Supplemental Figure 2. Pre- and post-LITT images of patient gliomas

Supplemental Figure 1. Surgical workflow for tumor biopsy and placement of minibolt

Intracranial tumors present unique challenges for immunotherapy, which can include both local and systemic immune suppression whose mechanistic underpinnings are incompletely understood. Here, we reveal that tumors harbored intracranially elicit systemic increases in catecholamines and chronic sympathetic hyperactivity that drives T cell dysfunction and limits immunotherapeutic success. Conversely, treatment with β-adrenergic receptor blockade increases NF-κB activity in immune cells, restores T cell polyfunctionality, modifies the tumor microenvironment, and licenses immune-based therapies in murine models of GBM to extend survival. Extended survival was also observed in GBM patients receiving β-blockers for any indication, as well as in patients with melanoma and lung cancer brain metastases who received concomitant immune checkpoint inhibition and β-adrenergic blockade compared to immune checkpoint inhibition alone. These data suggest roles for increased adrenergic activity in facilitating systemic immune dysfunction in the setting of intracranial tumors, specifically, and advance a role for β-adrenergic blockade in licensing immunotherapeutic responses within the intracranial compartment.### Competing Interest StatementThe authors have declared no competing interest.

PURPOSE:Laser interstitial thermal therapy (LITT) is a minimally invasive surgical intervention permitting thermal ablation of intracranial targets such as tumors, radiation necrosis, or epileptogenic brain, including lesions that are deep, difficult to access, or recurrent that would otherwise have few viable surgical options. Despite its advantages, LITT has several limitations, including a restricted effective treatment zone (approximately 3 cm) and a limited ability to distinguish tumor margins from healthy brain tissue. Few viable animal models of appropriate size exist for studying LITT's impact on these disorders or for optimizing the technology and obviating its current limitations. Pet dogs develop these same disorders at similar rates to humans. We hypothesized that LITT could be made feasible in dogs, creating a unique model for in vivo LITT research and development. EXPERIMENTAL DESIGN:Canine cadaveric specimens and live dogs, including canine patients with spontaneously occurring intracranial gliomas, were used in this study. Commercially available equipment was used for neuronavigation (Curve, Brainlab) and to perform LITT (NeuroBlate, Monteris Medical). RESULTS:Canine cadavers and two end-of-life laboratory dogs allowed adaptation of the neuronavigation and LITT systems to dogs, with successful targeting and ablation of intracranial targets. Four canine patients with intracranial gliomas were subsequently successfully treated with these same technologies. CONCLUSIONS:This work establishes a unique canine model for in vivo LITT research and development using commercially available systems, as well as creating a viable cutting-edge therapeutic intervention for pet dogs with intracranial lesions.

While the central nervous system (CNS) has long been known to regulate global physiologic processes, its role in regulating immune responses has only relatively recently been appreciated. Specifically, CNS input via the autonomic nervous system (ANS) is increasingly emerging as a crucial modulator of immune responses in numerous pathologies, though understanding of the role of these pathways in malignancy is limited. Herein, we provide an overview of CNS-immune signaling pathways, outline the evidence of ANS inputs to immune organs, provide a detailed description of the impact of ANS signaling on immune cell functions, and consider the implications of ANS-immune regulation for the antitumor immune response and CNS inflammation, with a specific focus on how these factors coalesce to impact the antitumor immune response in intracranial malignancies. This review concludes by highlighting the need to better understand cancer neuro-immunology, the tripartite interactions of malignancy and immune cells within the unique niche of the nervous system.

Whereas terminally exhausted T (Tex_term) cells retain anti-tumor cytotoxic functions, the frequencies of stem-like progenitor-exhausted T (Tex_prog) cells better reflect immunotherapeutic responsivity. Here, we examined the intratumoral cellular interactions that govern the transition to terminal T cell exhaustion. We defined a metric reflecting the intratumoral progenitor exhaustion-to-terminal exhaustion ratio (PETER), which decreased with tumor progression in solid cancers. Single-cell analyses of Tex_prog cells and Tex_term cells in glioblastoma (GBM), a setting of severe T cell exhaustion, revealed disproportionate loss of Tex_prog cells over time. Exhaustion concentrated within tumor-specific T cell subsets, with cognate antigen exposure requisite for acquisition of the Tex_term phenotype. Tumor-associated macrophages (TAMs)—not tumor cells—were the primary source of antigenic exposure governing the Tex_prog to Tex_term transition. TAM depletion increased frequencies of Tex_prog cells in multiple tumor models, increased PETER, and promoted responsiveness to αPD1 immunotherapy. Thus, targeting TAM-T cell interactions may further license checkpoint blockade responses.

Abstract Checkpoint inhibitors have been successful in various tumors. However, these treatments have failed in glioblastoma. Approaches harnessing the immune response are hindered by multiple factors, including T cell exhaustion. TOX is particularly important for the transcriptional and epigenetic reprogramming of exhausted T cells. While it is known that NFAT is upstream of TOX, our understanding of the upregulation of TOX remains incomplete. We hypothesized that tumor necrosis factor (TNF) could be involved in the upregulation of TOX as TNF can lead to the translocation of NFAT into the nucleus. We observed significant upregulation of the anti-inflammatory TNF receptor type II (TNFR2) in tumor-infiltrating T cells. This local upregulation mimics expression patterns of canonical exhaustion markers. TNFR2 expression is correlated with markers of exhaustion, including PD1, TIM3, and TOX. Furthermore, TNFR2 knock out (KO) CD8 T cells have significantly lower TOX expression, without the concomitant decrease of TIM3. Whereas previous studies have linked TIM3 and TOX expression, these data suggest that TIM3 and TOX are regulated independently. We utilized bulk RNA-sequencing to assess transcriptional regulation of WT and KO T cells and detected a significant reduction of T cell exhaustion and immune checkpoint pathways in TNFR2 KO T cells. Various exhaustion-related transcription factors and coinhibitory markers were significantly reduced. In contrast, a significant increase in AP1 transcription factors, commonly associated with T cell effector functions, was detected. Given this reduced exhaustion profile, we subsequently investigated the influence of TNFR2 on tumor burden. TNFR2 KO mice had significantly lower tumor burdens following subcutaneous tumor challenges. We subsequently treated mice with a TNFR2 antagonist. While the antagonist alone was not sufficient to improve tumor control, combination with anti-PD1 significantly reduced tumor volumes. These data provide evidence for a novel marker of exhaustion that could result in a unique therapeutic strategy.

INTRODUCTION: Immune checkpoint blockade (ICB) has gained acceptance as a life-extending therapy in a variety of solid tumor types. For patients with brain metastases, combination stereotactic radiosurgery (SRS) and ICB remains an area of clinical equipoise. Similarly, for recurrent or difficult-to-access intracranial lesions, safety and synergy of laser interstitial thermal therapy (LITT) and ICB has yet to be explored. METHODS: In accordance with an IRB-approved protocol, all patients undergoing LITT at a tertiary center from 2015-2022 were retrospectively reviewed. Patients who received ICB within 6 weeks of LITT were included in the LITT + ICB cohort. Demographic, clinical, and survival data were collected. RESULTS: Combination LITT and ICB occurred in 25 patients with clinically recurrent tumors. The median cohort age was 62 (40-78) and 12 (48%) were female. The median KPS was 80 (50-100). The most common primary pathology was non-small cell lung cancer (NSCLC) in 15 patients (60%), followed by melanoma in 3 (12%); two high-grade gliomas were additionally treated. The majority of the cohort (24 patients, 96%) recieved single agent ICB in combination with LITT, with pembrolizumab (12, 48%) and nivolumab (6, 24%) most common. Median duration between ICB dosing and LITT was 2.85 (0.85-5.84) weeks. Ten patients (40%) had evidence of immune-related adverse events attributable to ICB, with only 1 event > grade 3 according to Common Terminology Criteria; 17 (68%) had a pre-LITT steroid requirement. Intracranial adverse events related to LITT were rare, with median overall survival for the cohort of 8.6 months (1.4-26.4 months). CONCLUSIONS: Combination LITT and ICB appears safe and feasible. No prospective studies have compared cytoreduction with LITT to LITT + ICB; construction of a matched NSCLC cohort is ongoing. Exploration of the immune consequences of LITT + ICB is needed.

INTRODUCTION: Cytoreductive surgery is a cornerstone of management for intracranial tumors. However, such treatment may remove an important source of neoantigen. In the case of laser interstital thermal therapy (LITT), thermally-ablated tumor remans in situ, allowing for a potential anti-tumor immune response. METHODS: Brain tumor cell lines (CT-2A) were stereotactically implanted into C57BL/6 mice. A 1064 nm Nd:YAG laser (Neuroblate, Monteris Medical) was used to ablate normal brain and tumor tissue. RESULTS: To establish anatomical boundaries for our ablation, we characterized our CT-2A model on T2 MRI, selecting 10 days post-implantation for LITT based on lesion size. We simultaneously implanted a 400 μm laser fiber and thermocouple probe into the center and edge of the target volume, respectively, for simultaneous ablation and temperature monitoring. To characterize the laser's effects, we first performed a dose titration of laser power/time in normal mouse brains, finding that doses above 2 W for 30 s produced cavitary lesions with charring that resulted in death at 24 h post-LITT. We therefore selected 1 W for 60 s as a safe laser power dose in our tumor model. Ablations at this setting rapidly heated the lesion edge to a peak temperature of mean 43.79 ± 2.993 °C, sparing the surrounding brain. MRI imaging at 1, 3, and 7 days post-LITT revealed an expansile T2 hyperintense lesion, with hemmorhage and necrosis on H&E. LITT increased survival to 28 from 25.5 days (vs sham, p = 0.0172). CONCLUSIONS: Using a clinical laser system, we were able to model the essential clinicopathological effects of LITT in mice: a cytoreductive survival benefit; lesion expansion on MRI; and focal necrosis with hemorrhagic vasculature on H&E. This model allows for detailed immunological studies of LITT and use with existing brain metastasis cell lines.

Abstract Intracranial tumors present unique challenges for immunotherapy. These can include both local and systemic modes of immune suppression, the mechanistic underpinnings of which are incompletely understood. Our work reveals that intracranial tumors elicit systemic increases in circulating catecholamine levels, and that this chronic sympathetic hyperactivity results in T cell dysfunction that impedes immunotherapeutic efficacy. We show that treatment with b-adrenergic blockade can partially overcome the negative impacts of chronic sympathetic hyperactivity by increasing NF-kB activity in immune cells, restoring T cell polyfunctionality, favorably modifying the tumor microenvironment, and extending survival in murine models of glioblastoma treated with immune-based therapies. Furthermore, we demonstrate that extended survival is also observed in glioblastoma patients receiving b-adrenergic blockade, as well as in patients with melanoma and lung cancer brain metastases who received b-blockade alongside concomitant immune checkpoint inhibition. Importantly, while local sympathetic hyperactivity in the tumor microenvironment and b-blockade also impact the anti-tumor immune response in the context of extracranial disease, these effects are markedly more pronounced in intracranial disease. Taken together, these data reveal that sympathetic hyperactivity facilitates systemic immune dysfunction in the setting of intracranial tumors and highlight a novel role for the application of b-adrenergic blockade to license immunotherapy in intracranial malignancies.

SUMMARYWhile terminally exhausted T cells (Tex_term) retain important anti-tumor cytotoxic function, it is the relative preservation of renewable, stem-like progenitor exhaustion (Tex_prog) that better indicates immunotherapeutic responsivity. Although restraining the progression from Tex_prog to Tex_term thus takes on clinical significance, the cellular interactions in a tumor microenvironment (TME) governing such progression remain less established. Employing glioblastoma (GBM) and other solid tumors as models of severe exhaustion, we provide a detailed characterization of the progression from Tex_prog to Tex_term within the TME, where we observe a striking and disproportionate loss of Tex_prog over time, leading to a low progenitor exhaustion to terminal exhaustion ratio (PETER). We find exhaustion concentrated within tumor-specific T cell subsets, with cognate antigenic exposure requisite for acquisition of the Tex_term phenotype. However, we implicate tumor-associated macrophages (TAM), and not tumor cells, as the source of antigenic exposure governing the Tex_prog to Tex_term transition. Using cell – cell interaction analysis, we additionally highlight candidate receptor–ligand communications that may be specifically mediating the progression to Tex_term and resultant decline in PETER within the TME.GRAPHICAL ABSTRACT

There is an unmet need for new treatments for many central nervous system tumors. An expanding body of research supports the use of laser interstitial thermal therapy (LITT) in the treatment of gliomas, recurrent brain metastases, and radiation necrosis. In this review, we highlight emerging indications for LITT, including its use adjacent to eloquent structures, in the posterior fossa, and for meningioma and tumors of the vertebral column. We conclude by providing an overview of current research into post-LITT response assessment and adjunctive therapies. Evidence has continued to accumulate regarding the safety of LITT in locations as varied as the motor cortex, posterior fossa, and vertebral column, as well as for novel pathologies such as meningioma. Regardless of disease histology, most patients leave the hospital within 12–48 h of LITT and can rapidly return to systemic and radiation therapies. Emerging data has allowed for a characterization of post-LITT imaging findings, and receipt of LITT should not preclude subsequent clinical trial enrollment, especially as hyperthermia modulates blood-brain barrier permeability and may synergize with immunotherapies. As LITT is incorporated into neurosurgical oncology practice, novel use cases will continue to emerge. Given that laser ablation is associated with shortened length of stay and decreased debility relative to open resection, development of radiographic response assessment criteria for LITT-treated lesions is urgently needed so that patients may more rapidly receive definitive management or proceed to clinical trial enrollment. Prospective evaluation of LITT and adjunctive combination therapies is ongoing.

Abstract Immunotherapy is less effective against intracranial metastases compared to extracranial metastases and ineffective against primary brain tumors such as glioblastoma. Brain tumors present a unique challenge to in the context of immunotherapy, as these patients are immunosuppressed not just locally at the tumor, but also outside the CNS. Importantly, systemic immunosuppression observed in brain tumor patients limits the capacity of the immune system to respond to immunotherapy. Through this work we present a novel axis of immunosuppression in patients with intracranial tumors and demonstrate that overactive adrenergic signaling is a major barrier to immunotherapeutic success. Our data indicate that combining beta-adrenergic blockade with immunotherapy provides a survival benefit in the setting of brain tumors, where immunotherapy alone has proven ineffective. We demonstrate that this survival benefit is driven by a remodeling of the tumor microenvironment, increasing NF-kB activity and resulting in an increase in cDC1s and CD8+ T cells, as well as an increase in CD40-CD40L signaling, suggesting the immune system is poised to respond to immunotherapy. Further, we demonstrate that beta-adrenergic blockade can overcome systemic immunosuppression to restore the capacity of T cells to respond to an immune stimulus. Using the SEER-Medicaid database, we retrospectively examined outcomes in patients with melanoma and lung adenocarcinoma brain metastasis who received checkpoint blockade therapy alone vs those concurrently receiving beta-blocker therapy. We found that patients receiving combination therapy showed increased overall survival compared to those receiving checkpoint blockade alone. We then validated these findings in preclinical models of glioma, demonstrating that combining immunotherapy and propranolol, a widely prescribed FDA-approved beta-blocker, extended survival. Moving forward, we proffer that combination therapy with beta-blocker and immunotherapy, particularly checkpoint blockade, represents a promising translational treatment platform for patients with primary or metastatic intracranial malignancies. Citation Format: Selena Lorrey, Lucas Wachsmuth, Mackenzie Price, Corey Neff, Quinn Ostrom, Peter Fecci. Beta-adrenergic blockade licenses the use of immunotherapy in primary brain tumors and brain metastases [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-001.

Abstract BACKGROUND Laser interstitial thermal therapy (LITT) is a minimally invasive surgical treatment being employed frequently for radiographically progressive brain metastases (BM). Considerable interest exists in combining LITT-mediated in situ vaccination to license immune checkpoint blockade (ICB) and activate an anti-tumor immune response. However, LITT also disrupts the blood-brain barrier, causing transient peritumoral edema. Accordingly, information on safety and feasibility of this combination in BM is needed. METHODS All patients receiving LITT for radiographically progressive non-small cell lung carcinoma (NSCLC) BM at a single center from 2015 – 2023 were retrospectively reviewed. Combination therapy was defined as ICB within 6 weeks of LITT. Clinical data, post-LITT freedom from local progression (FFLP), and overall survival (OS) were collected. Adverse events (AEs) were evaluated according to Common Terminology Criteria. RESULTS Eighteen patients received LITT + ICB to a total of 19 lesions. Median time between therapies was 2.29 weeks (range 0.85 – 5.98). In comparison to NSCLC patients receiving LITT alone (n = 25), there was no decrement in % ablation (98 vs 95%, P = 0.1), length of stay (1 vs 1 days, P = 0.91), home discharge (100 vs 92%, P = 0.5), or 30-day readmissions (15.8 vs 16%, P = 0.99). Despite decreased preoperative steroid use (P = 0.0098), patients receiving LITT + ICB discontinued steroids at a median of 11 (4 – 147) days post-LITT vs. 24 (3 – 242) days for patients receiving LITT alone (P = 0.62). At study cutoff, 18/19 (94.7%) lesions in the LITT + ICB group and 22/25 (88.0%) in the LITT only group were locally controlled. There were 3 and 5 AEs ≥ Grade 3 in the LITT + ICB and LITT alone group, respectively. CONCLUSIONS Combination LITT and ICB does not compromise procedural outcomes and may favorably impact local control in NSCLC. Prospective studies are needed to assess biomarkers of immune response.

Abstract The long-accepted paradigm for both cellular and antitumor immunity relies upon tumor cell kill by CD8+ T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex class I (MHC I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, in contrast to the decades old model of T cell immunity, we instead report that CD8+ T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent instead on interactions between T cell NKG2D and tumor NKG2D ligands (NKG2DL), the latter of which are highly expressed on MHC-loss variants. Necessarily, tumor cell kill in these instances is antigen-independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumor cells. In this manner, adaptive priming can beget innate killing. These mechanisms are active in vivo in mice, as well as in vitro in human tumor systems, and are obviated by NKG2D knockout or blockade. These studies challenge the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape, and instead identify the NKG2D/NKG2DL axis as a therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC loss variants.

Abstract Brain tumors present a unique challenge to in the context of immunotherapy, as these patients are immunosuppressed not just at the tumor but also outside the CNS. The systemic immunosuppression observed in brain tumor patients limits the capacity of the immune system to respond to immunotherapy. Brain tumor patients, particularly those with metastatic disease, represent a patient population that is on the rise and in need of targeted treatment approaches. Through this work we present a novel axis of immunosuppression in brain tumor patients and demonstrate that overactive adrenergic signaling is a major barrier to immunotherapeutic success. Our data indicate that combining beta-adrenergic blockade with immunotherapy provides a survival benefit in the setting of brain tumors, where immunotherapy alone has proven ineffective. Using the SEER-Medicaid database, we retrospectively examined outcomes in patients with melanoma and lung adenocarcinoma brain metastasis who received checkpoint blockade therapy alone vs those concurrently on beta-blocker therapy. We found that patients receiving combination therapy showed increased overall survival compared to those receiving checkpoint blockade alone. We then validated these findings in preclinical models of both glioma and melanoma brain metastases, demonstrating that combining immunotherapy and propranolol, a widely-prescribed FDA-approved beta-blocker, extended survival. Moving forward, we suggest that combination therapy with propranolol and immunotherapy, particularly checkpoint blockade, represents a promising translational treatment platform for patients with primary or metastatic intracranial malignancies.

Background Laser interstitial thermal therapy (LITT) is a minimally invasive surgical treatment being employed frequently for radiographically progressive brain metastases. Considerable interest exists in combining LITT-mediated in situ vaccination to license immune checkpoint blockade (ICB). No studies have examined the clinical feasibility of this combination in brain metastases.Methods All patients receiving LITT for radiographically progressive non-small cell lung carcinoma (NSCLC) brain metastases at a single center from 2015 to 2023 were retrospectively reviewed. Combination therapy was defined as ICB within 6 weeks of LITT. Clinical data, post-LITT freedom from local progression, and overall survival (OS) were collected. Adverse events (AEs) were evaluated according to Common Terminology Criteria.Results Eighteen patients received LITT + ICB for a total of 19 lesions. The median time between therapies was 2.29 weeks (range 0.85-5.98). In comparison to NSCLC patients receiving LITT alone or with targeted therapy (LITT only) (n = 25), there was no decrement in procedural outcomes. Patients receiving LITT + ICB discontinued steroids at a median of 11 (4-147) days post-LITT vs. 24 (3-242) days for patients receiving LITT only (P = .62). At study cutoff, the local control rate was 18/19 (94.7%) lesions in the LITT + ICB group and 22/25 (88.0%) in the LITT only group. There were 3 and 5 AEs >= Grade 3 in the LITT + ICB and LITT-only groups, respectively.Conclusions Combination of LITT and ICB does not compromise procedural outcomes or time to steroid discontinuation in NSCLC. Prospective studies are needed to assess biomarkers of immune response.

Abstract Malignant gliomas are devastating intracranial tumors with dismal prognoses that impose unique therapeutic challenges. Treatment options include surgical resection, radiation, and chemotherapy, but efficacy is limited and carries associated morbidity. Promise persists for newer modalities, such as immune-based platforms, but the blood-brain barrier (BBB) restricts intracranial therapeutic access, limiting success. Laser interstitial thermal therapy (LITT) is a minimally invasive surgical intervention permitting thermal ablation of brain tumors and other intracranial lesions. During LITT, a laser probe is stereotactically introduced through a small skull burr hole into the lesion. Continuous MRI is used to conduct real-time temperature monitoring via software-based calculation of cumulative thermal dosage zones. LITT is not only capable of ablating neoplastic tissue, but can also open the BBB in peritumoral regions, and thus may synergize with other emerging therapies. We adapted a commercially available LITT system (Monteris Medical) for use in dogs with intracranial lesions. Canine cadavers were used to optimize LITT procedures before employing this platform in live dogs. Our approach consists of 1. obtaining volumetric, T1-weighted, MRI studies to plan trajectories to intracranial targets, 2. fixing canine patients to a surgical bed and registering them for surface matching to a volume rendered image, 3. using an integrated instrument holder (Varioguide, Brainlab) to guide drilling of 4.5 mm skull burr holes for placement of self-tapping titanium “mini-bolts” (Monteris Medical), and 4. introducing the laser catheter probe through the mini-bolts for lesion ablation in the MRI suite. This method allows for rigid stereotaxy, successful neuronavigation, and a minimally invasive approach. We have successfully performed LITT on four canine patients with spontaneously occurring intracranial gliomas and plan to treat additional canine patients with intracranial lesions. Future studies will explore combination therapeutic platforms, including immunotherapy and gold nanoparticles to improve the efficiency and specificity of tumor ablation.