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Intravital microscopy of fever-range hyperthermia as supporting strategy for cancer immunotherapy (#26)
1 University of Tübingen, Preclinical Imaging and Radiopharmacy, Tübingen, Germany
Adoptive transfer of ex vivo activated tumor-specific cytotoxic T cells (CTL) is a promising strategy to increase anti-tumor immunity, but its capacity to control tumor growth is often insufficient. External application of heat in the fever-range (38 – 40 °C) has been shown to activate and enhance immune effector function in tumors. However, the rational design of hyperthermia application schemes to support immunotherapy is currently hampered by an incomplete mechanistic understanding of how both therapies synergize.
By using time-lapse microscopy of tumor cells embedded in 3D collagen matrices and optical reporters, we monitored structural damage induced by OVA-specific CTL to the cellular and nuclear membranes and DNA double-strand breaks in B16F10/OVA melanoma cells under hyperthermia treatment. To evaluate the efficacy of adoptive CTL transfer in combination with whole-body hyperthermia (WBH) in the context of the melanoma microenvironment, we used intravital multiphoton microscopy combined with an imaging window for longitudinal monitoring of CTL effector function. Following intradermal tumor injection and adoptive CTL transfer, we applied WBH of 39.5 °C for 1 or 2 h and repeated the treatment every other day for one week.
CTL-mediated damage was predominantly sub-lethal and followed by rapid recovery of the tumor cell. Enhanced killing was observed already after treatments of 38.5 °C for 1 h on two consecutive days, while repeated, prolonged exposure to 40.5°C affected CTL viability and, consequently, killing capacity. Quantification of single CTL contacts with tumor cells derived from 48 h time-lapse recordings revealed that fever-range hyperthermia stabilized CTL–tumor cell contacts and impaired recovery of melanoma cells from CTL-mediated damage. In vivo imaging directly after treatment revealed an immediate block of tumor cell proliferation and increased apoptosis rates. Time-lapse microscopy showed enhanced CTL killing activity while CTL-tumor cell interaction dynamics remained unchanged, ranging from long-lasting to highly dynamic contacts. The combination of ACT and WBH further induced the infiltration of phagocytic cells which was absent in tumors of mice treated with either therapy alone.
Thus, kinetic imaging and intravital microscopy were successfully applied to deepen the mechanistic understanding of immune cell function during fever-range WHB which forms the basis for improved, rationale design of combination therapies.
Keywords: intravital microscopy, adoptive t cell transfer, hyperthermia
Imaging microglia/macrophages in vivo from microscale to macroscale in a murine model of ischemic stroke (#14)
Violaine Hubert1, Ines Hristovska2, Szilvia Karpati3, Frederic Lerouge3, Maelle Monteil4, Emmanuel Brun5, Naura Chounlamountri2, Chantal Watrin2, Fabien Chauveau6, Marc Lecouvey4, Stephane Parola3, Olivier Pascual2, Marlène Wiart1
1 Université de Lyon, CarMeN Inserm U1060, Bron, France
Tissue-resident microglia and infiltrated macrophages (M/M) are important mediators of tissue damage after ischemic stroke and thus represent therapeutic targets. In the present study, our aim was to investigate the potential of MRI coupled with the injection of a novel nanoparticle (NP), NanoGd , to image M/M phagocytic activity at the acute stage of ischemic stroke. To understand the biological substrates of NanoGd-induced MR signals, we performed two-photon intravital microscopy back-to-back with MRI in CX3CR1-GFP mice submitted to permanent middle cerebral artery occlusion (pMCAO).
To evaluate NanoGd M/M internalization, microglial primary cultures were incubated with NanoGd at [0-1.5 mM] during 24h and imaged with confocal microscopy. To evaluate NanoGd biodistribution and pharmacokinetic, dynamic MRI of the abdomen was performed in 4 mice before, during and after i.v. bolus injection of NanoGd at 2 mmol Gd/kg.
At day 0 (D0), 22 mice underwent pMCAO. Baseline MRI was performed at day 1 (D1). NanoGd was then administered to 16 of the 22 operated mice at 2 mmol Gd /kg (Group I). The 6 other operated mice did not receive NanoGd and served as controls (Group II). Three nonoperated mice (sham) received NanoGd at the same dose (Group III). A subgroup of 11 mice were imaged with intravital two-photon microscopy at D1 and D2. All mice had follow-up MRI at day 3 (D3).
Confocal microscopy of microglial cultures incubated with NanoGd showed an internalization by Iba-1+ cells (Fig 1A). Abdominal MRI showed a strong uptake of NanoGd in liver and spleen at 1h post-injection (Fig 1B) and a prolonged vascular remanence (blood half-life estimated > 6h). These data were used to design the in-vivo study (Fig 1C): post-NanoGd MRI was scheduled at 48h to allow time for NanoGd to be eliminated from the vascular bed at the time of follow-up MRI.
Baseline MRI showed blood brain barrier disruption in the ischemic lesion of all pMCAO mice (Fig 2A-B). Follow-up T2WI and T2*WI showed strong hypointense MR signals in the ischemic lesion of pMCAo mice, not found in control groups (Fig 2C-D). Intravital two-photon images of the same mice confirmed NanoGd extravasation in brain parenchyma and internalization by CX3CR1+ cells in the ischemic lesion but not in the extralesional area (Fig 2E-F). Post-mortem analyses of perfused brain corroborated these results (Fig 2E-F).
The present study builds upon our former works using NP-enhanced MRI to monitor M/M in vivo at the acute stage of stroke [2-4]. We confirm and extend our previous findings by performing the very first study using MRI and intravital bi-photon microscopy back-to-back in a murine model of ischemic stroke, thanks to a new multimodal NP. Our on-going work aims at investigating the immunomodulatory effects of simvastatin with this bimodal approach.
1. Halttunen N, Lerouge F, Chaput F, Vandamme M, Karpati S, Si-Mohamed S, et al. Hybrid Nano-GdF3 contrast media allows pre-clinical in vivo element-specific K-edge imaging and quantification. Scientific reports. 2019;9(1):12090.
2. Desestret V, Brisset JC, Moucharrafie S, Devillard E, Nataf S, Honnorat J, et al. Early-stage investigations of ultrasmall superparamagnetic iron oxide-induced signal change after permanent middle cerebral artery occlusion in mice. Stroke. 2009;40(5):1834-41.
3. Marinescu M, Chauveau F, Durand A, Riou A, Cho TH, Dencausse A, et al. Monitoring therapeutic effects in experimental stroke by serial USPIO-enhanced MRI. Eur Radiol. 2013;23(1):37-47.
4. Wiart M, Davoust N, Pialat JB, Desestret V, Moucharaffie S, Cho TH, et al. MRI monitoring of neuroinflammation in mouse focal ischemia. Stroke. 2007;38(1):131-7.
The authors thank Radu Bolbos and Jean-Baptiste Langlois of the Animage platform (CERMEP, Lyon) for their technical assistance. This research was funded by the French national research agency (ANR) project NanoBrain (grant # ANR-15-CE18-0026-01) and was performed within the framework of the RHU MARVELOUS (ANR16-RHUS-0009) of University Claude Bernard Lyon (UCBL), within the program “Investissements d’Avenir”. We also thank ESRF for allocation of beamtime.
Figure 1- NanoGd characterization and study design.
A. Confocal images of Iba-1 stained microglial cells incubated without NanoGd (A1) or with NanoGd (A2, 0.5 mmol/L). Scale bars: 50 µm for overview images; 10 µm for magnified insets. B. NanoGd biodistribution assessed by abdominal dynamic MRI. Left images were acquired before NanoGd injection and right images after NanoGd injection, at the end of the dynamic sequence (1h post-injection). Green arrows point out the hypointense signals induced by NanoGd in the organs of interest. C. In-vivo study design. pMCAO: permanent middle cerebral artery occlusion. 2γ µscopy: two-photon microscopy.
Figure 2- Spatiotemporal pattern of NanoGd distribution following pMCAo.
Pre- (A-B) and post-NanoGd (C-D) MRI: T2-WI shows the presence of an ischemic lesion in pMCAo mice (dotted white lines), but not in the sham mouse. Blood brain barrier breakdown was assessed using a T1-WI post-Gd (white arrowheads). Hypointense signals are observed with T2-WI (C) and T2*-WI (D) 48h post-NanoGd injection (red arrowheads). Scale bars: 1mm. (E-F) Representative images of two-photon microscopy sessions at D1 and D2 in the extralesional area (E) and ischemic core (F). Scale bar: 20µm. Post mortem examination, scale bars: 50 µm for overview images; 20 µm for magnified insets.
Keywords: MRI, intravital two-photon microscopy, pathophysiology, ischemic stroke, nanotechnology