Skip to main content

CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, targets cancer stem cells and impedes resistance to antiangiogenic therapy in mouse models of breast cancer


Antiangiogenic therapies inhibit the development of new tumor blood vessels, thereby blocking tumor growth. Despite the advances in developing antiangiogenic agents, clinical data indicate that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenics, which is attributed in part to the induction of intra-tumoral hypoxia and stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. Here, we tested whether inhibiting HIF-1α can reverse the stimulatory effects of antiangiogenic-induced hypoxia on breast CSCs. Breast cancer cells grown under hypoxic conditions were treated with the dual topoisomerase-1 (TOPO-1) and HIF-1α inhibitor camptothecin and assessed for their CSC content. In a preclinical model of breast cancer, treatment with bevacizumab was compared to the combination treatment of bevacizumab with CRLX101, an investigational nanoparticle-drug conjugate with a camptothecin payload or CRLX101 monotherapy. While exposure to hypoxia increased the number of breast CSCs, treatment with CPT blocked this effect. In preclinical mouse models, concurrent administration of CRLX101 impeded the induction of both HIF-1α and CSCs in breast tumors induced by bevacizumab treatment. Greater tumor regression and delayed tumor recurrence were observed with the combination of these agents compared to bevacizumab alone. Tumor reimplantation experiments demonstrated that the combination therapy effectively targets the CSC populations. The results from these studies support the combined administration of dual TOPO-1- and HIF-1α-targeted agents like CRLX101 with antiangiogenic agents to increase the efficacy of these treatments.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6(4):273–286. doi:10.1038/nrd2115

    Article  CAS  PubMed  Google Scholar 

  2. Mackey JR, Kerbel RS, Gelmon KA, McLeod DM, Chia SK, Rayson D, Verma S, Collins LL, Paterson AH, Robidoux A, Pritchard KI (2012) Controlling angiogenesis in breast cancer: a systematic review of anti-angiogenic trials. Cancer Treat Rev. doi:10.1016/j.ctrv.2011.12.002

    PubMed  Google Scholar 

  3. Rose S (2011) FDA pulls approval for avastin in breast cancer. Cancer Discov 1 (7):OF1–2. doi:10.1158/

  4. Hayes DF (2011) Bevacizumab treatment for solid tumors: boon or bust? JAMA 305(5):506–508. doi:10.1001/jama.2011.57

    Article  CAS  PubMed  Google Scholar 

  5. Rapisarda A, Melillo G (2012) Overcoming disappointing results with antiangiogenic therapy by targeting hypoxia. Nat Rev Clin Oncol 9(7):378–390. doi:10.1038/nrclinonc.2012.64

    Article  CAS  PubMed  Google Scholar 

  6. Wilson WR, Hay MP (2011) Targeting hypoxia in cancer therapy. Nat Rev Cancer 11(6):393–410. doi:10.1038/nrc3064

    Article  CAS  PubMed  Google Scholar 

  7. Hill RP, Marie-Egyptienne DT, Hedley DW (2009) Cancer stem cells, hypoxia and metastasis. Semin Radiat Oncol 19(2):106–111. doi:10.1016/j.semradonc.2008.12.002

    Article  PubMed  Google Scholar 

  8. Koch U, Krause M, Baumann M (2010) Cancer stem cells at the crossroads of current cancer therapy failures–radiation oncology perspective. Semin Cancer Biol 20(2):116–124. doi:10.1016/j.semcancer.2010.02.003

    Article  PubMed  Google Scholar 

  9. Conley SJ, Gheordunescu E, Kakarala P, Newman B, Korkaya H, Heath AN, Clouthier SG, Wicha MS (2012) Antiangiogenic agents increase breast cancer stem cells via the generation of tumor hypoxia. Proc Natl Acad Sci USA 109(8):2784–2789. doi:10.1073/pnas.1018866109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA (1966) Plant Antitumor Agents. I. The Isolation and Structure of Camptothecin, a Novel Alkaloidal Leukemia and tumor inhibitor from Camptotheca acuminata1,2. J Am Chem Soc 88(16):3888–3890

    Article  CAS  Google Scholar 

  11. Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, Melillo G (2002) Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 62(15):4316–4324

    CAS  PubMed  Google Scholar 

  12. Rapisarda A, Zalek J, Hollingshead M, Braunschweig T, Uranchimeg B, Bonomi CA, Borgel SD, Carter JP, Hewitt SM, Shoemaker RH, Melillo G (2004) Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts. Cancer Res 64(19):6845–6848. doi:10.1158/0008-5472.can-04-2116

    Article  CAS  PubMed  Google Scholar 

  13. Kehrer DF, Soepenberg O, Loos WJ, Verweij J, Sparreboom A (2001) Modulation of camptothecin analogs in the treatment of cancer: a review. Anticancer Drugs 12(2):89–105

    Article  CAS  PubMed  Google Scholar 

  14. Svenson S, Wolfgang M, Hwang J, Ryan J, Eliasof S (2011) Preclinical to clinical development of the novel camptothecin nanopharmaceutical CRLX101. J Control Release 153(1):49–55. doi:10.1016/j.jconrel.2011.03.007

    Article  CAS  PubMed  Google Scholar 

  15. Eliasof S, Lazarus D, Peters CG, Case RI, Cole RO, Hwang J, Schluep T, Chao J, Lin J, Yen Y, Han H, Wiley DT, Zuckerman JE, Davis ME (2013) Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle. Proc Natl Acad Sci USA 110(37):15127–15132. doi:10.1073/pnas.1309566110

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10(6):515–527. doi:10.1016/j.ccr.2006.10.008

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Hu Y, Smyth GK (2009) ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347(1–2):70–78. doi:10.1016/j.jim.2009.06.008

    CAS  PubMed  Google Scholar 

  18. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567. doi:10.1016/j.stem.2007.08.014

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, Hur MH, Diebel ME, Monville F, Dutcher J, Brown M, Viens P, Xerri L, Bertucci F, Stassi G, Dontu G, Birnbaum D, Wicha MS (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 69(4):1302–1313. doi:10.1158/0008-5472.can-08-2741

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Puppo M, Battaglia F, Ottaviano C, Delfino S, Ribatti D, Varesio L, Bosco MC (2008) Topotecan inhibits vascular endothelial growth factor production and angiogenic activity induced by hypoxia in human neuroblastoma by targeting hypoxia-inducible factor-1alpha and -2alpha. Mol Cancer Ther 7(7):1974–1984. doi:10.1158/1535-7163.mct-07-2059

    Article  CAS  PubMed  Google Scholar 

  21. Rapisarda A, Uranchimeg B, Sordet O, Pommier Y, Shoemaker RH, Melillo G (2004) Topoisomerase I-mediated inhibition of hypoxia-inducible factor 1: mechanism and therapeutic implications. Cancer Res 64(4):1475–1482

    Article  CAS  PubMed  Google Scholar 

  22. Burkitt K, Chun SY, Dang DT, Dang LH (2009) Targeting both HIF-1 and HIF-2 in human colon cancer cells improves tumor response to sunitinib treatment. Mol Cancer Ther 8(5):1148–1156. doi:10.1158/1535-7163.mct-08-0944

    Article  CAS  PubMed  Google Scholar 

  23. Rapisarda A, Hollingshead M, Uranchimeg B, Bonomi CA, Borgel SD, Carter JP, Gehrs B, Raffeld M, Kinders RJ, Parchment R, Anver MR, Shoemaker RH, Melillo G (2009) Increased antitumor activity of bevacizumab in combination with hypoxia inducible factor-1 inhibition. Mol Cancer Ther 8(7):1867–1877. doi:10.1158/1535-7163.mct-09-0274

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Merritt WM, Nick AM, Carroll AR, Lu C, Matsuo K, Dumble M, Jennings N, Zhang S, Lin YG, Spannuth WA, Kamat AA, Stone RL, Shahzad MM, Coleman RL, Kumar R, Sood AK (2010) Bridging the gap between cytotoxic and biologic therapy with metronomic topotecan and pazopanib in ovarian cancer. Mol Cancer Ther 9(4):985–995. doi:10.1158/1535-7163.mct-09-0967

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Hashimoto K, Man S, Xu P, Cruz-Munoz W, Tang T, Kumar R, Kerbel RS (2010) Potent preclinical impact of metronomic low-dose oral topotecan combined with the antiangiogenic drug pazopanib for the treatment of ovarian cancer. Mol Cancer Ther 9(4):996–1006. doi:10.1158/1535-7163.mct-09-0960

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Pham E, Birrer MJ, Eliasof S, Garmey EG, Lazarus D, Lee CR, Man S, Matulonis UA, Peters CG, Xu P, Krasner C, Kerbel RS (2014) Translational Impact of Nanoparticle-Drug Conjugate CRLX101 with or without Bevacizumab in Advanced Ovarian Cancer. Clin Cancer Res. doi:10.1158/1078-0432.ccr-14-2810

    PubMed Central  Google Scholar 

  27. Samanta D, Gilkes DM, Chaturvedi P, Xiang L, Semenza GL (2014) Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells. Proc Natl Acad Sci USA. doi:10.1073/pnas.1421438111

    PubMed Central  Google Scholar 

  28. Keefe SM, Hennessey M, Robinson J, Mykulowicz K, Gunnarsson O, Mamtani R, Vaughn DJ, Hoffman-Censits JH, Nathanson KL, Pryma DA, Eliasof S, Garmey EG, Cohen RB, Haas NB (2014) HIF inhibition in mRCC: Planned interim analysis of CRLX101 with bevacizumab (bev), a phase 1b/2a. ASCO Meeting Abstracts 32 (15_suppl):e15611

Download references


This study was supported by Cerulean Pharma Inc.

Conflict of interest

MSW has financial holding and is a scientific advisor for OncoMed Pharmaceuticals, Verastem, Paganini, and MedImmune and receives research support from Paganini, Dompe Pharmaceuticals, and MedImmune. SE, DL, and CGP have ownership interest (including patents) in Cerulean Pharma Inc. SJC is an employee of MedImmune.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Max S. Wicha.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (TIFF 38428 kb)

Supplementary material 2 (TIFF 151905 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Conley, S.J., Baker, T.L., Burnett, J.P. et al. CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, targets cancer stem cells and impedes resistance to antiangiogenic therapy in mouse models of breast cancer. Breast Cancer Res Treat 150, 559–567 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Cancer stem cell
  • Hypoxia
  • CRLX101
  • Antiangiogenic
  • Camptothecin
  • HIF-1α