Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound

Abstract

Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.

Keywords: RNA sequencing; blood-tumor barrier; focused ultrasound; immune cells; inflammation.

Figure 1

Characterization of FUS + MBs-induced BTB/BBB opening by contrast enhanced MRI. A. Representative images showing enhanced contrast Post-FUS. B. Quantification of pre- and post-FUS grayscale intensity for FUS+MBs treated tumors. Significance assessed by paired t test.

Figure 2

Bulk RNA sequencing reveals that FUS+MBs mediated BBB/BTB opening elicits increased proinflammatory gene expression. A,B: Volcano plots showing significantly upregulated and downregulated genes in FUS+MBs treated tumors compared to sham at (A) 6 and (B) 24 h post treatment. C,D,E: Log2 fold change of FUS+MBs treated vs. sham tumors at 6 hours and 24 hours post treatment. Data is displayed for selected mRNA transcripts related to (C) inflammatory cytokines, chemokines, and vascular cell adhesion molecules (D) pattern recognition receptors and signaling molecules and (E) MHC class I antigen presentation and processing. *adj p < 0.05. n = 3 for 24 h FUS+MBs group, n = 4 for all other groups.

Figure 3

Gene set enrichment analysis of RNA sequencing data indicates that FUS+MBs mediated BBB/BTB opening enriches proinflammatory gene sets. Normalized enrichment scores for selected pathways of FUS+MBs treated relative to sham treated tumors at 6 and 24 h post FUS+MBs. *adj p < 0.05. n = 3 for 24 h FUS+MBs group, n = 4 for all other groups.

Figure 4

Expression of the DC activation marker CD86 as assessed by flow cytometry 2 days post treatment. A,C,E: Percentage of dendritic cells expressing CD86 in the (A) tumor (C) meninges and (E) superficial cervical lymph nodes of FUS+MBs treated and sham animals 2 d post FUS+MBs or sham treatment. B,D,F: Geometric mean fluorescence of CD86 in CD86 positive DCs in the (B) tumor (D) meninges and (F) superficial cervical lymph nodes of FUS treated and sham animals 2 d post FUS+MBs or sham treatment. Significance assessed by unpaired t tests.

Figure 5

ZsGreen antigen uptake and OVA peptide presentation by dendritic cells as assessed by flow cytometry 2 Days following treatment. A,C: Percentage of ZsGreen positive dendritic cells in the (A) tumor and (C) meninges two days following treatment. B,D: Geometric mean fluorescence of ZsGreen in ZsGreen positive dendritic cells in the (B) tumor and (D) meninges two days following treatment. E,G,I: Percentage of MHC-SIINFEKL positive dendritic cells in the (E) tumor (G) meninges and (I) superficial cervical lymph nodes. F,H,J: Geometric mean fluorescence of MHC-SIINFEKL in MHC-SIINFEKL positive dendritic cells in the (F) tumor (H) meninges and (J) superficial cervical lymph nodes. Significance assessed by unpaired t tests.

Figure 6

Flow cytometry analysis of endothelial cell adhesion molecule expression on the vasculature 6 hours post-treatment. A,B,C,D: Percentage of endothelial cells expressing (A) E-selectin (B) P-selectin (C) VCAM-1 and (D) ICAM-1 from FUS treated and sham B16F1cOVA tumors. Significance assessed by unpaired t tests.

Figure 7

Assessment of adoptively transferred activated T cell populations in the tumor and meninges by flow cytometry. A,C: Number of transferred OT-1 T cells in the (A) tumor or (C) meninges of FUS+MBs treated or sham animals bearing intracranial B16F1cOVA tumors. B,D: Ratio of transferred OT-1 T cells in the (B) tumor or (D) meninges to the number in the spleen of FUS+MBs treated or sham animals bearing intracranial B16F1cOVA tumors. Significance assessed by unpaired t-tests.