Intravital 2-photon imaging reveals distinct morphology and infiltrative properties of glioblastoma-associated macrophages

Two-Photon Imaging Permits Direct, Longitudinal Observation of TAMs In Vivo.

Advantages of 2-photon microscopy over traditional confocal microscopy include reduced autofluorescence and photobleaching effects, increased imaging depth, and minimal photodamage to surrounding brain tissue (19). Consequently, we used 2-photon microscopy for the in vivo analysis of individual TAM populations. To minimize breathing artifacts during imaging, a custom acrylic adapter that attaches to the cranium after skull window placement was manufactured in-house. This adapter attaches to a stainless-steel stage that holds the mouse in place throughout image acquisition (Fig. 1A). Superficial tumors are easily visualized through the skull window and can be distinguished from adjacent normal brain tissue by eye or at low magnification with a stereoscope (Fig. 1B and SI Appendix, Fig. S1). To avoid visual artifacts induced from skull window placement, such as glial scarring and leukocyte accumulation, all image stacks were analyzed at a depth of 50 μm or deeper from the pial surface, typically between 100 and 500 μm (Fig. 1 C and D). High-resolution image stacks with approximate dimensions of 400 μm × 400 μm × 400 μm were typically acquired and analyzed. Although not performed in this study, this methodology also allows for the visualization of other cell types and cellular components tagged with fluorescent molecules, such as tumor cells, lymphocytes, microtubules, and others (18, 20, 21).

Visualization of TAM Populations in Chimeric Mice.

Using a Cx3cr1^GFP/WT;Ccr2^RFP/WT mouse model, we previously demonstrated that infiltrating peripheral monocytes show decreased CCR2-RFP expression as they mature in the GBM microenvironment. Since we are interested in quantifying both newly infiltrating monocytes and differentiated macrophages, this model is not optimal for our studies, as we would inevitably miss a large population of cells as they mature. To remedy this, we used reciprocal bone marrow chimera mouse models with 1 allele of the Cx3cr1 gene replaced with GFP (Fig. 2A). These models allow us to discern differences between infiltrating BMDMs and brain-resident microglia. CX3CR1 is the sole receptor for the chemokine CX3CL1 (fractalkine), which is widely expressed throughout the central nervous system. Replacement of 1 allele with GFP (Cx3cr1^GFP/WT) has been demonstrated to have no effect on the migration capacity or infiltration of TAMs (22, 23).

To generate mice with GFP-expressing BMDMs and WT microglia (i.e., no GFP expression in microglia), bone marrow from Cx3cr1^GFP/WT mice was mixed in a 1:1 mixture with bone marrow from Cx3cr1^WT/WT mice. This mixture was then injected into whole-body–irradiated mice to reconstitute the bone marrow with 50% GFP-expressing BMDMs and 50% WT BMDMs (SI Appendix, Fig. S2). This scheme allows for single-cell resolution of infiltrating BMDMs, whereas a nondiluted GFP signal poses difficulties for resolving individual cells due to the high density of these cells in the tumor. Non–tumor-bearing mice had few to no GFP-expressing BMDMs in the brain parenchyma, as these cells do not readily cross the BBB (Fig. 2B and SI Appendix, Fig. S3 A–D); however, imaging of tumor-bearing mice revealed a significant infiltration of GFP-expressing BMDMs (Fig. 2 B–D). These cells were seen to widely populate tumor bulk, perinecrotic areas, and infiltrative tumor margins, while their presence was absent in adjacent nontumor tissue (SI Appendix, Fig. S4B).

To generate mice with WT BMDMs and GFP-expressing microglia, donor bone marrow isolated from Cx3cr1^WT/WT mice was injected into whole-body–irradiated Cx3cr1^GFP/WT recipients. Microglia populate the brain during embryogenesis and are found consistently dispersed throughout the tissue (24). In tumor-bearing brains, microglia are sparse in tumor bulk but often accumulate in clusters at the periphery of tumor margins (Fig. 2 B and E and SI Appendix, Figs. S3B and S4A). As previously demonstrated by our laboratory (5), we did not observe an increase in the microglial population in tumor-bearing brains but did find a significant increase in the BMDM population. We also observed a slight increase in GFP intensity in tumor-associated microglia compared with nontumor microglia (SI Appendix, Fig. S3F). In addition, we found a decrease in microglial branching with increases in surface area and volume in tumor-bearing mice, depicting the accepted notion of microglia adopting an amoeboid morphology in pathological conditions as they phagocytose dead or damaged cells (SI Appendix, Fig. S5) (12, 13, 25, 26).

TAMs Dynamically Alter Their Phenotype in the Tumor Microenvironment.

After image acquisition, XYZ stacks were imported into Imaris for creation of 3D representations (Fig. 3A). Images acquired via 2-photon microscopy were highly amenable for the detection and quantification of morphology, whereas traditional confocal microscopy was unable to resolve the fine microglial processes due to GFP signal loss and reduced Z stack depth (SI Appendix, Fig. S4 C and D). After image processing and quality control measures, morphology analysis was performed on both BMDMs and microglia (SI Appendix, Fig. S6). Microglia were found to be larger in mean surface area (5,516 ± 267.3 μm²) and volume (4,729 ± 258.5 μm³) compared with BMDMs (970.5 ± 24.32 μm² and 2,154 ± 94.24 μm³ respectively) (Fig. 3 B and C). Microglia also had significantly more primary branches per cell (6 vs. 1), causing them to assume a less spherical morphology with numerous terminal points (28 vs. 1) (Fig. 3 D and E).

Time-lapse images were analyzed to determine migratory differences between the two cell types. BMDMs were found to be migratory, consisting of two phenotypically distinct populations (Fig. 4A). There were low–GFP-expressing BMDMs that were highly mobile (0.07826 μm/s), reflecting the phenotype of newly infiltrating inflammatory monocytes (Fig. 4B) (5). These cells had an average directional track “straightness” of 0.45 (0, nonstraight; 1, perfectly straight), and were visually observed to migrate through the tissue in a nonspecific fashion. High-GFP-expressing BMDMs were observed to be stationary (0.003478 μm/s), reflecting the phenotype of differentiated macrophages (Fig. 4 B and C and Movies S1 and S2).

Microglia were found to be stationary; however, their processes were continuously extending and retracting in both tumor and nontumor brains (Fig. 4D). These results reinforce previous findings that even in the absence of a stimulus, these cells sense their microenvironment through the continuous extension and retraction of ramified projections (27, 28). Although no instances of microglial cell migration were observed, phagocytic behavior was seen in 1 imaging volume (Movies S3 and S4). The complex branching phenotype coupled with the sensing behavior is likely how these cells detect pathogenic insults in the local microenvironment (27).

Anti-VEGFA Treatment Reduces Monocyte Infiltration.

Given their localization and protumor behavior, TAMs have been suggested to promote resistance to antiangiogenic agents, such as bevacizumab, but the underlying mechanisms remain unknown (29–31). Evidence suggests that resistance may be due to changes in the tumor microenvironment and the presence of myeloid-derived TAMs (32, 33). Mechanisms of TAM infiltration may include passive migration due to a leaky BBB or active migration via chemokine gradients; however, which of these mechanisms is most responsible remains unclear. To address this, we treated mice with a neutralizing anti-VEGFA antibody (B20-4.1.1) and performed 2-photon imaging (Fig. 5A). We previously found that B20-4.1.1 reduced tumor vessel area and vascular leakiness in a PDGF-B–driven mouse model (34); therefore, it can be inferred that any reduction observed in the infiltration of TAMs is due in part to normalized vasculature (20, 35, 36). We found increased postinjection survival in tumor-bearing mice treated with B20-4.1.1 compared with vehicle-treated mice (42.5 d vs 30 d) (Fig. 5B). In addition, the number of infiltrating monocytes/macrophages was significantly lower in the B20-4.1.1–treated mice, as observed on 2-photon imaging (8,619 cells/mm³ vs. 21,317 cells/mm³) (Fig. 5 C and D). A morphological switch also occurred in the B20-4.1.1–treated mice. Compared with vehicle-treated mice, the cells assumed a more highly branched phenotype (2.46 vs. 0.99 branches per cell) and significantly increased total cell volume (2,661.96 ± 72.82 vs. 2,154 ± 94.24 μm³) and surface area (1,503 ± 41.78 vs. 970.5 ± 24.41 μm²), causing their sphericity to decrease (0.668 ± 0.005 vs. 0.734 ± 0.004) (Fig. 5 E–J). Interestingly, we did not observe any significant difference in cell motility or cell directionality with B20-4.1.1 treatment (Fig. 5 K and L). This treatment also appeared to normalize the tumor vasculature; however, due to the leaky nature of our vehicle-treated tumors, the TRITC signal was either too weak or too diffuse to enable a reliable quantitative vascular comparison. These changes suggest that B20-4.1.1 treatment partially prevents the infiltration of circulating BMDMs and induces the differentiation of existing monocytes into tissue macrophages.

Mice.

All experimental procedures utilizing live mice were approved by Emory University’s Institutional Animal Care and Use Committee (Protocol 2003253). Details are provided in SI Appendix.

Bone Marrow Chimera.

Bone marrow transplants were performed as reported previously (5) and described in detail in SI Appendix.

Orthotopic Cell Injections.

We used the RCAS/tv-a system to generate de novo primary murine GBM as reported previously (5, 53) and described in detail in SI Appendix. Once tumors were formed in donor transgenic mice, tumor cells were freshly dissociated, and 2.5 × 10⁴ cells were superficially injected into the right frontal striatum of recipient mice at 8 to 10 wk after bone marrow transplantation using the following coordinates: bregma AP (anterior/posterior), 0.2 mm; ML (medial/lateral), 1.0 mm, over which a burr hole was made with a sterile 0.7-mm-diameter drill bit (19007-7; Fine Science Tools). A Hamilton syringe affixed to a stereotactic apparatus was then lowered to a depth of 1.0 mm, and 1 µL of cell suspension was injected through a 30-gauge needle.

Skull Window Placement and 2-Photon Imaging.

At approximately 3 wk after tumor transplantation, mice began to show neurologic signs of brain tumor. A 5-mm-diameter sterile circular coverslip (Electron Microscopy Sciences) was carefully placed to facilitate visual access to the tumor bulk and tumor margin. At the time of imaging, TRITC-dextran (2.5 mg/mL; Sigma-Aldrich) was injected i.v. to outline the blood vessels. Mice were fixed to a custom-built stage to minimize breathing artifact during image acquisition. Images were acquired with a 25× water immersion objective (Leica; NA 0.95) on a Leica SP8 confocal microscope equipped with a tunable pulsed chameleon infrared multiphoton laser (Coherent) and 2 high-sensitivity hybrid-PMT (HyD) detectors (Leica). High-resolution XYZ stack images (1,024 × 1,024 pixels per Z step) were taken with a step size of 2.5 μm, and images of XYZT stacks were taken every 2.5 min for approximately 3 h on average. At the end of each imaging session, the mice were euthanized and their brains processed for confocal imaging as described previously (5). More details are provided in SI Appendix.

B20-4.1.1 Treatment.

At 15 d after tumor cell injection, mice were injected i.p. with either anti-VEGFA (B20-4.1.1, 5 mg/kg; Genentech) or vehicle control (bacteriostatic 0.9% sodium chloride) every 4 d until terminal neurologic symptoms appeared.

Image and Data Analysis.

High-resolution image stacks were imported into Imaris version 9.0.2 (Bitplane). Total cell numbers within each XYZ stack were quantified using the spots function. Morphology analysis of BMDMs and microglia were done using the surfaces and filaments functions, respectively. Additional details are provided in SI Appendix.

Statistical Analysis.

Graphs were created using GraphPad Prism 6. Results are expressed as mean ± SD. Comparisons between 2 groups were performed using the unpaired 2-tailed t test or nonparametric Mann–Whitney U test, as indicated in the figure legends. One-way ANOVA was used to compare the means between 3 or more independent groups. Kaplan–Meier survival analysis was performed using the log-rank test. A P value < 0.05 was considered to indicate statistical significance.