Fc-enhanced anti-CTLA-4, anti-PD-1, doxorubicin, and ultrasound-mediated BBB opening: A novel combinatorial immunotherapy regimen for gliomas.

BACKGROUND: Glioblastoma is a highly aggressive brain cancer that is resistant to conventional immunotherapy strategies. Botensilimab, an Fc-enhanced anti-CTLA-4 antibody (FcE-aCTLA-4), has shown durable activity in "cold" and immunotherapy-refractory cancers. METHOD: We evaluated the efficacy and immune microenvironment phenotype of a mouse analogue of FcE-aCTLA-4 in treatment-refractory preclinical models of glioblastoma, both as a monotherapy and in combination with doxorubicin delivered via low-intensity pulsed ultrasound and microbubbles (LIPU/MB). Additionally, we studied 4 glioblastoma patients treated with doxorubicin, anti-PD-1 with concomitant LIPU/MB to investigate the novel effect of doxorubicin modulating FcγR expressions in tumor associated macrophages/microglia (TAMs). RESULTS: FcE-aCTLA-4 demonstrated high-affinity binding to FcγRIV, the mouse ortholog of human FcγRIIIA, which was highly expressed in TAMs in human glioblastoma, most robustly at diagnosis. Notably, FcE-aCTLA-4 mediated selective depletion of intra-tumoral regulatory T cells (Tregs) via TAM-mediated phagocytosis, while sparing peripheral Tregs. Doxorubicin, a chemotherapeutic drug with immunomodulatory functions, was found to upregulate FcγRIIIA on TAMs in glioblastoma patients who received doxorubicin and anti-PD-1 with concomitant LIPU/MB. In murine models of immunotherapy-resistant gliomas, a combinatorial regimen of FcE-aCTLA-4, anti-PD-1, and doxorubicin with LIPU/MB, achieved a 90% cure rate, that was associated robust infiltration of activated CD8+ T cells and establishment of immunological memory as evidenced by rejection upon tumor rechallenge. CONCLUSION: Our findings demonstrate that FcE-aCTLA-4 promotes robust immunomodulatory and anti-tumor effects in murine gliomas and is significantly enhanced when combined with anti-PD-1, doxorubicin, and LIPU/MB. We are currently investigating this combinatory strategy in a clinical trial (clinicaltrials.gov NCT05864534).

Ultrasound-mediated delivery of doxorubicin to the brain results in immune modulation and improved responses to PD-1 blockade in gliomas.

Given the marginal penetration of most drugs across the blood-brain barrier, the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication, respectively. Similarly, LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab, respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice, which rely on myeloid cells and lymphocytes for their efficacy. Overall, this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM.

Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression 1-6 . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic 7-9 . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach 10-12 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.

Minimizing pneumocephalus during deep brain stimulation surgery.

BACKGROUND: Deep brain stimulation (DBS) surgery is an effective treatment for movement disorders. Introduction of intracranial air following dura opening in DBS surgery can result in targeting inaccuracy and suboptimal outcomes. We develop and evaluate a simple method to minimize pneumocephalus during DBS surgery. METHODS: A retrospective analysis of prospectively collected data was performed on patients undergoing DBS surgery at our institution from 2014 to 2022. A total of 172 leads placed in 89 patients undergoing awake or asleep DBS surgery were analyzed. Pneumocephalus volume was compared between leads placed with PMT and leads placed with standard dural opening. (112 PMT vs. 60 OPEN). Immediate post-operative high-resolution CT scans were obtained for all leads placed, from which pneumocephalus volume was determined through a semi-automated protocol with ITK-SNAP software. Awake surgery was conducted with the head positioned at 15-30°, asleep surgery was conducted at 0°. RESULTS: PMT reduced pneumocephalus from 11.2 cm3±9.2 to 0.8 cm3±1.8 (P<0.0001) in the first hemisphere and from 7.6 cm3 ± 8.4 to 0.43 cm3 ± 0.9 (P<0.0001) in the second hemisphere. No differences in adverse events were noted between PMT and control cases. Lower rates of post-operative headache were observed in PMT group. CONCLUSION: We present and validate a simple yet efficacious technique to reduce pneumocephalus during DBS surgery.

Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity.

DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.

RNA fusion transcript panel identifies diverse repertoire of fusions in adult glioma patients with therapeutic implications.

Background: Recurrent gliomas are therapeutically challenging diseases with few treatment options available. One area of potential therapeutic vulnerability is the presence of targetable oncogenic fusion proteins. Methods: To better understand the clinical benefit of routinely testing for fusion proteins in adult glioma patients, we performed a retrospective review of 647 adult patients with glioma who underwent surgical resection at our center between August 2017 and May 2021 and whose tumors were analyzed with an in-house fusion transcript panel. Results: Fifty-two patients (8%) were found to harbor a potentially targetable fusion with 11 (21%) of these patients receiving treatment with a fusion-targeted inhibitor. The targetable genes found to be involved in a fusion included FGFR3, MET, EGFR, NTRK1, NTRK2, BRAF, ROS1, and PIK3CA. Conclusions: This analysis demonstrates that routine clinical testing for gene fusions identifies a diverse repertoire of potential therapeutic targets in adult patients with glioma and can offer rational therapeutic options for patients with recurrent disease.

Repeated blood-brain barrier opening with an implantable ultrasound device for delivery of albumin-bound paclitaxel in patients with recurrent glioblastoma: a phase 1 trial.

BACKGROUND: Low-intensity pulsed ultrasound with concomitant administration of intravenous microbubbles (LIPU-MB) can be used to open the blood-brain barrier. We aimed to assess the safety and pharmacokinetics of LIPU-MB to enhance the delivery of albumin-bound paclitaxel to the peritumoural brain of patients with recurrent glioblastoma. METHODS: We conducted a dose-escalation phase 1 clinical trial in adults (aged ≥18 years) with recurrent glioblastoma, a tumour diameter of 70 mm or smaller, and a Karnofsky performance status of at least 70. A nine-emitter ultrasound device was implanted into a skull window after tumour resection. LIPU-MB with intravenous albumin-bound paclitaxel infusion was done every 3 weeks for up to six cycles. Six dose levels of albumin-bound paclitaxel (40 mg/m2, 80 mg/m2, 135 mg/m2, 175 mg/m2, 215 mg/m2, and 260 mg/m2) were evaluated. The primary endpoint was dose-limiting toxicity occurring during the first cycle of sonication and albumin-bound paclitaxel chemotherapy. Safety was assessed in all treated patients. Analyses were done in the per-protocol population. Blood-brain barrier opening was investigated by MRI before and after sonication. We also did pharmacokinetic analyses of LIPU-MB in a subgroup of patients from the current study and a subgroup of patients who received carboplatin as part of a similar trial (NCT03744026). This study is registered with ClinicalTrials.gov, NCT04528680, and a phase 2 trial is currently open for accrual. FINDINGS: 17 patients (nine men and eight women) were enrolled between Oct 29, 2020, and Feb 21, 2022. As of data cutoff on Sept 6, 2022, median follow-up was 11·89 months (IQR 11·12-12·78). One patient was treated per dose level of albumin-bound paclitaxel for levels 1 to 5 (40-215 mg/m2), and 12 patients were treated at dose level 6 (260 mg/m2). A total of 68 cycles of LIPU-MB-based blood-brain barrier opening were done (median 3 cycles per patient [range 2-6]). At a dose of 260 mg/m2, encephalopathy (grade 3) occurred in one (8%) of 12 patients during the first cycle (considered a dose-limiting toxicity), and in one other patient during the second cycle (grade 2). In both cases, the toxicity resolved and treatment continued at a lower dose of albumin-bound paclitaxel, with a dose of 175 mg/m2 in the case of the grade 3 encephalopathy, and to 215 mg/m2 in the case of the grade 2 encephalopathy. Grade 2 peripheral neuropathy was observed in one patient during the third cycle of 260 mg/m2 albumin-bound paclitaxel. No progressive neurological deficits attributed to LIPU-MB were observed. LIPU-MB-based blood-brain barrier opening was most commonly associated with immediate yet transient grade 1-2 headache (12 [71%] of 17 patients). The most common grade 3-4 treatment-emergent adverse events were neutropenia (eight [47%]), leukopenia (five [29%]), and hypertension (five [29%]). No treatment-related deaths occurred during the study. Imaging analysis showed blood-brain barrier opening in the brain regions targeted by LIPU-MB, which diminished over the first 1 h after sonication. Pharmacokinetic analyses showed that LIPU-MB led to increases in the mean brain parenchymal concentrations of albumin-bound paclitaxel (from 0·037 µM [95% CI 0·022-0·063] in non-sonicated brain to 0·139 µM [0·083-0·232] in sonicated brain [3·7-times increase], p<0·0001) and carboplatin (from 0·991 µM [0·562-1·747] in non-sonicated brain to 5·878 µM [3·462-9·980] µM in sonicated brain [5·9-times increase], p=0·0001). INTERPRETATION: LIPU-MB using a skull-implantable ultrasound device transiently opens the blood-brain barrier allowing for safe, repeated penetration of cytotoxic drugs into the brain. This study has prompted a subsequent phase 2 study combining LIPU-MB with albumin-bound paclitaxel plus carboplatin (NCT04528680), which is ongoing. FUNDING: National Institutes of Health and National Cancer Institute, Moceri Family Foundation, and the Panattoni family.

Checkpoint kinase 1/2 inhibition potentiates anti-tumoral immune response and sensitizes gliomas to immune checkpoint blockade.

Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cell mediated killing are less understood. Kinases are potentially druggable targets that drive tumor progression and might influence immune response. Here, we perform an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cell recognition. The screen reveals checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are currently being evaluated in clinical trials, in combination with PD-1 or PD-L1 blockade, lead to survival benefit in multiple preclinical glioma models. Mechanistically, loss of Chek2 enhances antigen presentation, STING pathway activation and PD-L1 expression in mouse gliomas. Analysis of human GBMs demonstrates that Chek2 expression is inversely associated with antigen presentation and T-cell activation. Collectively, these results support Chek2 as a promising target for enhancement of response to immune checkpoint blockade therapy in GBM.

Management of pediatric appendicitis during the COVID-19 pandemic: A nationwide multicenter cohort study.

BACKGROUND: The COVID-19 pandemic has impacted timely access to care for children, including patients with appendicitis. This study aimed to evaluate the effect of the COVID-19 pandemic on management of appendicitis and patient outcomes. METHODS: A multicenter retrospective study was performed including 19 children's hospitals from April 2019-October 2020 of children (age≤18 years) diagnosed with appendicitis. Groups were defined by each hospital's city/state stay-at-home orders (SAHO), designating patients as Pre-COVID (Pre-SAHO) or COVID (Post-SAHO). Demographic, treatment, and outcome data were obtained, and univariate and multivariable analysis was performed. RESULTS: Of 6,014 patients, 2,413 (40.1%) presented during the COVID-19 pandemic. More patients were managed non-operatively during the COVID-19 pandemic compared to before the pandemic (147 (6.1%) vs 144 (4.0%), p < 0.001). Despite this change, there was no difference in the proportion of complicated appendicitis between groups (1,247 (34.6%) vs 849 (35.2%), p = 0.12). COVID era non-operative patients received fewer additional procedures, including interventional radiology (IR) drain placements, compared to pre-COVID non-operative patients (29 (19.7%) vs 69 (47.9%), p < 0.001). On adjusted analysis, factors associated with increased odds of receiving non-operative management included: increasing duration of symptoms (OR=1.01, 95% CI: 1.01-1.012), African American race (OR=2.4, 95% CI: 1.3-4.6), and testing positive for COVID-19 (OR=10.8, 95% CI: 5.4-21.6). CONCLUSION: Non-operative management of appendicitis increased during the COVID-19 pandemic. Additionally, fewer COVID era cases required IR procedures. These changes in the management of pediatric appendicitis during the COVID pandemic demonstrates the potential for future utilization of non-operative management.

Glioblastoma modeling with 3D organoids: progress and challenges.

Glioblastoma (GBM) is the most aggressive adult primary brain tumor with nearly universal treatment resistance and recurrence. The mainstay of therapy remains maximal safe surgical resection followed by concurrent radiation therapy and temozolomide chemotherapy. Despite intensive investigation, alternative treatment options, such as immunotherapy or targeted molecular therapy, have yielded limited success to achieve long-term remission. This difficulty is partly due to the lack of pre-clinical models that fully recapitulate the intratumoral and intertumoral heterogeneity of GBM and the complex tumor microenvironment. Recently, GBM 3D organoids originating from resected patient tumors, genetic manipulation of induced pluripotent stem cell (iPSC)-derived brain organoids and bio-printing or fusion with non-malignant tissues have emerged as novel culture systems to portray the biology of GBM. Here, we highlight several methodologies for generating GBM organoids and discuss insights gained using such organoid models compared to classic modeling approaches using cell lines and xenografts. We also outline limitations of current GBM 3D organoids, most notably the difficulty retaining the tumor microenvironment, and discuss current efforts for improvements. Finally, we propose potential applications of organoid models for a deeper mechanistic understanding of GBM and therapeutic development.

αvß1 integrin is enriched in extracellular vesicles of metastatic breast cancer cells: A mechanism mediated by galectin-3.

Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote metastasis. To identify EV-bound proteins related to metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that integrins, including αv and ß1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that integrin αv is associated with disease progression in breast cancer patients. Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that integrins αv and ß1 are enriched in the CD63+ subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the galectin family as potential shuttlers of integrin αvß1 into EVs. In particular, knockdown of galectin-3, but not galectin-1, causes a reduction in the levels of cell surface integrins ß1 and αv, and decreases the colocalization of these integrins with CD63. Importantly, knockdown of galectin-3 leads to a decrease of integrin αvß1 export into the EVs concomitant with a decrease in the metastatic potential of breast cancer cells. Moreover, inhibition of the integrin αvß1 complex leads to a reduction in the binding of EVs to fibronectin, suggesting that integrin αvß1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound integrin αvß1 with breast cancer metastasis and provide additional insights into the export of integrin αvß1 into EVs in the context of metastasis.

Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma.

PURPOSE: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. EXPERIMENTAL DESIGN: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. RESULTS: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. CONCLUSIONS: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

Milk-Derived Proteins and Peptides in Head and Neck Carcinoma Treatment.

Research investigating milk-derived proteins has brought to light the potential for their use as novel anticancer agents. This paper aims to systematically review studies examining the effectiveness of milk-derived proteins in the treatment of head and neck cancer. A systematic literature search of Medline, Evidence-Based Medicine, and Web of Science databases including papers published from all dates was completed. Inter-rater reliability was high during the title, abstract, and full-text screening phases. Inclusion criteria, exclusion criteria, and data extraction were based on the PICOS tool and research questions. Reporting followed the Preferred Reporting Items for Systematic Review and Meta-Analysis criteria. Eligible in vitro and in vivo studies (n = 8/658) evaluated lactoferrin, α-lactalbumin, and its complexes, such as HAMLET, BAMLET and lactalbumin-oleic acid complexes, as well as lactoperoxidase, whey, and casein. Their effectiveness in the treatment of head and neck cancer cells lines found that these compounds can inhibit tumour growth modulate cancer gene expression, and have cytotoxic effects on cancer cells. However, the exact mechanisms by which these effects are achieved are not well understood. Systematically designed, large, optimally controlled, collaborative studies, both in vitro and in vivo, will be required to gain a better understanding of their potential role in the treatment of head and neck cancer.

Extracellular vesicles as natural therapeutic agents and innate drug delivery systems for cancer treatment: Recent advances, current obstacles, and challenges for clinical translation.

As cancer poses a significant threat to the well-being of humans on a global scale, many researchers have embarked on the search for effective anticancer therapeutic agents. In recent years, many drugs have been shown to have extraordinary anticancer effects. However, in a lot of cases the treatment is accompanied by undesirable side effects due to some intrinsic properties linked to the therapeutic agents, such as poor targeting selectivity and short half-life in the circulation. In this regard, extracellular vesicles (EVs), a diverse family of natural cell-derived vesicles, steal the show as potential anticancer immunotherapy or delivery vectors of anticancer agents since they are an innate mechanism of intercellular communication. Here, we describe some of the most hotly-debated issues regarding the use of EVs as anticancer therapeutics. First, we review the biology of EVs providing the most up-to-date definition of EVs as well as highlighting their circulation kinetics and homing properties. Next, we share our views on popular methods reported for EV isolation, characterization, and functional analysis. Pioneering and innovative reports along with emerging challenges in the field of EV imaging and EV drug loading strategies are then discussed. Finally, we examine in detail the therapeutic application of EVs in cancer treatment, including their role in cancer immunotherapy and as natural delivery systems for anticancer agents including natural compounds such as paclitaxel and doxorubicin. We consider standardised protocols and proper analytical approaches to be crucial in improving the reproducibility and rigor in EV research and ensuring the successful translation of EVs as anticancer therapeutics.

Low neoantigen expression and poor T-cell priming underlie early immune escape in colorectal cancer.

Immune evasion is a hallmark of cancer, and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (e.g., those with microsatellite instability (MSI)). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor potentially immunogenic neoantigens remains unclear. Here, we show that tumors from all patients with microsatellite stable (MSS) colorectal cancer (CRC) express clonal predicted neoantigens despite low TMB. Unexpectedly, these neoantigens are broadly expressed at lower levels compared to those in MSI CRC. Using a versatile platform for modulating neoantigen expression in CRC organoids and transplantation into the distal colon of mice, we show that low expression precludes productive cross priming and drives immediate T cell dysfunction. Strikingly, experimental or therapeutic rescue of priming rendered T cells capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression level in immune evasion and therapy response.