Tumor stroma-containing 3D spheroid arrays: A tool to study nanoparticle penetration.
Nanoparticle penetration through tumor tissue after extravasation is considered as a key issue for tumor distribution and therapeutic effects. Most tumors possess abundant stroma, a fibrotic tissue composed of cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM), which acts as a barrier for nanoparticle penetration. There is however a lack of suitable in vitro systems to study the tumor stroma penetration of nanoparticles. In the present study, we developed and thoroughly characterized a 3D co-culture spheroidal array to mimic tumor stroma and investigated the penetration of silica and PLGA nanoparticles in these spheroids. First, we examined human breast tumor patient biopsies to characterize the content and organization of stroma and found a high expression of alpha-smooth muscle actin (α-SMA; 40% positive area) and collagen-1 (50% positive area). Next, we prepared homospheroids of 4T1 mouse breast cancer cells or 3T3 mouse fibroblasts alone as well as heterospheroids combining 3T3 and 4T1 cells in different ratios (1:1 and 5:1) using a microwell array platform. Confocal live imaging revealed that fibroblasts distributed and reorganized within 48h in heterospheroids. Furthermore, immunohistochemical staining and gene expression analysis showed a proportional increase of α-SMA and collagen in heterospheroids with higher fibroblast ratios attaining 35% and 45% positive area at 5:1 (3T3:4T1) ratio, in a good match with the clinical breast tumor stroma. Subsequently, we studied the penetration of high and low negatively charged fluorescent silica nanoparticles (30nm; red and 100 or 70nm; green; zeta potential: -40mV and -20mV) and as well as Cy5-conjugated pegylated PLGA nanoparticles (200nm, -7mV) in both homo- and heterospheroid models. Fluorescent microscopy on spheroid cryosections after incubation with silica nanoparticles showed that 4T1 homospheroids allowed a high penetration of about 75-80% within 24h, with higher penetration in case of the 30nm nanoparticles. In contrast, spheroids with increasing fibroblast amounts significantly inhibited NP penetration. Silica nanoparticles with a less negative zeta potential exhibited lesser penetration compared to highly negative charged nanoparticles. Subsequently, similar experiments were conducted using Cy5-conjugated pegylated PLGA nanoparticles and confocal laser scanning microscopy; an increased nanoparticle penetration was found in 4T1 homospheroids until 48h, but significantly lower penetration in heterospheroids. Furthermore, we also developed human homospheroids (MDA-MB-231 or Panc-1 tumor cells) and heterospheroids (MDA-MB-231/BJ-hTert and Panc-1/pancreatic stellate cells) and performed silica nanoparticle (30 and 100nm) penetration studies. As a result, heterospheroids had significantly a lesser penetration of the nanoparticles compared to homospheroids. In conclusion, our data demonstrate that tumor stroma acts as a strong barrier for nanoparticle penetration. The 30-nm nanoparticles with low zeta potential favor deeper penetration. Furthermore, the herein proposed 3D co-culture platform that mimics the tumor stroma, is ideally suited to systematically investigate the factors influencing the penetration characteristics of newly developed nanomedicines to allow the design of nanoparticles with optimal penetration characteristics.
Targeted Therapeutics, Department of Biomaterials Science and Technology, MIRA Institute, University of Twente, Enschede, The Netherlands.
Dwi L
DL
Priwitaningrum
Applied Microfluidics for BioEngineering Research, MIRA Institute, University of Twente, Enschede, The Netherlands.
Jean-Baptiste G
JG
Blondé
Applied Microfluidics for BioEngineering Research, MIRA Institute, University of Twente, Enschede, The Netherlands.
Adithya
A
Sridhar
Laboratorium Pathologie Oost-Nederland (LabPON), Hengelo, The Netherlands.
Joop
J
van Baarlen
Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands.
Wim E
WE
Hennink
Targeted Therapeutics, Department of Biomaterials Science and Technology, MIRA Institute, University of Twente, Enschede, The Netherlands; Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands.
Gert
G
Storm
Applied Microfluidics for BioEngineering Research, MIRA Institute, University of Twente, Enschede, The Netherlands.
Séverine
S
Le Gac
Targeted Therapeutics, Department of Biomaterials Science and Technology, MIRA Institute, University of Twente, Enschede, The Netherlands. Electronic address: j.prakash@utwente.nl.
Jai
J
Prakash
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D005260:Female; D006801:Humans; D019344:Lactic Acid / Q000008:administration & dosage*; D051379:Mice; D041681:NIH 3T3 Cells; D053758:Nanoparticles / Q000008:administration & dosage*; D011100:Polyglycolic Acid / Q000008:administration & dosage*; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D012822:Silicon Dioxide / Q000008:administration & dosage*; D018874:Spheroids, Cellular / Q000378:metabolism*
D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D012822:Silicon Dioxide
3D in vitro model; Nanoparticles; Tumor penetration; Tumor stroma