Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane‐bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not normal cells and therefore provides a tumor‐specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells. Herein, it is demonstrated that GrB functionalized SPIONs act as a contrast enhancement agent for magnetic resonance imaging and induce specific tumor cell apoptosis. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting are found to further enhance the therapeutic efficacy of GrB‐SPIONs in different tumor mouse models.

Over the past several decades, nanocarriers have demonstrated diagnostic and therapeutic (i.e., theranostic) potencies in translational oncology, and some agents have been further translated into clinical trials. However, the practical application of nanoparticle-based medicine in living organisms is limited by physiological barriers (blood–tissue barriers), which significantly hampers the transport of nanoparticles from the blood into the tumor tissue. This review focuses on several approaches that facilitate the translocation of nanoparticles across blood–tissue barriers (BTBs) to efficiently accumulate in the tumor. To overcome the challenge of BTBs, several methods have been proposed, including the functionalization of particle surfaces with cell-penetrating peptides (e.g., TAT, SynB1, penetratin, R8, RGD, angiopep-2), which increases the passing of particles across tissue barriers. Another promising strategy could be based either on the application of various chemical agents (e.g., efflux pump inhibitors, disruptors of tight junctions, etc.) or physical methods (e.g., magnetic field, electroporation, photoacoustic cavitation, etc.), which have been shown to further increase the permeability of barriers.

Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane-bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not on normal cells and therefore provides a tumor-specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells inducing the process of cellular apoptosis. Decoration of the nanoparticles surface with GrB increased the targeting properties of the nanocarriers as was shown by confocal and electron microscopies. In series of in vivo studies it was demonstrated that GrB functionalized SPIONs act simultaneously as a contrast enhancement agent when being intravenously administered for magnetic resonance imaging and induce specific tumor cell apoptosis (human U87 glioblastoma in NMRI nu/nu mice, rat C6 glioma in Wistar rats, B16 melanoma in C57/Bl6 mice). Continuous prolonged administration of GrB-SPIONs via programmable subcutaneously implanted mini-pump system iPrecio further enhanced the therapeutic benefit of magnetic conjugates. Moreover, significant therapeutic response was achieved in immunocompromised mice models (advanced U87 brain tumor, small lung cancer H1339 with brain metastasis) with a high tumor burden. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting with immune checkpoint inhibitors (anti-CTLA-4 monoclonal antibodies) are found to further enhance the therapeutic efficacy of GrB-SPIONs in different tumor mouse models. The study was funded by RFBR, project № 19-08-00024; DFG grant SFB824/3, Germany, TaGoNaX DFG project № 336532926, STA1520/1-1. Citation Format: Maxim Shevtsov, Susanne Kaesler, Stefan Stangl, Luidmila Yakovleva, Ruslana Tagaeva, Yaroslav Marchenko, Boris Nikolaev, William Khachatryan, Oleg Galibin, Emil Pitkin, Gabriele Multhoff. Granzyme B functionalized nanocarriers targeting membrane-bound Hsp70 for multimodal cancer therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2878.

Biomedical nanotechnologies based on superparamagnetic iron oxide nanoparticles (SPIONs) represent a promising instrument for malignant brain tumor theranostics. Due to their unique physico-chemical properties, they can be used for multimodal cancer therapy (including hyperthermia), diagnostics, and targeted delivery of therapeutic molecules. To enhance the tumor-homing properties of nanoparticles the latter were conjugated with bioligands (i.e., monoclonal antibodies, Fab-fragments, peptides, and proteins such serine protease granzyme B) that recognize 72 kDa membrane-bound heat shock protein (mHsp70) that is expressed on the plasma membrane of tumor cells, including multiforme glioblastoma (GBM), but not on normal corresponding cells. Therapeutic potential of magnetic conjugates can be attenuated by binding to the particles surface of granzyme B (GrB-SPIONs) that exerts pro-apoptotic activity. Functionalized particles significantly contrast-enhanced the orthotopic glioblastoma (U87 glioma in NMRI nu/nu mice, GL261 glioma in C57/Bl6 mice) in preclinical animal models when high-field (11 ) magnetic resonance tomography (Bruker) was employed. Furthermore, when GrB-SPIONs were applied, we detected a statistically significant delay of tumor progression and increase of overall animal survival. Anti-tumor activity of GrB-SPIONs was further synergistically potentiated by stereotactic radiotherapy combined with immune check-point inhibitors (i.e., CTLA-4). In conclusion, synthesized superparamagnetic nanoparticles targeting mHsp70-positive could be employed for theranostics of malignant brain tumors and translated into further clinical trials.This research was funded by the Ministry of Science and Higher Education of the Russian Federation (Agreement № 075-15-2020-901). Citation Format: Ruslana Tagaeva, Vyacheslav Fedorov, Natalia Yudintceva, Luidmila Yakovleva, Boris Nikolaev, Veronica Osnach, Maxim Shevtsov. Functionalized superparamagnetic nanoparticles for theranostics of membrane-bound Hsp70 positive malignant brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5068.

Heat shock proteins (HSPs) constitute a large family of highly conserved proteins acting as molecular chaperones that play a key role in intracellular proteostasis, regulation of apoptosis, and protection from various stress factors (including hypoxia, thermal stress, oxidative stress). Apart from their intracellular localization, members of different HSPs families such as Hsp70 have been found to be localized on the plasma membrane of malignantly transformed cells, including multiforme glioblastoma (GBM). However, the role of the membrane-bound mHsp70 has not yet been elucidated in the pathophysiology of GBM. We have assessed the mHsp70 expression in biopsies obtained from neuro-oncological patients employing inverted confocal microscopy (Leica Microsystems) and CellVoyager CQ1 Benchtop High-Content Analysis System (Yokogawa). Live-cell imaging of the patient-derived biopsy revealed the increased cellular density of mHsp70-positive tumor cells particularly in the tissue along the tumor-brain margin (biopsy was derived 3 mm from contrast-enhancing margin and also within hyperintense FLAIR). Time-lapse analysis of the tumor sample further demonstrated the migration of mHsp70-positive cells into the surrounding culture medium. Subsequent high-powered multiplex immunofluorescence analysis (Akoya Biosystems Inc.) confirmed markedly increased single-cell infiltration of mHsp70(+) tumor cells (that were also co-immunofluorescently stained for SOX2, Nestin, and Oct4) in the normal brain tissues. In conclusion, expression of mHsp70 on the plasma membrane of tumor cells corresponds to the highly invasive potential of the GBM cells and recurrence of these tumors in neuro-oncological patients. This research was funded by the Ministry of Science and Higher Education of the Russian Federation (Agreement № 075-15-2020-901). Citation Format: Maxim Shevtsov, Ruslana Tagaeva, Natalia Yudintceva, Danila Bobkov, Darya Goncharova, Alexander Kim, Evgeniy Fedorov, Anastasia Nechaeva, Anastasia Mihrina, Anna Patruhina, Vyacheslav Fedorov, Konstantin Samochernykh. Membrane-associated heat shock protein mHsp70 is involved in cancer cell invasion and tumor recurrence in malignant brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 504.