumab-induced increases in HIF1a. The reduced MVD observed forcombination-treated tumors did not affect tumoral accumula-tion of CRLX101 (Supplementary Fig. S2). Interestingly, con-current bevacizumab did not further increase the extent ofapoptosis observed compared with CRLX101 monotherapies.

We next evaluated whether the reduction in MVD by bev-acizumab correlated with a change in functional tumor perfu-sion using in vivo contrast-enhanced ultrasound (CEUS) imag-ing, which uses gas-filled microbubbles to noninvasively mon-itor changes in blood flow and volume within tumors follow-ing therapy (Fig. 4; ref. 30). Tumors from mice treated withvehicle or bevacizumab alone showed decreased tumor bloodvolume and flow rate over the course of two weeks of therapy.In contrast, tumors from mice treated with CRLX101 mono-therapy showed greatly improved perfusion, whereas perfusionin tumors treated with the combination remained unchanged.Parametric maps showing regions of high, low, or no perfusionconfirm that tumors from mice treated with CRLX101 hadsmaller necrotic cores compared with vehicle and bevacizumabtreatment. In the combination group, while a less perfused corewas present, the surrounding "viable rim" remained well per-fused. Similarly, CEUS imaging of PDX tumors showedCRLX101 maintained higher tumor perfusion and reducedtumor hypoxia, also confirmed by pimonidazole staining (Sup-plementary Fig. S4).

In addition tomeasuring perfusion using CEUS, photoacousticimaging was used to measure oxygen saturation (Fig. 4; ref. 30).Short laser pulses are directed at the tumor, generating thermo-elastic expansion to create acoustic waves detected by an ultra-sound transducer. Different absorption spectra of deoxygenatedand oxygenated hemoglobin are then used to noninvasivelyestimate the spatial distribution of oxygen saturation in vivo(30). Consistent with CEUS data showing improved perfusionin CRLX101-treated tumors, these tumors had relatively highertissue oxygenation levels compared with vehicle- and bevacizu-mab-treated tumors (Fig. 4C).

Bevacizumab with CRLX101 to Treat Metastatic TNBC

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CRLX101 caused regression of orthotopic primary breast tumors. A, SCIDmice bearing LM2-4 tumors were treated with CRLX101 monotherapies (2, 4, and 8mg/kg)and combined with bevacizumab (Bev). Therapy was started day 18 after tumor cell injection when tumors were approximately 200 to 250 mm3. Therapywas stopped after 6 months for mice treated with 8 mg/kg CRLX101 monotherapy or combination with bevacizumab. All other mice were treated until endpoint.Error bars, SD. n ¼ 5 mice per group. B, all doses of CRLX101 significantly extended survival of mice, whereas bevacizumab monotherapy did not. ns, notsignificant.

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CRLX101 suppressed tumor growth of the PDX,HCI-002.A,SCIDmice bearingorthotopicHCI-002primary tumorswere treatedwith 4and8mg/kgCRLX101 as singleagent or combined with bevacizumab (Bev). Therapy was started day 51 after implantation when tumors were approximately 200 to 250 mm3 and stoppedafter 5 months. Error bars, SD. n ¼ 5 mice per group. B, all therapies, including bevacizumab monotherapy, significantly extended survival of mice.






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CRLX101 therapy maintained low levels of HIF1a and significantly increased tumor cell apoptosis. After two weeks of therapy, tumors were stained for CD31 (A),processed for Western blot analysis to quantify HIF1a protein levels (n ¼ 3 tumors/group; B), and stained for HIF1a (C) and CAIX (D). Bev, bevacizumab. Ki67 (E)and cleaved caspase-3 stainings (F) were used to assess changes in tumor cell proliferation and apoptosis, respectively. Error bars, SEM. � , P < 0.05;�� , P < 0.01 compared with vehicle. n ¼ 5 tumors per group for all stainings.

Pham et al.





Improved perfusion with CRLX101 treatment suggests amore functional tumor vasculature, as results from Fig. 3Ashow CRLX101 does not result in an increase in the numberof vessels. Tumor cells rapidly growing within a confined spacecauses compression (or "solid stress") of tumoral blood vessels(31, 32). One possible explanation for the overall improvedperfusion following CRLX101 is the drug causes extensivetumor cell apoptosis, which may then relieve compression oftumor blood vessels. To assess whether this may be the case, weevaluated whether CRLX101 resulted in more vessels with anopen lumen, which would allow better blood flow within thetumor (Fig. 4D). Indeed, we observed that CRLX101 mono-therapy resulted in a higher proportion of tumor blood vesselswith open lumen compared with both vehicle and bevacizu-mab treatment. It is worth noting that in tumors treated withthe drug combination, overall number of tumor vessels wasreduced, but more of the remaining vessels had open lumens.These results were similarly confirmed in PDX tumors (Sup-plementary Fig. S5).

To obtain confirmation that CRLX101 relieves compressionof tumor blood vessels by killing tumor cells, we evaluatedwhether tumor cell-packing density, that is, the number of cellsfound within a given area, changed following therapy (Fig.5A). Although the average number of cells present within animaged field was similar between vehicle- and bevacizumab-

treated tumors, all doses of CRLX101 caused a significantreduction in cell-packing density. Fewer tumor cells denselypacked within a tumor thus may result in fewer compressedtumor blood vessels and hence improved perfusion andoxygenation.

Paradoxically, a more functional vasculature could result in ahigher degree of tumor cell dissemination and metastasis todistant organs, such as the lungs. We therefore analyzed lungsfrom mice still bearing primary tumors for the presence ofmicrometastases or tumor cells that previously shed from theprimary tumor and seeded in the lungs (Fig. 5B).Mice treatedwithbevacizumab monotherapy showed more micrometastases thatwere also larger in cluster size than those present in vehicle-treatedmice. This is consistentwith someprevious preclinical reports thatantiangiogenic agents used as monotherapies may cause anincrease in metastasis and promote disease progression in micedespite an initial antitumor effect (33, 34). In stark contrast, micetreatedwithCRLX101 (particularly at higher doses) either showedno micrometastases or only singly dispersed tumor cells withinthe lungs, evenwhen combinedwith bevacizumab. CRLX101wastherefore able to counteract potential prometastatic effects ofbevacizumab. This effect is similar to published data wherepaclitaxel counteracted the proinvasive and prometastatic effectsof DC101, the VEGFR-2 antibody (20), and metronomic topo-tecan reduced metastatic spread elicited by sunitinib (35).

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CEUS and photoacoustic imaging of primary LM2-4 tumors.A, changes in blood flowvolume (peak enhancement) and blood flow rate (wash-in rate)weremeasuredusing CEUS imaging. Bev, bevacizumab. Photoacoustic imaging was used to monitor changes in average tumor oxygen saturation. B, representative CEUSimages of one tumor from each therapy group overlaid with parametric color mapping to show areas of high (red), low (blue), or no (black) perfusion. C,representative photoacoustic images of the same tumors overlaidwith colormapping to indicate areas of high (red) and low (blue) tissue oxygenation.D, the numberof vessels with open lumens was counted and reported as a percentage of the average number of vessels present per field. Vessels were counted in five randomlyselected fields from five tumors per group (bracketed numbers, average number of vessels counted per field). Error bars, SEM. n ¼ 4 to 6 tumors per group.� , P < 0.05; #, P ¼ 0.07; �� , P < 0.01 compared with vehicle.






CRLX101 in a postsurgical metastatic breast cancer modelshrinks existing metastases and prevents the emergence of newmetastases

Given the potency of CRLX101 in both primary tumor modelsand evidence showing CRLX101was able to prevent formation ofmicrometastases, CRLX101 alone and with bevacizumab wereevaluated in a postsurgical model of advanced metastatic breastcancer. Following surgical resection of established primary LM2-4tumors, distant metastases were allowed to develop before treat-ment was initiated. Metastases were monitored by biolumines-cence imaging and can manifest as lymph node metastases(causing mobility issues), lung metastases (leading to laboredbreathing), primary tumor regrowth or local metastases at thesurgical site, as well as liver metastases and ascites (causingdistended abdomens). Given the inherent variability of whenand where metastases appear, every treatment group had ran-domized cohorts of mice with apparent distant metastases, localmetastases or regrowths, as well as mice with no signs of metas-tases at the start of therapy (Fig. 6). Within 1.5months of therapy,all mice treated with vehicle and bevacizumab monotherapysuccumbed to disease, even if no apparent metastases wereobserved at the start of treatment. In contrast, bioluminescenceimaging showed that in mice that had apparent metastases at thestart of therapy, treatment with CRLX101 or CRLX101 plusbevacizumab caused these existing metastases to regress. In micewith no apparent metastases at the start of treatment, CRLX101alone or combined with bevacizumab was able to prevent theemergence of new metastases both while mice were on-therapy(for 7 months) and then off-therapy (for 2 months). It should benoted, however, that if mice had a very heavy metastatic load inthe lungs or ascites at the start of therapy, CRLX101 therapy wasnot as effective; we hypothesize this may be due to the need for

some initial time for CRLX101 to accumulate within tumor cellsand release its drug payload.

CRLX101 thus significantly extended the survival of micewith metastatic disease (Fig. 6B). Although mice treated withvehicle or bevacizumab succumbed to metastases after 4 to 6weeks, survival of mice treated with 4 mg/kg CRLX101 mono-therapy was extended to 15 weeks. CRLX101 at 4 mg/kg plusbevacizumab was effective at durably suppressing metastasis.Notably, MTD CRLX101 was highly efficacious, so much so thatany added benefit of combining it with bevacizumab was notdetected. Upon necropsy, 3 of 9 mice (treated with 4 mg/kgCRLX101 plus bevacizumab), 5 of 8 mice (treated with 8 mg/kgCRLX101 alone), and 6 of 9 mice (treated with 8 mg/kgCRLX101 plus bevacizumab) were still alive and free of mac-roscopic metastases at the end of the experiment (7 months on-therapy and 2 months off-therapy).

DiscussionWe recently reported metronomic topotecan plus pazopanib

delayed tumor growth of primary TNBC tumors and prolongedsurvival ofmice with advancedmetastatic disease (36). Given thispromising therapy of combining a topoisomerase-I inhibitor(topotecan) and an antiangiogenic drug (pazopanib) for treatingTNBC in mice, here, we evaluated CRLX101 and bevacizumab.There are several reasons why this alternative drug combinationmay be superior to that of topotecan and pazopanib. Single-agenttopotecan had no antitumor efficacy and resulted in increasedHIF1a. In contrast, single-agent CRLX101 is potently effective innot only delaying tumor growth, but causing marked and sus-tained tumor regressions. Furthermore, CRLX101 whether aloneor combined with bevacizumab durably suppressed HIF1a.

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Figure 5.

CRLX101 reduced tumor cell density and resulted in fewer, smaller lungmicrometastases.A, average number of nuclei countedwithin an imaged field (n¼ 5 tumors/group). Error bars, SEM. � , P < 0.05; �� , P < 0.01 compared with vehicle. Bev, bevacizumab. B, lungs from mice still bearing primary tumors were collectedafter twoweeks of therapy and stained for vimentin, used to specifically stain for human tumor cells. Lungswere classified as havingno vimentin staining (and thus notumor cells) present, only singly dispersed tumor cells, small clusters (<20 cells), or large clusters (>20 cells). For each therapy group, lungs from 5 mice wereevaluated. Five serial sections were assessed for each lung, sectioned with 100 mm separation. Scale bar, 150 mm.


Pham et al.




Finally, one important concern of combining topotecan andpazopanib is the tolerability and toxicity of both drugs, especiallythe combination, in the clinic (13, 14, 37). In contrast, currentclinical data suggest that CRLX101 is tolerable in combinationwith standard doses of bevacizumab in patients (38, 39). Thecombination of CRLX101 andbevacizumabwould thus appear tobe more promising considering its lesser toxicity and betterpreclinical efficacy, an improved therapeutic index comparedwith the pazopanib/topotecan combination.

Limited efficacy successes of approved VEGF pathway–target-ing antiangiogenic drugs may be due to several possible factors,including reduced intratumoral delivery of concurrently admin-istered drugs, such as chemotherapy, as well as elevated hypoxia(and hence HIF1a), which contributes to resistance and maypromote metastases (24, 40–42). Given these considerations, acomplementary chemotherapy partner for combination with anantiangiogenic agentwould be ahighly potent agent that is able toimprove tumor perfusion and reduce HIF1a without increasingtumor dissemination and/or metastases. Here, we showed thatCRLX101 may be such a drug.

A number of strategies have been proposed, including vesselnormalization (43) and "vascular promotion" therapy (31, 44),to improve intratumoral chemotherapy drug delivery. Alterna-tively, impaired perfusion, whether caused by antiangiogenictherapy or due to mechanical factors, such as tumor cells com-

pressing vessels (31, 32), would be expected to reduce intratu-moral delivery of chemotherapy drugs, such as CRLX101, in solidtumors (8, 45). However, CRLX101 maintained tumor perfusiondespite concurrent antiangiogenic therapy.We report here that thepotent antitumor activity of CRLX101 alleviated the solid stress byrapidly targeting tumor cells, in effect leading to major tumorregressions and decompressing tumor blood vessels to improvetumor perfusion ["tumor priming" (46)]. We and others havereported that metronomic dosing of a gemcitabine prodrugresulted in increased perfusion (47, 48). Paclitaxel-loadedtumor-penetratingmicroparticles have also been used to enhancesiRNA delivery into solid tumors (46). Clinically, a similar effectwas observed, where weekly paclitaxel reduced interstitial fluidpressure and improved tumor oxygenation (49). Interestingly,results from these studies and our work here with CRLX101suggest chronic exposure of tumor cells to a cytotoxic drug, eitherby administering chemotherapy drugs in a frequent, metronomicfashion, or using drug formulations with intrinsically long half-lives, such as CRLX101, may be required to maintain sufficientdurable tumor cell kill to persistently reduce compression oftumoral vessels and thus potentially improve perfusion. Thisimprovement in tumor perfusion significantly reduces tumorhypoxia, which contributes to the ability of CRLX101 to effec-tively anddurably suppressHIF1a. Even thoughHIF1a is elevatedby tumor hypoxia, reduction in tumor hypoxia by improving

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Figure 6.

CRLX101 prevented the emergence of new metastases and caused regression of existing metastases, thus greatly extending mice survival. A, bioluminescenceimages of 7 to 9 mice per group. All therapies were initiated 25 days after primary tumor resection and stopped after 7 months. B, all doses of CRLX101significantly extended survival of mice, whereas bevacizumab monotherapy did not. ns, not significant.






tumor perfusion alonemay not completely account for theHIF1asuppression by CRLX101. Rapisarda and colleagues have shownin vitro that metronomic topotecan was able to suppress HIF1aprotein accumulation even when cells were maintained in nor-moxic conditions (50), suggesting HIF1a inhibition may occureven in the absence of elevated hypoxia. Taken together, we haveshown that CRLX101maintains or improves tumor perfusion andis able to durably suppress HIF1a protein levels even in thepresence of bevacizumab, where tumors are more hypoxic, thusmaking CRLX101 an effective chemotherapy partner to pair withan antiangiogenic agent, such as bevacizumab.

One limitation of our results is the use of two primary tumormodels and one metastatic model. Nevertheless, given our pre-clinical results of CRLX101 showing potent antitumor activity,particularly when treating advanced metastatic TNBC, and itscomplementary mode of action for combining with bevacizu-mab, CRLX101 plus bevacizumab may be a promising treatmentstrategy for breast cancer and other solid tumors.

Disclosure of Potential Conflicts of InterestC.G. Peters is a scientist at Cerulean Pharma. S. Eliasof has ownership interest

(including patents) in Cerulean Pharma, Inc. F.S. Foster reports receiving acommercial research grant from and is a consultant/advisory boardmember forVisualSonics. R.S. Kerbel reports receiving other commercial research supportfrom Cerulean and is a consultant/advisory board member for Cerulean andTriphase Accelerator. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: E. Pham, M. Yin, L. Jayaraman, R.S. KerbelDevelopment of methodology: E. Pham, M. Yin, L. Jayaraman, E. Rohde,D. Lazarus, F.S. Foster

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Pham, M. Yin, C.G. Peters, C.R. Lee, S. Man,E. Rohde, R.S. KerbelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Pham, M. Yin, C.G. Peters, L. Jayaraman,R.S. KerbelWriting, review, and/or revision of the manuscript: E. Pham, M. Yin,C.G. Peters, P. Xu, L. Jayaraman, E. Rohde, D. Lazarus, S. Eliasof, F.S. Foster,R.S. KerbelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Pham, C.R. Lee, S. Man, A. ChowStudy supervision: L. Jayaraman, S. Eliasof, F.S. Foster, R.S. KerbelOther (bioanalysis of study samples): D. Brown

AcknowledgmentsThe authors sincerely thank Dr. Marta Paez-Ribes for her assistance with

animal surgeries and technical advice. The authors also thank Cassandra Chengfor outstanding administrative assistance and their student volunteer, TaylorVanVeen. Virtual slide scanning and image analysis consultation was providedby Taha Rashed at the Biomarker Imaging Research Laboratory (BIRL, Sunny-brook Research Institute) under the direction of Dr. Martin Yaffe. HCI-002 wasgenerously provided by Dr. Alana Welm.

Grant SupportThis work was supported by grants from the Canadian Institutes of Health

Research (CIHR) and the Canadian Breast Cancer Foundation (R.S. Kerbel).Financial support was also provided by Cerulean Pharma Inc. R.S. Kerbel andF.S. Foster were recipients of Tier I Canada Research Chairs during the course ofthese studies. E. Pham is a recipient of a CIHR Post-Doctoral Fellowship Award.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 24, 2015; revised March 11, 2016; accepted March 23,2016; published OnlineFirst June 20, 2016.

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2016;76:4493-4503. Published OnlineFirst June 20, 2016.Cancer Res Elizabeth Pham, Melissa Yin, Christian G. Peters, et al. Metastatic Triple-Negative Breast Cancer

Drug Conjugate, in Treatment of−Investigational Nanoparticle Preclinical Efficacy of Bevacizumab with CRLX101, an

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