Reactive astrocytes potentiate tumor aggressiveness in a murine glioma resection and recurrence model

Abstract

Background: Surgical resection is a universal component of glioma therapy. Little is known about the postoperative microenvironment due to limited preclinical models. Thus, we sought to develop a glioma resection and recurrence model in syngeneic immune-competent mice to understand how surgical resection influences tumor biology and the local microenvironment.

Methods: We genetically engineered cells from a murine glioma mouse model to express fluorescent and bioluminescent reporters. Established allografts were resected using image-guided microsurgery. Postoperative tumor recurrence was monitored by serial imaging, and the peritumoral microenvironment was characterized by histopathology and immunohistochemistry. Coculture techniques were used to explore how astrocyte injury influences tumor aggressiveness in vitro. Transcriptome and secretome alterations in injured astrocytes was examined by RNA-seq and Luminex.

Results: We found that image-guided resection achieved >90% reduction in tumor volume but failed to prevent both local and distant tumor recurrence. Immunostaining for glial fibrillary acidic protein and nestin showed that resection-induced injury led to temporal and spatial alterations in reactive astrocytes within the peritumoral microenvironment. In vitro, we found that astrocyte injury induced transcriptome and secretome alterations and promoted tumor proliferation, as well as migration.

Conclusions: This study demonstrates a unique syngeneic model of glioma resection and recurrence in immune-competent mice. Furthermore, this model provided insights into the pattern of postsurgical tumor recurrence and changes in the peritumoral microenvironment, as well as the impact of injured astrocytes on glioma growth and invasion. A better understanding of the postsurgical tumor microenvironment will allow development of targeted anticancer agents that improve surgery-mediated effects on tumor biology.

Keywords: glioblastoma; glioma; migration; reactive astrocytosis; recurrence.

Fig. 1.

TRP cells engineered to express mCherry and luciferase proliferate in vitro and form GBM allografts in vivo. Cultured TRP glioma cells transduced with lentiviral vectors encoding TRP-mcF express mCherry and luciferase in vitro as determined by (A and C) white light and (B and D) fluorescence imaging. TRP-mcF cells (doubling time 1.1 days) showed a small but statistically significant decrease in growth compared with uninfected TRP cells (doubling time 0.9 days, P = .01) (E). TRP-mcF cell number showed linear correlation with Fluc activity (R² = .998, P < .0001) (F). TRP-mcF allografts showed exponential growth with a doubling time of 3.3 days in vivo by BLI (G). Representative H&E staining (H and J–M) and mCherry fluorescence imaging (I) of brain sections show large intracortical tumors 14 days after injection of TRP-mcF cells. Tumors showed histopathological features of GBM, including microvascular proliferation (black arrowheads), an invasive brain-tumor interface (white arrow), and elevated mitotic activity (white arrowheads). Original magnifications: 15x (H and I), 40x (A and B), 100x (C, D, and J), 200x (K), 400x (L), and 600x (M). Scale bars, 100 µm (A–D) and 200 µm (J–M).

Fig. 2.

Fluorescence-guided microsurgical resection reduces volumes of orthotopic TRP-mcF allografts that redevelop to induce death. A scalp incision (A) was made 14 days after injection of TRP-mcF cells. A craniotomy was performed (B) to visualize the underlying mCherry+ tumors (C). Fluorescence-guided microsurgery significantly reduced tumor burden as determined by intraoperative fluorescence imaging (D). Postoperative BLI showed a 91% reduction in mean tumor burden (N = 6 mice per group, P < .0001) (E). Representative BLIs pre- and postresection are shown. Representative mCherry fluorescence (F) and H&E images (G) of brain sections taken 1 day postresection showed residual tumor (arrows) within the resection cavity (RC). Recurrent TRP-mcF allografts (H) grew faster than their pre-resection counterparts (data from Fig. 1G), with doubling times of 1.9 vs 3.3 days, respectively (P = .0003). Microsurgical resection extended survival, as resected mice survived significantly longer than nonresected mice (31 vs 21 days, log-rank P = .001) (I). Fluorescence (J) and H&E images (K) of brain sections taken 7 days postresection show recurrent tumor near the RC. Original magnifications: 15x (J and K) and 100x (F and G).

Fig. 3.

Local and distant tumor recurrences develop following microsurgical resection of TRP-mcF allografts. Representative H&E stained sections of mouse brains harvested 1 (A–D), 3 (E–L), 5 (M–P), and 7 (Q–X) days after resection of TRP-mcF allografts. Panels A–H show near-complete resection of tumor around the surgical cavity. However, extensive tumor invasion was evident in the deep ipsilateral cortex (I–L) of the mouse brain shown in E–H. Panels M–X show local recurrence of tumor within the resection cavity, but the mouse brain in Q–X also shows extensive perivascular invasion (U and V) and recurrent tumor near the ventricle (Q, arrowhead). Black arrowheads indicate the invasive brain-tumor interface (L), reactive astrocytes (P), mitoses (J, S, and T), and foamy macrophages (W) present in recurrent tumors. White (P) and black (X) arrowheads indicate hemosiderin-laden macrophages. Asterisk indicates necrosis (R, S, and T). Original magnifications: 40x (A, E, I, M, and Q), 100x (U), 200x (B, F, K, N, and R), 400x (C, G, H, J, O, S, W, and V), 600x (D, L, P, T, and X). Scale bars = 200 µm.

Fig. 4.

Microsurgical resection induces dynamic changes in reactive astrocytes surrounding TRP-mcF allografts. Reactive astrocytes were abundant in the peritumoral brain parenchyma in mCherry+ (red) TRP-mcF allografts established for 7 days (A), and their density increased further in tumors established for 14 days (B). Tumor cells, but not reactive astrocytes, expressed nestin (C). Over the course of 7 days following microsurgical tumor resection, a gradual decrease in the density of reactive astrocytes in the peritumoral microenvironment is highlighted by GFAP immunostaining (D–H). However, over this time course, reactive astrocytes gain expression of the stem cell marker nestin and their density peaks 3 days postresection (I–M). Double immunostaining (N–P, merge, Q) of brains 3 days postresection shows that reactive astrocytes express both GFAP (green, O) and nestin (red, P). Nuclei are stained with Hoechst (blue, I–L, N, and Q). All pairwise comparisons in panels H and M were significant except days 5 and 7 in (H) (P < .0001). Original magnifications: 100x. Scale bars = 100 µm.

Fig. 5.

Astrocyte injury promotes TRP migration and proliferation in vitro. Compared with cultured, uninjured astrocytes (A), scratch wounding induces significant increases in nestin (green) expression at 24 h (2.5-fold increase, P < .0001) (B). Astrocytes and TRP were seeded approximately 500 µm apart and groups were constructed with and without scratch injury of each cell population (C). Time-lapse images were captured every 20 min for 24 h and used to construct movies that revealed the migration of TPRs in real time. Injuring astrocytes by scratch induced significantly increased migration of TRP-mcF tumor cells at 24 h (P < .0001) (D and E). Astrocytes isolated from the in vivo injury model (ExAC uninjured) increased the migration of TRP cells (P = .0002) and injury in vitro (ExAC injured) did not alter migration further (P = .3) (E). Single cell imaging analysis showed that injury of cocultured astrocytes (F) increased the velocity (P < .0001) (G) of individual TRP-mcF tumor cells. Astrocyte injury induced increased migration in established GL261 (P < .0001), U251 (P < .0001), and U87 (P < .0001) GBM cell lines (H). Compared with uninjured astrocytes, scratch injury of immortalized astrocytes increased proliferation of cocultured TRP-mcF tumor cells 32% as measured by EdU incorporation at 24 h (P < .0001) (I). TRP-mcF cell proliferation was significantly increased when cultured in scratch injured-astrocyte conditioned media (SACM) compared with uninjured astrocyte conditioned media (ACM) as measured by BLI (doubling time: 1.07 vs 1.19 days, P = .02) (J). Scratch injury of astrocytes significantly increased proliferation of GL261 (P = .0006), U251 (P < .0001), and U87 (P = .034) GBM cell lines (K). Original magnification: 100x. Scale bars = 100 µm.

Fig. 6.

Astrocyte injury induces transcriptome and secretome changes. Differential expression analysis (A) and hierarchical clustering showed that scratch injury of astrocytes (S1–S4) induced significant transcriptome alterations (2093 genes, q < 0.001) compared with uninjured (C1–C4) astrocytes (B). Ontology analysis of upregulated genes revealed enrichment in genes associated with cytokine production and response (C). Compared with uninjured astrocytes, astrocyte injury increased secretion of Cxcl5 (P = .0074) (D) as measured by Luminex analysis.