Nanoparticle interactions with immune cells dominate tumor retention and induce T cell-mediated tumor suppression in models of breast cancer
Authors: Mohammad Hedayati
Todd D Armstrong
Brian W Simons
James D Barnett
Cordula Gruettner
Elizabeth Jaffee
Xian C Zhou
Julia S Caserto
Wei Fu
Jacqueline Stewart
Chun-Ting Yang
Saraswati Sukumar
Shu-Han Yu
Sri Kamal Kandala
Elizabeth T Henderson
Anirudh Sharma
Preethi Korangath
Chen Hu
Robert Ivkov
Journal: Science advances (2020)
Discipline: Multidisciplinary
Keywords: nanoparticle fate; active targeting; passive targeting; cancer-specific antibody ligand; intratumor retention
Affiliations: Department of Radiation Oncology and Molecular Radiation Sciences, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA., Department of Mechanical Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore 21218, USA., National Taiwan University, Taipei 10617, Taiwan., Department of Oncology, Sidney Kimmel Comprehensive Cancer Centre, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA., micromod Partikeltechnologie GmbH, Rostock, Germany., Department of Biostatistics and Bioinformatics, Sidney Kimmel Comprehensive Cancer Centre, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA., Department of Urology, James Buchanan Brady Urological Institute, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA., Department of Materials Science and Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore 21218, USA., Institute for NanoBioTechnology, Whiting School of Engineering, Johns Hopkins University, Baltimore 21218, USA.

Abstract:  The factors that influence nanoparticle fate in vivo following systemic delivery remain an area of intense interest. Of particular interest is whether labeling with a cancer-specific antibody ligand ("active targeting") is superior to its unlabeled counterpart ("passive targeting"). Using models of breast cancer in three immune variants of mice, we demonstrate that intratumor retention of antibody-labeled nanoparticles was determined by tumor-associated dendritic cells, neutrophils, monocytes, and macrophages and not by antibody-antigen interactions. Systemic exposure to either nanoparticle type induced an immune response leading to CD8+ T cell infiltration and tumor growth delay that was independent of antibody therapeutic activity. These results suggest that antitumor immune responses can be induced by systemic exposure to nanoparticles without requiring a therapeutic payload. We conclude that immune status of the host and microenvironment of solid tumors are critical variables for studies in cancer nanomedicine and that nanoparticle technology may harbor potential for cancer immunotherapy. 

Retracted: No

PubPeer comments:
 Below are notes from a journal club presentation: - Despite advances in nanotechnology and cancer biology, the success of nano translation is modest, most are liposomal lipid based; no targeted therapy exists - is there a fundamental flaw in our approach to develop nanoparticle therapies for cancer? - nanoparticles are engineered foreign particles, they actively interact with immune system, the fate may be determined by tumour microenvironment and host immune status - iron oxide nanoparticles: good MRI contrast agents, provides hyperthermia applications - BNF-Plain (100 nm iron oxide nanoparticle with starch shell) - BNF-H (100 nm core iron oxide nanoparticle with herceptin antibodies) - Fig 1: HER2 expression quantification in different cell lines - panel D: in vitro, only cells with overexpression of HER2 have taken up iron oxide nanoparticles at 3 hours after administration - Both nude and NSG cells still have phagocytic innate immune cells, but other immune cell populations are different - Figure 1E: mouse model is orthotopic human xenografts with a variety of HER2- and HER2+ cell lines - Figure 2: BNF-P particles not well taken up in all mouse models used - BNF-H particles are more taken up in all mouse models, irrespective of HER2 status (this was unexpected because by active targeting, you would assume only HER2+ tumours would have more BNF-H particles) - There is difference in uptake between NSG and athymic nude mice; it s always higher in NSG mice - Figure 3: Vascularity and HER2 status did not seem to correlate with NP uptake in tumour in both NSG and nude mice - There is colocalization between IBA-1+ phagocytic cells with prussian blue staining of iron oxide nanoparticles; and not much association with CD31 endothelial cells or HER2+ tumour cells - Figure 3FG: Herceptin is hIgG1, using an isotype IgG control on the iron oxide nanoparticle, the uptake is similar to herceptin-conjugated iron oxide nanoparticle - this suggests that the uptake is due to the IgG interaction with immune cells and not the specificity of the IgG to an antigen to the tumour cells - Figure 4: humanized mouse model - now syngeneic transgenic HER2+ spontaneous tumour; and then allograft into different mice - iron oxide nanoparticles are accumulating in the stromal areas of the tumour and less in cancer cell-rich regions - Figure 5: in vitro and in vivo experiments showed that there was preferential uptake of nanoparticles into immune cells, dependent on the M1-M2 polarization of the tumour-associated macrophages - there was more uptake of herceptin-iron oxide nanoparticles with M1 macrophages - in vivo, NK cells, monocytes, TAMs, neutrophils, and DCs take up more nanoparticles than tumour cells - FIgure 7: nanoparticles stimulate T cell infiltration into the tumour microenvironment to inhibit tumour growth (in athymic nude mice, there are no T cells, and the herceptin nanoparticles cannot cause tumour regression) Questions: - Dose normalization not by weight - this is for ICPMS considerations so that the iron concentration is the same (equivalent to 250 mg kg roughly) There has been no observed toxicity for up to 1000mg kg - Is the ICPMS result affected by background heme content in the organ: we use PBS injected animals as an iron signal control for each experiment, dilution is required to bring the iron concentration into the dynamic range of the ICPMS - Are there any differences in in vitro uptake of BP vs BH with hepatocytes or Kupffer cells? No, we did not look at hepatocytes, but there is definitely differences in the Kupffer cells. - Of the tumour models, are the Her2+ cells mainly distributed outside the tumour on the periphery? BT474 tumours have HER2+ signal distributed everywhere; some tumours only have HER2+ cells in the periphery; very dependent on the expression of the HER2+ cell type itself (endogenous expression vs induced overexpression) as well as the tumour it forms itself) - What is the half-life of the different particle types used in this study? BNF-P eliminated fully from blood after 24 hours; they didn t look at BNF-H, data may be available in a previous paper. (To be sent by Ivkov - PK is quite low for both; and there does not seem to be a correlation between half-life and accumulation into tumour) - Does the colloidal stability of these particles correlate with distribution data? Herceptin content is slightly higher than IgG in particles; will this affect stability? (see references below) - Does the orientation of the antibody matter? The Fab vs Fc exposed regions? Answer: (1) we did try Fab fragments at the beginning to test the HER2 epitope, in vitro we couldn t find good specificity of Fab-iron oxide nanoparticles in vitro. Took 4-5 years to get the chemistry right for Herceptin-iron oxide nanoparticles (see Figure 1). (2) Fc should interact with Fc-receptors in immune cells, we are currently looking into it. (3) There is a paper comparing Fab fragments vs Fc antibodies nanoparticle binding; they show that the binding of those antibodies between in vitro and in vivo is different (see references below) - In this study, NPs were injected 5 days after tumour induction when the tumours are fairly small; how big are the tumours on day 5 and was there a reason why you didn t wait until it grew 120-200 mm^3 like you had done before? The tumour growth is very fast and heterogeneous between tumours; that is why we used time as standardization; the timeline is optimized so that we are able to see a reduction in tumour size in the control treatment group to compare treatments. The allograft experiment are also graft transfers, so we can t control how many cells are in the tumour as opposed to cell number standardization of xenograft injections. - Do you expect any of the effects observed here to be dose-relaed? 5mg seems to be a significant amount of NP? We do not know about the dose effect; if it is indeed immune system mediated as we have shown here, a repeated dose might lead to non-linear effects; and likewise, a lower dose might not lead to any effect. - HER2 is a humanized anitbody and the IgG is also human, do you expect to see similar results with mouse IgG? Ans: we expect that the mouse IgG will lead to less of an effect because it is  less foreign - Do you know if the infiltrating immune cells are picking up NPs locally when they arrive at the tumour, or if they arrived at the tumour with NPs inside already (that they picked up elsewhere)? We think it s picking up locally in the tumour microenvironment. - Do you think this relates to iron oxide only or other antibody-conjugated nanoparticle core types as well? We are not sure, we are doing ongoing experiments for this. - For translation, how would this treatment change since patients undergoing chemotherapy are immunocompromised? The host immune status will for sure make a difference, as shown in the supplementary information where we used clodronate-liposomes. - Thoughts on the 2016 Zanganeh Nature Nano paper, they show that bare iron oxide nanoparticles could remodel macrophages into M1 macrophages and lead to cancer tumour regression. Answer: Our data shows something similar to their observations. - Some other papers show that M2 macrophages actually take up more nanoparticles, and there is a lot of controversy and disagreements in literature, what are your thoughts? Ans: Hypothesize might be related to the coating of the nanoparticle. - Do you expect certain receptor-mediated uptake? Ans: We are doing more TEM in vitro and in vivo to see how these nanoparticles are accumulating in cells and which cell compartments are involved. Reference papers: - NanoFerrite Particle Based Radioimmunonanoparticles: Binding Affinity and In Vivo Pharmacokinetics (Natarajan et al, Bioconjugate Chemistry, 2008) - Internal Magnetic Structure of Nanoparticles Dominates Time-Dependent Relaxation Processes in a Magnetic Field (Dennis et al, Adv. Func. Materials., 2015) ,