Phenotyping of Macrophages After Radiolabeling and Safety of Intra-arterial Transplantation Assessed by SPECT/CT and MRI

Abstract

Cell therapy is an integral modality of regenerative medicine. Macrophages are known for their sensitivity to activation stimuli and capability to recruit other immune cells to the sites of injury and healing. In addition, the route of administration can impact engraftment and efficacy of cell therapy, and modern neuro-interventional techniques provide the possibility for selective intra-arterial (IA) delivery to the central nervous system (CNS) with very low risk. The effects of radiolabelling and catheter transport on differentially activated macrophages were evaluated. Furthermore, the safety of selective IA administration of these macrophages to the rabbit brain was assessed by single-photon emission computed tomography/computed tomography (SPECT/CT) and ultra-high-field (9.4 T) magnetic resonance imaging (MRI). Cells were successfully labeled with (¹¹¹In)In-(oxinate)₃ and passed through a microcatheter with preserved phenotype. No cells were retained in the healthy rabbit brain after IA administration, and no adverse events could be observed either 1 h (n = 6) or 24 h (n = 2) after cell administration. The procedure affected both lipopolysaccharide/gamma interferon (LPS/IFNγ) activated cells and interleukin 4 (IL4), interleukin 10 (IL10)/transforming growth factor beta 1 (TGFβ1) activated cells to some degree. The LPS/IFNγ activated cells had a significant increase in their phagocytotic function. Overall, the major impact on the cell phenotypes was due to the radiolabeling and not passage through the catheter. Unstimulated cells were substantially affected by both radiolabeling and catheter administration and are hence not suited for this procedure, while both activated macrophages retained their initial phenotypes. In conclusion, activated macrophages are suitable candidates for targeted IA administration without adverse effects on normal, healthy brain parenchyma.

Keywords: MRI; SPECT/CT; endovascular; intra-arterial; rabbit.

Figure 1.

A step-by-step graphic chart over the in vivo validation process of the radiolabeling and catheter passage of unstimulated monocytes, LPS/IFNγ or IL4, IL10/TGFβ1 activated macrophages.

Figure 2.

Phenotype analysis and phagocytosis function of (A) M0, (B) LPS/IFNγ activated cells, and (C) IL4, IL10/TGFβ1 activated cells measured during each step of the radiolabeling procedure with (¹¹¹In)In-(oxinate)₃ and catheter passage. An antibody panel of CD14, CD16, CD86, CD163, and CD206 for phenotype and Dextran Alexa 640 for phagocytosis was analyzed by flow cytometry. Statistical analysis was calculated with the Student t-test or rm-ANOVA. Data were considered statistically significant at a p-value <0.05, values under 0.05 are marked as *, and those under 0.001 are marked as <0.001**. P-values are presented in Table 3.

Figure 3.

Flow cytometry parameters sideway scatter (SSC) and forward scatter (FSC). SSC represents the intracellular structures providing information on the internal complexity of the cells. The FSC represents the cell size. Combined they provide the basic information of the cell morphology of unstimulated, LPS/IFNγ, or IL4/IL10/TGFβ1 activated cells. Values are presented as the mean SSC/FSC value for each step in the process; pre-catheter or post-catheter passage for controls and (¹¹¹In)In-(oxinate)₃ labeled cells.

Figure 4.

A representative digital subtraction angiogram taken 15 min after administration of LPS/IFNγ activated cells. Images are taken in the following order: the right internal carotid artery (ICA) (arrow, A) followed by selective ICA arteriograms in lateral (B) and frontal (C) planes.

Figure 5.

Volumetric MRI images, (A) T2- and (B) Diffusion-weighted, were taken 24 h post-cell administration of LPS/IFNγ activated cells through the catheter to rabbit brain. The illustration shows no adverse events caused by the procedure.

Figure 6.

SPECT coronal maximum intensity projections for the full rabbit scan 24 h following administration of LPS/IFNγ activated cells (A) and IL4, IL10/TGFβ1 activated cells (C). Liver indicated by L, spleen by S, pancreas by P, kidneys by K and in panel C, and the top of the bladder marked by B. Coronal SPECT-CT fusion images demonstrating the colocalization of signal for LPS/IFNγ activated cells (B) and IL4, IL10/TGFβ1 activated cells (D) to organs. Axial SPECT-CT fusion images demonstrate the lack of signal from the brain parenchyma for both LPS/IFNγ activated cells (E) and IL4, IL10/TGFβ1 activated cells (F), and the visible signals are due to accumulations in glands.