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Lymphatic Transport and Lymph Node Location of Microspheres Subcutaneously Injected in the Vicinity of Tumors in a Rabbit Model of Breast Cancer

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Abstract

Purpose

To assess the lymphatic transport of microparticles of 100 nm, 1 μm and 10 μm subcutaneously injected into the breast area of healthy and tumor-bearing rabbits, and to analyze their location in lymph node (LN) in relation to malignant cells.

Methods

Female rabbits (n = 9) bearing a VX2 tumor in one thoracic mammary gland were subcutaneously injected at D15 with polystyrene fluorescent particles around the nipple, on the tumor and on the healthy sides. The tumor and the LN measured by ultrasound at D9, D15 and D20 were explanted at D20. The LN metastases were evaluated by cytokeratin staining. LN uptake of the particles was measured by quantifying the green fluorescence surface in hot spot regions of healthy and pathologic LN.

Results

All animals developed mammary tumors. Metastases were found in 39% of LN from the tumor side. LN invasion was significantly lower for the 10 μm group versus the 100 nm group (p < 0.0348). The fully invaded area of metastatic LN contained significantly less 100 nm and 1 μm particles compared to the low and non-invaded regions and to the healthy LN. In the invaded LN, the 1 μm MS occupied more surface than the 100 nm particles.

Conclusions

1 μm MS arrived numerously into the areas low-invaded and non-invaded by the tumoral cells of the pathologic LN, but they were very rare in the fully invaded regions. Compared to the 100 nm nanospheres, the 1 μm were better retained (20 times) into the sentinel LN, showing the advantage of micrometric particles for lymph-targeted chemotherapy when injected before complete invasion by metastases.

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Abbreviations

DDS:

Drug delivery system

KW:

Kruskal Wallis test

LN:

Lymphnode

mAb:

Monoclonal antibody

MS:

Microspheres

MW:

Mann-Whitney U test

NP:

Nanoparticles

PBS:

Phosphate buffered saline

PLGA:

Poly(lactic-co-glycolic acid)

SLN:

Sentinel lymph node

References

  1. Swartz MA, Skobe M. Lymphatic function, lymphangiogenesis, and cancer metastasis. Microsc Res Tech. 2001;55(2):92–9.

    Article  PubMed  CAS  Google Scholar 

  2. Proulx ST, Luciani P, Dieterich LC, Karaman S, Leroux J-C, Detmar M. Expansion of the lymphatic vasculature in cancer and inflammation: new opportunities for in vivo imaging and drug delivery. J Control Release Off J Control Release Soc. 2013;172(2):550–7.

    Article  CAS  Google Scholar 

  3. Hirakawa S, Brown LF, Kodama S, Paavonen K, Alitalo K, Detmar M. VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood. 2007;109(3):1010–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Ashikaga T, Krag DN, Land SR, Julian TB, Anderson SJ, Brown AM, et al. Morbidity results from the NSABP B-32 trial comparing sentinel lymph node dissection versus axillary dissection. J Surg Oncol. 2010;102(2):111–8.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ryan GM, Kaminskas LM, Porter CJH. Nano-chemotherapeutics: maximising lymphatic drug exposure to improve the treatment of lymph-metastatic cancers. J Control Release Off J Control Release Soc. 2014;193:241–56.

    Article  CAS  Google Scholar 

  6. Trevaskis NL, Kaminskas LM, Porter CJH. From sewer to saviour - targeting the lymphatic system to promote drug exposure and activity. Nat Rev Drug Discov. 2015;14(11):781–803.

    Article  PubMed  CAS  Google Scholar 

  7. Shackney SE, McCormack GW, Cuchural GJ. Growth rate patterns of solid tumors and their relation to responsiveness to therapy: an analytical review. Ann Intern Med. 1978;89(1):107–21.

    Article  PubMed  CAS  Google Scholar 

  8. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008;38(5):1404–13.

    Article  PubMed  CAS  Google Scholar 

  9. Randolph GJ, Angeli V, Swartz MA. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol. 2005;5(8):617–28.

    Article  PubMed  CAS  Google Scholar 

  10. Wang P, Xie X, Wang J, Shi Y, Shen N, Huang X. Ultra-small superparamagnetic iron oxide mediated magnetic hyperthermia in treatment of neck lymph node metastasis in rabbit pyriform sinus VX2 carcinoma. Tumour Biol. 2015;36(10):8035–40.

    Article  PubMed  CAS  Google Scholar 

  11. Johnson L, Pinder SE, Douek M. Deposition of superparamagnetic iron-oxide nanoparticles in axillary sentinel lymph nodes following subcutaneous injection. Histopathology. 2013;62(3):481–6.

    Article  PubMed  Google Scholar 

  12. Oussoren C, Storm G. Liposomes to target the lymphatics by subcutaneous administration. Adv Drug Deliv Rev. 2001;50(1–2):143–56.

    Article  PubMed  CAS  Google Scholar 

  13. Kaminskas LM, Ascher DB, McLeod VM, Herold MJ, Le CP, Sloan EK, et al. PEGylation of interferon α2 improves lymphatic exposure after subcutaneous and intravenous administration and improves antitumour efficacy against lymphatic breast cancer metastases. J Control Release Off J Control Release Soc. 2013;168(2):200–8.

    Article  CAS  Google Scholar 

  14. Luboldt W, Pinkert J, Matzky C, Wunderlich G, Kotzerke J. Radiopharmaceutical tracking of particles injected into tumors: a model to study clearance kinetics. Curr Drug Deliv. 2009;6(3):255–60.

    Article  PubMed  CAS  Google Scholar 

  15. Lu H, Li B, Kang Y, Jiang W, Huang Q, Chen Q, et al. Paclitaxel nanoparticle inhibits growth of ovarian cancer xenografts and enhances lymphatic targeting. Cancer Chemother Pharmacol. 2007;59(2):175–81.

    Article  PubMed  CAS  Google Scholar 

  16. Lu B, Xiong S-B, Yang H, Yin X-D, Zhao R-B. Mitoxantrone-loaded BSA nanospheres and chitosan nanospheres for local injection against breast cancer and its lymph node metastases. II: Tissue distribution and pharmacodynamics. Int J Pharm. 2006;307(2):175–81.

    Article  PubMed  CAS  Google Scholar 

  17. Liu R, Gilmore DM, Zubris KAV, Xu X, Catalano PJ, Padera RF, et al. Prevention of nodal metastases in breast cancer following the lymphatic migration of paclitaxel-loaded expansile nanoparticles. Biomaterials. 2013;34(7):1810–9.

    Article  PubMed  CAS  Google Scholar 

  18. Yang Q, Wang X, Chen J, Tian C, Li H, Chen Y-J, et al. A clinical study on regional lymphatic chemotherapy using an activated carbon nanoparticle-epirubicin in patients with breast cancer. Tumour Biol. 2012;33(6):2341–8.

    Article  PubMed  CAS  Google Scholar 

  19. Barenholz Y. Doxil®--the first FDA-approved nano-drug: lessons learned. J Control Release Off J Control Release Soc. 2012;160(2):117–34.

    Article  CAS  Google Scholar 

  20. Chen J-H, Ling R, Yao Q, Li Y, Chen T, Wang Z, et al. Effect of small-sized liposomal Adriamycin administered by various routes on a metastatic breast cancer model. Endocr Relat Cancer. 2005;12(1):93–100.

    Article  PubMed  CAS  Google Scholar 

  21. Niu C, Wang Z, Lu G, Krupka TM, Sun Y, You Y, et al. Doxorubicin loaded superparamagnetic PLGA-iron oxide multifunctional microbubbles for dual-mode US/MR imaging and therapy of metastasis in lymph nodes. Biomaterials. 2013;34(9):2307–17.

    Article  PubMed  CAS  Google Scholar 

  22. Damgé C, Aprahamian M, Marchais H, Benoit JP, Pinget M. Intestinal absorption of PLAGA microspheres in the rat. J Anat. 1996;189(Pt 3):491–501.

    PubMed  PubMed Central  Google Scholar 

  23. Lemperle G, Lappin PB, Stone C, Lemperle SM. Urethral bulking with polymethylmethacrylate microspheres for stress urinary incontinence: tissue persistence and safety studies in miniswine. Urology. 2011;77(4):1005.e1–7.

    Article  Google Scholar 

  24. Ueno H, Hihara J, Shimizu K, Osaki A, Yamashita Y, Yoshida K, et al. Experimental study on fluorescent microspheres as a tracer for sentinel node detection. Anticancer Res. 2005;25(2A):821–5.

    PubMed  Google Scholar 

  25. De S, Miller DW, Robinson DH. Effect of particle size of nanospheres and microspheres on the cellular-association and cytotoxicity of paclitaxel in 4T1 cells. Pharm Res. 2005;22(5):766–75.

    Article  PubMed  CAS  Google Scholar 

  26. Chakravarthi SS, De S, Miller DW, Robinson DH. Comparison of anti-tumor efficacy of paclitaxel delivered in nano- and microparticles. Int J Pharm. 2010;383(1–2):37–44.

    Article  PubMed  CAS  Google Scholar 

  27. Narita K, Ikuta H, Hamabe Y, Kuroda Y. Experimental study on paratumoral injection of cisplatin-loaded microspheres for gastric cancer. Kobe J Med Sci. 1999;45(5):229–43.

    PubMed  CAS  Google Scholar 

  28. Nakamura Y, Hamabe Y, Ikuta H, Hyon S-H, Kuroda Y. An experimental study of regional chemotherapy using CDDP-loaded microspheres for esophageal cancer. Surg Today. 2002;32(4):335–42.

    Article  PubMed  CAS  Google Scholar 

  29. Liu J, Meisner D, Kwong E, Wu XY, Johnston MR. Translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer. Cancer Res. 2009;69(3):1174–81.

    Article  PubMed  CAS  Google Scholar 

  30. Hawley AE, Illum L, Davis SS. The effect of lymphatic oedema on the uptake of colloids to the lymph nodes. Biopharm Drug Dispos. 1998;19(3):193–7.

    Article  PubMed  CAS  Google Scholar 

  31. Hawley AE, Illum L, Davis SS. Lymph node localisation of biodegradable nanospheres surface modified with poloxamer and poloxamine block co-polymers. FEBS Lett. 1997;400(3):319–23.

    Article  PubMed  CAS  Google Scholar 

  32. Barone R. Système lymphatique du lapin dans “Anatomie comparée des mamiferes domestiques”, page 845. Vigot Frères. Vol. Angiologie. Paris; 1996. 903 p.

  33. Kidd JG, Rous P. A transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form. J Exp Med. 1940;71(6):813–38.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Pouw JJ, Grootendorst MR, Bezooijen R, Klazen CAH, De Bruin WI, Klaase JM, et al. Pre-operative sentinel lymph node localization in breast cancer with superparamagnetic iron oxide MRI: the SentiMAG multicentre trial imaging subprotocol. Br J Radiol. 2015;88(1056):20150634.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Pascale F, Bedouet L, Baylatry M, Namur J, Laurent A. Comparative Chemosensitivity of VX2 and HCC cell lines to drugs used in TACE. Anticancer Res. 2015;35(12):6497–503.

    PubMed  CAS  Google Scholar 

  36. Yoshiji H, Gomez DE, Shibuya M, Thorgeirsson UP. Expression of vascular endothelial growth factor, its receptor, and other angiogenic factors in human breast cancer. Cancer Res. 1996;56(9):2013–6.

    PubMed  CAS  Google Scholar 

  37. Pascale F, Ghegediban S-H, Bonneau M, Bedouet L, Namur J, Verret V, et al. Modified model of VX2 tumor overexpressing vascular endothelial growth factor. J Vasc Interv Radiol JVIR, 2012 Jun. 23(6):809–817.e2.

  38. Chao P, Deshmukh M, Kutscher HL, Gao D, Rajan SS, Hu P, et al. Pulmonary targeting microparticulate camptothecin delivery system: anticancer evaluation in a rat orthotopic lung cancer model. Anti-Cancer Drugs. 2010;21(1):65–76.

    Article  PubMed  CAS  Google Scholar 

  39. Junping W, Tongguo S, Yunting Z, Chunshui Y, Renju B. Discrimination of axillary metastatic from nonmetastatic lymph nodes with PROPELLER diffusion-weighted MR imaging in a metastatic breast cancer model and its correlation with cellularity. J Magn Reson Imaging JMRI. 2012;36(3):624–31.

    Article  PubMed  Google Scholar 

  40. Veronesi U, Viale G, Paganelli G, Zurrida S, Luini A, Galimberti V, et al. Sentinel lymph node biopsy in breast cancer: ten-year results of a randomized controlled study. Ann Surg. 2010;251(4):595–600.

    Article  PubMed  Google Scholar 

  41. Ji R-C. Lymph nodes and Cancer metastasis: new perspectives on the role of Intranodal lymphatic sinuses. Int J Mol Sci. 2016;28:18(1).

    Google Scholar 

  42. Ivanov S, Scallan JP, Kim K-W, Werth K, Johnson MW, Saunders BT, et al. CCR7 and IRF4-dependent dendritic cells regulate lymphatic collecting vessel permeability. J Clin Invest. 2016;126(4):1581–91.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments and Disclosures

We thank Celine Urien from Molecular Virology and Immunology Laboratory and Pierre Adenot from the Microscopy and Imaging of the Microrganismes, Animals and Aliments Platform (MIMA2) from the French National Institut of Agricultural Research (INRA) Jouy en Josas for the technical support in confocal imaging acquisition.

Author information

Authors and Affiliations

  1. ArchimMed SARL, 12, rue Charles de Gaulle, 78350, Jouy en Josas, France

    Florentina Pascale, Julien Namur & Saida Homayra Ghegediban

  2. Occlugel, 12, rue Charles de Gaulle, 78350, Jouy en Josas, France

    Laurent Bédouet

  3. Hôpital Lariboisière, 2, Rue Ambroise Paré, 75010, Paris, France

    Afchine Fazel, Michel Wassef & Alexandre Laurent

  4. Laboratoire de Virologie et Immunologie Moleculaires, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy en Josas, France

    Isabelle Schwartz Cornil

  5. Institut Galien Paris Sud, CNRS, Université Paris-Saclay, 5, rue Jean-Baptiste Clément, 92296, Châtenay-Malabry Cedex, France

    Laurence Moine

  6. Université Paris Diderot-Paris 7, 75205, Paris Cedex 13, France

    Alexandre Laurent

  7. Cr2i, INRA, Domaine de Vilvert, 78352, Jouy en Josas, France

    Alexandre Laurent

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  1. Florentina Pascale
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Correspondence to Florentina Pascale.

Electronic supplementary material

Fig. S1

-supplementary data. The intensity of the fluorescence and the diameter of the particles was analyzed with Zen Lite 2.3 Karl Zeiss Microscopy GmBH,2011 Software on 5 isolated 1 μm (A, B) and 10 μm (C,D) microspheres on confocal images from healthy lymphnodes stained with TO-PRO3 for nuclei. The size of the particles measured was close to the size indicated by the manufacturer (1.015 ± 0.072 μm for 1 μm MS and 10.35 ± 0.19 μm for 10 μm MS) (B). The intensity of the fluorescence was not different between 1 μm and 10 μm particles (252 ± 6,8 vs 255)(p < 0,1100, MW) (D). (PNG 1014 kb)

High resolution image (TIF 1412 kb)

Fig. S2

-supplementary data. The nanoparticles were found into the LN only as clusters and not as single particles (A).The intensity of the fluorescence and the diameter of clusters were analyzed with Zen Lite 2.3 Karl Zeiss Microscopy GmBH, 2011 Software on 5 isolated clusters of 0.1 μm (A,B) and 1 μm microspheres (C,D) on confocal images from healthy lymphnodes stained with TO-PRO3 for nuclei. The diameter of the 0.1 μm clusters was 1.79 ± 0.42 and that corresponded to almost 17 nanoparticles, each particle visualized on the intensity profile as peak of fluorescence (white arrow, Fig. A, B). The diameter of the 1 μm clusters was 5 ± 3.19 μm and corresponded to clusters formed by 3–8 particles, each particle visualized on the fluorescence intensity profile as peak of fluorescence (white arrow, Fig. B,C). The intensity of the fluorescence was not different between the clusters of 0.1 μm and 1 μm (222.6 ± 28 vs 215.8 ± 33)(p < 0.1116, MW) (D). (PNG 1137 kb)

High resolution image (TIF 1577 kb)

Fig. S3

At day 5 the skin was sampled and epifluorescence analysis was performed to observe the presence of the particles at the site of injection. Slides were stained with DAPI for nuclei and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined under ×100 magnification for particle detection. The particles were still observed at the sites of injection surrounding the depot (Inj). Many nuclei were observed around the clusters of particles (arrowhead), they probably belong to the inflammatory cells that infiltrate the injection site. The nanoparticles were rather distributed as large patches (white star) than small patches (white asterisk) at the site of injection. Huge clusters of 1 μm MS (white star) were observed nearby the injection site. The small clusters and 1 μm single particles (arrows) were numerously and were scattered more distant from the injection site. The particles of 10 μm were distributed around the depot as single particle (arrow) or small clusters formed by 2 MS (empty arrow). (PNG 2733 kb)

High resolution image (TIF 1349 kb)

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Pascale, F., Bédouet, L., Fazel, A. et al. Lymphatic Transport and Lymph Node Location of Microspheres Subcutaneously Injected in the Vicinity of Tumors in a Rabbit Model of Breast Cancer. Pharm Res 35, 191 (2018). https://doi.org/10.1007/s11095-018-2474-6

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  • DOI: https://doi.org/10.1007/s11095-018-2474-6

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