19Fluorine-MRI Based Longitudinal Immuno-Microenvironment-Monitoring for Pancreatic Cancer

Abstract

Background: Pancreatic cancer has a poor prognosis. Targeting Kirsten Rat Sarcoma (KRAS) mutation and its related pathways may enhance immunotherapy efficacy. While in vivo monitoring of therapeutic response and immune cell migration remains challenging, Fluorine-19 MRI (¹⁹F MRI) may allow noninvasive longitudinal imaging of immune cells.

Purpose: Evaluating the potential of ¹⁹F MRI for monitoring changes in the tumor immune microenvironment, in response to combined SHP2/MEK inhibition.

Study type: Pre-clinical animal study.

Animal model: Murine genetically engineered pancreatic cancer model (N = 20, both sexes).

Field strength/sequence: 9.4-T, two-dimensional multi-slice Rapid Acquisition with Relaxation Enhancement sequence. Intravenous injection of ¹⁹F-perfluorocarbon (PFC) nanoparticles.

Assessment: Upon tumor detection by conventional ¹H MRI screening, ¹⁹F MRI was performed in mice 24 hours after PFC nanoparticle administration. Animals were randomly assigned to four treatment groups: allosteric Src-homology-2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor SHP099, the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor Trametinib, the combination of both, or a vehicle control (4 to 6 mice each group), administered every other day per oral gavage. ¹H and ¹⁹F MRI was repeated 7 days and 14 days later. Pancreatic immune cell infiltrates were analyzed by flow cytometry and multiplex immunohistofluorescence (mIHF) upon sacrifice.

Statistical tests: Independent t-tests and one-way analysis of variance.

Results: ¹⁹F MRI revealed continuous decrease of PFC-signals in tumors from vehicle controls (100%, 80%, and 74% on days 0, 7, and 14, respectively), contrasting with stable or increasing signals under KRAS-pathway-directed treatment. MEK inhibition showed 100%, 152%, and 84% and dual SHP2/MEK1/2 inhibition demonstrated signals of 100%, 134%, and 100% on days 0, 7, 14, respectively. mIHF analyses indicated CD11b⁺ macrophages/monocytes as primary contributors to the observed ¹⁹F MRI signal differences.

Data conclusion: ¹⁹F MRI might provide non-invasive longitudinal estimates for abundance and spatial distribution of CD11b⁺ macrophages/monocytes in pancreatic cancer.

Evidence level: 1 TECHNICAL EFFICACY: Stage 2.

Keywords: 19Fluorine‐MRI; KRAS; immuno‐microenvironment‐monitoring; pancreatic cancer.

FIGURE 1

¹⁹F MRI allows the noninvasive imaging of PDAC‐associated inflammation. (a) ¹H MRI T2 transverse section of a KPC mouse. Marked by color are the pancreas, harboring multifocal PDAC (purple), the spleen (green), the liver (blue) and the ¹⁹F reference tube, attached to the animal (orange). (b) Composite ¹⁹F/¹H overlay image featuring the corresponding ¹⁹F signal (24 hours after injection of the PFC‐NP‐emulsion) of the animal and image shown in (a). (c) Pancreatic volume changes in response to treatment over 14 days. Left: individual mice; right: mice grouped by treatment, shown are the means and SEMs. (d) Quantification of the ¹⁹F signal over the course of treatment. Measurements at any given time point were normalized to the externally attached ¹⁹F reference. Left: ¹⁹F signal from the pancreas areas (tumor); middle: ¹⁹F signal from the spleens; right: ¹⁹F signal from the livers. Means and SEMs are shown. Measured volumetric data and ¹⁹F signal intensities are displayed as changes (in %) vs. the baseline, which is set at 100%. *P < 0.05; **P < 0.01; ***P < 0.001; one‐way ANOVA with Dunnett's correction for multiple comparisons.

FIGURE 2

PFC‐nanoparticle accumulation predominantly occurs in myeloid cells. Immunohistofluorescence analysis of PFC uptake and myeloid cell colocalization. A red fluorescent version of the PFC‐nanoemulsion (V‐Sense) was used to visualize cellular uptake following intravenous administration. Frozen section samples were used for colocalization of PFC fluorescent signal with various myeloid cell markers (CD11b, CD11c, F4/80, or CD103) in single‐staining. Nuclear counterstaining was done with DAPI. Representative images for (a) pancreatic tissue (tumor), (b) spleen, and (c) liver with single signal and merge are shown. All scale bars: 50 μm. For representative images for colon and lungs, see Fig. S1 in the Supplemental Material.

FIGURE 3

Flow cytometric quantification of myeloid cells in the KPC‐PDAC‐TiME following SHP2/MEK1/2 inhibition. CD45⁺ cells from treated KPC‐PDAC tumors were isolated by tissue digestion and enrichment via positive selection by magnetic bead separation. The summary of cell percentage (of CD45⁺) obtained from 6 to 10 independent experiments per treatment arm is presented. Left: overall CD11b⁺ myeloid cells; middle: CD11b⁺F4/80⁺ cells (representing resident macrophages); right: CD11b⁺CD11c⁺ cells (representing the majority of dendritic cells, i.e., all monocyte‐derived dendritic cells, CD11b⁺ conventional migratory dendritic cells, and CD4⁺ conventional lymphoid resident dendritic cells).

FIGURE 4

CD11b⁺ cells contribute most significantly to ¹⁹F signal alteration following SHP2/MEK1/2 inhibition. (a) Image of a KPC‐PDAC‐TiME stained with a myeloid marker panel, including Arg1 (Opal520, green), Ly6g (Opal570, yellow), F4/80 (Opal620, orange), Ly6c (Opal690, red), CD11b (Opal780, purple) and DAPI (blue). Scale bar: 100 μm. (b) Quantification results given as cell numbers per area (one whole slide/section per treated tumor). Left: overall CD11b⁺ myeloid cells; middle left: CD11b⁺F4/80⁺ cells (representing resident macrophages); middle right: F4/80⁺Arg1⁺ cells (representing resident macrophage derived M2‐like TAMs); right: CD11b⁺Ly6c⁺ cells (representing mostly monocytes and immigrant monocyte‐derived macrophages). (c) Image of a KPC‐PDAC‐TiME staining with a dendritic cell (DC) marker panel, including CD8a (Opal520, green), CD11c (Opal570, yellow), CD103 (Opal620, orange), MHCII (Opal690, red), CD11b (Opal780, purple), and DAPI (blue). Scale bar: 100 μm. (d) Cell numbers per area (quantification of one whole slide/section per treated tumor). Left: overall CD11c⁺ dendritic cells; middle: CD11c⁺MHCII⁺CD103⁺ cells (representing the subset of CD103⁺ conventional migratory DCs); right: CD11c⁺MHCII⁺CD8a⁺ cells (representing the subset of CD8a⁺ conventional lymphoid resident DCs). Results were obtained from 4 to 6 independent experiments/animals per treatment group. Individual values and SEMs are depicted. *P < 0.05; one‐way ANOVA with Dunnett's correction for multiple comparisons.