Inhalant Cannabidiol Inhibits Glioblastoma Progression Through Regulation of Tumor Microenvironment

Abstract

Introduction: Glioblastoma (GBM) is the most common invasive brain tumor composed of diverse cell types with poor prognosis. The highly complex tumor microenvironment (TME) and its interaction with tumor cells play important roles in the development, progression, and durability of GBM. Angiogenic and immune factors are two major components of TME of GBM; their interplay is a major determinant of tumor vascularization, immune profile, as well as immune unresponsiveness of GBM. Given the ineffectiveness of current standard therapies (surgery, radiotherapy, and concomitant chemotherapy) in managing patients with GBM, it is necessary to develop new ways of treating these lethal brain tumors. Targeting TME, altering tumor ecosystem may be a viable therapeutic strategy with beneficial effects for patients in their fight against GBM. Materials and Methods: Given the potential therapeutic effects of cannabidiol (CBD) in a wide spectrum of diseases, including malignancies, we tested, for the first time, whether inhalant CBD can inhibit GBM tumor growth using a well-established orthotopic murine model. Optical imaging, histology, immunohistochemistry, and flow cytometry were employed to describe the outcomes such as tumor progression, cancer cell signaling pathways, and the TME. Results: Our findings showed that inhalation of CBD was able to not only limit the tumor growth but also to alter the dynamics of TME by repressing P-selectin, apelin, and interleukin (IL)-8, as well as blocking a key immune checkpoint-indoleamine 2,3-dioxygenase (IDO). In addition, CBD enhanced the cluster of differentiation (CD) 103 expression, indicating improved antigen presentation, promoted CD8 immune responses, and reduced innate Lymphoid Cells within the tumor. Conclusion: Overall, our novel findings support the possible therapeutic role of inhaled CBD as an effective, relatively safe, and easy to administer treatment adjunct for GBM with significant impacts on the cellular and molecular signaling of TME, warranting further research.

Keywords: CBD; IDO; P-selectin; glioblastoma; immune checkpoint; tumor microenvironment.

FIG. 1.

Inhalant CBD inhibits tumor growth and improved survival in GBM. (a) Optical imaging (bioluminescence imaging after injecting luciferin) showed the establishment and growth of GBM tumor on day 8 post-implantation. (b) Tumor growth was inhibited after eight consecutive daily treatments of inhalant CBD compared to the placebo-treated group as optical bioluminescence images were quantified (*p>0.05) (c). (d) Mice were treated with CBD/placebo using inhaler ApelinDx. (e) H&E staining suggested significant decrease in tumor size after CBD treatment compared to placebo. The difference in tumor size between CBD- and placebo-treated groups (p<0.05) was quantified using ImageJ Java-based image processing program (*p>0.05) (f). (g) Survival was evaluated (n=6/group), showed that, while all animals in CBD-treated group were alive at 16 days after orthotopic implantation of the tumor, however, 50% of animals from placebo group (3/6) died by day 13 post-implantation, suggesting beneficial effects of CBD treatment. GBM, glioblastoma; H&E, hematoxylin and eosin. Color images are available online.

FIG. 2.

Immunohistochemical staining of paraffin-embedded GBM tumor tissues showed inhalant CBD decreased expression of angiogenic factors: (a) P-selectin, (b) apelin, and (c) IL-8 significantly compared to the placebo treated group as quantified (*p>0.05; **p>0.01) (d). All images have been obtained using bright field Zeiss (AXIO Imager M2) light microscope, magnifications of 200×and 630×. IL, interleukin; TME, tumor microenvironment. Color images are available online.

FIG. 3.

Inhalant CBD modulates immune checkpoints within TME in GBM, altering the intratumoral immune profile. Immunohistochemical staining of paraffin-embedded GBM tumor tissues showed inhalant CBD blocked the immune checkpoint, IDO (a), while enhancing CD8 (b) and CD103 (c) expression compared to the placebo-treated group as quantified (*p > 0.05; **p > 0.01) (d). All images have been obtained using bright field Zeiss (AXIO Imager M2) light microscope, magnifications of 200× and 630×. CD, cluster of differentiation; IDO, indoleamine 2,3-dioxygenase. Color images are available online.

FIG. 4.

Inhalant CBD decreased ILC frequencies in GBM, regulating local proliferation and activation of ILCs within TME. Flow cytometry analysis showed that inhalant CBD was able to reduce frequencies of ILCs within TME of GBM significantly (p<0.001), potentially improving antitumor immunity. ILCs were characterized by live gating of total tumor cells (a) based on FSC/SSC, and further gating (b) as CD45⁺ lineage negative (CD3e, CD11b, CD24, CD5, CD11c, CD19, NK1.1, Gr-1, TER119, and gd TCR), followed by additional gating (*p > 0.05) (c) as Lin-CD45⁺Thy1⁺. A pie chart (d) displays the representation of the ratio of ILC counts of CBD-treated (green 33%) vs placebo group (red 67%). FSC, forward scatter; ILC, innate lymphoid cell; NK, natural killer; SSC, side scatter. Color images are available online.